Preparation And Identification Of HepG2 Cells Inhibitory Peptides From Mung Bean

Tumor inhibitory activity of peptides has been attracting more and more attention.Tumor inhibition of small peptides has gradually become a research hotspot.In order to screen out the mung bean bioactive peptides with HepG2 cells proliferation inhibitory activity,mung bean protein isolates were used as raw materials to prepare mung bean active peptides using different enzymes for single and double enzymatic hydrolysis.The degree of hydrolysis and HepG2 cells proliferation inhibition rate were used as indicators.The inhibition rate was determined by CCK-8 method,and the relationship between degree of hydrolysis and inhibition rate were examined.The active peptides with higher inhibition rate were further separated and purified by using ultrafiltration and optimized Sephadex G-15 gel chromatography conditions.The structure of the inhibitory peptides of HepG2cells with the highest inhibition rate were identified by LC-MS/MS.The main results are as follows:?1?Examine the degree of hydrolysis and the inhibition rate of HepG2 cell proliferation of four hydrolysates.The degree of hydrolysis were:double enzyme hydrolysates>alcalase hydrolysates>papain hydrolysates>neutral protease hydrolysates,and the inhibition rate was:papain hydrolysates>double enzyme hydrolysates>neutral protease hydrolysates>alcalase hydrolysates.The results showed that the degree of hydrolysis of mung bean peptides was not positively correlated with its inhibitory rate,and papain was selected as the best enzyme for the preparation of mung bean active peptides.The mung bean peptide has significant proliferation inhibitory effect on HepG2 human hepatoma cells,and exhibits a certain dose effect,at the doses of 16 mg/mL,the inhibitory rate was78.74%and the IC₅₀0 value at 48 h was 2.99 mg/mL.?2?Two fractions of>3 kDa and<3 kDa obtained from ultrafiltration of mung bean bioactive peptides.The inhibition rate of<3 kDa fraction was higher than that of>3 kDa fraction,and the inhibition rate was 62.28%.Fraction of<3 kDa were separated by Sephadex G-15 gel chromatography,and the best elution conditions were determined:eluent:0.01 mol/L acetic acid solution,flow rate:0.3 mL/min,one tube per 8min,and 70tubes were collected.Under this condition,five fractions of A-E are obtained.Among them,the fraction A had a significant proliferation inhibition effect on HepG2 cells,with a inhibition rate of 86.35%.?3?Detected by HPLC method,the molecular weight of fraction A was mainly under1000 Da,accounting for 89.52%of the total.The molecular weight above 1000 Da only10.4%;the molecular weight of mung bean peptides were focused on the below 2000 Da,accounting for 90.28%of the total.?4?Five short peptides were identified by Q Exactive Plus mass spectrometry,and the amino acid sequences of four short peptide segments were:LAFGINAENNQRN,VEGINKIVTGNL,EGAPLEDIAEEEEQ and PQGEVSTSVAADQ,which mainly contained the amino acids such as Ala,Asn,Gly,Gln,Ile.The bioactive peptides containing these amino acids may play a major role in inhibiting the proliferation of HepG2 cells.