Isolation and characterization of activators of ERK/MAPK from citrus plants

Abstract

Extracellular signal-regulated kinases 1/2 (ERK1/2), components of the mitogen-activated protein kinase (MAPK) signaling cascade, have been recently shown to be involved in synaptic plasticity and in the development of long-term memory in the central nervous system (CNS). We therefore examined the ability of Citrus compounds to activate ERK1/2 in cultured rat cortical neurons, whose activation might have a protective effect against neurodegenerative neurological disorders. Among the samples tested, extracts prepared from the peels of Citrus grandis (Kawachi bankan) were found to have the greatest ability to activate ERK1/2. The active substances were isolated by chromatographic separation, and one of them was identified to be 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF). HMF significantly induced the phosphorylation of cAMP response element-binding protein (CREB), a downstream target of activated ERK1/2, which appears to be a critical step in the signaling cascade for the structural changes underlying the development of long-term potentiation (LTP). In addition, the administration of HMF into mice treated with NMDA receptor antagonist MK-801 restored the MK-801-induced deterioration of spatial learning performance in the Morris mater-maze task. Taken together, these results suggest that HMF is a neurotrophic agent for treating patients with memory disorders.

Keywords: 3,5,6,7,8,3′,4′-heptamethoxyflavone; Citrus grandis; ERK1/2; Kawachi bankan; MK-801; methoxyflavone; neurons.

Figure 1

Effects of ethyl acetate (Ac-OEt), n-hexane and H₂O (H₂O) extracts of Citrus grandis (Kawachi bankan) on ERK1/2 activation in rat cortical neurons. Cells were treated with 100 μg/mL of each extract for 30 min or 50 ng/mL brain-derived neurotrophic factor (BDNF) for 10 min, and then equal amounts of protein were analyzed by immnoblot analysis.

Figure 2

Chemical structure of 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF).

Figure 3

Time- and dose-dependent phosphorylation of ERK2 after HMF treatment of cultured rat cortical neurons. (A) The cells were treated for 30 min with various concentrations (0, 0.1, 1.0, 10, and 100 μM) of HMF or BDNF (50 ng/mL) for 10 min, and cell lysates were then prepared and applied to immunoblot analysis; (B) Cells were treated with 100 μM HMF for various times (0, 10, 30, 60, and 90 min) or with BDNF (50 ng/mL) for 10 min, and the cell lysates were then prepared for immunoblot analysis. The density ratio of pERK2 to total ERK2 in untreated cultures was expressed as 1 arbitrary unit. Results represent mean ± SEM (n = 4, different cultures). Significant difference in values between the compound-treated and non-treated cells: * P < 0.05; ** P < 0.01; *** P < 0.01 (Student’s t test).

Figure 4

Effects of MEK inhibitors on HMF-induced ERK2 activation in cultured rat cortical neurons. Cells were sequentially pretreated for 30 min with 10 μM PD980059 (A) or 10 μM U0126 (B) and then incubated for 30 min with or without 50 μM HMF. Thereafter they were subjected to the immunoblot analysis. The ratios of the value for the drug-treated cells to that value for the control cells were calculated, and are shown on the ordinate. Values are presented as the mean ± SEM (n = 4). Significant difference in values between the HMF-treated and non-treated cells (** P < 0.01, *** P < 0.001; Student’s t test) and in those indicated by the brackets (^# P < 0.05, ^## P < 0.01; Student’s t test) are shown.

Figure 5

Effects of HMF, nobiletin (NBT) and tangeretin (TGN) on ERK2 activation in rat cortical neurons. Cells were treated with a 50 μM concentration of each compound for 30 min, and harvested for immunoblot analysis using antibodies against phosphorylated ERK1/2 and unphosphorylated ERK1/2. When cells were treated with 50 ng/mL BDNF, the incubation time was 10 min. The density ratio of pERK2 to total ERK2 in untreated cultures was expressed as 1 arbitrary unit. Results represent the mean ± SEM (n = 4, different cultures). Significant difference in values between the compound-treated and nontreated cells: * P < 0.05; ** P < 0.01 (Student’s t test).

Figure 6

Effects of HMF on cAMP response element-binding protein (CREB) activation in rat cortical neurons. Cells were incubated with or without HMF at the concentration of 100 μM for 30 min, and the cell lysates were prepared and applied to immunoblot analysis. Values are presented as the mean ± SEM (n = 4). Significant difference in values between the HMF-treated and non-treated cells: ** P < 0.01 (Student’s t test).

Figure 7

Effects of HMF on deterioration of spatial learning performance in MK-801-treated mice. (A) The time schedule of injection of HMF/MK-801 and the MWM test are shown; (B) Columns indicate the mean ± SEM (n = 5). Significant differences in values between the MK-801-treated and vehicle-treated (* P < 0.05; followed by Bonferroni’s Multiple Comparison Test), and HMF-treated (^# P = 0.05).