Inhibition of Oxidative Neurotoxicity and Scopolamine-Induced Memory Impairment by γ-Mangostin: In Vitro and In Vivo Evidence

Abstract

Among a series of xanthones identified from mangosteen, the fruit of Garcinia mangostana L. (Guttifereae), α- and γ-mangostins are known to be major constituents exhibiting diverse biological activities. However, the effects of γ-mangostin on oxidative neurotoxicity and impaired memory are yet to be elucidated. In the present study, the protective effect of γ-mangostin on oxidative stress-induced neuronal cell death and its underlying action mechanism(s) were investigated and compared to that of α-mangostin using primary cultured rat cortical cells. In addition, the effect of orally administered γ-mangostin on scopolamine-induced memory impairment was evaluated in mice. We found that γ-mangostin exhibited prominent protection against H₂O₂- or xanthine/xanthine oxidase-induced oxidative neuronal death and inhibited reactive oxygen species (ROS) generation triggered by these oxidative insults. In contrast, α-mangostin had no effects on the oxidative neuronal damage or associated ROS production. We also found that γ-mangostin, not α-mangostin, significantly inhibited H₂O₂-induced DNA fragmentation and activation of caspases 3 and 9, demonstrating its antiapoptotic action. In addition, only γ-mangostin was found to effectively inhibit lipid peroxidation and DPPH radical formation, while both mangostins inhibited β-secretase activity. Furthermore, we observed that the oral administration of γ-mangostin at dosages of 10 and 30 mg/kg markedly improved scopolamine-induced memory impairment in mice. Collectively, these results provide both in vitro and in vivo evidences for the neuroprotective and memory enhancing effects of γ-mangostin. Multiple mechanisms underlying this neuroprotective action were suggested in this study. Based on our findings, γ-mangostin could serve as a potentially preferable candidate over α-mangostin in combatting oxidative stress-associated neurodegenerative diseases including Alzheimer's disease.

Figure 1

Chemical structures of α-mangostin (a) and γ-mangostin (b) isolated from G. mangostana.

Figure 2

Effects of α- and γ-mangostins on neuronal cell viability in primary cultured rat cortical cells. Cells were exposed to the indicated concentrations of α- or γ-mangostin for 24 h. The control cells were treated with vehicle (DMSO) only. Cell viability was determined by the MTT reduction assay, as described in Materials and Methods. The viability of control cells treated with vehicle only was considered to be 100%, and the data were expressed as percentages of the control. Each point represents the mean ± S.E.M. from at least three independent experiments, performed in duplicate.

Figure 3

Effects of α- and γ-mangostins on H₂O₂- or X/XO-induced oxidative neurotoxicity and ROS generation in primary cultured rat cortical cells. (a and d) The cells were exposed to 100 μM H₂O₂ for 5 min (a) or 0.5 mM X in combination with 10 mU/ml XO for 10 min (d) in the absence or presence of either α- or γ-mangostin at various concentrations as indicated. Cell viability was determined by the MTT reduction assay at 18-20 h after exposure, as described in Materials and Methods. The cell survival was expressed as percentages of the control treated with vehicle only. (b and e) The cells were preincubated with 10 μM DCFH-DA for 30 min at 37°C in the dark, then treated with 100 μM H₂O₂ for 2 h (b) or 0.5 mM X in combination with 10 mU/ml XO for 2 h (e) in the absence or presence of either α- or γ-mangostin at various concentrations as indicated. The generation of intracellular ROS was measured as described in Materials and Methods. The ROS levels were expressed as percentages of the control treated with vehicle only. Each data point represents the mean ± S.E.M. from at least three independent experiments, performed in duplicate (^# P < 0.05 vs. vehicle-treated control cells without α- or γ-mangostin treatment; ^∗ P < 0.05 vs. H₂O₂- or X/XO-treated cells). (c) Fluorescence microscopic images showing the inhibition of H₂O₂-induced ROS generation by γ-mangostin in primary cultured rat cortical cells. The cells were preincubated with 10 μM DCFH-DA for 30 min at 37°C in the dark and treated with 100 μM H₂O₂ in the absence (B) or presence of 10 μM α-mangostin (C) or γ-mangostin (D) for 2 h. The control cells were treated with vehicle only without α- or γ-mangostin (A). Following the desired treatment, ROS levels were imaged using epifluorescence microscopy as described in Materials and Methods. Representative photomicrographs from three independent experiments are shown.

