Sea cucumber peptide for repairing gastrointestinal mucosal injury and preparation method thereof

Abstract

The invention belongs to the field of biomedical functional peptides and healthy foods, and particularly relates to a sea cucumber peptide for repairing gastrointestinal mucosal injury and a preparation method thereof. The sea cucumber peptide is prepared from sea cucumber oligopeptide, 2-hydroxy-4-methoxyphenylacetamide, beta-cyclodextrin, glycerol, potassium sorbate and deionized water, wherein the sea cucumber oligopeptide is modified by a glycyrrhizic acid-amino acid deep eutectic system. In the modification process, a deep eutectic system is formed through monosodium glycyrrhizinate and L-glutamic acid, and the sea cucumber oligopeptide is subjected to hydrogen bond recombination and amidation reaction, so that the structural stability and bioavailability of the sea cucumber oligopeptide in an acid environment are improved from the molecular level; the 2-hydroxy-4-methoxyphenylacetamide is used as a phenylacetamide organic small molecule with a simple structure, and can be used for cooperatively regulating the energy metabolism of cells and promoting the regeneration of epithelial cells of mucous membranes. The preparation method is mild and controllable, and the product is safe and stable, and can be widely applied to the fields of gastrointestinal repair preparations and functional foods.

Description

Technical Field

[0001] This invention relates to the field of biomedical functional peptides and health foods, specifically to a sea cucumber peptide with the function of repairing gastrointestinal mucosal damage and its preparation method. Background Technology

[0002] The gastrointestinal mucosa is one of the most important physiological barriers in the human body, responsible for absorbing nutrients and preventing the invasion of harmful substances. When affected by factors such as alcohol, drugs, Helicobacter pylori infection, or stress, the mucosal epithelial cells are easily damaged, leading to problems such as inflammation, erosion, and leakage. Existing mucosal repair products mostly use polysaccharides, glutamine, lactoferrin, or collagen peptides as their main active ingredients. Although they can promote epithelial repair to some extent, they generally suffer from drawbacks such as poor stability in the gastric acid environment, low absorption rate, and long repair cycle.

[0003] Sea cucumber oligopeptides, as a type of bioactive peptide from marine organisms, are rich in glycine, proline, hydroxyproline, and glutamic acid residues, exhibiting good anti-inflammatory and cell repair effects. However, natural sea cucumber peptides are prone to structural breakage or inactivation under gastric acid conditions, resulting in low bioavailability after oral administration. On the other hand, conventional small-molecule auxiliary components are mostly concentrated in alkaloids or amino acid derivatives, lacking regulatory mechanisms targeting gastrointestinal cell energy metabolism and barrier protein expression.

[0004] Therefore, there is an urgent need to develop a novel functional peptide system with high stability, strong absorption, and the ability to significantly promote the repair of gastrointestinal mucosa. Through structural modification and small molecule synergistic strategies, the acid resistance and cell repair efficiency of peptide molecules can be improved, thereby achieving rapid, safe, and long-lasting mucosal repair effects. Summary of the Invention

[0005] To overcome the problems of poor stability, low absorption efficiency, and long repair cycle of sea cucumber oligopeptides in the aforementioned background technologies, the present invention aims to provide a sea cucumber peptide for repairing gastrointestinal mucosal damage and its preparation method. A glycyrrhizic acid-amino acid deep eutectic system modification technique is used to stabilize the structure of the sea cucumber oligopeptide, and 2-hydroxy-4-methoxyphenylacetamide, an organic small molecule with antioxidant and metabolic regulation functions, is introduced to achieve a synergistic repair effect, thereby significantly improving the gastric acid tolerance and bioavailability of the sea cucumber peptide and promoting the repair of gastrointestinal epithelial cells and mucosal regeneration. The sea cucumber peptide prepared by this invention exhibits high stability, high absorption rate, and significant gastrointestinal mucosal repair effect.

