Artichoke antioxidant peptide for protecting the liver and improving gastrointestinal function and preparation method thereof

Abstract

The present invention relates to the fields of medicine and health foods, and specifically to an artichoke antioxidant peptide that protects the liver and improves gastrointestinal function, and a method for preparing the same. The artichoke antioxidant peptide is extracted after temperature stress treatment, and the bioactive peptide segments are selectively extracted using trypsin enzymatic hydrolysis. The artichoke antioxidant peptide of the present invention can be used to develop drugs and functional foods for treating liver diseases, helping to scavenge free radicals, protect liver cells, and enhance the body's antioxidant capacity. Furthermore, the artichoke antioxidant peptide of the present invention can be used as a supplement and functional food to regulate intestinal flora, thereby improving overall gastrointestinal health.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and health food, and in particular to an artichoke antioxidant peptide capable of protecting the liver and improving gastrointestinal function, and a preparation method thereof. The antioxidant peptide is obtained by temperature stress extraction, has significant biological activity, and can be used to develop new health products and therapeutic drugs. Background Art

[0002] The liver is a crucial metabolic organ in the human body, responsible for detoxification, nutrient storage, and the synthesis of a variety of biological substances. However, factors such as modern lifestyles, dietary habits, and environmental pollution have led to a significant increase in the incidence of liver diseases such as fatty liver disease, hepatitis, and cirrhosis. These liver diseases not only severely impact patients' quality of life but can also lead to more serious health problems. Furthermore, oxidative stress is considered one of the primary mechanisms underlying many liver diseases and tissue damage. Gastrointestinal health is also crucial, directly impacting nutrient absorption and overall immune function. Many gastrointestinal diseases, such as inflammatory bowel disease and gastritis, are closely linked to insufficient dietary antioxidants. Therefore, the search for natural ingredients that can simultaneously protect the liver and improve gastrointestinal function has become increasingly important.

[0003] Artichoke (Cynara scolymus L.) is a widely consumed vegetable rich in various bioactive components, such as polyphenols, amino acids, and cellulose. Currently, research on artichoke antioxidant peptides is relatively limited, with most studies focusing on extraction and preliminary analysis, lacking systematic bioactivity validation and application development. Therefore, the development of novel artichoke antioxidant peptides and their application in protecting the liver and improving gastrointestinal health not only holds significant scientific research value but also provides a promising foundation for the development of new drugs and healthcare products. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides a novel antioxidant peptide obtained by enzymatic hydrolysis of artichokes subjected to high temperature stress using trypsin, which has excellent biological activity and application prospects.

[0005] The present invention relates to an artichoke antioxidant peptide that protects the liver and improves gastrointestinal function, named AA-peptide, whose amino acid sequence is Lys-Thr-His-Glu-Arg-Met-Ala-Gly-Thr-Val-Cys-Arg-Lyss (SEQ ID NO.1: KTHERMAGTVCRK). The antioxidant peptide has significant biological activity, a molecular weight of 1515.78 g / mol, and a molecular formula of C 61 H 109 N 23 O 18 S2.

[0006] In one aspect of the present invention, the nucleic acid sequence of the antioxidant peptide AA-peptide is provided, as shown in SEQ ID NO.2.

[0007] SEQ ID NO.2:

[0008] AAGACCCACGAGCGAATGGCCGGAACCGTCTGCCGAAAG.

[0009] On the other hand, the present invention also provides a method for preparing the above-mentioned antioxidant peptide, comprising the following steps:

[0010] (1) Place fresh artichoke samples in an incubator at 40°C for 3 hours under high temperature stress.

[0011] (2) The treated artichoke samples were mixed with trypsin for fermentation to promote the release and enrichment of peptides.

[0012] (3) The fermentation broth is treated to inactivate enzymes, remove the effects of enzymes, and adjust the pH value.

[0013] (4) The supernatant was filtered using an ultrafiltration membrane to selectively extract peptides with high antioxidant activity, which were further separated and identified to obtain AA-peptide.

