Bioactive peptide derived from trypsin hydrolysis of unicellular cyanobacterium synechococcus sp.

Abstract

A free radicals can promote denaturing reactions in many cellular components, which create oxidative stress and it results to damage in body tissue. The free radicals can causing many diseases including heart diseases, Alzheimer, inflammation and cancer. This research aimed to investigate the effect of bioactive peptides derived from Synechococcus sp. cultured for 21 days on antioxidant, anti-inflammation and anticancer activities. The Synechococcus sp. protein hydrolysate was prepared by trypsin digest and purified by ultrafiltration with molecular weight cut off membranes of 10, 5 and 3 kDa. The MW <3 kDa fraction showed the highest NO, ABTS and DPPH radical scavenging activities with IC50 values of 34.51±9.8 µg protein/mL, IC50 11.54±0.3 µg protein/mL and IC50 13.63±0.15 µg protein/mL, respectively. The F2 fraction from gel filtration chromatography showed the strongest NO and ABTS radical scavenging activities with IC50 values of 7.68±0.64 µg protein/mL and 9.74±0.2 µg protein/mL, respectively. The F2 was purified by RP-HPLC to yield four fractions. The 30 – 40 min sub-fraction (F2-4) was selected for further analysis by mass spectroscopy. Five isolated peptides with amino acid sequences of AILQSYSAGKTK; 1,265.69 Da, ALNKTHLIQTK; 1,265.74 Da, LLVHAPVK; 875.55 Da, IPDAHPVK; 875.48 Da and VVVLRDGAVQQLGTPR; 1,706.97 Da were identified. The F2-4 had higher DPPH and NO radical scavenging activity compared to the synthetic peptide. Moreover, AILQSYSAGKTK had the highest ABTS radical scavenging activity. Furthermore, the F2 fraction protected oxidation-induced DNA damage in pBR322, pKS and pUC19 cells. F2 fraction was selected to study anti-inflammatory and anticancer properties. Anti-inflammatory effect, F2 fraction showed no cytotoxicity toward RAW264.7 macrophage cells. RT-PCR and qRT-PCR results showed that F2 reduced gene expression of pro-inflammatory cytokines iNOS, TNF-α, COX-1, COX-2 and IL-6. For anticancer activity, F2 fraction showed high anticancer activities in the human colon cancer cells (SW620) according to cytotoxic activity (MTT assay), with IC50 values of 106.58±21.46 µg protein/mL. The F2 fraction activated the apoptotic pathway in SW620 cells after treatment for 24, 48 and 72 hours. The highest activities of caspases 3, 8 and 9 were observed after treatment for 72 hours. These findings suggested that unicellular cyanobacterium Synechococcus sp. may be used to develop for natural anti-inflammation and natural anticancer drugs.