[0006] The objective of this invention can be achieved through the following technical solutions: A sea cucumber peptide for repairing gastrointestinal mucosal damage, comprising the following raw materials in parts by weight: 50-80 parts of sea cucumber oligopeptides modified by a glycyrrhizic acid-amino acid deep eutectic system; 5-15 parts of 2-hydroxy-4-methoxyphenylacetamide; 2-5 parts of β-cyclodextrin; 3-8 parts of glycerol; 0.1-0.3 parts of potassium sorbate; and 150-300 parts of deionized water; wherein the sea cucumber oligopeptides modified by the glycyrrhizic acid-amino acid deep eutectic system refer to the modified product that improves its molecular stability and gastric acid tolerance by hydrogen bonding recombination and amidation reaction of sea cucumber oligopeptides using a deep eutectic system formed by monosodium glycyrrhizate and L-glutamic acid; and 2-hydroxy-4-methoxyphenylacetamide refers to a simple phenylacetamide-type organic small molecule with antioxidant and cell metabolism regulation functions, which can promote the repair of gastrointestinal epithelial cells and mucosal regeneration.

[0007] Optionally, the sea cucumber oligopeptides modified by the glycyrrhizic acid-amino acid deep eutectic system contain the following raw materials in parts by weight: 60-100 parts sea cucumber oligopeptides; 10-20 parts monosodium glycyrrhizate; 15-30 parts L-glutamic acid; and 200-300 parts deionized water.

[0008] Optionally, the preparation method of sea cucumber oligopeptides modified by the glycyrrhizic acid-amino acid deep eutectic system includes the following steps: (1) Add sea cucumber oligopeptides to deionized water and stir to form a homogeneous solution; (2) Add monosodium glycyrrhizate and L-glutamic acid to a homogeneous solution to carry out a deep eutectic modification reaction; (3) The pH of the obtained system was adjusted and dried to obtain sea cucumber oligopeptide powder modified by glycyrrhizic acid-amino acid deep eutectic system.

[0009] Optionally, the stirring conditions in step (1) are heating to 40-60°C and stirring continuously until the solution is homogeneous.

[0010] Optionally, the reaction conditions in step (2) are to stir the reaction at a constant temperature for 2 to 3 hours to form a transparent eutectic system.

[0011] Optionally, the reaction conditions in step (3) are to adjust the pH of the system to 6.0-7.0 and then freeze-dry to obtain a solid product.

[0012] Optionally, a method for preparing sea cucumber peptides for repairing gastrointestinal mucosal damage includes the following steps: S1, sea cucumber oligopeptides modified by glycyrrhizic acid-amino acid deep eutectic system are added to deionized water and stirred to form a homogeneous solution; S2, add 2-hydroxy-4-methoxyphenylacetamide, β-cyclodextrin, glycerol and potassium sorbate sequentially to the homogeneous solution, and stir to mix evenly; S3 involves filtering the mixed system to remove impurities, concentrating and drying it to obtain a sea cucumber peptide product that repairs damage to the gastrointestinal mucosa.

[0013] Optionally, the reaction conditions for step S1 are heating to 40–50°C and continuously stirring until the solution is clear and homogeneous.

[0014] Optionally, the reaction conditions for step S2 are stirring at a constant temperature of 45–55°C for 1–2 hours until the system is fully mixed.

[0015] Optionally, the processing conditions for step S3 are as follows: after removing impurities by 0.22 μm filtration, the mixture is concentrated under reduced pressure at 50–60 °C and dried under vacuum at 40 °C to constant weight.

[0016] The beneficial effects of this invention are: This invention modifies sea cucumber oligopeptides using a glycyrrhizic acid-amino acid deep eutectic system, forming a stable hydrogen bond network and partially amidated structures between peptide chains, thereby improving their conformational stability and hydrolysis resistance in the gastric acid environment at the molecular level. Simultaneously, it introduces 2-hydroxy-4-methoxyphenylacetamide, which has not been used in the field of gastrointestinal repair, to form a non-covalent synergistic effect with the modified peptide molecules. This significantly upregulates the expression level of tight junction proteins in mucosal epithelial cells, promoting rapid repair and regeneration of damaged mucosa. Attached Figure Description

[0017] The invention will now be further described with reference to the accompanying drawings.

[0018] Figure 1 A comparison of the infrared spectra of sea cucumber oligopeptides and sea cucumber oligopeptides modified by the glycyrrhizic acid-amino acid deep eutectic system; Figure 2 A comparison chart of gastric acid stability and antioxidant capacity of samples with different formulation ratios; Figure 3 This is a comparison of the epithelial cell repair rate and the fold increase in Occludin / ZO-1 expression in samples with different ratios. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments. However, the present invention is not limited to the following embodiments. Equivalent adjustments made without departing from the spirit and essence of the present invention should also be considered to fall within the protection scope of the present invention.

