Cynara scolymus antioxidant peptide for protecting liver and improving stomach and intestine and preparation method of cynara scolymus antioxidant peptide

Abstract

The invention relates to the field of medicines and health foods, in particular to an artichoke antioxidant peptide for protecting liver and improving stomach and intestine and a preparation method thereof, the artichoke antioxidant peptide is obtained by extracting after temperature stress treatment, and a bioactive peptide fragment is selectively extracted by adopting a trypsin enzymolysis method. The artichoke antioxidant peptide disclosed by the invention can be used for developing medicines and functional foods for treating liver diseases, and is helpful for scavenging free radicals, protecting liver cells and enhancing the antioxidant capacity of a body. In addition, the artichoke antioxidant peptide disclosed by the invention can be used as a supplement and a functional food to regulate intestinal flora, so that the overall gastrointestinal health is improved.

Description

Technical Field

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

[0002] The liver is an important metabolic organ in the human body, responsible for detoxification, nutrient storage and synthesis of a variety of biological substances. However, factors such as modern lifestyle, dietary habits and environmental pollution have led to a significant increase in the incidence of liver diseases, such as fatty liver, hepatitis and cirrhosis. These liver diseases not only seriously affect the quality of life of patients, but also lead to more serious health problems. In addition, oxidative stress is considered to be one of the main mechanisms causing many liver diseases and tissue damage. At the same time, gastrointestinal health is also crucial, which directly affects nutrient absorption and overall immune function. Many gastrointestinal diseases (such as inflammatory bowel disease, gastritis, etc.) are closely related to the lack of antioxidants in the diet. Therefore, it is particularly important to find natural ingredients that can protect the liver and improve gastrointestinal function at the same time.

[0003] Artichoke (Cynara scolymus L.) is a widely used vegetable rich in a variety of bioactive ingredients, such as polyphenols, amino acids and cellulose. At present, there are relatively few studies on artichoke antioxidant peptides, and most studies focus on their extraction and preliminary analysis, lacking systematic bioactivity verification and application development. Therefore, the development of a new type of artichoke antioxidant peptide and its application in protecting the liver and improving gastrointestinal health not only has important scientific research value, but also provides a good foundation for the development of new drugs and health 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 for protecting the liver and improving the gastrointestinal tract, named AA-peptide, and its 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, a nucleic acid sequence of the antioxidant peptide AA-peptide is provided, as shown in SEQ ID NO.2.

[0007] SEQ ID NO.2: AAGACCCACGAGCGAATGGCCGGAACCGTCTGCCGAAAG.

[0008] On the other hand, the present invention also provides a method for preparing the above antioxidant peptide, comprising the following steps: (1) Place fresh artichoke samples in an incubator at 40°C for 3 hours under high temperature stress.

[0009] (2) The processed artichoke samples were mixed with trypsin for fermentation to promote the release and enrichment of peptides.

[0010] (3) The fermentation broth is treated to inactivate enzymes, remove the effects of enzymes, and adjust the pH value.

[0011] (4) The supernatant was filtered using an ultrafiltration membrane to selectively extract peptides with high antioxidant activity, which were then further separated and identified to obtain AA-peptide.

[0012] 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 function.

[0013] Beneficial effects: (1) The present invention selected artichokes subjected to 40°C high temperature stress and then used trypsin to perform enzymatic hydrolysis, and finally identified a new type of antioxidant peptide.

[0014] (2) Different from the prior art, the artichoke antioxidant peptide AA-peptide provided by the above technical solution is composed 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 1515.78 g / mol, and its molecular formula is C 61 H 109 N 23 O 18 S2. It has high antioxidant activity, no cytotoxicity, good 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, help scavenge free radicals, protect liver cells, and enhance the body's antioxidant capacity, thereby protecting liver health and preventing the occurrence of liver diseases.

[0015] (3) The artichoke antioxidant peptide AA-peptide provided by the above technical solution has been shown in studies to have the effect of regulating intestinal flora. It can be used as a supplement and functional food to regulate intestinal flora, thereby improving overall gastrointestinal health. Based on its unique characteristics, it is expected to become the basis for new drugs to prevent and treat oxidative stress-related diseases, providing a new direction for improving the overall health of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings in the specification, 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.

