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Antioxidant Peptides and Their Genes of Frog Rana and Their Application in Pharmaceuticals

A technology of antioxidant peptides and drugs, applied in the field of biomedicine

Active Publication Date: 2020-07-10
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is based on the above technical background, aiming at the shortage of natural antioxidants and the safety problems of artificially synthesized antioxidants, to provide a new antioxidant peptide and its gene and its Application in the preparation of drugs and food additives with antioxidant effect

Method used

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  • Antioxidant Peptides and Their Genes of Frog Rana and Their Application in Pharmaceuticals
  • Antioxidant Peptides and Their Genes of Frog Rana and Their Application in Pharmaceuticals
  • Antioxidant Peptides and Their Genes of Frog Rana and Their Application in Pharmaceuticals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Cloning of Antioxidant Peptide Gene of Frog

[0031] I. Extraction of total RNA from the skin of Huaji frog: Wash the living Huaji frog with water, put it in liquid nitrogen and quick-freeze for 4h, take the skin tissue, weigh it, take 300mg skin tissue, add 10ml total RNA extraction buffer (Trizol solution, USA GIBCOBRL company product), homogenized in a 20ml glass homogenizer for 30 minutes. Add an equal volume of phenol / chloroform solution, mix vigorously, place at room temperature for 10 minutes, centrifuge at 4°C, 12000 rpm for 10 minutes, and discard the precipitate. Add an equal volume of isopropanol to the supernatant, place it at room temperature for 10 minutes, centrifuge at 4°C, and 12000 rpm for 10 minutes. Wash the pellet once with 75% ethanol and air dry. The sediment at the bottom of the tube is the total RNA of the skin of the frog.

[0032] II. Purification of the skin mRNA of the flower frog skin: the separation and purification of the skin mRNA...

Embodiment 2

[0044] Example 2, Preparation of Antioxidant Peptides from the Flowering Frog

[0045] Ⅰ. Preparation method of Huaji frog antioxidant peptide: According to the gene of Huaji frog antioxidant peptide, the amino acid sequence encoding the functional mature secretion peptide is inferred and the polypeptide is synthesized by an automatic peptide synthesizer. The formation of disulfide bonds adopts the air oxidation method, specifically, the polypeptide is dissolved in a 0.1 mg / ml 0.1% acetic acid solution in a flask, then titrated with ammonium hydroxide to pH 7.8, and then stirred at room temperature overnight. Desalting and purifying by HPLC reversed-phase C18 column chromatography. During purification, liquid A is 0.05%TFA+2%CH 3 CN, B solution is 0.05%TFA+90%CH 3 The gradient of CN, B solution is 30-50% in 20min, the detection wavelength is 220nm, and the peptide appears in 12.962 minutes.

[0046] Ⅱ. The molecular weight is determined by fast atom bombardment mass spectrometry (...

Embodiment 3

[0049] Example 3, the activity experiment of the anti-oxidant peptide of Hua Ji frog

[0050] Ⅰ. Determination of antioxidant capacity

[0051] 1) Determination of DPPH free radical scavenging ability

[0052] Using DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging rate determination method to study antioxidant peptides. Prepare a DPPH ethanol solution with a concentration of 1×10-5mol / L and store in the dark. Add 2ml, 0.1mM DPPH anhydrous ethanol solution to a clean test tube containing 2ml samples of different enzymatic digestion, and mix. After being placed at room temperature for 30 minutes, the absorbance was measured at 517 nm. The smaller the absorbance value, the stronger the free radical scavenging ability.

[0053] Clearance rate (%) = [1-(A i -A j ) / A 0 】*100%

[0054] In the formula, A 0 2ml, 0.1mM DPPH absolute ethanol solution + 2ml sample reagent, blank control, A i 2ml, 0.1mM DPPH absolute ethanol solution + 2ml sample, A j It is a sample of 2ml absolute e...

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Abstract

The invention relates to active polypeptide and a gene thereof, and application of the active polypeptide to pharmacy. Microhyla pulchra antioxidation peptide is cyclopeptide consisting of 12 pieces of amino acid, wherein the molecular weight is 1334.91 daltons; the isoelectric point is 8.002; the amino acid sequence is SEQ ID NO.1; and the third-position cysteine and the eleventh-position cysteine of the polypeptide form intramolecular disulfide bonds. The gene sequence of the microhyla pulchra antioxidation peptide consists of SEQ ID NO.4, and the gene encoded with the functional mature microhyla pulchra antioxidation peptide is the 187th to the 222nd position nucleotide. The gene of the microhyla pulchra antioxidation peptide deduces the mature functional polypeptide amino acid sequence, and the synthesized microhyla pulchra antioxidation peptide has strong erythrocyte agglutination and anti-oxidation functions.

Description

Technical field: [0001] The invention relates to the field of biomedicine, in particular to a protein obtained from animal tissues and its use in biopharmaceuticals. Background technique: [0002] The oxidation of biomolecules is a process mediated by free radicals, which will cause many adverse effects on food and biological systems. In aerobic organs, free radicals related to various diseases such as arteriosclerosis and cancer are inevitably produced along with the process of oxygen metabolism. In addition, oxidative stress is believed to be related to various diseases such as Alzheimer’s disease and Parkinson’s disease. This pathology is caused by diabetes, complications caused by diabetes, rheumatoid arthritis and amyotrophic lateral sclerosis. The induced neurodegeneration is related (Food Chemistry, 2008, 107, 1485–1493). In food, the oxidation of nutrients will produce peroxides, which will not only affect the nutritional value of the food, cause the decline of food qua...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C12N15/12A61K38/10A61K8/64A61K47/42A61P39/06A61P25/16A61P1/04A61P1/18A61Q19/08
CPCA61K8/64A61K38/00A61K47/42A61Q19/08C07K7/08
Inventor 徐学清陈新曾白霜张贝
Owner SOUTHERN MEDICAL UNIVERSITY
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