Antioxidant polypeptide scp2 and application thereof

Abstract

The application provides an antioxidant polypeptide SCP2 and application thereof, and belongs to the technical field of functional polypeptides. The antioxidant polypeptide SCP2 provided by the application has good solubility, and has ideal antioxidant activity, and has a high clearance rate on ABTS free radicals, DPPH free radicals and hydroxyl free radicals. Therefore, the polypeptide SCP2 provided by the application can be applied to various fields of food, cosmetics and medicine.

Description

Technical Field

[0001] This invention belongs to the field of functional peptide technology, specifically relating to an antioxidant peptide SCP2 and its applications. Background Technology

[0002] With changes in the living environment and people's lifestyles, in addition to breathing, people are exposed to factors such as food and raw material pollution, radiation exposure, etc., which also generate a large number of oxidative free radicals (ROS) in the human body. Excessive production of ROS can cause oxidative damage, which can lead to diseases such as tumors, cardiovascular diseases, and diabetes.

[0003] Antioxidants are widely used in food additives, health products, cosmetics, and drugs for treating cardiovascular and cerebrovascular diseases and cancer. As people’s concerns about the safety of chemically synthesized compounds increase, the use of natural antioxidants is also increasing year by year.

[0004] In recent years, antioxidant peptides have received widespread attention due to their small molecular weight, easy absorption, and safety. Antioxidant peptides are widely found in animals and possess significant antioxidant and anti-aging functions. Various antioxidant peptides have been discovered in frog skin, which can scavenge the vast majority of free radicals within seconds, much faster than commonly used antioxidant additives. These antioxidant peptides' rapid and powerful free radical scavenging ability can maximize the protection of frogs from oxidative free radical damage. Because of their safety and easy absorption by the human body, they are often used as raw materials in food, added directly to effectively delay oxidative spoilage. They are also added as important ingredients in cosmetics and pharmaceuticals to inhibit diseases caused by oxidative reactions in the human body and slow down aging.

[0005] Sea cucumber is not only a delicacy but also a nourishing health food. Records of its nourishing effects can be found in Xie Zhaozhe's *Wuzazu* from the Wanli period of the Ming Dynasty. Zhao Xuemin's *Compendium of Materia Medica Supplement* from the Qing Dynasty states: "Sea cucumber is warm and nourishing, comparable to ginseng, hence its name; it tastes sweet and salty, nourishes the kidneys, benefits essence, eliminates phlegm, controls urination, strengthens yang, treats impotence, and kills sores and parasites." *Pharmacopoeia* and *Pharmacopoeia* provide more detailed records of its medicinal value. Modern pharmacological research shows that sea cucumber possesses various active ingredients with anti-tumor, immunomodulatory, and antioxidant effects. However, research and discovery regarding the antioxidant activity of sea cucumber are still insufficient. The applicant's previous research discovered the presence of polypeptide fragments with antioxidant activity in sea cucumber, but their poor solubility significantly limits their application. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an antioxidant polypeptide SCP2, which has good solubility and high antioxidant capacity.

[0007] This invention provides an antioxidant polypeptide SCP2, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] The present invention provides an antioxidant comprising the antioxidant polypeptide SCP2 and excipients.

[0009] Preferably, the auxiliary material includes water.

[0010] This invention provides an anti-aging cosmetic, comprising the antioxidant polypeptide SCP2 or the antioxidant and cosmetic excipients.

[0011] This invention provides an antioxidant health food, which is prepared by combining the antioxidant polypeptide SCP2 or the antioxidant and food ingredients.

[0012] The present invention provides a pharmaceutical product with antioxidant activity, comprising the antioxidant polypeptide SCP2 or the antioxidant and pharmaceutically acceptable excipients.

[0013] This invention provides the use of the antioxidant polypeptide SCP2 or the antioxidant in the preparation of medicaments for treating diseases related to oxidative damage caused by excessive free radicals.

[0014] Preferably, the oxidative free radicals include at least one of the following: ABTS free radicals, DPPH free radicals, and hydroxyl free radicals.

[0015] Preferably, the oxidative damage-related diseases caused by excessive free radicals include at least one of the following: tumors, cardiovascular diseases, and diabetes.

[0016] This invention provides the application of the antioxidant polypeptide SCP2 or the antioxidant in the preparation of antioxidant health foods or cosmetics with anti-aging effects.

[0017] This invention provides an antioxidant polypeptide SCP2, the amino acid sequence of which is shown in SEQ ID NO:1. The antioxidant polypeptide SCP2 provided by this invention exhibits good solubility, with a solubility exceeding 12.28 mg / ml, which is beneficial for expanding its practical application areas and directions. Furthermore, the embodiments of this invention also measured the scavenging effects of polypeptide SCP2 on ABTS free radicals, DPPH free radicals, and hydroxyl free radicals, as well as its reducing power. The results show that SCP2 polypeptide possesses ideal antioxidant activity, with a scavenging rate of 64.5% for ABTS free radicals, 55.4% for DPPH free radicals, and 58.5% for hydroxyl free radicals, and a reducing power of Fe... 3+ The reducing power is 0.22. It is evident that the polypeptide SCP2 provided by this invention possesses both good solubility and strong ability to remove oxidative free radicals, making it applicable to various fields such as food, cosmetics, and pharmaceuticals.

