An antioxidant peptide CATH1, its prepared products and applications

Abstract

This invention provides an antioxidant peptide CATH1, its prepared products, and its applications, belonging to the field of functional peptide technology. The antioxidant peptide CATH1 provided by this invention has the amino acid sequence shown in SEQ ID NO:1. The antioxidant peptide CATH1 provided by this invention exhibits good solubility and ideal antioxidant activity, showing high scavenging rates against ABTS and DPPH free radicals. Therefore, the peptide CATH1 provided by this invention can be applied in various fields such as food, skincare products, and pharmaceuticals.

Description

Technical Field

[0001] This invention belongs to the field of functional peptide technology, specifically relating to an antioxidant peptide CATH1 and its prepared products and applications. Background Technology

[0002] ROS are a class of oxygen-containing substances containing unpaired electrons that can exist independently and possess strong oxidizing properties. Various environmental factors such as pollution, radiation exposure, and nutrient deficiencies can disrupt the balance between oxidation and antioxidation in the body, leading to the production of large amounts of free radicals (ROS). Excessive ROS production in the body can cause various diseases, such as arthritis, muscle dystrophism, pulmonary dysfunction, tumors, cardiovascular disease, and diabetes.

[0003] Antioxidants have a wide range of applications, including food additives, livestock and poultry farming, skincare products, health supplements, and various drugs for treating cardiovascular and cerebrovascular diseases. Common antioxidants on the market include sandoquinone, tert-butylhydroxyanisole, butylated hydroxytoluene, propyl gallic acid, and tert-butylhydroquinone (TBHQ). Although antioxidants prepared through chemical synthesis are less expensive and more effective, their potential toxicity limits their application in skincare products, livestock and poultry farming, and the pharmaceutical industry.

[0004] In recent years, antioxidant peptides have attracted widespread attention due to their advantages such as high activity, low molecular weight, simple structure, easy absorption, efficient ROS scavenging, simple acquisition, and low toxicity, and are gradually becoming alternatives to synthetic antioxidants. However, the antioxidant activity of currently reported antioxidant peptides is often not ideal. In addition, the application of peptides often requires certain water solubility, and poor solubility greatly affects the multi-field application of peptides. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an antioxidant polypeptide CATH1, which has good solubility and high antioxidant capacity.

[0006] This invention provides an antioxidant polypeptide CATH1, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] The present invention provides an antioxidant comprising the antioxidant polypeptide CATH1 and pharmaceutical excipients.

[0008] The present invention provides a drug with antioxidant activity, comprising the antioxidant peptide CATH1 and pharmaceutically acceptable excipients.

[0009] This invention provides an anti-aging skincare product, comprising the antioxidant peptide CATH1 and skincare excipients.

[0010] This invention provides a health food with antioxidant properties, comprising the antioxidant polypeptide CATH1 and food additives.

[0011] This invention provides the use of the antioxidant polypeptide CATH1 or the antioxidant in the preparation of medicaments for the prevention and / or treatment of diseases related to oxidative damage caused by excessive free radicals.

[0012] Preferably, the oxidative free radicals include ABTS free radicals and / or DPPH free radicals.

[0013] Preferably, the oxidative damage-related diseases caused by excessive free radicals include at least one of the following diseases: tumors, cardiovascular diseases, and diabetes.

[0014] This invention provides the application of the antioxidant polypeptide CATH1 or the antioxidant in the preparation of skin care products or health foods with anti-aging effects or antioxidant effects.

[0015] This invention provides an antioxidant peptide, CATH1, with the amino acid sequence shown in SEQ ID NO:1. The antioxidant peptide CATH1 provided by this invention exhibits good solubility, with a solubility exceeding 11.59 mg / ml, which is beneficial for expanding its practical application areas and directions. This invention also evaluated the scavenging effects and reducing power of peptide CATH1 on ABTS and DPPH free radicals. The results show that peptide CATH1 possesses ideal antioxidant activity, with a scavenging rate of over 65.13% for ABTS free radicals and over 61.46% for DPPH free radicals. Therefore, the peptide CATH1 provided by this invention not only has good solubility but also strong performance in scavenging oxidative free radicals, and can be applied in various fields such as food, skincare products, and pharmaceuticals.

