Fatty acid modified antioxidant peptide and application thereof

Abstract

The invention belongs to the field of medicine, and relates to an antioxidant peptide and application thereof in tumor treatment, anti-inflammatory and antibacterial related diseases, in particular to a fatty acid modified antioxidant peptide and application thereof, the antioxidant peptide is a novel peptide G-peptide, has remarkable antioxidant activity, and can effectively scavenge free radicals so as to protect cells from oxidative damage. The G-peptide shows the potential application value in antioxidant treatment by improving the cell activity, enhancing the antioxidant capacity of cells and improving the metabolic state of the cells. In addition, the fatty acid modified antioxidant peptide is named as G-peptide-RC18: 2, and the fatty acid modified antioxidant peptide is named as G-peptide-RC18: 2. Through the modification of the linoleic acid, the G-peptide-RC18: 2 shows a stronger anti-oxidation characteristic and an effect of inducing the apoptosis of tumor cells. The G-peptide-RC18: 2 has wide application prospects in anti-tumor, anti-inflammatory, antibacterial and the like related to anti-oxidation prevention and treatment, and provides a new direction for developing novel anti-tumor, anti-inflammatory and antibacterial drugs based on natural products.

Description

Technical Field

[0001] This patent relates to the field of medicine, and particularly to an antioxidant peptide and its applications in tumor treatment, anti-inflammatory and antibacterial related diseases. In particular, the present invention aims to provide a fatty acid-modified antioxidant peptide and its applications, by modifying with fatty acids to improve its biological activity and stability, so as to be widely used in the development of antioxidants. Background Art

[0002] Oxidative stress plays an important role in the pathogenesis of various diseases, such as cardiovascular diseases, diabetes, cancer, and the process of aging. Antioxidants can neutralize free radicals, reduce oxidative stress, and protect cells from damage, so they have received extensive attention in the medical and health care fields. In nature, peptides, as an important bioactive component, exhibit good antioxidant activity. However, peptides from natural sources have certain limitations in terms of stability, bioavailability, and production cost. In addition, natural antioxidant peptides often cannot meet the growing market demand. Recently, as an excellent bioactive molecule, fatty acids have been widely used to modify various bioactive molecules. The introduction of fatty acids not only helps to improve the water solubility and biocompatibility of molecules, but also can improve their antioxidant performance, thus enhancing their application potential. Therefore, the development of a fatty acid-modified synthetic antioxidant peptide has become a current research hotspot.

[0003] In the prior art, although various antioxidant peptides have been studied, there is still a lack of a fatty acid-modified antioxidant peptide that can simultaneously possess high antioxidant ability, good biocompatibility, and excellent stability. Therefore, searching for novel fatty acid-modified antioxidant peptides and exploring their applications will provide new ideas and directions for the development of antioxidants. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a novel antioxidant peptide obtained by fatty acid modification, which is derived from the oligopeptide sequence of marine microorganisms. The antioxidant ability of this antioxidant peptide is significantly improved after fatty acid modification, and it has excellent biological activity and application prospects.

[0006] On the one hand, the present invention provides an antioxidant peptide named G-peptide. The amino acid sequence of the antioxidant peptide G-peptide is Gly-Ile-Arg-Pro-Leu-Gly-Gln-Arg-Ala-Glu (SEQ ID NO.1: GIRPLGQRAE), which is a novel peptide with a new sequence. Its molecular weight is 1211.25 g / mol, and the molecular formula is C 52 H 87 N 19 O 8 .

[0007] One aspect of the present invention provides the nucleic acid sequence of the oxidized peptide G-peptide, as shown in SEQ ID NO.2.

[0008] SEQ ID NO.2: GGTATTCGTCCTCTTGGTCAACGTGCTGAA.

[0009] On the other hand, the present invention also provides a method for preparing fatty acid-modified antioxidant peptides using DCC (N,N'-dicyclohexylcarbodiimide), fatty acids, and polypeptides, which is characterized by including the following steps: (1) Dissolve fatty acids, DCC, and the catalyst for esterification reaction (DMAP) in a dichloromethane solvent, and add the antioxidant peptide to the reaction mixture; (2) React at room temperature for 12 h, and terminate by adding ammonia water (NaOH / EtOH) to dilute the compound or by cooling the reaction mixture.

