Neuroprotective Effects of Euonymus alatus Extract on Scopolamine-Induced Memory Deficits in Mice

Abstract

Euonymus alatus is considered to elicit various beneficial effects against cancer, hyperglycemia, menstrual discomfort, diabetic complications, and detoxification. The young leaves of this plant are exploited as food and also utilized for traditional medicine in East Asian countries, including Korea and China. Our preliminary study demonstrated that ethanolic extract from the Euonymus alatus leaf (EAE) exhibited the strongest antioxidant enzyme-inducing activity among more than 100 kinds of edible tree leaf extracts. This study investigated whether EAE could attenuate the cognitive deficits caused by oxidative stress in mice. Oral intubation of EAE at 100 mg/kg bw or higher resulted in significant improvements to the memory and behavioral impairment induced via i.p. injection of scopolamine. Furthermore, EAE enhanced the expression levels of hippocampal neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor in mice, activated the Nrf2, and the downstream heme oxygenase-1 (HO-1) a quintessential antioxidant enzyme. As rutin (quercetin-3-O-rutinose) was abundantly present in EAE and free quercetin was able to induce defensive antioxidant enzymes in an Nrf2-dependent manner, our findings suggested that quercetin derived from rutin via the intestinal microflora played a significant role in the protection of the mouse hippocampus from scopolamine-induced damage through BDNF-mediated Nrf2 activation, thereby dampening cognitive decline.

Keywords: BDNF; Euonymus alatus; HT22 cells; animal model; antioxidant; cognition improvement; neuroprotective.

Figure 1

DPPH and the ABTS radical scavenging capacities of Euonymus alatus leaf (EAE). (A) DPPH and (B) ABTS radical scavenging activities. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed using a one-way ANOVA, followed by Duncan’s multiple range test. Values with different alphabetical letters (a–d) represent significant differences from each other (p < 0.05).

Figure 2

Protective effect of the EAE on glutamate-induced cell growth inhibition in HT22 cells. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed using a one-way ANOVA, followed by Duncan’s multiple range test. Values not sharing common alphabetical letters (a–d) represent significant differences from each other (p < 0.05).

Figure 3

Effect of EAE on the scopolamine-induced memory deficit tested by the passive avoidance task (A), Morris water maze test (B), and Y-maze test (C). Results are expressed as mean ± SD (n = 5). Statistical analysis was performed using one-way ANOVA, following Duncan’s multiple range test. Bars not sharing common alphabetical letters (a–c) represent significant differences from each other (p < 0.05).

Figure 3

Effect of EAE on the scopolamine-induced memory deficit tested by the passive avoidance task (A), Morris water maze test (B), and Y-maze test (C). Results are expressed as mean ± SD (n = 5). Statistical analysis was performed using one-way ANOVA, following Duncan’s multiple range test. Bars not sharing common alphabetical letters (a–c) represent significant differences from each other (p < 0.05).

Figure 4

Supplementation with EAE decreased the oxidative damage on lipid and DNA in scopolamine-treated mice. (A) The MDA levels in the liver homogenate and (B) 8-OHdG levels in the serum. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA, following Duncan’s multiple range test. Bars not sharing common alphabetical letters (a–c) represent significant differences from each other (p < 0.05).

Figure 5

Effects of EAE on the mRNA expression of the BDNF (A), GDNF (B), and NMDA receptor (C) genes in the mouse hippocampus. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA, following Duncan’s multiple range test. Bars not sharing common alphabetical letters (a,b) represent significant differences from each other (p < 0.05).

Figure 6

Effects of EAE on the protein levels of nuclear Nrf2 (A) and protein expression of HO-1 (B) and PSD-95 (C) in the mouse hippocampus. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA, following Duncan’s multiple range test. Bars not sharing common alphabetical letters (a,b) represent significant differences from each other (p < 0.05).

Figure 6

Effects of EAE on the protein levels of nuclear Nrf2 (A) and protein expression of HO-1 (B) and PSD-95 (C) in the mouse hippocampus. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA, following Duncan’s multiple range test. Bars not sharing common alphabetical letters (a,b) represent significant differences from each other (p < 0.05).

Figure 7

Prevention of scopolamine-induced neuronal damage in the hippocampal CA1 region by oral supplementation of EAE. The Vehicle, no treatment; Sco, i.p injection with scopolamine (5 mg/kg bw) alone, Sco + Don, treatment with donepezil followed by i.p. injection with scopolamine; Sco + EAE 50, treatment with EAE (50 mg/kg bw) followed by i.p. injection with scopolamine, Sco + EAE 100, treatment with EAE (100 mg/kg bw) followed by i.p. injection with scopolamine, Sco + EAE 150, treatment with EAE (150 mg/kg bw) followed by i.p. injection with scopolamine. A representative picture for each experimental group is depicted (magnification: 100× or 400×).

Figure 8

Effects of the EAE and EA fractions such as rutin and quercetin on ARE-mediated transcriptional activation in the HT22-ARE cells. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA, followed by Duncan’s multiple range test. Values not sharing common alphabetical letters (a–h) represent significant differences from each other (p < 0.05).

Figure 9

Acetylcholine-esterase-inhibitory activities of EAE.