[0014] The present invention also provides an application of the artichoke antioxidant peptide in developing drugs for protecting the liver, improving gastrointestinal function, and having antioxidant functions.

[0015] Beneficial effects:

[0016] (1) The present invention selected artichokes subjected to high temperature stress at 40°C and then used trypsin to perform enzymatic hydrolysis, ultimately identifying a new type of antioxidant peptide.

[0017] (2) Different from the existing technology, the artichoke antioxidant peptide AA-peptide provided by the above technical solution consists of 13 amino acids, the amino acid sequence is Lys-Thr-His-Glu-Arg-Met-Ala-Gly-Thr-Val-Cys-Arg-Lys, its molecular weight is 1515.78g / mol, and its molecular formula is C 61 H 109 N 23 O 18 S2. It has high antioxidant activity, is non-cytotoxic, and exhibits excellent digestive stability and liver cell protection. The artichoke antioxidant peptide provided by the above technical solution can be used to develop drugs and functional foods for the treatment of liver diseases. It helps scavenge free radicals, protect liver cells, and enhance the body's antioxidant capacity, thereby protecting liver health and preventing the occurrence of liver diseases.

[0018] (3) The artichoke antioxidant peptide AA-peptide provided by the above technical solution has been shown in studies to have an intestinal flora-regulating effect. It can be used as a supplement and functional food to regulate intestinal flora, thereby improving overall gastrointestinal health. Based on its unique properties, it is expected to become the basis for new drugs to prevent and treat oxidative stress-related diseases, providing a new direction for improving patients' overall health. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 This is a comparison chart of the scavenging rate of hydroxy DPPH free radicals by trypsin fermentation of artichoke samples;

[0021] Figure 2 This is the result diagram of chromatographic separation and purification of antioxidant peptide AA-peptide;

[0022] Figure 3 This is a comparison chart of the scavenging rate of antioxidant peptide AA-peptide on hydroxy DPPH free radicals;

[0023] Figure 4 This is a comparison chart of the effects of antioxidant peptide AA-peptide pretreatment on ROS levels in human hepatocytes;

[0024] Figure 5 The effect of antioxidant peptide AA-peptide on the Chao1 index of intestinal diversity;

[0025] Figure 6 The effect of antioxidant peptide AA-peptide on the composition of intestinal flora. DETAILED DESCRIPTION

[0026] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, the protection scope of the present invention is not limited to the following embodiment.

[0027] In the following examples, the chemical reagents used were purchased from regular chemical reagent suppliers and were of analytical grade.

[0028] Example 1 Trypsin fermentation of artichoke samples with different treatments

[0029] High temperature stress group: artichoke samples were placed in an incubator at 40°C for three hours;

[0030] Low temperature treatment group: artichoke samples were treated in a refrigerated environment at 4°C for 24 hours;

[0031] Control group: artichoke samples at room temperature (about 25°C).

[0032] Artichoke samples were prepared as follows: fresh artichokes were rinsed thoroughly with running water and then soaked in 70% ethanol or bleach for several minutes to disinfect the surface. Rinse again with clean water to remove any residue. Wearing sterile gloves, use a clean, sharp knife to remove the outer leaves of the artichokes, leaving the tender inner leaves.

[0033] After the three groups were fermented for 4 hours, the enzyme activity was terminated by heating the liquid, and the pH was adjusted to 8.3 before use.

[0034] The treated artichoke samples, trypsin, and artichoke sample fermentation medium (with 3% cysteine ​​added to stimulate the synthesis of antioxidant enzymes) were fermented for 4 hours. The enzyme activity was terminated by heating the liquid and the pH was adjusted to 8.3.

