Quantitative Structure-Activity Relationship Model to Predict Antioxidant Effects of the Peptide Fraction Extracted from a Co-Culture System of Chlorella pyrenoidosa and Yarrowia lipolytica

Abstract

In this study, the antioxidant components in co-culture of Chlorella pyrenoidosa and Yarrowia lipolytica (3:1 ratio) were confirmed as trypsin-hydrolyzed peptides (EHPs). The EHPs were composed of 836 different peptides with molecular weights ranging from 639 to 3531 Da and were mainly composed of hydrophobic amino acids (48.1%). These peptides showed remarkable protective effects against oxidative stress in HepG2, which may be attributed to their structures. Furthermore, the mRNA and protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were significantly lower in the peptide-treated group than in the control group, suggesting that the antioxidant enzyme-coding genes were not activated. The EC₅₀ value of three peptides in the EHPs were in the order of AGYSPIGFVR (0.04 ± 0.002 mg/mL) > VLDELTLAR (0.09 ± 0.001 mg/mL) > LFDPVYLFDQG (0.41 ± 0.03 mg/mL); these results agreed with the prediction of the model (R² > 0.9, Q² > 0.5). Thus, EHPs show potential as potent new antioxidant agents.

Keywords: Chlorella pyrenoidosa; HepG2; Yarrowia lipolytica; enzymatic hydrolysis peptides; quantitative structure–activity relationship model.

Figure 1

Antioxidative abilities of trypsin-hydrolyzed peptide (EHP) in vitro and in HepG2 cells. The 2,2-azobis(2-amidinopropane) dihydrochloride radical scavenging power of different concentrations of EHP (A). Reducing power of different concentrations of EHP (B). Effects of EHP on the viability of HepG2 cells (C). Effects of different concentrations of EHP on 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced changes in intracellular malondialdehyde (MDA) (D), glutathione (GSH) (E), and glutathione (GSSG) (F) levels in HepG2 cells. Data are the mean ± standard deviations of three independent experiments. Intracellular reactive oxygen species (ROS) scavenging capacities of blank group, AAPH group, Trolox, 0.375 mM EHP, 0.75 mM EHP, and 1.5 mM EHP at 180 min of incubation (G).

Figure 2

Effect of EHP on catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) activities and mRNA expression. Effects of EHP on AAPH-induced changes in cellular antioxidant enzyme levels in HepG2 cells. CAT (A), SOD (B), and GSH-Px (C). The mRNA levels of AAPH-induced oxidative stress metabolism-related genes, including CAT (D), SOD (E), and GSH-PX (F), in HepG2 cells pretreated with different concentrations of EHP. Each mRNA level was normalized to those of GAPDH and expressed relative to the control. Data are the mean ± standard deviations of three independent experiments.

Figure 3

Antioxidant mechanism. The mRNA expression levels of antioxidant signaling pathway-related genes, including encoding nuclear factor erythroid 2-related factor 2 (Nrf2) (A) and Kelch-like ECH-associated protein-1 (Keap1) (B) in HepG2 cells pretreated with different concentrations of EHP. Each mRNA level was normalized to that of GAPDH and expressed relative to the control. Hepatic expression of oxidative stress metabolism-related proteins and antioxidant signaling pathway-related proteins (C,D) in HepG2 cells pretreated with different concentrations of EHP. Each protein expression level was normalized to that of GAPDH and expressed relative to the control and positive control levels. A possible hypothesis to explain these effects is that AAPH-induced oxidative stress increased reactive oxygen species (ROS) generation and caused Keap1 oxidation, which released Nrf2, thereby activating the antioxidant enzymes. EHP can provide electrons, which allows it to directly quench free radicals and act as an antioxidative (E). Data are the mean ± standard deviations of three independent experiments.

Figure 4

Quantitative structure–activity relationships (QSAR) model: Plot showing the comparison of observed and predicted linear relationship in the QSAR model (A). Prediction of antioxidant model of peptide sequence using the QSAR model. Three peptides were synthesized to verify the accuracy of the model, including AGYSPIGFVR (B), VLDELTLAR (C), and LFDPVYLFDQG (D). Effects of the three peptides on the viability of HepG2 cells (E), and the concentration required for 50% of the maximal AAPH-induced oxidative stress in HepG2 cells (F). Data are the mean ± standard deviations of three independent experiments.