Protective effect of myricetin derivatives from Syzygium malaccense against hydrogen peroxide-induced stress in ARPE-19 cells

Abstract

Purpose: Oxidative stress is implicated in the etiology of diabetes and its debilitating complications, such as diabetic retinopathy (DR). Various flavonoids have been reported to be useful in reducing DR progression. Myricetin derivatives (F2) isolated from leaf extract of Syzygium malaccense have the potential to serve as functional food as reported previously. The present study was performed with the aim of determining the antioxidant potential and protective effect of myricetin derivatives (F2) isolated from leaf extract of S. malaccense against glucose oxidase (GO)-induced hydrogen peroxide (H₂O₂) production that causes oxidative stress in ARPE-19 (RPE) cells.

Methods: Antioxidant properties were assessed through various radical (DPPH, ABTS, and nitric oxide) scavenging assays and determination of total phenolic content and ferric reducing antioxidant power level. ARPE-19 cells were preincubated with samples before the addition of GO (to generate H₂O₂). Cell viability, change in intracellular reactive oxygen species (ROS), H₂O₂ levels in cell culture supernatant, and gene expression were assessed.

Results: F2 showed higher antioxidant levels than the extract when assessed for radical scavenging activities and ferric reducing antioxidant power. F2 protected the ARPE-19 cells against GO-H₂O₂-induced oxidative stress by reducing the production of H₂O₂ and intracellular reactive oxygen species. This was achieved by the activation of nuclear factor erythroid 2-related factor 2 (Nrf2/NFE2L2) and superoxide dismutase (SOD2), as well as downregulation of nitric oxide producer (NOS2) at the transcriptional level.

Conclusions: The results showed that myricetin derivatives from S. malaccense have the capacity to exert considerable exogenous antioxidant activities and stimulate endogenous antioxidant activities. Therefore, these derivatives have excellent potential to be developed as therapeutic agents for managing DR.

Figure 1

Cytoprotective effects of myricitrin, myricetin, leaf extract of Syzygium malaccense, and F2 (myricetin derivatives) when pretreated for 2 h against glucose oxidase-H₂O₂ (GO-H₂O₂)-induced oxidative stress in ARPE-19 cells. The concentration of GO used is 12 mUnit/ml. Control (100%) is the untreated cells grown in basal medium. Data are expressed in mean ± standard deviation (SD); n=3. The paired t test between the GO-treated cells and control showed statistical significance at ^###p<0.001. The one-way ANOVA Tukey’s post hoc test showed statistical significance at *p<0.05, ** p<0.01, and *** p<0.001 when the test samples were compared to the GO-treated cells. S. mal = Syzygium malaccense.

Figure 2

Intracellular reactive oxygen species (ROS) levels in ARPE-19 cells when pretreated with myricitrin, myricetin, leaf extract of Syzygium malaccense, and F2 (myricetin derivatives) for 2 h before exposure to GO-H₂O₂ induced stress. The concentration of glucose oxidase (GO) used is 12 mUnit/ml. Intracellular ROS that was generated during the incubation period was monitored using the 2′,7′-dichlorofluorescin diacetate (DCFH-DA) fluorescent probe. Control (100%) is the untreated cells grown in basal medium. The fold change was normalized to 10,000 cells for all GO-treated cells with and without pretreatment of samples by considering the number of viable cells after the treatment. Data are expressed in mean ± standard deviation (SD), n=3. The paired t test between the GO-treated cells and control showed statistical significance at ^###p<0.001. The one-way ANOVA Tukey’s post hoc test showed statistical significance at *p<0.05, ** p<0.01, and *** p<0.001 when the test samples were compared to the GO-treated cells. S. mal = Syzygium malaccense.

Figure 3

Hydrogen peroxide (H₂O₂) levels in ARPE-19 cell culture supernatants when pretreated with myricitrin, myricetin, leaf extract of Syzygium malaccense, and F2 (myricetin derivatives) for 2 h before exposure to GO-H₂O₂ induced stress. The concentration of GO used is 12 mUnit/ml. Control (100%) is the untreated cells grown in basal medium. Data are expressed in mean ± standard deviation (SD); n=3. The paired t test between the GO-treated cells and control showed statistical significance of ^###p<0.001. The one-way ANOVA Tukey’s post hoc test showed statistical significance at *** p<0.001 for all the test samples and the GO-treated cells when compared to the GO-treated cells only. S. mal = Syzygium malaccense.

Figure 4

Gene expression profile of selected markers in glucose oxidase-hydrogen peroxide (GO-H₂O₂) –induced stress treated ARPE-19 cells that were subjected to a 2 h pretreatment with samples (M and F2). M (40 µg/ml) and F2 (200 µg/ml) represent myricetin or myricetin derivative–rich fraction, respectively. The concentration of glucose oxidase (GO) used is 12 mUnit/ml. Quantitative real-time reverse transcriptase-PCR was performed using a custom RT² profiler PCR array. y-axis represents the n-fold of regulation (mean ± standard deviation (SD) of two separate experiments) against control (untreated cells grown in basal media) after normalization with human 18SrRNA, the endogenous control. The dotted line represents the fold regulation of the untreated control (1.00). The paired t test between the GO-treated cells and control showed statistical significance at ^#p<0.05, ^##p<0.01, and ^###p<0.001, respectively. The one-way ANOVA Tukey’s post hoc test showed statistical significance at *p<0.05, ** p<0.01, and *** p<0.001 when the test samples (M and F2) were compared to the GO-treated cells only.