[0018] Meanwhile, the polypeptide SCP2 is derived from natural polypeptides, has good biocompatibility, and is easy to synthesize and has low production cost. It is also easily absorbed by the body, making it convenient to obtain and use, and suitable for promotion and application in various industries. Attached Figure Description

[0019] Figure 1 The graph shows the results of the assay for the good ABTS free radical scavenging ability of the SCP2 peptide;

[0020] Figure 2 The graph shows the results of the determination of the strong DPPH free radical scavenging ability of the SCP2 peptide.

[0021] Figure 3 The graph shows the results of the determination of the strong hydroxyl radical scavenging ability of the SCP2 peptide. Detailed Implementation

[0022] This invention provides an antioxidant polypeptide SCP2, the amino acid sequence of which is shown in SEQ ID NO:1(KVARCDWEQLQN).

[0023] In this invention, the antioxidant polypeptide SCP2 is derived from a sea cucumber-derived polypeptide with a molecular weight of 1489.63 Da and an isoelectric point of 6.06. The polypeptide SCP2 exhibits good solubility and dispersibility in water, with a solubility increased to over 12.28 mg / ml, which is 11-12 times higher than the solubility of the original polypeptide (1.36 mg / ml), significantly expanding the application range of this polypeptide.

[0024] In this invention, the method for obtaining the polypeptide SCP2 preferably includes obtaining it through solid-phase polypeptide synthesis and purification or through recombinant protein expression.

[0025] In this invention, the polypeptide SCP2 has ideal antioxidant properties, which is beneficial for scavenging free radicals in the body and has good antioxidant activity.

[0026] The present invention provides an antioxidant comprising the antioxidant polypeptide SCP2 and excipients.

[0027] In this invention, the excipients preferably include water. The antioxidant peptide SCP2 has been verified to have good dispersibility and solubility in water, greatly facilitating the subsequent preparation of various products using water as a solvent.

[0028] This invention provides an anti-aging cosmetic, comprising the antioxidant polypeptide SCP2 or the antioxidant and cosmetic excipients.

[0029] In this invention, excessive accumulation of free radicals in the body often leads to cellular or systemic aging. Given that the antioxidant peptide SCP2 has excellent antioxidant properties and can remove free radicals, it can thus delay aging. This invention does not impose special limitations on the excipients used in the cosmetics; any cosmetic raw materials well-known in the art can be used. Furthermore, this invention preferably includes the antioxidant peptide SCP2 in combination with other antioxidant ingredients as an antioxidant or in combination with other active ingredients (such as those for removing blemishes, whitening, or firming the skin) to prepare the cosmetics. The preferred mass percentage of the antioxidant peptide SCP2 in the cosmetics is 0.01–5%, more preferably 0.1%.

[0030] This invention provides an antioxidant health food, which is prepared by combining the antioxidant polypeptide SCP2 or the antioxidant and food ingredients.

[0031] In this invention, the antioxidant polypeptide SCP is added as a food additive during food processing. The health food preferably includes functional beverages, solid beverages, etc.

[0032] This invention provides the application of the antioxidant polypeptide SCP2 or the antioxidant in the preparation of antioxidant health foods or cosmetics with anti-aging effects.

[0033] This invention provides a pharmaceutical product with antioxidant activity, comprising the antioxidant polypeptide SCP2 or the antioxidant and pharmaceutically acceptable excipients. The preferred mass percentage of the antioxidant polypeptide SCP2 in the pharmaceutical product is 0.01–5%, more preferably 0.2%. This invention does not impose any particular limitation on the preparation method of the pharmaceutical product; any method well-known in the art for preparing polypeptide pharmaceutical products may be used.

[0034] This invention provides the use of the antioxidant polypeptide SCP2 or the antioxidant in the preparation of medicaments for treating diseases related to oxidative damage caused by excessive free radicals.

[0035] In this invention, the oxidative damage-related diseases caused by excessive free radicals preferably include at least one of the following: tumors, cardiovascular diseases, and diabetes. The free radicals preferably include at least one of the following: ABTS free radicals, DPPH free radicals, and hydroxyl free radicals. Experiments show that although the antioxidant activity of the antioxidant peptide SCP2 is slightly inferior to that of Vitamin C, it has a good removal effect on several free radicals, with a removal rate of 55% to 65%, among which the removal effect on ABTS free radicals is the best. Furthermore, the antioxidant peptide SCP2 also has reducing power, Fe... 3+ The reducing power is 0.22, while the Fe of Vitamin C is... 3+ The reducing power is 0.3.