[0035] The artichoke sample fermentation medium is as follows: 10 g of artichoke extract, 20 g of glucose, 2 g of ammonium nitrate (NH4NO3), 5 g of yeast extract, 1 g of potassium dihydrogen phosphate (KH2PO4), 1 g of dipotassium hydrogen phosphate (K2HPO4), 0.5 g of magnesium sulfate (MgSO4·7H2O), 0.5 g of sodium chloride (NaCl), 0.01 g of FeSO4, 0.01 g of ZnSO4, 0.01 g of CuSO4 and 0.01 g of MnSO4; the pH of the culture medium is adjusted to 6.5 and the volume is adjusted to 1 L.

[0036] Artichoke extract: Fresh artichokes are selected, cleaned, cut into small pieces, heated at 50°C for 30 minutes, and then filtered to remove solids. The filtered liquid is collected and freeze-dried to obtain the extract.

[0037] The artichoke samples after the above treatment were subjected to trypsin fermentation treatment, and the specific steps were as follows:

[0038] The fermentation broth with a concentration of 1.0 mg / mL was taken and the changes in antioxidant activity in the culture broth were detected using a chromatographic method (2,2-diphenyl-1-pyridinylhydrazine free radical scavenging rate test, i.e., DPPH scavenging rate test). Figure 1 , both low temperature and high temperature stress can improve the antioxidant capacity of the fermentation broth, among which high temperature stress has the highest antioxidant activity.

[0039] Example 2 Preparation of mixed polypeptides

[0040] Collect the fermentation broth from the heat-stressed sample and inactivate the enzyme in a 90°C water bath for 10 minutes. Adjust the pH to 8.3 and lyophilize to obtain a freeze-dried sample for later use. Weigh a certain amount of freeze-dried sample and dissolve it in deionized water. Centrifuge at 8000 rpm / min at 4°C for 15 minutes, and collect the supernatant.

[0041] Ultrafiltration was performed using 10 KDa and 3 KDa filter membranes to separate and purify the supernatant into three different components, including a component larger than 10 KDa (M3), a component between 3 KDa and 10 KDa (M2), and a component smaller than 3 KDa (M1).

[0042] The specific steps are as follows: 10 mL of the supernatant is placed in a 10 kDa ultrafiltration tube, and the experimental conditions are set at 4°C to maintain the biological activity of the sample. Subsequently, ultrafiltration is performed using a centrifuge at a speed of 3500 rpm / min for 20 minutes. After centrifugation, the ultrafiltrate with a molecular weight less than 10 kDa is collected. This portion of the liquid contains smaller molecular weight components and will be used for the next step. The above ultrafiltrate is again centrifuged and ultrafiltered using a 3 kDa ultrafiltration tube under the same conditions. The settings are also set at 4°C and 3500 rpm / min, and the ultrafiltration time is maintained for 20 minutes. Through the above steps, the ultrafiltrate is further separated, and finally three components are obtained: a component with a molecular weight greater than 10 kDa (M3), a component between 3 kDa and 10 kDa (M2), and a component less than 3 kDa (M1).

[0043] After the ultrafiltration step, the filtrates from each fraction were collected and concentrated to approximately 50 mL using a rotary evaporator to reduce the volume and prepare for subsequent analysis. The concentrated samples were then freeze-dried to ensure the stability of the active ingredient.

[0044] Finally, DPPH scavenging rate was tested using chromatography to evaluate the antioxidant activity of each fraction. The DPPH test was performed on each fraction, and the experimental results showed that fraction M1 had the greatest antioxidant activity among the three fractions, indicating that antioxidant peptides less than 3 kDa may be the components with the highest biological activity.

[0045] Fraction M1 was isolated and purified by Sephadex G-15 chromatography: a certain amount of Sephadex G-15 powder was placed in five times the volume of distilled water and allowed to swell at room temperature for 3 hours to fully dissolve the G-15. The suspended gel particles were then discarded. After the chromatography column was secured, the pretreated gel was slowly poured into the column along the upper end, allowing it to naturally settle to two-thirds of the column height. Before injection, the gel was continuously equilibrated with distilled water until there were no bubbles or stratification. The loading volume was 1 mL, the eluent was distilled water, the loading concentration was 200 mg / mL, the flow rate was 1 mL / min, and the UV detection wavelength was 280 nm using an AKTA protein chromatography system. Peak fractions were collected to yield three components: G1, G2, and G3. After freeze-drying, the antioxidant activity of each component was assessed by DPPH scavenging chromatography. When the concentration was 1 mg / mL, component G2 had the highest antioxidant activity of 63.10%, and its inhibitory activity was significantly higher than that of other components.

