Identification Of Bio-Active-Peptides From Apricot Kernel Inhibiting Nitric Oxide Production Induced By Lipopolysaccharide

Apricot (Prunus armeniaca L. var. ansu Maxim.) kernel protein was hydrolyzed separately by five different proteases. The sequential purification of the hydrolysates was carried out by ultra-filtration, to isolate and identify the nitric oxide inhibitory peptide, Nano-LC-ESI-MS/MS technique and molecular docking technique were emoloyed.The main results were as follows:(1) Apricot kernel protein was hydrolyzed by five different proteases respectively. The effect of apricot protein hydrolysates on LPS-stimulated RAW264.7 macrophage cell was investigated by Griess assay. The hydrolysates produced by papain showed the significant growth inhibitory activity on the LPS-stimulated RAW264.7 macrophage cell (when the concentration was 500μg/mL, the unwanted NO production dropped from 31.2 μM to12.0 μM, dropped by 75%,While the other four different enzymatic protein hydrolysates showed no obvious inhibitory effect.). Therefore the follow researches focused on apricot kernel papain hydrolysates, apricot hydrolysates for short.(2) The condition prepareing apricot kernel protein hydrolysates was optimized by single factor experiment and an orthogonal test design (L9)(3)4). The optimized conditions were 5% substrate concentration (S) and an enzyme-substrate ratio (E/S) of 10% at temperature 60℃ for 3h incubation time.(3) Utrafiltration membrane with MWCO 1 kDa, MWCO 3kDa, MWCO 10kDa were used to separate the walnut protein hydrolysate into three fractions,10kDa>MW>3kDa,3kDa>MW>1kDa, MW<1kDa. Fraction MW<3kDa showed the highest NO production inhibitory activity among the three fractions (when the concentration was 500μg/mL, NO production was inhibited from 31.2μM. to 6.1 μM,110% the blank group.)(4) After employing Nano-LC-ESI-MS/MS and NCBI database searching,16 apricot kernel originated peptide were characterized.2 of them were picked for subsequent observation due to their prominent binding energy with iNOS in silico. The amino acid sequence of the 2 fractions were identified as DMTEEF and FEAYEF by N-amino acid sequencing assay(5) DMTEEF and FEAYEF showed greater NO inhibitory effect by Griess assay in LPS-stimulated RAW264.7 macrophage cell model at concentration level of 50-100μg/mL.(6)DMTEEF and FEAYEF also showed great anti-oxidative effect in vitro.·OH、MDA、SOD and GSH-Px were all enhanced.(7)It is indicated that DMTEEF and FEAYEF have both NO inhibitory effect and anti-oxidative effect.’OH was inhibited from abnormal 153% to 98% and 101% separately by DMTEEF and FEAYEF, MDA was inhibited from abnormal 142% to 100% and 93% separately by DMTEEF and EAYEF, SOD was enhanced from abnormal 52% to 99% and 99.5% separately by DMTEEF and FEAYEF; GSH-Px was enhanced from 66% to 90% and 81% by DMTEEF and FEAYEF.As shown above, apricot kernel protein papain hydrolysates produced under T 60℃, time 3h, S 5%, E/S 10% situation exhibit strong ability inhibiting LPS-stimulated NO production enhancement, and the mechanism is highly related to its anti-oxidative ability.