Bioassay-guided fractionation of okra seed peptide on antioxidant activities and inhibition of oxidative stress in LPS stimulated RAW 264.7 cells

Okra is the well-known vegetable, green pods, within the Asian cuisine. This study aimed to evaluate the antioxidant activities of okra seed protein hydrolysate produced by combining three enzymes (trypsin, chymotrypsin, and pepsin). The hydrolysate was filtered through an ultrafiltration membrane with a selective molecular weight of 3 kDa and the % yield was calculated. Subsequently, it was desalted by solid phase extraction (SPE), and peptides were fractionated by hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The results indicated that the protein extracted from defatted okra seed showed a % yield of about 24.25 ± 0.25 %. The % yield of ultrafiltered peptides (≤ 3 kDa) was 14.39 ± 0.85 %. These peptides (after SPE desalting) showed DPPH radical scavenging properties (IC 50 0.37 ± 0.79 mg/mL). Moreover, after HILIC fractionation, the HILIC 70 % ACN+0.1 %FA fraction exhibited the most potent DPPH radical scavenging activity (IC 50 0.56 mg/mL). This fraction underwent additional separation with RP-HPLC, revealing that fraction 3 (RP-HPLC_3) had the highest DPPH radical scavenging activity, and LC-MS/MS identified its pure peptides. 11 peptides sequence contained hydrophobic, hydrophilic, basic amino acids and aromatic acids residues, which play a crucial role in the antioxidant activity of peptides. According to the cell model, non-cytotoxic concentrations (0.0001 – 1 mg/mL) of peptides from okra seed protein hydrolysates effectively inhibited intracellular ROS generation by LPS-induced oxidative stress in macrophage RAW 264.7 cells, as monitored by DCFH-DA fluorescence. Furthermore, the peptides from okra seed protein hydrolysates at 0.0001 – 1 mg/mL concentrations also strongly suppressed nitric oxide (NO) production. Therefore, the study suggests that peptides from okra seed protein hydrolysates are a potential source of natural antioxidants with pharmaceutical properties worthwhile developing into natural chemo-preventive or nutraceutical products in the future.