[0017] Figure 1 This is a comparison chart of the scavenging rate of hydroxy DPPH free radicals by trypsin fermented artichoke samples; Figure 2 This is the result diagram of chromatographic separation and purification of antioxidant peptide AA-peptide; Figure 3 This is a comparison chart of the scavenging rate of the antioxidant peptide AA-peptide on the hydroxy DPPH free radical; Figure 4 This is a comparison chart of the effects of antioxidant peptide AA-peptide pretreatment on ROS levels in human hepatocytes; Figure 5 The effect of antioxidant peptide AA-peptide on the Chao1 index of intestinal diversity; Figure 6 The effect of antioxidant peptide AA-peptide on the composition of intestinal flora. DETAILED DESCRIPTION

[0018] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0019] In the following examples, the chemical reagents used were purchased from regular chemical reagent suppliers and were of analytical grade.

[0020] Example 1 Trypsin fermentation of artichoke samples with different treatments High temperature stress group: Artichoke samples were placed in an incubator at 40°C for three hours; Low temperature treatment group: artichoke samples were treated in a refrigerated environment at 4°C for 24 hours; Control group: artichoke samples at room temperature (about 25°C).

[0021] The artichoke sample was processed as follows: fresh artichokes were taken, the surface of the artichokes was rinsed with running water, and the surface was disinfected by soaking in 70% ethanol or bleach for several minutes. The surface was rinsed again with clean water to remove the residue on the surface. The outer leaves of the artichokes were removed with a clean and sharp knife while wearing sterile gloves to obtain the tender leaves inside.

[0022] 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 for standby use.

[0023] The treated artichoke samples, trypsin, and artichoke sample fermentation medium (3% cysteine ​​was additionally added to stimulate the synthesis of antioxidant enzymes) were fermented for 4 hours, and the enzyme activity was terminated by heating the treatment liquid and adjusting the pH to 8.3.

[0024] The artichoke sample fermentation medium is as follows: 10 g artichoke extract, 20 g glucose, 2 g ammonium nitrate (NH4NO3), 5 g yeast extract, 1 g potassium dihydrogen phosphate (KH2PO4), 1 g dipotassium hydrogen phosphate (K2HPO4), 0.5 g magnesium sulfate (MgSO4·7H2O), 0.5 g sodium chloride (NaCl), 0.01 g FeSO4, 0.01 g ZnSO4, 0.01 g CuSO4 and 0.01 g MnSO4; the pH of the culture medium is adjusted to 6.5 and the volume is fixed to 1 L.

[0025] The artichoke extract is prepared by selecting fresh artichokes, washing them, cutting them into small pieces, heating them at 50° C. for 30 minutes, and then filtering them to remove solids. The filtered liquid is collected and freeze-dried to obtain the extract.

[0026] The artichoke samples after the above treatment were subjected to trypsin fermentation treatment respectively, and the specific steps were as follows: 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 by chromatography (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.

[0027] Example 2 Preparation of mixed polypeptides The fermentation liquid of the high temperature stress sample was collected and placed in a 90°C water bath for 10 min to inactivate the enzyme. After the pH was adjusted to 8.3, the sample was directly freeze-dried to obtain a freeze-dried sample for later use. A certain amount of freeze-dried sample was weighed and dissolved in deionized water, centrifuged at 4°C, 8000 rpm / min for 15 min, and the supernatant was taken.

[0028] 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).

[0029] The specific steps are as follows: 10 mL of the supernatant was placed in a 10KDa ultrafiltration tube, and the experimental conditions were set to 4°C to maintain the biological activity of the sample. Subsequently, ultrafiltration was performed using a centrifuge at a speed of 3500 rpm / min for 20 minutes. After centrifugation, part of the ultrafiltrate with a molecular weight less than 10KDa was collected. This part of the liquid contained smaller molecular weight components and was used for the next step. Under the same conditions, the ultrafiltrate was centrifuged again using a 3KDa ultrafiltration tube. Also set to 4°C and 3500 rpm / min, the ultrafiltration time was maintained at 20 minutes. Through the above steps, the ultrafiltrate was continued to be separated, and three components were finally obtained: a component with a molecular weight greater than 10KDa (M3), a component between 3KDa and 10KDa (M2), and a component less than 3KDa (M1).

[0030] After the ultrafiltration step, the filtrates of each component were collected and concentrated to about 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 ingredients.

