Ethanolic Extract of Orthosiphon stamineus Improves Memory in Scopolamine-Induced Amnesia Model

Abstract

Alzheimer's disease (AD) is a chronic neurodegenerative brain disease which is characterized by impairment in cognitive functioning. Orthosiphon stamineus (OS) Benth. (Lamiaceae) is a medicinal plant found around Southeast Asia that has been employed as treatments for various diseases. OS extract contains many active compounds that have been shown to possess various pharmacological properties whereby in vitro studies have demonstrated neuroprotective as well as cholinesterase inhibitory effects. This study, therefore aimed at determining whether this Malaysian plant derived flavonoid can reverse scopolamine induced learning and memory dysfunction in the novel object recognition (NOR) test and the elevated plus maze (EPM) test. In the present study, rats were treated once daily with OS 50 mg/kg, 100 mg/kg, 200 mg/kg and donepezil 1 mg/kg via oral dosing and were given intraperitoneal (ip) injection of scopolamine 1 mg/kg daily to induce cognitive deficits. Rats were subjected to behavioral analysis to assess learning and memory functions and hippocampal tissues were extracted for gene expression and immunohistochemistry studies. All the three doses demonstrated improved scopolamine-induced impairment by showing shortened transfer latency as well as the higher inflexion ratio when compared to the negative control group. OS extract also exhibited memory-enhancing activity against chronic scopolamine-induced memory deficits in the long-term memory novel object recognition performance as indicated by an increase in the recognition index. OS extract was observed to have modulated the mRNA expression of CREB1, BDNF, and TRKB genes and pretreatment with OS extract were observed to have increased the immature neurons against hippocampal neurogenesis suppressed by scopolamine, which was confirmed by the DCX-positive stained cells. These research findings suggest that the OS ethanolic extract demonstrated an improving effect on memory and hence could serve as a potential therapeutic target for the treatment of neurodegenerative diseases like AD.

Keywords: Alzheimer’s disease; Orthosiphon stamineus; animal model; learning and memory; scopolamine.

Figure 1

Schematic representation of the experimental procedure.

Figure 2

Behavioral analysis for novel object recognition (NOR) and elevated plus maze (EPM). (A) represent the graph plot for the recognition indices in NOR for the nootropic model (B) represents the graph plot for the inflection ratios in EPM for the nootropic model. The behavioral analysis for (A, B) were compared to control group. Data are expressed as Mean ± SEM, n = 8 and statistical analysis by one-way ANOVA followed by Dunnett test *P < 0.05 and **P < 0.01.

Figure 3

Behavioral analysis for NOR and EPM. (A1 and A2) represents the graph plot for the recognition indices in NOR for both the acute and chronic scopolamine model respectively (B1 and B2) represents the graph plot for inflection ratios in EPM for both the acute and chronic scopolamine model. All the behavioral analysis was compared to the negative control (SCP 1 mg/kg). Data are expressed as Mean ± SEM, n = 8 and statistical analysis by one-way ANOVA followed by Dunnett test **P < 0.01 and ****P < 0.0001.

Figure 4

Gene expression in the rat hippocampi determined by real time-PCR. The genes included are (A) BDNF, (B) CREB1, and (C) TrKB. All changes in the expression levels were compared to the negative control group (SCP 1 mg/kg). Data are expressed as Mean ± SEM, n = 4 and statistical analysis by one-way ANOVA followed by Dunnett test *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 5

DCX immunohistochemical analysis of the effects of OS extract in improving scopolamine-induced suppression of neurogenesis in the dentate gyrus. (A) DCX-positive staining in immature neurons is shown in the SGZ of the dentate gyrus. Photomicrographs of the hippocampal section of treatment groups was (i) Control (ii) SCP 1 mg/kg alone (iii) DPZ 1 mg/kg + SCP 1 mg/kg (iv) 50 mg/kg OS + SCP 1 mg/kg (v) 100 mg/kg + SCP 1 mg/kg (vi) 200 mg/kg + SCP 1 mg/kg. Representative photomicrographs were taken at magnifications of 40× and 200×. (B) Quantification of DCX population. Data are expressed as means Mean ± SEM, n = 4 – 6 and statistical analysis by one-way ANOVA followed by Dunnett test **P < 0.01, ***P < 0.001 and ****P < 0.0001.