[0010] Preferably, the fatty acid is oleic acid, linoleic acid, or sulfuric acid.

[0011] The present invention also provides the application of the fatty acid-modified antioxidant peptide in the development of drugs related to tumor treatment, anti-inflammation, and anti-bacterial.

[0012] Preferably, the fatty acid-modified antioxidant peptide can be used for the research and development of new anti-tumor drugs. By utilizing its antioxidant properties and the ability to induce apoptosis of tumor cells, it is expected to significantly inhibit tumor growth and improve the efficacy of existing treatment methods.

[0013] Preferably, the fatty acid-modified antioxidant peptide can also be used as a key component of anti-inflammatory drugs. For the inflammatory reaction caused by oxidative stress, it can alleviate related symptoms and provide effective support for improving the overall health of patients.

[0014] Preferably, the fatty acid-modified antioxidant peptide shows good application prospects in the development of anti-bacterial drugs. It can effectively inhibit bacterial infections related to oxidative stress and enhance the effect of existing anti-bacterial treatment regimens.

[0015] Beneficial effects: (1) The present invention selects the potential strain - deep-sea actinomycete Micromonospora sp. AF14 with high-yield antioxidant peptides screened in the laboratory, induces the synthesis of antioxidant enzymes by amino acid stimulation (medium supplemented with 3% cysteine), and promotes the expression of antioxidant peptides. Further, the culture product is separated and purified, and finally a novel antioxidant peptide is identified.

[0016] (2) Different from the prior art, the antioxidant peptide G-peptide provided by the above technical solution is composed of 10 amino acids, and the amino acid sequence is Gly-Ile-Arg-Pro-Leu-Gly-Gln-Arg-Ala-Glu, with a molecular weight of 1211.25 g / mol and a molecular formula of C 52 H 87 N 19 O 8 . It has high antioxidant activity, no cytotoxicity, good digestive stability and cytoprotective effect. The antioxidant peptide GIRPLGQRAE provided by the above technical solution is expected to play a positive role in the production and preparation of drugs for preventing or treating oxidative stress-related diseases, and the development of drugs related to tumor treatment, anti-inflammation and anti-bacterial. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments and descriptions thereof of the invention are used to explain the invention and do not constitute an improper limitation to the invention.

[0018] Figure 1 It is a result diagram of chromatographic separation and purification of the antioxidant peptide G-peptide; Figure 2 It is a comparison diagram of the scavenging rate of antioxidant peptide G-peptide on hydroxyl DPPH free radicals; Figure 3 It is a comparison diagram of the scavenging rate of linoleic acid-modified antioxidant peptide G-peptide-RC18:2 on hydroxyl DPPH free radicals; Figure 4 It is a comparison diagram of the effect of antioxidant peptide G-peptide pretreatment on the intracellular ROS level of human hepatocytes; Figure 5 It is a comparison diagram of the effect of linoleic acid-modified antioxidant peptide G-peptide-RC18:2 pretreatment on the intracellular ROS level of human hepatocytes; Figure 6 It is a comparison diagram of the apoptosis effect of linoleic acid-modified antioxidant peptide G-peptide-RC18:2 on liver cancer cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The embodiments of the present invention will be described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and the detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0020] In the following embodiments, the chemical reagents used are all purchased from regular chemical reagent suppliers and the purity is analytical pure.

[0021] Example 1 Induction and Identification of Antioxidant Polypeptides By culturing deep-sea actinomycetes Micromonospora sp. AF14 and adopting the amino acid stimulation method, the synthesis of its antioxidant enzymes was further induced to promote the expression of antioxidant peptides. First, seawater agar medium was selected as the Micromonospora basic medium for sp. AF14, and 3% (w / v) cysteine was added as a stimulant. The addition of cysteine was aimed at enhancing the antioxidant capacity of the bacteria and promoting the biosynthesis of antioxidant enzymes. The medium was adjusted to an appropriate pH value (6.5 to 7.5) and sterilized at a temperature controlled at 30°C to ensure aseptic conditions.