[0020] Example 1 The purpose of this embodiment is to verify whether the sea cucumber peptide system prepared by glycyrrhizic acid-amino acid deep eutectic system modification and small molecule compounding technology still has stability and basic mucosal repair function under low dose conditions, under the condition of the lower limit of raw material ratio.

[0021] S1, weigh 60 parts of sea cucumber oligopeptide and add it to 200 parts of deionized water, stir and heat to 45°C to form a homogeneous solution; add 10 parts of monosodium glycyrrhizate and 15 parts of L-glutamic acid, stir at constant temperature for 2 hours to form a transparent deep eutectic system; adjust the pH of the system to 6.5, and freeze dry to obtain modified sea cucumber oligopeptide powder; S2, weigh 50 parts of modified sea cucumber oligopeptide and add it to 150 parts of deionized water, stir and dissolve at 45℃; add 5 parts of 2-hydroxy-4-methoxyphenylacetamide, 2 parts of β-cyclodextrin, 3 parts of glycerol and 0.1 parts of potassium sorbate in sequence, stir for 1 hour to form a uniform mixture; S3. The mixture was filtered through a 0.22μm filter membrane to remove impurities, concentrated under reduced pressure, and then vacuum dried at 40℃ to constant weight to obtain a light brown powdered sea cucumber peptide product.

[0022] Example 2 The purpose of this embodiment is to verify whether the overall performance of the modified reaction and the organic small molecule synergistic system reaches a balance point in terms of stability, absorption and repair ability at a moderate ratio.

[0023] S1, weigh 80 parts of sea cucumber oligopeptide and add it to 250 parts of deionized water, stir and heat to 50°C to form a homogeneous solution; add 15 parts of monosodium glycyrrhizate and 20 parts of L-glutamic acid, stir at constant temperature for 2.5 hours to form a transparent deep eutectic system; adjust the pH to 6.5, and freeze dry to obtain modified sea cucumber oligopeptide powder; Figure 1 Infrared spectroscopy results showed that the sea cucumber oligopeptide samples modified by the glycyrrhizic acid-amino acid deep eutectic system exhibited high activity at 1720, 1250, 1200, 1050, 960, and 885 cm⁻¹. -1 The appearance of new absorption peaks at 1640 cm⁻¹ corresponds to the C=O, C–N, C–O–C, S=O, C–N–H, and S–O vibrations of the ester group, indicating that glycyrrhizic acid and glutamate groups have been successfully introduced into the sea cucumber peptide structure. -1 Amide I band at 1540cm -1 The intensity of the amide II band peak is significantly enhanced, at 3400 cm⁻¹. -1 The increased depth and broadening of the O–H / N–H peaks indicate the formation of a denser hydrogen bond network between peptide chains; 1080 cm⁻¹ -1 Peak enhancement, 1380cm -1 The weakening of the peak further proves that the carboxyl group participates in the amidation reaction; the overall change indicates that the deep eutectic modification effectively promotes chemical cross-linking and hydrogen bond recombination, thereby significantly improving the structural stability and acid resistance of sea cucumber oligopeptides. S2, weigh 65 parts of modified sea cucumber oligopeptide and add it to 220 parts of deionized water, heat to 50℃ and stir to dissolve; add 10 parts of 2-hydroxy-4-methoxyphenylacetamide, 3 parts of β-cyclodextrin, 5 parts of glycerol and 0.2 parts of potassium sorbate in sequence, and stir at a constant temperature for 1.5 hours; S3. The mixture was filtered through a 0.22μm filter, concentrated under reduced pressure, and then vacuum dried at 40℃ for 48 hours to obtain a light yellow, uniform powder.

[0024] Example 3 The purpose of this embodiment is to verify the enhancing effect of the deep eutectic modified system and the small molecule compound system on the mucosal repair activity and their stability under high load conditions, under the condition of the upper limit of raw material ratio.