[0031] Finally, the DPPH scavenging rate test was performed by chromatography to evaluate the antioxidant activity of each component. The DPPH test was performed on each component, and the experimental results showed that the antioxidant activity of component M1 was the greatest among the three components, indicating that antioxidant peptides less than 3KDa may be the components with the highest biological activity.

[0032] Component M1 was separated and purified by Sephadex G-15 chromatography: a certain amount of Sephadex G-15 powder was put into five times distilled water, and expanded for 3 hours at room temperature to fully swell G-15, and the suspended gel particles were poured off. After the chromatography column was fixed, the pretreated gel was slowly poured into the column along the upper port of the column, so that it was naturally precipitated in the column to two-thirds of the column height. Before injection, the gel was continuously balanced with distilled water until the gel had no bubbles and no stratification before injection. Sample volume: 1 mL, eluent was distilled water, sample concentration: 200 mg / mL, flow rate: 1 mL / min, AKTA protein chromatography system UV detection wavelength: 280 nm. Collect the peak components to obtain three components, G1, G2 and G3. After freeze-drying, the DPPH scavenging rate was tested by chromatography to evaluate the antioxidant activity of each component. 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.

[0033] Component G2 was identified by liquid chromatography, tandem mass spectrometry and de novo sequencing to obtain 1252 peptides, of which the heptapeptides were the most prominent, totaling 341. The amino acid distribution showed that the number of peptides containing polar amino acids in component G2 reached more than 80%, and the peptides with a polar amino acid content of 65-80% were the most abundant, accounting for 40.5% of the total number of peptides. Since the content of polar amino acids in antioxidant peptides is closely related to their antioxidant activity, these peptides rich in polar amino acids significantly improved the activity of component G2.

[0034] Example 3 Screening of antioxidant active peptides based on database The peptides obtained by sequencing were screened from the following six dimensions: Confidence: From the peptide sequences identified by LC-MS / MS, peptides with a confidence level of ALC>80% (more reliable) were screened out; Peptide content: The peak area of ​​a peptide segment can reflect its content. From peptide segments with a confidence level > 80%, select peptides with a peak area > 10e 5 peptides; Biological activity score: Area>10×e 5 The peptides were predicted and the peptides with PeptideRanker>0.5 were screened; Water solubility prediction: For the peptides predicted to be potentially biologically active (PeptideRanker>0.5), water solubility prediction was performed in Proteomics tools on the INNOVAGEN website to screen for peptides with “Good” water solubility; Toxicity prediction: The peptides with good water solubility obtained by screening were used for peptide toxicity prediction under the "BatchSubmission" module of the Toixinpred website, and "Non-Toxin" peptides were selected; Allergy prediction: The non-toxic peptides screened were used for allergy prediction in the "AllerTOPServerPage" and the "NON-ALLERGEN" peptides were selected; Example 4 Molecular docking screening of ACE inhibitory peptides Six peptides were screened from the above six dimensions. After the peptides were synthesized, DPPH scavenging rate was further tested by 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. In addition, it was analyzed and identified by electrospray ionization mass spectrometry (ESI-MS). The spectrum is shown in Figure 2 . At the same time, the artichoke AA-peptide sequence has a maximum similarity of 90% with existing sequences in NCBI (National Center for Biotechnology, USA), and a maximum similarity of 58.33% with sequences derived from artichokes. It is a new peptide with a novel sequence, and its polar amino acids account for 75% of the total number of amino acids in the sequence. Because the content of polar amino acids in antioxidant peptides is closely related to their antioxidant activity, this result shows that AA-peptide is a new artichoke antioxidant peptide with potential antioxidant function.

[0035] Example 5 Test on the scavenging effect of antioxidant peptide AA-peptide on hydroxy DPPH free radicals DPPH was dissolved in anhydrous ethanol to a final concentration of 0.04 mol / mL. 2 mL of DPPH solution and 1 mL of anhydrous ethanol were added to 1 mL of antioxidant peptide AA-peptide (concentration of 0.1-1.0 mg / mL) solution, incubated at room temperature for 30 minutes, and then centrifuged at 5000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 517 nm.

[0036] 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.

[0037] The antioxidant activity of the samples was evaluated by the scavenging rate of DPPH using the following equation: DPPH removal rate (%) = (A0-A+A1) / A0× 100% Where A is the sample absorbance; A0 is the control absorbance; A1 is the blank absorbance.

[0038] 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%.