[0016] Meanwhile, the peptide CATH1 exhibits good cell safety and low hemolytic activity. Furthermore, the peptide CATH1 has a small molecular weight, making it easily absorbed by the body. It is also convenient to synthesize and has low production costs, making it easy to obtain and use, and suitable for promotion and application in various industries. Attached Figure Description

[0017] Figure 1 The results show the determination of the scavenging ability of the peptide CATH1 against ABTS free radicals.

[0018] Figure 2 The results show the determination of the scavenging ability of the peptide CATH1 against DPPH free radicals.

[0019] Figure 3 The results are for the hemolytic activity assay of peptide CATH1;

[0020] Figure 4The results are from the cytotoxicity assay of the peptide CATH1. Detailed Implementation

[0021] This invention provides an antioxidant polypeptide CATH1, the amino acid sequence of which is shown in SEQ ID NO:1(RITKQPWAPPQAARICQFVLIRVCR).

[0022] In this invention, the antioxidant peptide CATH1 has a molecular weight of 2,951.59 Da and an isoelectric point of 11.54. The peptide CATH1 exhibits good solubility and dispersibility in water, with a solubility of 11.59 mg / ml. The C-terminus of the antioxidant peptide CATH1 is modified with an amino group (-NH2) to maintain the structural stability of the antimicrobial peptide CATH2.

[0023] In this invention, the antioxidant polypeptide CATH1 is derived from the submandibular gland of goats, specifically from submandibular gland tissues of 1-month-old, 6-month-old, and 12-month-old female goats, and obtained by transcriptomic analysis of differentially expressed genes.

[0024] The present invention does not specifically limit the preparation method of the antimicrobial peptide CATH2, and preferably includes direct synthesis, prokaryotic expression, and eukaryotic expression. In the embodiments of the present invention, the antioxidant peptide CATH1 is preferably prepared by peptide solid-phase synthesis and purification or by recombinant protein expression.

[0025] The present invention provides an antioxidant comprising the antioxidant polypeptide CATH1 and pharmaceutical excipients.

[0026] In this invention, the formulation excipient is preferably water or a buffer solution using water as a solvent. The water is preferably distilled water, double-distilled water, deionized water, etc. Experimental verification shows that the antioxidant peptide CATH1 has good dispersibility and solubility in water, greatly facilitating the subsequent preparation of various products using water as a solvent. The working concentration of the antioxidant peptide CATH1 in the antioxidant is preferably 0.6 mM or higher, more preferably 0.8–5.0 mM, and most preferably 1.0 mM. This invention does not impose any special limitations on the preparation method of the antioxidant; any method well-known in the art can be used, such as fully dissolving the antioxidant peptide CATH1 in a solvent.

[0027] The present invention provides a drug with antioxidant activity, comprising the antioxidant peptide CATH1 and pharmaceutically acceptable excipients.

[0028] In this invention, the mass percentage of the antioxidant polypeptide CATH1 in the drug is preferably 0.01% to 5%, more preferably 0.05% to 4%, further preferably 0.1% to 3%, and most preferably 0.2%. This invention does not impose any special limitations on the preparation method of the drug; any method for preparing polypeptide drugs well-known in the art can be used.

[0029] This invention provides an anti-aging skincare product, comprising the antioxidant peptide CATH1 and skincare excipients.

[0030] This invention does not impose any special restrictions on the types of excipients used in the skincare products; any skincare excipients well-known in the art can be used. This invention also does not impose any special restrictions on the preparation method of the anti-aging skincare products; any preparation method for peptide-containing skincare products well-known in the art can be used. In the anti-aging skincare products, the mass percentage of the antioxidant peptide CATH1 is preferably 0.01% to 5%, more preferably 0.05% to 2%, and most preferably 0.1%.

[0031] This invention provides a health food with antioxidant properties, comprising the antioxidant polypeptide CATH1 and food additives.

[0032] In this invention, the health food is preferably a functional beverage, a solid beverage, etc. The antioxidant polypeptide CATH1 is added as a food additive during food processing. The mass concentration of the antioxidant polypeptide CATH1 in the health food is preferably 0.01% to 5%, more preferably 0.1% to 1%, and most preferably 0.2%.

[0033] This invention provides the application of the antioxidant polypeptide CATH1 or the antioxidant in the preparation of skin care products or antioxidant health foods with anti-aging effects.

[0034] This invention provides the use of the antioxidant polypeptide CATH1 or the antioxidant in the preparation of medicaments for the prevention and / or treatment of diseases related to oxidative damage caused by excessive free radicals.