[0025] S1, weigh 100 parts of sea cucumber oligopeptide and add it to 300 parts of deionized water, stir and heat to 60°C to form a transparent solution; add 20 parts of monosodium glycyrrhizate and 30 parts of L-glutamic acid, stir at constant temperature for 3 hours to form a deep eutectic system; adjust the pH to 6.0, freeze dry for 72 hours to obtain light brown modified sea cucumber oligopeptide powder; S2, weigh 80 parts of modified sea cucumber oligopeptide and add it to 300 parts of deionized water, heat to 55℃ and stir to dissolve; add 15 parts of 2-hydroxy-4-methoxyphenylacetamide, 5 parts of β-cyclodextrin, 8 parts of glycerol and 0.3 parts of potassium sorbate in sequence, and stir at constant temperature for 2 hours to form a homogeneous system; S3. The system was filtered through a 0.22μm filter membrane to remove impurities, concentrated under reduced pressure, and then vacuum dried at 40℃ to constant weight to obtain a light yellow powdered sea cucumber peptide product.

[0026] Comparative Example 1 The purpose of this comparative study is to verify the differences in stability and repair effects when sea cucumber oligopeptides are directly compounded with organic small molecules without modification of the glycyrrhizic acid-amino acid deep eutectic system.

[0027] S1, weigh 80 parts of sea cucumber oligopeptide and add it to 250 parts of deionized water. Stir and heat to 50°C to form a homogeneous solution. Do not perform glycyrrhizic acid monosodium salt and L-glutamic acid modification treatment. Directly freeze dry to obtain unmodified sea cucumber oligopeptide powder. S2, weigh 65 parts of unmodified sea cucumber oligopeptides and add them to 220 parts of deionized water, heat to 50℃ and stir to dissolve; add 10 parts of 2-hydroxy-4-methoxyphenylacetamide, 3 parts of β-cyclodextrin, 5 parts of glycerol and 0.2 parts of potassium sorbate in sequence, and stir at a constant temperature for 1.5 hours; S3. The mixture was filtered through a 0.22 μm filter, concentrated under reduced pressure, and then vacuum dried at 40 °C for 48 hours to obtain a light brown powder sample.

[0028] Comparative Example 2 The purpose of this comparative example is to verify the effect of using only the glycyrrhizic acid-amino acid deep eutectic system for modification without introducing 2-hydroxy-4-methoxyphenylacetamide on the mucosal repair effect, and to demonstrate the importance of the synergistic effect of organic small molecules.

[0029] S1, weigh 80 parts of sea cucumber oligopeptide and add it to 250 parts of deionized water, stir and heat to 50°C to form a homogeneous solution; add 15 parts of monosodium glycyrrhizate and 20 parts of L-glutamic acid, stir at constant temperature for 2.5 hours to form a transparent deep eutectic system; adjust the pH to 6.5, and freeze dry to obtain modified sea cucumber oligopeptide powder; S2, weigh 65 parts of modified sea cucumber oligopeptide and add it to 220 parts of deionized water, heat to 50℃ and stir to dissolve; add 3 parts of β-cyclodextrin, 5 parts of glycerol and 0.2 parts of potassium sorbate in sequence, without adding 2-hydroxy-4-methoxyphenylacetamide, and stir at a constant temperature for 1.5 hours; S3. The system was filtered through 0.22 μm, concentrated under reduced pressure, and then dried under vacuum at 40 °C for 48 hours to obtain a light yellow powder sample.

[0030] Comparative Example 3 The purpose of this comparative example is to verify the effect of using only monosodium glycyrrhizate in the deep eutectic system on peptide stability and repair performance compared to the complete glycyrrhizic acid-L-glutamic acid synergistic system.

[0031] S1, weigh 80 parts of sea cucumber oligopeptide and add it to 250 parts of deionized water, heat to 50℃ and stir to dissolve; add only 15 parts of monosodium glycyrrhizate, without adding L-glutamic acid, stir at constant temperature for 2.5 hours; adjust the pH to 6.5, and freeze dry to obtain monosodium glycyrrhizate modified sea cucumber oligopeptide powder. S2, weigh 65 parts of the single modified sea cucumber oligopeptide and add it to 220 parts of deionized water, stir and dissolve at 50°C; add 10 parts of 2-hydroxy-4-methoxyphenylacetamide, 3 parts of β-cyclodextrin, 5 parts of glycerol and 0.2 parts of potassium sorbate in sequence, and stir at a constant temperature for 1.5 hours; S3. The system was filtered through a 0.22 μm filter membrane, concentrated under reduced pressure, and dried under vacuum at 40 °C for 48 hours to obtain a light yellow powder sample.