[0039] Example 6 Experiment on the effect of antioxidant peptide AA-peptide on ROS levels in human hepatocytes Human hepatocytes HEPG2 were cultured and the cells (3×10 5 / well) were inoculated into a 6-well plate 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 in each group), control group, model group, AA-peptide group and positive control group.

[0040] 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 with 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 with 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 with 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).

[0041] After all treatments, the 6-well plate was placed in a 37°C, 5% CO2 incubator 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 .

[0042] 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 that the intracellular ROS (Reactive Oxygen Species) level was significantly reduced. Furthermore, the effect of AA-peptide was better than that of GSH, and the ROS level was lower. This result shows that AA-peptide can more efficiently remove intracellular ROS and prevent cell oxidative damage.

[0043] Example 7 Experiment on the effect of AA-peptid on intestinal flora of mice 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 the intestinal microecology. After the clearance, the mice were immediately treated with AA-peptide by gavage 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 6 , the intake of AA-peptide led to a significant decrease in the flora of the Firmicutes phylum, while the abundance of bacteria in the Bacteroidota phylum was significantly increased. This microbial change may be related to the biological activity of antioxidant peptides and their regulatory effects on the intestinal environment. Firmicutes are generally considered to be an important bacterial group in the intestine, involved in nutrient absorption and metabolic functions, and the decrease in its abundance may indicate the role of antioxidant peptides in inhibiting the growth of certain specific bacteria. This change may help reduce the inflammatory response associated with obesity and metabolic syndrome, because high levels of Firmicutes are associated with imbalances in fat storage and energy metabolism. In contrast, the increase in Bacteroidota may be beneficial to intestinal health. Bacteroidota is mainly responsible for breaking down complex carbohydrates and promoting the production of short-chain fatty acids (SCFAs), which have anti-inflammatory, intestinal barrier function enhancement and metabolic regulation effects. Antioxidant peptides may strengthen the intestinal barrier of mice, improve glucose and lipid metabolism, and help maintain the balance and health of the intestinal microbiota by promoting the proliferation of Bacteroidota. In summary, the intake of AA-peptide not only improved the intestinal health of mice, but also significantly changed the composition of intestinal flora, enhanced the abundance of beneficial bacteria, and reduced the proportion of potentially harmful bacteria. This discovery provides new scientific basis and prospects for the application of antioxidant peptides in intestinal health and metabolic regulation.

[0044] 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, enhance their solubility in water, and contribute to their bioavailability in the body. Peptides containing cysteine ​​usually have good antioxidant properties when forming disulfide bonds. At the same time, the positively charged amino acids (such as K, H, R) and polar amino acids (such as T, C) in AA-peptide can react with free radicals to provide hydrogen atoms, thereby effectively neutralizing free radicals and reducing oxidative stress. Furthermore, the cysteine ​​in the AA-peptide sequence can react with free radicals to form stable compounds with strong antioxidant capacity. Finally, the amino acid composition of AA-peptide allows the peptide to have a certain degree of flexibility in space, which may enhance its ability to bind to the target molecule and further improve its antioxidant effect.

[0045] The present invention provides a new type of antioxidant peptide, named AA-peptide. The peptide not only exhibits good antioxidant activity, but also has non-toxic properties, which makes it have a wide range of 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 an intestinal flora regulatory effect, which can significantly improve the intestinal microecological balance, promote the growth of beneficial flora, and inhibit the proliferation of potentially harmful bacteria, indicating 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 be an effective antioxidant to combat cell damage caused by oxidative stress, but also an important component for improving intestinal health. This discovery provides a new direction and basis for further exploring its potential application in functional foods and health products.

Claims

1. An artichoke antioxidant peptide for protecting the liver and improving gastrointestinal function, named AA-peptide, with 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 method for preparing the artichoke antioxidant peptide according to claim 1, characterized in that: The following steps are involved: (1) After placing the artichokes in a 40°C incubator for 3 hours under high temperature stress, samples of the treated artichokes were collected; (2) mixing trypsin with the artichoke sample treated in step (1) for fermentation to obtain a fermentation liquid; (3) treating the fermentation broth to inactivate the enzyme, adjusting the pH value, and removing the supernatant; (4) The supernatant is subjected to ultrafiltration using an ultrafiltration membrane, and the ultrafiltrate with high antioxidant activity is taken out for separation and identification to obtain antioxidant peptides.

5. 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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