Pequi Fruit Extract Increases Antioxidant Enzymes and Reduces Oxidants in Human Coronary Artery Endothelial Cells

Abstract

Reactive oxygen species (ROS) imbalance results in endothelial cell function impairment. Natural phenolic antioxidant compounds have been investigated as therapeutic alternatives. The fruit bark of Brazilian-native pequi (Caryocar brasiliense, Camb.) is rich in polyphenols. The HPLC-MS (High-Performance Liquid Chromatography coupled with Mass Spectrometry) analyses identified gallic acid and catechin in six out of seven ethanolic extract samples prepared in our lab. In this study, we examined the effects of ethanolic pequi extract on ROS levels in human coronary artery endothelial cells (HCAEC) subjected to hypoxia or oxidative stress. We first confirmed the oxidant scavenging capacity of the extract. Then, HCAEC pre-incubated with 10 or 25 μg/mL of extract were subjected to hypoxia for 48 h or 100 μM H₂O₂ for six hours and compared to the normoxia group. Total and mitochondrial ROS levels and cell proliferation were measured. Pequi significantly reduced cytosolic HCAEC ROS levels in all conditions. Mitochondrial ROS were also reduced, except in hypoxia with 10 μg/mL of extract. HCAEC proliferation increased when treated with 25 μg/mL extract under hypoxia and after H₂O₂ addition. Additionally, pequi upregulated oxidative stress defense enzymes superoxide dismutase (SOD-)1, SOD-2, catalase, and glutathione peroxidase. Together, these findings demonstrate that pequi bark extract increases antioxidative enzyme levels, decreases ROS, and favors HACEC proliferation, pointing to a protective effect against oxidative stress.

Keywords: cardiovascular disease; ethnopharmacology; human coronary artery endothelium; phenols; reactive oxygen species.

Figure 1

Pequi (Caryocar brasiliense, Camb.). The tree (left) is native to the Brazilian Cerrado (A), a savanna-like biome, with marked dry and wet seasons. The fruit (B) is edible and oily and has a thick, dense mesocarp and epicarp (asterisk) that form the bark, which is dried (C), macerated (D), and percolated in 95% ethanol to extract phenols. After percolation, the ethanol is removed by rotative evaporation (40 °C) and the final product (E) is obtained.

Figure 2

Qualitative high-efficiency liquid chromatography coupled to high-resolution mass spectrometry of the ethanolic extract of pequi bark. Representative chromatographic profiles of components detected in six of seven samples: (A) gallic acid; (B) catechin. RT—retention time.

Figure 3

Pequi bark ethanolic extract is rich in phenolic compounds and scavenges free radicals in vitro: (A) graphic representation of DPPH reagent discoloration (92.62 ± 0.23%) as a measure for free radical neutralization by pequi extract; (B) gallic acid standard curve and concentration of total phenol in the extract based on gallic acid equivalence are shown. DPPH: 2,2-diphenyl-1-picrylhydrazyl.; GAE: gallic acid equivalent; dw: dry weight.

Figure 4

Cytosolic (A–C) and mitochondrial (D–F) Reactive Oxygen Species (ROS) production in human coronary artery endothelial cells (HCAEC) subjected to normoxia, hypoxia, and H₂O₂. HCAEC (1 × 10⁴ cells/well) were pre-treated with 0 (vehicle control), 10, or 25 μg/mL of pequi extract for 24 h, followed by 48 h of normoxia (A,D), hypoxia (B,E), or six-hour exposure to 100 μM of H₂O₂ (C,F). Pequi extract decreased cytosolic ROS production in all treatments and concentrations as compared to controls (p < 0.05). The trend was similar for mitochondrial ROS, except for the pequi 10 μg/mL treatment in hypoxia conditions, where mitochondrial ROS were not significantly different from vehicle control without extract. Vehicle = Dimethyl sulfoxide (DMSO); Pequi 10 = 10 μg/mL; Pequi 25 = 25 μg/mL. Statistical analyses were performed using Mann–Whitney’s test. The results shown represent n = 6 (A–C) or n = 8 (D–F) independent experiments, using 5 replicates for each experiment/condition. MitoSox: Mitochondrial Superoxide Indicator.

Figure 5

Human coronary artery endothelial cell proliferation assay. HCAEC were pre-treated for 24 h with 0 (Control), 10, or 25 μg/mL of Pequi extract. Cells (1 × 10⁴ cells/well) were subjected to normoxia (A) or hypoxia (B) for 48 h or 100 μM H₂O₂ for six hours (C). Cell proliferation significantly increased in hypoxia (25 μg/mL group) as compared to vehicle-only (DMSO) control. Although proliferation decreased after addition of H₂O₂ in HCAEC without extract, an increase in proliferation was observed when cells were pre-treated with pequi extract. Variance analysis revealed differences between the treatments (C). Experiments were carried out using six (n = 6) independent experiments, each consisting of 5 replicates per condition/group. Statistical analysis was carried out using Mann–Whitney’s test.

Figure 6

Pequi extract increases expression of antioxidant enzymes in HCAEC. Cells were cultured, pre-treated with pequi extract (25 μg/mL) for 24 h under normoxia conditions, and total protein content was extracted for Western blots analysis for Superoxide dismutase (SOD)-1 (A), SOD-2 (B), catalase (C), glutathione peroxidase—GPx (D), sirtuin 1—SIRT-1 (E), peroxisome-proliferator-activated receptor gamma coactivator 1-alpha (PCG1-α) (F), glycogen synthase kinase 3 beta (GSK3β) (G), and microtubule-associated protein 1A/1B-light chain 3 (LC3) (H). Western blots were normalized using α-tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as loading control. Five independent Western blot experiments were carried for each condition. Representative images are shown here. Statistical analysis was performed using Student’s t-test (p < 0.05). OD: Optical Density.

Figure 7

Proposed mechanisms of reduction in ROS in human coronary endothelial cells (HCAEC) by pequi extract. Hypoxia and H₂O₂ increase cytosolic and mitochondrial ROS in HCAEC. Polyphenol-rich pequi extract induces expression of antioxidant enzymes as a first line of defense against ROS, possibly by activating the Nrf2/ARE pathway and may reduce ROS and induce HCAEC proliferation. LDL: Low-density Lipoprotein; EC: Endothelial Cells; ARE: Antioxidant Responsive Element; Nrf-2: Nuclear factor-erythroid factor 2-related factor 2; HO-1: Heme Oxygenase-1; SOD-1: superoxide dismutase-1; SOD-2: superoxide dismutase-2; ROS: Reactive Oxygen Species.