[0036] The following detailed description, in conjunction with embodiments, illustrates an antioxidant polypeptide SCP2 and its applications provided by the present invention, but these should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] Determination of the ability of SCP2 peptide to scavenge ABTS free radicals

[0039] Mix 5 mL of 7 mM ABTS solution with 88 μL of 140 mM potassium persulfate solution, and let stand at room temperature in the dark for 12–16 h to obtain ABTS. + Stock solution. Dilute ABTS with phosphate buffer (10 mM). + The absorbance of the stock solution at a wavelength of 734 nm is 0.7 ± 0.2, which is considered ABTS. + The working solution was prepared by adding 20 μL of samples at different concentrations (1, 0.5, 0.25, 0.125, 0.0625 mM) and 280 μL of ABTS working solution to a 96-well plate. The absorbance value A1 was measured at 734 nM, and the absorbance value A2 was obtained by replacing the sample with ddH2O. The sample used was SCP2 peptide (KVARCDWEQLQN) as the experimental group, ascorbic acid (Vitamin C) as the positive control, and H2O2 as the negative control. The experiment was repeated 3 times and the average value was taken. The ABTS free radical scavenging rate was calculated using formula II:

[0040] Clearance rate = (A2-A1) / A2×100% Formula II.

[0041] The results are shown in Table 1 and... Figure 1 Compared to ascorbic acid, SCP2 peptide exhibits better ABTS free radical scavenging ability, with a scavenging rate of 64.5%.

[0042] Table 1. Results of the scavenging ability of SCP2 peptide against ABTS free radicals.

[0043]

[0044] Example 2

[0045] Assay of the ability of SCP2 peptide to scavenge DPPH free radicals

[0046] The antioxidant capacity of the SCP2 peptide was determined by chelating or consuming the stable free radical 2,2-diphenyl-1-picryl-hydrazila (DPPH). DPPH consumption was determined by absorbance assay.

[0047] 50 μL of different concentrations of SCP2 peptide (1, 0.5, 0.25, 0.125, 0.0625, and 0 mM) were placed in 96-well plates. 50 μL of solvent (ddH2O) and 100 μL of DPPH solution (30 mg / mL, using 95% ethanol as solvent) were added to each well, and this was repeated three times. Ascorbic acid was used as a positive control. 50 μL of different concentrations of ascorbic acid (1, 0.5, 0.25, 0.125, 0.0625, and 0 mM, Vitamin C) were added to each well, followed by 50 μL of solvent (ddH2O) and 100 μL of DPPH solution, and this was repeated three times. After mixing the samples, they were precipitated at room temperature in the dark for 30 min. The absorbance at 517 nm was measured using an ELISA reader, and the antioxidant activity of the SCP2 peptide was calculated and statistically analyzed. The scavenging rate was calculated using formula III as follows:

[0048] Clearance rate (%) = [1 - (A1 - A2) / A0] × 100% Formula III

[0049] Where: A1—absorbance of the sample solution + DPPH ethanol solution;

[0050] A2 – Absorbance of the sample solution + 95% ethanol solution (by volume);

[0051] A0 – Absorbance of ultrapure water + DPPH ethanol solution.

[0052] The results are shown in Table 2 and Figure 2 Compared with the control group, the SCP2 peptide exhibited better DPPH free radical scavenging ability, with a scavenging rate of 55.4%.

[0053] Table 2. Results of the scavenging ability of SCP2 peptide against DPPH free radicals.

[0054]

[0055] Example 3

[0056] Determination of the scavenging ability of SCP2 peptides to remove hydroxyl radicals

[0057] Take 50 μL of SCP12 peptide sample solutions of different concentrations (1, 0.5, 0.25, 0.125, 0.0625 mM), add 50 μL of FeSO4 (6 mM) and 100 μL of H2O2 (6 mM) respectively, shake thoroughly and incubate at room temperature for 10 min; add 50 μL of salicylic acid (6 mM, dissolved in ethanol), shake thoroughly and incubate at room temperature for 30 min. Detect the absorbance at 510 nm using an ELISA reader. Repeat three times and take the average value; ascorbic acid was used as a positive control. The clearance rate was calculated using formula IV as follows:

[0058] Clearance rate = (A0-A1) / A0×100% Formula IV.

[0059] In the formula: A0—replaces the absorbance value of the sample with ddH2O;

[0060] A1—Absorbance value of the sample to be tested.

[0061] See results Figure 3 Compared with the control group, the SCP2 peptide exhibited better scavenging ability of hydroxyl radicals, with a scavenging rate of 58.6%.

[0062] Example 4

[0063] SCP2 peptide reducing power measurement

[0064] Take 10 μL of 2 mg / ml SCP2 peptide solution, Vitamin E solution, and H2O2, 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.

[0065] The results are shown in Table 3.

[0066] Table 3. In vitro reducing power results of antioxidant peptide SCP12

[0067]

[0068] 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 SCP2, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. An antioxidant, characterized in that, Includes the antioxidant polypeptide SCP2 as described in claim 1 and excipients.

3. The antioxidant according to claim 2, characterized in that, The auxiliary material includes water.

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