[0046] Liquid chromatography, tandem mass spectrometry, and de novo sequencing identified 1,252 peptides in fraction G2, of which 341 were heptapeptides. Amino acid distribution revealed that over 80% of the peptides in fraction G2 contained polar amino acids. Peptides with a polar amino acid content of 65-80% were the most abundant, accounting for 40.5% of the total peptides. Since the polar amino acid content of antioxidant peptides is closely related to their antioxidant activity, these polar amino acid-rich peptides significantly enhanced the activity of fraction G2.

[0047] Example 3 Screening of Antioxidant Active Peptides Based on Database

[0048] The peptides obtained by sequencing were screened from the following six dimensions:

[0049] Confidence: From the peptide sequences identified by LC-MS / MS, peptides with a confidence level of ALC > 80% (relatively reliable) were selected;

[0050] Peptide content: The peak area of ​​the peptide segment can reflect its content. From the peptide segments with a confidence level > 80%, the peak area > 10e was screened. 5 peptides;

[0051] Biological activity score: Area>10×e 5 The peptides were predicted and peptides with PeptideRanker>0.5 were screened;

[0052] Water solubility prediction: For peptides predicted to have potential biological activity (PeptideRanker>0.5), water solubility prediction was performed in the Proteomics tools under the INNOVAGEN website to screen for peptides with "Good" water solubility;

[0053] Toxicity prediction: The water-soluble peptides obtained by screening were used for peptide toxicity prediction in the "Batch Submission" module of the Toixinpred website, and "Non-Toxin" peptides were selected;

[0054] Allergy prediction: For the non-toxic peptides obtained by screening, perform allergy prediction in the "AllerTOPServerPage" and select the "NON-ALLERGEN" peptide;

[0055] Example 4 Molecular docking screening of ACE inhibitory peptides

[0056] Six peptides were screened from the six dimensions above. After synthesis, the peptides were further tested for DPPH scavenging rates using chromatography. The artichoke AA-peptide with the highest antioxidant activity was obtained. Its amino acid sequence (sequence 1) is Lys-Thr-His-Glu-Arg-Met-Ala-Gly-Thr-Val-Cys-Arg-Lys. Furthermore, it was analyzed and identified using electrospray ionization mass spectrometry (ESI-MS). The spectrum is shown in Figure 1. Figure 2 The artichoke AA-peptide sequence also showed a maximum similarity of 90% with existing sequences in NCBI (National Center for Biotechnology Information), and a maximum similarity of 58.33% with artichoke-derived sequences. This peptide is a novel peptide with a novel sequence, with polar amino acids comprising 75% of the total amino acids in the sequence. Because the content of polar amino acids in antioxidant peptides is closely related to their antioxidant activity, this result suggests that AA-peptide is a novel artichoke antioxidant peptide with potential antioxidant properties.

[0057] Example 5: Test on the scavenging effect of antioxidant peptide AA-peptide on hydroxy DPPH free radicals

[0058] Dissolve DPPH in anhydrous ethanol to a final concentration of 0.04 mol / mL. Add 2 mL of DPPH solution and 1 mL of anhydrous ethanol to 1 mL of antioxidant peptide AA-peptide (concentration 0.1-1.0 mg / mL) solution. Incubate at room temperature for 30 minutes. Centrifuge at 5000 rpm for 5 minutes, and measure the absorbance of the supernatant at 517 nm.

[0059] A control group (containing 2 mL of DPPH solution, 1 mL of water, and 1 mL of ethanol) and a blank group (containing 1 mL of sample solution and 3 mL of ethanol) were set up.