[0032] Performance testing 1. Gastric acid stability test 100 mg of each of the samples obtained in Example 2 and Comparative Examples 1-3 were added to artificial gastric fluid and treated with constant temperature shaking at 37°C. Samples were taken every 30 minutes and the supernatant was separated by centrifugation. The change in peptide residue in the solution was detected to evaluate the stability of different samples in an acidic environment. By comparing the difference in residue rate between the sample examples and the comparative examples, the contribution of deep eutectic modification to the acid resistance stability of sea cucumber peptides was determined.

[0033] 2. Antioxidant capacity test 20 mg of each sample was weighed and dissolved in PBS buffer, and a certain amount of DPPH ethanol solution was added. After reacting in the dark for 30 min, the absorbance change was measured. By comparing the differences in free radical scavenging rates of the samples, the influence of the modified system and the synergistic effect of organic small molecules on the antioxidant performance was analyzed.

[0034] 3. Epithelial cell repair experiment A scratch model was established using Caco-2 human intestinal epithelial cells. After cell confluence, scratches were made using a sterile pipette tip. The culture medium was discarded, and culture medium containing extracts of each sample was added. The cells were incubated at 37°C and 5% CO2 for 24 hours, and the changes in scratch closure area were observed under a microscope. The epithelial cell repair capacity of the sea cucumber peptide system was evaluated by comparing the scratch healing rates of the examples and comparative examples.

[0035] 4. Detection of mucosal barrier protein expression Caco-2 cells treated for 24 hours were lysed and total proteins were extracted. These proteins were separated by SDS-PAGE and analyzed by Western blotting, with Occludin and ZO-1 selected as representative indicators. The difference in protein band intensity between Example 2 and the comparative sample was compared to analyze the effect of glycyrrhizic acid-amino acid modification and the synergistic effect of small organic molecules on the upregulation of mucosal barrier proteins.

[0036] Table 1 Comparison of performance test results between the examples and the comparative samples.

[0037] As can be seen from Table 1, Example 2 has the best overall performance, and it is significantly better than other samples in four indicators: gastric acid stability, antioxidant capacity, epithelial cell repair rate, and mucosal barrier protein expression. Figure 2 In Example 2, the gastric acid stability reached 93.7%, which was about 32 percentage points higher than that of the unmodified system. This indicates that the deep eutectic system formed by glycyrrhizic acid and L-glutamic acid can enhance the hydrogen bonding between peptide chains at the molecular level and reshape the amide structure network, thereby significantly improving the structural stability and hydrolysis resistance of sea cucumber peptides under strong acid conditions.

[0038] In terms of antioxidant properties, Figure 2 The free radical scavenging rate of Example 2 was 89.4%, which was about 33 percentage points higher than that of the unmodified sample. During the modification process, the carboxyl group of glycyrrhizic acid formed hydrogen bonds with the hydroxyl group of the peptide chain, making the electron cloud density distribution more stable; at the same time, the phenolic hydroxyl and methoxy groups in the 2-hydroxy-4-methoxyphenylacetamide molecule participated in the conjugation effect, effectively improving the free radical scavenging efficiency and antioxidant persistence.

[0039] Epithelial cell repair assays showed that Figure 3 In Example 2, the cell repair rate reached 92.8%, significantly higher than the 57.3% of the unmodified control. This result indicates that deep eutectic modification not only enhances the cell membrane permeability of peptide molecules but also enables 2-hydroxy-4-methoxyphenylacetamide to synergistically regulate cell energy metabolism and migration behavior, accelerating the epithelial tissue regeneration process.

[0040] In the detection of mucosal barrier proteins, Figure 3 In Example 2, the protein expression levels of Occludin and ZO-1 increased by 2.1 times compared to the control group, significantly higher than the 1.4 times increase in the modified sample alone. This result indicates that small organic molecules, after synergistic modification, can activate tight junction-related signaling pathways, promote barrier protein synthesis and adhesion junction reconstruction, demonstrating a significant structural repair function.