[0022] The Micromonospora sp. AF14 was inoculated into the above-prepared medium at an inoculation amount of 3% of the medium volume. Then, it was cultured on a shaker at a speed of 150 rpm for 3 days, and observations were made according to the growth of the bacteria. During the culturing process, samples were taken regularly to monitor cell growth and enzyme activity. Chromatography (DPPH scavenging rate test) was used to detect the changes in antioxidant activity in the culture solution. Over time, with the increase in cysteine concentration, the synthesis of antioxidant enzymes would also increase significantly.

[0023] After the culturing was completed, the culture solution was centrifuged at 12,000 rpm for 30 minutes, and the supernatant was separated using a 1 kDa ultrafiltration membrane. The fraction with Mw < 5 kDa was collected. This fraction was separated by chromatography on a Sephadex G-15 column (1×100 cm), and the fraction with the highest DPPH scavenging rate activity was selected for HPLC separation. Finally, the antioxidant peptide G-peptide was obtained. The N-terminal sequence was detected by an amino acid sequence analyzer, and the amino acid sequence (Sequence 1) of G-peptide was Gly-Ile-Arg-Pro-Leu-Gly-Gln-Arg-Ala-Glu. In addition, it was analyzed and identified by ESI-MS, and the spectrum is shown in Figure 1 .

[0024] Example 2 Preparation of linoleic acid-modified antioxidant peptide G-peptide-RC18:2 An appropriate amount of linoleic acid (1.0 g) was weighed in a beaker, and then DCM was used as a solvent to completely dissolve the oleic acid, with a concentration of about 0.5 M. 0.5 g of DCC was slowly added to the solution of linoleic acid, and the mixture was stirred and reacted for 30 minutes to form an acyl intermediate. After adding 0.1 g of DMAP, the stirring and reaction were continued for 1 hour to improve the reaction activity of the formed acyl intermediate.

[0025] Entrusted to Sangon Biotech (Shanghai) Co., Ltd., the antioxidant peptide G-peptide was synthesized by solid-phase chemical synthesis method with a purity of over 95%. The synthesized antioxidant peptide G-peptide was dissolved in the above solvent. The concentration of the peptide was adjusted to 0.1 M to ensure complete dissolution. The dissolved antioxidant peptide was slowly added to the activated oleic acid solution, and the reaction was carried out with stirring at room temperature for 4 hours while monitoring the reaction progress using thin-layer chromatography.

[0026] After the reaction, it was washed with saturated sodium chloride solution to remove unreacted oleic acid and DCC. The reaction mixture was evaporated to remove the organic solvent using a rotary evaporator to concentrate the product. The concentrated product was redissolved in the aqueous phase for phase separation. The aqueous phase was ultrafiltered using a 5 kDa ultrafiltration membrane, and the supernatant was collected. Further purification was carried out using a Sephadex G-15 column (1×100 cm), and appropriate fractions were collected according to the activity. The selected fractions were separated by HPLC to obtain highly purified oleic acid-modified antioxidant peptide. Reverse-phase high-performance liquid chromatography (RP-HPLC) was used for separation.

[0027] The separated peptide was collected and dried at low temperature to remove the residual solvent, obtaining the final linoleic acid-modified antioxidant peptide. Its mass and molecular structure were analyzed using mass spectrometry (ESI-MS). Further confirmation of its modification and sequence structure was carried out using an amino acid sequence analyzer.

[0028] Example 3 Scavenging test of antioxidant peptide G-peptide and linoleic acid-modified G-peptide-RC18:2 on hydroxyl DPPH radicals DPPH was dissolved in absolute ethanol with a final concentration of 00.04 mol / mL. 2 mL of DPPH solution and 1 mL of ethanol were added to 1 mL of the sample solution. The mixture was incubated at room temperature for 30 minutes. After centrifuging at 5000 rpm for 5 minutes, the absorbance of the supernatant was measured at 517 nm. 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. The antioxidant activity of the sample was evaluated by the scavenging rate of DPPH using the following equation: DPPH scavenging rate (%) = (A 0 - A + A 1 ) / A 0 × 100% where A is the absorbance of the sample; A 0 is the absorbance of the control; A 1 is the absorbance of the blank.

