Preparation and application of novel Arcainflata Reeve oxidation-resistant active peptide

A technology of antioxidant activity and active polypeptide, applied in the field of biomedicine, can solve problems such as unseen, rare, rare, etc., and achieve the effect of significant antioxidant activity and increased lifespan

Active Publication Date: 2016-12-07
于荣敏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Domestic scholars mainly focus on the study of cockles on the ecological habits of cockles, breeding technology and analysis of their nutritional components, and there are very few studies on the protein polypeptides of cockles. Protein active substances and their preparation methods and uses”; patent 201310619247.7 announced “the method and use of an anti-tumor polypeptide compound isolated from cockles”, and they all described in detail the extraction of active protein polypep...

Method used

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  • Preparation and application of novel Arcainflata Reeve oxidation-resistant active peptide
  • Preparation and application of novel Arcainflata Reeve oxidation-resistant active peptide
  • Preparation and application of novel Arcainflata Reeve oxidation-resistant active peptide

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Experimental program
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Effect test

Embodiment 1

[0012] A commercial protease is selected to hydrolyze the tissue of cockles, and the active polypeptide components in cockles are separated and prepared by using modern protein separation and purification technology.

[0013] Shell the clam, take its tissue mass, wash it three times with distilled water, weigh it after drying it, add double distilled water according to the ratio of 1:3 (w / v), use a high-speed tissue masher, at 3000 rpm, every Homogenize for 1 min at intervals of 30 sec, for a total of 3 min, place the homogenate in a low-frequency ultrasonic instrument and sonicate for 40 min, add 5% (g / 100 g protein substrate) neutral protease, maintain the temperature at 45°C, and react at pH 7.0 for 5 h. Adjust the pH with 1M NaOH, stop the reaction in a boiling water bath for 10 min, cool to room temperature, then centrifuge at 8000 rpm for 30 min at 4°C to remove the precipitate, and take the supernatant to freeze-dry; Centrifuge at 4°C and 3000 rpm for 60 min, and divide...

Embodiment 2

[0017] Three kinds of in vitro free radical scavenging experiments were used to analyze the antioxidant activity of the active polypeptide compounds in the hydrolyzate of cockles.

[0018] (1) Determination of DPPH free radical scavenging activity: Prepare sample solutions with concentrations of 1, 2, 4 and 8 mg / ml in double-distilled water, take 10 μL of sample solutions with different concentrations and 190 μL of 0.2 mM DPPH solution, and add them to 96 wells In the culture plate, react at room temperature on a horizontal shaker in the dark for 30 min, measure the absorbance value at a wavelength of 517 nm with a microplate reader, set three parallel experiments for each concentration, and take the average value of the results. Reduced glutathione is positive control.

[0019] (2) Determination of ABTS free radical scavenging activity: Prepare sample solutions with concentrations of 1, 2, 4 and 8 mg / ml in double distilled water, take 10 μL of sample solutions with different ...

Embodiment 3

[0026] The model animal Caenorhabditis elegans was used to analyze the anti-aging effect of active peptides in vivo.

[0027] The synchronized L4 stage Caenorhabditis elegans was selected for administration, and the dosages were 2, 4 and 8 mg / ml, respectively, and cultured at 20°C for 48 hours. E. coli Culture in fresh NGM petri dishes of OP50, transfer the surviving C. elegans to fresh NGM petri dishes every day, and accurately record the number of deaths and losses of C. The nematodes cannot respond to external stimuli, and the time when the last C. elegans dies in the blank group and the experimental group is the longest lifespan of the nematodes in this group; the parallel experiment is repeated at least twice, and the number of nematodes in the control group and the experimental group is equal to 50±2 strips.

[0028] surface 5 . The effect of polypeptide compounds on the lifespan of Caenorhabditis elegans ( ±S, n=3, *p<0.05)

[0029]

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Abstract

The invention relates separation preparation and application of an Arcainflata Reeve oxidation-resistant active peptide. The separation preparation method comprises the following steps: peeling and cleaning Arcainflata Reeve, carrying out tissue homogenization, carrying out hydrolysis technique screening and optimization, centrifugating, taking the supernate, and carrying out freeze-drying to obtain an active polypeptide component; and sequentially carrying out ultrafiltration interception concentration, ion-exchange chromatography, gel filtration and high-efficiency exclusion chromatography on the active component to finally obtain the oxidation-resistant active polypeptide with the purity of 99%. Multiple modern protein purification techniques are creatively utilized to separate the active polypeptide with definite material basis from Arcainflata Reeve hydrolysate; the molecular weight of the active polypeptide is 17578.31Da; and the active polypeptide contains the following secondary structures: 40.8% of alpha-spiral, 24.7% of beta-fold, 15.9% of beta-corner and 18.6% of random coil. The polypeptide has obvious oxidation-resistant activity; the in-vitro removal DPPH, ABTS and hydroxy free radical IC50 values are respectively 3.89, 0.22 and 10.77 mg/ml; and the polypeptide can prolong the average life of the Caenorhabditis elegans by 18.29%. Therefore, the active peptide can be used for development and application of antioxidants, medicinal cosmetics or health products.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a polypeptide compound with antioxidant activity in marine organism Arca inflata Reeve, a separation and preparation method thereof, and an antioxidant medical application of the compound. Background technique [0002] Arca inflata Reeve (Arca inflata Reeve) belongs to the Phylum Animals, Phyllobranches, Cockles, and Clamidae. It is a member of the Marine Molluscs Phylum. It is a large marine benthic economic shellfish, mainly distributed in Japan, the Korean Peninsula and China. The soft mud bottom of the Bohai Sea, the Yellow Sea and the East China Sea coast is also one of the important economic shellfish in my country. Its meat is delicious, the blood is bright red, and it has the characteristics of high protein, low fat, and rich vitamins. It has been regarded as a nourishing product since ancient times. , a famous dish with wine, it is recorded in ancient books that cockles ...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/36C07K1/34C07K1/18C07K1/16A61P39/06
Inventor 于荣敏高静静朱建华
Owner 于荣敏
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