Figure 4

Effects of α- and γ-mangostins on H₂O₂-induced apoptosis in primary cultured rat cortical cells. (a and b) Inhibition of H₂O₂-induced DNA fragmentation by γ-mangostin. Cells were treated with 100 μM H₂O₂ for 2 h with or without α- or γ-mangostin at the concentration of 10 μM, and the TUNEL assay was carried out as described in Materials and Methods. Representative microscopic images from at least three individual experiments are shown (a). (A and E) Control cells were treated with vehicle only; (B and F) cells were treated with 100 μM H₂O₂ for 2 h; (C and G) cells were treated for 2 h with either 10 μM α-mangostin (C) or γ-mangostin (G) in combination with 100 μM H₂O₂; (D and H) cells were treated with 10 μM α-mangostin (D) or γ-mangostin for 2 h without H₂O₂ (H). Scale bar = 10 μm. Quantitative analyses of the TUNEL-positive cells from at least three independent experiments are shown (b) (^# P < 0.05 vs. vehicle-treated control cells without α- or γ-mangostin treatment; ^∗ P < 0.05 vs. H₂O₂-treated cells without α- or γ-mangostin). (c and d) Inhibition of the H₂O₂-induced activation of caspases 3 and 9 by γ-mangostin. Cells were treated with 100 μM H₂O₂ for 2 h in the absence or presence of either α- or γ-mangostin at 3 and 10 μM. The expression of cleaved caspases 3 and 9 was assessed by Western blotting as described in Materials and Methods. Representative blots from at least three individual experiments are shown (c). The intensities of the bands from at least three independent experiments were quantified by densitometric analyses and normalised to β-actin (d) (^# P < 0.05 vs. vehicle-treated control cells without α- or γ-mangostin treatment; ^∗ P < 0.05 vs. H₂O₂-treated cells without α- or γ-mangostin).

Figure 5

Effects of α- and γ-mangostins on DPPH radical formation and lipid peroxidation (LPO). Inhibition of DPPH radical (a) and LPO induced by Fe²⁺ (10 μM) and L-ascorbic acid (100 μM) in rat forebrain homogenate (b) by α- or γ-mangostin at the indicated concentrations were measured as described in Materials and Methods. Each data point represents the mean ± S.E.M. from at least three independent experiments, performed in duplicate (^∗ P < 0.05 vs. vehicle-treated control without α- or γ-mangostin treatment). Vit. C and BHA were used as references to validate the assay procedures for DPPH radical scavenging activity and inhibition of LPO, respectively (grey bars). Vit. C: vitamin C; BHA: butylhydroxyanisole.

Figure 6

Effects of α- and γ-mangostins on β-secretase activity. The inhibitory effects of α- and γ-mangostins on the enzymatic activity of β-secretase were determined by the β-secretase FRET assay as described in Materials and Methods. The data were expressed as percentages of the control treated without α- or γ-mangostin. Each data point represents the mean ± S.E.M. from at least three independent experiments, performed in duplicate (^∗ P < 0.05 vs. vehicle-treated control without α- or γ-mangostin treatment).

Figure 7

Effect of γ-mangostin on the scopolamine-induced memory impairment in mice. Animals were randomly divided into 6 groups with 6-7 mice in each group. To the 3 groups of animals, γ-mangostin was orally administered at the respective dosages of 5, 10, or 30 mg/kg as indicated. For the reference drug-treated group, donepezil was administered at the dosage of 10 mg/kg. For the control group (the vehicle-treated group without scopolamine, γ-mangostin, or donepezil treatment) and the scopolamine group (the group treated with scopolamine injection, not with γ-mangostin or donepezil treatment), vehicle was only administered. After 30 min of each administration, memory impairment was induced by intraperitoneal injection of scopolamine (3 mg/kg in normal saline) in 5 groups as indicated above in the figure; for the control group, normal saline without scopolamine was injected. Following 30 min of scopolamine or saline injection, the acquisition trial was initiated by delivering a foot shock to the animals. Twenty-four hours after the acquisition trials, the retention trials were performed. The detailed experimental procedures are described in Materials and Methods. The time latency was calculated from three independent experiments. Each data point represents the mean ± S.E.M. (^# P < 0.05 vs. vehicle-treated control group without scopolamine, γ-mangostin, or donepezil treatment; ^∗ P < 0.05 vs. scopolamine group treated with scopolamine only without γ-mangostin or donepezil).