[0029] The results are shown in Figure 2 and Figure 3, it can be seen that the scavenging activities of antioxidant peptide G-peptide and linoleic acid-modified G-peptide-RC18:2 against DPPH increase with the increase of concentration. The scavenging activity of G-peptide-RC18:2 against DPPH is better than that of antioxidant peptide G-peptide. When the concentration of antioxidant peptide G-peptide reaches 1.0 mg / mL, the scavenging rate can reach more than 50%. After modification with fatty acid, the DPPH scavenging activity of antioxidant peptide G-peptide-RC18:2 is significantly enhanced, and the scavenging rate can reach more than 75% when the concentration is 1.0 mg / mL.

[0030] Example 4 Experiment on the effects of antioxidant peptide G-peptide and linoleic acid-modified G-peptide-RC18:2 on the level of ROS in human hepatocytes First, place the sterile cover glass on the 6-well plate. Then, inoculate the cells (3×10 5 cells / well) into the 6-well plate containing 2 mL of culture medium and incubate for 24 hours. After that, treat the cells with 2 mL of peptide solution (final concentrations are 25 μM respectively) and incubate for 24 hours. The peptide solution is considered to be replaced with 2 mL of PBS (0.01 M, pH 7.2 - 7.4) in the model group and the control group. After 24 hours, treat the sample groups with 2 mL of H 2 O 2 with a final concentration of 600 μM and incubate for 4 hours. The control group is treated with 2 mL of DMEM culture medium after hydrogen peroxide treatment. After that, incubate the cells with 50 μL of 2’,7 ’-dichlorodihydrofluorescein diacetate (DCFH-DA) in the cells at 37 °C for 20 minutes. Finally, remove the cover glass with forceps and observe under a fluorescence microscope. The results are shown in Figure 4 and Figure 5 .

[0031] The results show that compared with the model group induced by H 2 O 2 , the intracellular fluorescence intensity is significantly reduced after pretreatment with antioxidant peptide G-peptide and linoleic acid-modified G-peptide-RC18:2, indicating that the intracellular ROS level decreases significantly. Further, pretreatment with antioxidant peptide G-peptide-RC18:2 modified with fatty acid (linoleic acid) has a better effect than G-peptide and a lower ROS level. This result shows that the antioxidant peptide modified with fatty acid can more efficiently scavenge intracellular ROS and prevent cell oxidative damage.

[0032] Example 5 Experiment on the effects of antioxidant peptide G-peptide and linoleic acid-modified G-peptide-RC18:2 on the content of MDA and the activity of SOD The cells (3×105 Cells / well) were seeded into a 6-well plate containing 2 mL of medium and incubated for 24 hours, then treated with 2 mL of peptide solution (final concentration 25 μM) and incubated for another 24 hours. In the model group and the control group, 2 mL of PBS (0.01 M, pH 7.2 - 7.4) was considered to replace the peptide solution. After 24 hours, they were treated with 2 mL of H 2 O 2 to treat the model and sample groups and incubated for 4 hours. The control group was treated with 2 mL of DMEM medium after H 2 O 2 treatment. Cell morphology was observed under an inverted microscope. In addition, the levels of MDA and SOD were measured according to the instructions of the assay kit, and the results are shown in Table 1.

[0033] Table 1 Effects of oxidized peptide G-peptide and linoleic acid-modified G-peptide-RC18:2 on MDA content and SOD activity.

[0034] The results in Table 1 showed that compared with the model group treated only with H 2 O 2 pretreatment with antioxidant peptide G-peptide and linoleic acid-modified G-peptide-RC18:2 could significantly reduce the MDA content and increase the activity of antioxidant enzyme SOD. Moreover, G-peptide-RC18:2 after fatty acid modification showed better effects than G-peptide, indicating that the antioxidant ability of G-peptide was significantly improved after modification with fatty acid (linoleic acid).