[0060] The antioxidant activity of the samples was evaluated by the scavenging rate of DPPH using the following equation:

[0061] DPPH clearance rate (%) = (A0-A+A1) / A0× 100%

[0062] Where A is the absorbance of the sample; A0 is the absorbance of the control; A1 is the absorbance of the blank.

[0063] See the results Figure 3 It can be seen that the scavenging activity of antioxidant peptide AA-peptide on DPPH increases with the increase of concentration. When the concentration of antioxidant peptide AA-peptide reaches 1.0 mg / mL, the clearance rate can reach more than 75%.

[0064] Example 6 Experimental study on the effect of antioxidant peptide AA-peptide on ROS levels in human hepatocytes

[0065] Culture human hepatocytes HEPG2, collect the cells and place them (3×10 5 / well) were inoculated into 6-well plates filled with 2 mL of culture medium, with a total of 20 wells inoculated. After incubation for 24 hours, the cell samples were divided into four groups (5 replicates per group), control group, model group, AA-peptide group and positive control group.

[0066] The specific treatments were as follows: (1) Control group: only culture medium was added without any induction to serve as a baseline control. (2) Model group: treated with 2 mL of H2O2 at a final concentration of 600 μM and incubated for 4 hours to induce oxidative stress, followed by treatment with 2 mL of PBS (0.01 M, pH 7.2-7.4). AA-peptide group: treated with 2 mL of H2O2 at a final concentration of 600 μM and incubated for 4 hours to induce oxidative stress, followed by treatment with 2 mL of AA-peptide peptide solution (final concentration of 25 μM). Positive control group: treated with 2 mL of H2O2 at a final concentration of 600 μM and incubated for 4 hours to induce oxidative stress, followed by treatment with 2 mL of GSH solution (final concentration of 25 μM).

[0067] After all treatments, the 6-well plate was placed in a 37°C, 5% CO2 incubator and incubated for another 24 hours. Finally, the cells were incubated with 50 μL of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) in a 37°C cell culture incubator for another 20 minutes. Finally, the coverslip was removed with tweezers and observed under a fluorescence microscope. The results are shown in Figure 4 .

[0068] The results showed that compared with the H2O2-induced model group, the intracellular fluorescence intensity was significantly reduced after pretreatment with the antioxidant peptide AA-peptide and the positive control glutathione (GSH), indicating a significant decrease in intracellular ROS (Reactive Oxygen Species) levels. Furthermore, AA-peptide was more effective than GSH, with lower ROS levels. This result suggests that AA-peptide can more efficiently scavenge intracellular ROS and prevent oxidative damage.

[0069] Example 7 Experiment on the effect of AA-peptid on the intestinal flora of mice

[0070] Six-week-old male mice were selected as model organisms. First, a quadruple antibiotic (gentamicin, ampicillin, cephalothin, and metronidazole) was used to clear the intestinal flora. The drug was administered for one week to significantly reduce the bacterial richness in the mouse intestine and ensure that the mice were in a "sterile" state of intestinal microecology. After clearance, the mice were immediately gavaged with AA-peptide for two weeks to evaluate the effect of AA-peptide on the reconstruction of intestinal flora. During the experiment, the weight and behavioral activities of the mice were regularly monitored to ensure the safety and tolerability of AA-peptide. At the end of the experiment, the intestinal contents of the mice were collected by sterile methods, and high-throughput sequencing technology (16S rRNA gene sequencing) was used to analyze the composition and abundance of the intestinal microbiota. Figure 5 AA-peptide can significantly improve the Chao1 index and increase the diversity of intestinal flora. Figure 6Ingestion of AA-peptide resulted in a significant decrease in the Firmicutes phylum and a significant increase in the abundance of Bacteroidota bacteria. This microbial shift may be related to the bioactivity of antioxidant peptides and their regulatory effects on the intestinal environment. Firmicutes are generally considered an important bacterial group in the intestine, involved in nutrient absorption and metabolism. The decrease in their abundance may indicate a role for antioxidant peptides in inhibiting the growth of certain specific bacteria. This change may help reduce the inflammatory response associated with obesity and metabolic syndrome, as high levels of Firmicutes are associated with imbalances in fat storage and energy metabolism. Conversely, the increase in Bacteroidota may benefit intestinal health. Bacteroidota are primarily responsible for breaking down complex carbohydrates and promoting the production of short-chain fatty acids (SCFAs), which have anti-inflammatory, intestinal barrier-enhancing, and metabolic regulatory effects. By promoting the proliferation of Bacteroidota, antioxidant peptides may strengthen the intestinal barrier and improve glucose and lipid metabolism in mice, helping to maintain a balanced and healthy intestinal microbiome. In summary, AA-peptide intake not only improved the intestinal health of mice, but also significantly altered the composition of the intestinal flora, increasing the abundance of beneficial bacteria and reducing the proportion of potentially harmful bacteria. This finding provides new scientific evidence and prospects for the application of antioxidant peptides in intestinal health and metabolic regulation.

