Inhibition of oxidative stress and lipid peroxidation by anthocyanins from defatted Canarium odontophyllum pericarp and peel using in vitro bioassays

Abstract

Canarium odontophyllum, also known as CO, is a highly nutritious fruit. Defatted parts of CO fruit are potent sources of nutraceutical. This study aimed to determine oxidative stress and lipid peroxidation effects of defatted CO pericarp and peel extracts using in vitro bioassays. Cell cytotoxic effect of the CO pericarp and peel extracts were also evaluated using HUVEC and Chang liver cell lines. The crude extracts of defatted CO peel and pericarp showed cytoprotective effects in t-BHP and 40% methanol-induced cell death. The crude extracts also showed no toxic effect to Chang liver cell line. Using CD36 ELISA, NAD(+) and LDL inhibition assays, inhibition of oxidative stress were found higher in the crude extract of defatted CO peel compared to the pericarp extract. Hemoglobin and LDL oxidation assays revealed both crude extracts had significantly reduced lipid peroxidation as compared to control. TBARS values among defatted CO pericarp, peel, and cyanidin-3-glucoside showed no significant differences for hemoglobin and LDL oxidation assays. The protective effects of defatted CO parts, especially its peel is related to the presence of high anthocyanin that potentially offers as a pharmaceutical ingredient for cardioprotection.

Figure 1. Inhibition of t-BHP-induced cell death by defatted CO extracts.

HUVEC (A) and Chang liver cell line (B) were treated with different concentrations of defatted CO peel and pericarp (PP) extracts. Different upper case letters (X–Y) show significant differences between the extract concentrations and t-BHP-induced control (p≥0.05), while similar lower case letter (a or b) of the same extract concentration shows no significant difference between the peel and pericarp (PP) (p≥0.05).

Figure 2. Inhibition of 40% methanol-induced cell death by defatted CO extracts.

Chang liver cells were treated with different concentrations of defatted CO peel and pericarp (PP) extracts. Different upper case letters (X–Y) show significant differences between the extract concentrations and 40% methanol-induced control (p<0.05), while similar lower case letter (a) shows no significant difference between peel and pericarp (PP) (p≥0.05).

Figure 3. Percentages of cell viability of Chang liver cells by defatted CO extracts.

Chang liver cells were treated with different concentrations of defatted CO peel and pericarp (PP) extracts (A) and cyanidin-3-glucoside (B) for comparison. Different lower case letters (a, b) show a significant difference between peel and pericarp (PP) (p<0.05), while similar upper case letters (X–Z) show no significant difference between two different extract concentrations of C3G (p≥0.05).

Figure 4. Protective effect of defatted CO extracts on depletion of NAD⁺ in H₂O₂-induced Chang liver cells.

Values are expressed as % of control incubation (without H₂O₂). High, low, and IC₅₀ concentrations of defatted CO peel and pericarp (PP) extracts were tested. Cyanidin-3-glucoside (C3G–200 µg/ml) was for comparison. Similar lower case letters (a–d) show no significant differences between two different extracts or between extract and H₂O₂/C3G (p≥0.05).

Figure 5. Inhibition of CD36 binding to oxidized LDL by defatted CO extracts.

Values are expressed as % of inhibition. High, low and IC₅₀ of concentration of defatted CO peel and pericarp (PP) extracts were tested. Cyanidin-3-glucoside (C3G–10 µg/ml) was for comparison. Similar lower case letters (a–c) show no significant differences between two different extracts or between extract and C3G (p≥0.05).

Figure 6. Inhibition of LDL-binding to endothelial cells by defatted CO extracts.

IC₅₀ concentrations of the defatted CO pericarp and pericarp (PP) extracts were used. Endothelial cells were treated with 80 µg/ml of LDL protein and incubated together with defatted CO peel and pericarp (PP) at IC₅₀ extract concentration. Different lower case letters (a, b) show significant differences between two different extracts or between extract and control (p<0.05).

Figure 7. TBARS values of hemoglobin oxidation inhibition by defatted CO extracts.

IC₅₀ concentrations of the defatted CO pericarp and pericarp (PP) extracts were applied. Cyanidin-3-glucoside (C3G, 10 µg/ml) was for comparison. Red blood cells were obtained from a pool of blood from both normal healthy fasting and obese fasting rats (n = 5 each). No significant difference was found for the TBARS values between normal and obese rats. Different lower case letters (a–c) show significant differences between two different extracts or between extract and control/C3G for either normal or obese rats (p<0.05).

Figure 8. TBARS values of LDL oxidation inhibition by defatted CO extracts.

IC₅₀ concentrations of the defatted CO pericarp and pericarp (PP) extracts were applied. Cyanidin-3-glucoside (C3G, 10 µg/ml) was for comparison. Red blood cells were obtained from a pool of blood from both normal healthy fasting and obese fasting rats (n = 5 each). No significant difference was found for the TBARS values between normal and obese rats. Different lower case letters (a–c) show significant differences between two different extracts or between extract and control as well as C3G for either normal or obese rats (p<0.05).