[0035] Example 6 Experiment on the effects of antioxidant peptide G-peptide and linoleic acid-modified G-peptide-RC18:2 on apoptosis of hepatoma cells The human hepatoma cell line (HepG2 cells) was cultured until the cells grew to the logarithmic growth phase. The HepG2 cells were divided into a control group (untreated), a G-peptide group (25 μM), a G-peptide-RC18:2 group (25 μM), and a positive control group (cisplatin). At 24 hours after cell seeding, different concentrations of G-peptide and G-peptide-RC18:2 were added and the cells were cultured for another 48 hours. The Annexin V-FITC / PI double staining method was used for cell apoptosis detection. After the culture was completed, the cells were collected, washed with PBS, stained according to the instructions of the flow cytometry kit, and finally the cell apoptosis rate was analyzed by flow cytometry. The results are shown in Figure 6, Pretreatment with antioxidant peptide G-peptide and linoleic acid-modified G-peptide-RC18:2 can significantly promote tumor cell apoptosis. Moreover, G-peptide-RC18:2 after fatty acid modification showed better effects than G-peptide, indicating that the ability of G-peptide to promote apoptosis was significantly enhanced after modification with fatty acid (linoleic acid).

[0036] G-peptide has a mixture of polar and non-polar amino acids, which makes the peptide have good stability in water and can effectively interact with lipids in the aqueous phase and cell membranes. Further, the arginine (R) and glutamic acid (E) in the peptide segment result in the positive and negative charge balance of the peptide segment at physiological pH. This charge property helps the peptide bind to amino acid groups and free radicals, thereby enhancing its antioxidant capacity. With the presence of glutamine (Q) and arginine (R), the peptide segment can form hydrogen bonds, which helps to enhance the stability of its secondary structure and further improve the antioxidant activity. Finally, the combination of arginine (R) and glutamic acid (E) in the peptide chain can interact with free radicals, and the amino group in its side chain can form covalent bonds or hydrogen bonds with peroxides or free radicals, thereby neutralizing their activities. The antioxidant property of G-peptide can be achieved by regulating the expression of related enzymes (such as antioxidant enzymes like SOD, CAT, GPx, etc.), thereby enhancing the resistance of cells under oxidative stress.

[0037] G-peptide-RC18:2 is an antioxidant peptide modified with linoleic acid. The introduction of fatty acid (linoleic acid) modification increases the hydrophobicity of the peptide segment, making the interaction between G-peptide-RC18:2 and biological membranes such as cell membranes stronger. This enhanced membrane-binding ability helps the peptide enter the cell interior more effectively, thus more directly combating oxidative stress. At the same time, fatty acid modification can play an important role in the folding and structural stability of the peptide, forming a suitable spatial structure to make the peptide react with free radicals more efficiently.

Claims

1. An antioxidant peptide named G-peptide, whose amino acid sequence is Gly-Ile-Arg-Pro-Leu-Gly-Gln-Arg-Ala-Glu (SEQ ID NO.1: GIRPLGQRAE), whose molecular weight is 1211.25 g / mol, and whose molecular formula is C 52 H 87 N 19 O8.

2. The nucleic acid sequence of the antioxidant peptide G-peptide according to claim 1, as shown in SEQ ID NO.2, SEQ ID NO. 2: GGTATTCGTCCTCTTGGTCAACGTGCTGAA.

3. An application of a fatty acid-modified antioxidant peptide, characterized in that: The antioxidant peptide is the antioxidant peptide G-peptide described in claim 1 or claim 2.

4. The method for preparing the antioxidant peptide according to claim 1, characterized in that: The following steps are involved: (1) Dissolve fatty acids, DCC (N,N'-dicyclohexylcarbodiimide) and esterification catalyst (DMAP) in dichloromethane solvent, and add antioxidant peptides to the reaction mixture; (2) reacting at room temperature for 12 hours, and then terminating the reaction by adding aqueous ammonia (NaOH / EtOH) to dilute the compound or by cooling the reaction mixture; Preferably, the fatty acid is oleic acid, linoleic acid or sulfated acid.

5. Use of the antioxidant peptide G-peptide according to claim 1 or the fatty acid-modified antioxidant peptide according to claim 3 in the following fields: (1) Application of G-peptide-RC18:2 in the preparation of drugs for inhibiting tumor cell growth and promoting cell apoptosis; (2) Application of G-peptide-RC18:2 in the preparation of anti-inflammatory drugs; (3) Application of G-peptide-RC18:2 in the preparation of antibacterial drugs.

Images