[0071] AA-peptide is composed of a variety of polar and non-polar amino acids. Polar amino acids (such as K, T, H, E, R, and C) can form hydrogen bonds with water molecules, enhancing their solubility in water and aiding their bioavailability. Peptides containing cysteine ​​often exhibit excellent antioxidant properties when forming disulfide bonds. Furthermore, the positively charged amino acids (such as K, H, R) and polar amino acids (such as T and C) in AA-peptide can react with free radicals, donating hydrogen atoms, effectively neutralizing them and reducing oxidative stress. Furthermore, the cysteine ​​residues in the AA-peptide sequence react with free radicals to form stable compounds with strong antioxidant potential. Finally, the amino acid composition of AA-peptide allows for a certain degree of spatial flexibility, which may enhance its binding ability to target molecules and further improve its antioxidant efficacy.

[0072] The present invention provides a novel antioxidant peptide, named AA-peptide. This peptide not only exhibits excellent antioxidant activity but also possesses non-toxic properties, giving it broad application potential in the biomedical field. Studies have found that AA-peptide can help scavenge free radicals, protect liver cells, and enhance the body's antioxidant capacity. This provides strong support for the development of new drugs and functional foods for the treatment of liver diseases. In addition, AA-peptide exhibits intestinal flora regulatory effects, significantly improving the balance of intestinal microecology, promoting the growth of beneficial bacteria, and inhibiting the proliferation of potentially harmful bacteria. This indicates that the artichoke antioxidant peptide described in the present invention can be used as a supplement and functional food to regulate intestinal flora, thereby improving overall gastrointestinal health. In summary, AA-peptide is not only expected to serve as an effective antioxidant to combat cell damage caused by oxidative stress, but is also an important component for improving intestinal health. This discovery provides new directions and basis for further exploring its potential applications in functional foods and health products.

Claims

1. An artichoke antioxidant peptide that protects the liver and improves gastrointestinal function, named AA-peptide, has an amino acid sequence of Lys-Thr-His-Glu-Arg-Met-Ala-Gly-Thr-Val-Cys-Arg-Lys, SEQ ID NO.1: KTHERMAGTVCRK, a molecular weight of 1515.78 g / mol, and a molecular formula of C 61 H 109 N 23 O 18 S2.

2. The artichoke antioxidant peptide according to claim 1, characterized in that The nucleic acid sequence of the AA-peptide is shown in SEQ ID NO.

2. SEQ ID NO. 2: AAGACCCACGAGCGAATGGCCGGAACCGTCTGCCGAAAG.

3. An application of artichoke antioxidant peptide in anti-oxidation, characterized in that: The antioxidant peptide is the AA-peptide described in claim 1.

4. The use of the artichoke antioxidant peptide in anti-oxidation according to claim 3, characterized in that: The application areas are as follows: liver protection and antioxidant health products and improvement of gastrointestinal health.

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