[0035] In this invention, the oxidative free radicals preferably include ABTS free radicals and / or DPPH free radicals. Oxidative damage-related diseases caused by excessive oxidative free radicals include at least one of the following: cancer, cardiovascular disease, and diabetes.

[0036] In this embodiment of the invention, free radical scavenging experiments show that the antioxidant peptide CATH1 has a good removal effect on ABTS free radicals and DPPH free radicals, with a removal rate of 55% to 60%, among which the removal effect on ABTS free radicals is the best.

[0037] In this embodiment of the invention, hemolytic activity assays and cytotoxicity assays showed that peptide CATH1 at different concentrations did not exhibit cytotoxicity, indicating that peptide CATH1 has high safety; furthermore, the hemolytic activity of peptide CATH1 was low at different concentrations. This suggests that peptide CATH1 can be used in the preparation of antibacterial drugs or skincare products, especially medical aesthetic products.

[0038] The following detailed description, in conjunction with embodiments, illustrates an antioxidant polypeptide CATH1 provided by the present invention, its prepared products, and its applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Determination of the ability of peptide CATH1 to scavenge ABTS free radicals

[0041] Determination principle: 2,2-Azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) can be oxidized to generate a relatively stable blue-green free radical cation ABTS. + ABTS exhibits a maximum absorption peak at 734 nm and, in the presence of an antioxidant with hydrogen-donating capacity, [the following is likely a separate, unrelated sentence: ABTS has a maximum absorption peak at 734 nm]. + It reacts to form colorless ABTS, and the change in absorbance at 734 nm can reflect the antioxidant's ability to scavenge ABTS+.

[0042] PBS buffer preparation: Dissolve 8.18g NaCl, 0.27g KH2PO4, 3.58g NaHPO4·11H2O, and 0.15g KCl in 1L of deionized water, and adjust the pH to 7.4 with 0.1M NaOH.

[0043] Preparation of ABTS stock solution: Dissolve ABTS in PBS buffer to a final concentration of 2 mM.

[0044] ABTS + Solution preparation: Mix ABTS stock solution and 70 mM potassium persulfate (K2S2O8) aqueous solution at a volume ratio of 250:1, and let stand at room temperature in the dark for 15–16 hours. Before the experiment, prepare ABTS... + The solution was diluted with PBS until the absorbance at 734 nm was 0.80 ± 0.03.

[0045] Preparation of CATH1 solution samples of different concentrations: Using water as solvent, CATH1 aqueous solution samples of 0.2, 0.4, 0.6, 0.8 and 1.0 mM were prepared respectively.

[0046] Take 2 μL and 48 μL of CATH1 samples at different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mM) and the above-corrected ABTS.+ After mixing the solutions and allowing them to stand at room temperature for 10 minutes, the absorbance of the reaction solution was measured at a wavelength of 734 nm. The sample dissolution medium served as a blank control, and ascorbic acid of the same concentration served as a positive control. ABTS was calculated according to Formula I. + Clearance rate.

[0047] ABTS + Clearance rate (%) = (AB - AA) / AB × 100% Formula I

[0048] Where AB: absorbance value of blank control; AA: absorbance value of sample. Each sample is tested in triplicate, and the results are mean ± SD.

[0049] See results Figure 1 Compared with ascorbic acid, CATH1 peptide has a better ABTS free radical scavenging ability, with a scavenging rate of 65.13%.

[0050] Example 2

[0051] Assay of the ability of CATH1 peptide to scavenge DPPH free radicals

[0052] Measurement principle: The antioxidant capacity of CATH1 peptides is determined by chelating or consuming the stable free radical 2,2-diphenyl-1-picryl-hydrazila (DPPH). DPPH consumption is measured by absorbance detection.

[0053] Weigh a certain amount of DPPH, dissolve it in methanol, and prepare a solution of 6×10 -5 Solution M was prepared fresh for immediate use. 48 μL of DPPH solution was mixed with 2 μL of CATH1 samples of different concentrations, ensuring a final mass ratio of DPPH to M solution of 3:1. The mixture was allowed to stand at room temperature in the dark for 30 min, and then the absorbance was measured at 517 nm. The UV spectrophotometer was zeroed using methanol. The blank control group consisted of sterile ultrapure water as the sample dissolution medium, with ascorbic acid of the same concentration used as a positive control. Each sample was tested in triplicate, and the results are expressed as mean ± SD. The DPPH clearance rate was calculated according to Formula II.