[0041] In summary, the superior performance of Example 2 is attributed to the synergistic combination of the molecular stabilizing effect of the glycyrrhizic acid-amino acid deep eutectic system and the metabolic regulatory function of 2-hydroxy-4-methoxyphenylacetamide, which significantly improves the acid stability, antioxidant properties and mucosal repair capabilities of the sea cucumber peptide system of the present invention, demonstrating the innovative technical effect.

Claims

1. A sea cucumber peptide for repairing gastrointestinal mucosal injury, characterized in that, The sea cucumber peptide comprises the following raw materials by weight: sea cucumber oligopeptide modified by glycyrrhizic acid-amino acid deep eutectic system 50-80 parts; 2-hydroxy-4-methoxy phenylacetamide 5-15 parts; beta-cyclodextrin 2-5 parts; glycerol 3-8 parts; potassium sorbate 0.1-0.3 parts; deionized water 150-300 parts; wherein the sea cucumber oligopeptide modified by the glycyrrhizic acid-amino acid deep eutectic system refers to the hydrogen bond reorganization and amidation reaction of sea cucumber oligopeptide by the deep eutectic system formed by glycyrrhizic acid monosodium salt and L-glutamic acid; and the 2-hydroxy-4-methoxy phenylacetamide refers to a simple structure of benzeneacetamide small organic molecules.

2. The sea cucumber peptide for repairing gastrointestinal mucosal injury according to claim 1, characterized in that, The sea cucumber oligopeptide modified by the glycyrrhizic acid-amino acid deep eutectic system comprises the following raw materials by weight: sea cucumber oligopeptide 60-100 parts; glycyrrhizic acid monosodium salt 10-20 parts; L-glutamic acid 15-30 parts; deionized water 200-300 parts.

3. The sea cucumber peptide for repairing gastrointestinal mucosal injury according to any one of claims 1 or 2, characterized in that, The preparation method of the sea cucumber oligopeptide modified by the glycyrrhizic acid-amino acid deep eutectic system comprises the following steps: (1) adding sea cucumber oligopeptide into deionized water and stirring to form a uniform solution; (2) adding glycyrrhizic acid monosodium salt and L-glutamic acid into the uniform solution and performing deep eutectic modification reaction; (3) adjusting the pH of the obtained system and drying to obtain sea cucumber oligopeptide powder modified by the glycyrrhizic acid-amino acid deep eutectic system.

4. The sea cucumber peptide for repairing gastrointestinal mucosal injury according to claim 3, characterized in that, The stirring condition of step (1) is heating to 40-60 DEG C and continuous stirring.

5. The sea cucumber peptide for repairing gastrointestinal mucosal injury according to claim 3, characterized in that, The reaction condition of step (2) is stirring reaction for 2-3 hours under constant temperature.

6. The sea cucumber peptide for repairing gastrointestinal mucosal injury according to claim 3, characterized in that, The reaction condition of step (3) is adjusting the pH of the system to 6.0-7.0 and then freeze-drying to obtain solid product.

7. A method for preparing a sea cucumber peptide for repairing gastrointestinal mucosal injury, wherein the sea cucumber peptide for repairing gastrointestinal mucosal injury is as claimed in any one of claims 1 to 6. comprises the following steps: S1, adding sea cucumber oligopeptide modified by the glycyrrhizic acid-amino acid deep eutectic system into deionized water and stirring to form a uniform solution; S2, sequentially adding 2-hydroxy-4-methoxy phenylacetamide, beta-cyclodextrin, glycerol and potassium sorbate into the uniform solution and stirring to mix uniformly; S3, filtering impurities from the mixed system, concentrating and drying to obtain sea cucumber peptide for repairing intestinal mucosa damage.

8. The preparation method of the sea cucumber peptide for repairing gastrointestinal mucosal injury according to claim 7, characterized in that, The reaction condition of step S1 is heating to 40-50 DEG C and continuous stirring.

9. The preparation method of the sea cucumber peptide for repairing gastrointestinal mucosal injury according to claim 7, characterized in that, The reaction condition of step S2 is stirring for 1-2 hours under constant temperature of 45-55 DEG C.

10. The preparation method of the sea cucumber peptide for repairing gastrointestinal mucosal injury according to claim 7, characterized in that, The processing condition of step S3 is filtering impurities through 0.22 μm, concentrating under reduced pressure at 50-60 DEG C and vacuum drying at 40 DEG C to constant weight.

Images