[0054] DPPH clearance rate (%) = (AB - AA) / AB × 100% (Formula II)

[0055] AB: Absorbance of blank control; AA: Absorbance of sample.

[0056] See results Figure 2 Compared with the control group, the CATH1 peptide showed better DPPH free radical scavenging ability, with a scavenging rate of 61.46%.

[0057] Example 3

[0058] CATH1 peptide reducing power assay

[0059] Take 10 μL of 2 mg / mL CATH1 peptide solution and Vitamin C solution, respectively, and mix them with 50 μL of sodium phosphate buffer and 50 μL of 1% K3Fe(CN)6. Incubate at 50 °C for 20 min. Then add 50 μL of 10% trichloroacetic acid and centrifuge at 3000 rpm for 10 min. Take 50 μL of the supernatant and add 50 μL of deionized water and 10 μL of 1% FeCl3. Measure the absorbance at 700 nm. The stronger the absorbance, the stronger the reducing power.

[0060] The results are shown in Table 1.

[0061] Table 1. In vitro reducing power results of antioxidant peptide SCP12

[0062]

[0063] Example 4

[0064] The specific steps for determining the hemolytic activity and cytotoxicity of peptide CATH1 are as follows:

[0065] 1. Adjust the dilution of the washed red blood cells to 10 with physiological saline. 7 ~10 8 The red blood cell suspension was collected at a concentration of [number] cells / mL, and then mixed with peptide samples of different concentrations. The mixture was incubated at 37°C for 30 min. After centrifugation at 1000 rpm for 5 min, the supernatant was transferred to a 96-well plate, and the UV absorbance was measured at 540 nm. The negative control was physiological saline, and the positive control was Triton X-100. As a positive control, hemolytic activity was directly proportional to the absorbance at 540 nm; hemolytic activity is shown in [details omitted]. Figure 3 ;

[0066] Depend on Figure 3 It can be seen that the light absorption value of the peptide CATH1 at 540 nm is significantly lower than that of the positive control Triton X-100, but very close to that of the negative control. Therefore, it can be concluded that the hemolytic activity of the peptide CATH1 is low at different concentrations.

[0067] 2. Activated HaCAT (human keratinocytes), HepG2 (liver cancer cells), and 4T1 (mouse breast cancer cells) cells were seeded separately into culture dishes. When the cells covered approximately 80% of the bottom area of ​​the dish, they were digested with 0.25% trypsin. The cells were gently scraped off, pipetted until single cells were formed, and then transferred to sterile centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in RPMI 1640 cell culture medium. After rehydration, the cells were counted using a hemocytometer. After counting, the cells were diluted to the required concentration for the experiment, and these were then used as seed cells.

[0068] Take a sterile 96-well plate and add 10,000 seed cells to each well. Adjust the total volume of each well to 100 μL using RPMI 1640. After the cells adhere, add peptide samples of various concentrations, and set up a plate control and a positive control group. Incubate at 37℃ for 24 h, then add 20 μL of LTT solution (5 mg / mL, freshly prepared and stored in the dark), and continue incubation for 4 h. Then discard the cell culture medium in each well, add 150 μL of DMSO (dimethyl sulfoxide), and gently shake to dissolve the crystals. Measure the absorbance at 490 nm using a full-wavelength microplate reader. The results are as follows. Figure 4 As shown.

[0069] Depend on Figure 4 As can be seen, compared with the control group NC, the peptide CATH1 at different concentrations did not exhibit cytotoxicity, indicating that the peptide CATH1 described in this invention has high safety.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antioxidant polypeptide CATH1, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. An antioxidant, characterized in that, It includes the antioxidant peptide CATH1 as described in claim 1 and pharmaceutical excipients.

3. A drug with antioxidant activity, characterized in that, It includes the antioxidant peptide CATH1 as described in claim 1 and pharmaceutically acceptable excipients.

4. An anti-aging skincare product, characterized in that, It includes the antioxidant peptide CATH1 as described in claim 1 and excipients for skin care products.

5. A health food product with antioxidant properties, characterized in that, Includes the antioxidant peptide CATH1 as described in claim 1 and food additives.

6. The use of the antioxidant polypeptide CATH1 of claim 1 or the antioxidant of claim 2 in the preparation of skin care products or health foods with anti-aging effects or antioxidant effects.

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