Effects of Methoxyflavone and their Derivatives on Memory Deficits Induced by Scopolamine in Zebrafish Models of Alzheimer’s Disease

Received: 21-Oct-2025, Manuscript No. JDAR-26-176236; Editor assigned: 28-Oct-2025, Pre QC No. JDAR-26-176236 (PQ); Reviewed: 22-Dec-2025, QC No. JDAR-26-176236; Revised: 24-Dec-2025, Manuscript No. JDAR-26-176236 (R); Published: 31-Dec-2025, DOI: 10.4303/JDAR/236475

Introduction

Neurodegenerative diseases are progressive neurological disorders characterized by the loss of neurons that is irreversible, causing profound impairments in cognition, movement, and behavior. Dementia is the leading disease burden; about 60-70% of all dementia cases are due to Alzheimer’s disease [1]. Dysfunction within the cholinergic system is widely recognized as an important factor in the early stage of AD development, as supported by various findings of disrupted cholinergic functions in both patients with AD and animal models thereof. One of the early pathophysiological events common to AD is the deficit of acetylcholine, also known as ACh, which is a neurotransmitter important for memory and learning. The need for such a deficit is primarily the enzymatic destruction of ACh via acetylcholinesterase, AChE, and to a lesser degree, butyrylcholinesterase [2-4]. Considering the involvement of cholinesterase enzymes in the pathology of AD, inhibitors of cholinesterases became the first therapeutic strategy developed against the disease. However, despite the detection of other molecular targets for AD, cholinesterase inhibitors are still the most prescribed drugs for the treatment of mild to moderate stages of the disease. They act by promoting synaptic plasticity and enhancing acetylcholine availability. Further to cholinergic dysfunction, the accumulation of amyloid beta plaques and the presence of abnormal tau protein are among other important factors in AD progression [5,6].

Although several animal models exist that are used for screening neurodegenerative disorders, many of these models only partially emulate the pathology of AD. zebrafish, Danio rerio, have recently become an increasingly encouraging model in the study of such diseases, offering a wider range of molecular and behavioral features related to Alzheimer’s disease.

Zebrafish genes are 70% homologous with human genes; 84% of human disease-causing genes are reported to have their zebrafish counterparts, which underscores their value and relevance as translational models. The general structure of the zebrafish brain is significantly conserved compared to that of mammals. Both the zebrafish and mammalian brains are similarly organized into the hindbrain, midbrain, and forebrain, further divided into the telencephalon, mesencephalon, metencephalon, and myelencephalon. Many specific areas, such as the olfactory bulbs, hypothalamus, and cerebellum, are also similarly defined within these regions [7-9]. The BBB of a zebrafish also structurally and functionally resembles the mammalian BBB, making it useful for permeability studies of new neurodrugs [10].

Moreover, the cognitive abilities of zebrafish can be evaluated through several paradigms for testing their learning and memory, many of which parallel those used in rodent studies [11-13].

Besides that, Kaempferia parviflora has demonstrated promising potential in cholinesterase inhibition. In the case of compounds isolated from the plant, 5,7,4’- trimethoxyflavone and 5,7-dimethoxyflavone have demonstrated the most potent inhibiting activity against AChE and BChE enzymes, whose inhibition activity has been reported to lie in the range of 43–85% according to Pattara Sawasdee, et al. [14]. In addition, a structureactivity relationship identified methoxy groups at the 5, 7, and 4’ position as important for AChE inhibition. As only limited studies exist concerning AD, this study evaluates the impact of different doses of three related synthetic compounds 7-methoxyflavone, 7,4’-dimethoxyflavone, and 7,2’,4’-trimethoxyflavone on the cognitive functions of zebrafish through behavioural tests like T-maze and lightand- dark chamber tests.

Materials and Methods

An eight-month-old male adult Danio rerio will be used in this neurocognitive function study.

The zebrafish will be kept in a 10-liter tank with an aerator to maintain enough oxygen. The tank’s ambient temperature will be maintained at 25 ± 2°C, with a photoperiod of 11 to 12 hours of light and 11 to 12 hours of dark.

The adult male zebrafish were divided into 12 groups by average body weight in such a manner that each group consisted of 10 fish, which were housed in a 2-liter circulating tank. The body weight for the fish in each group ranged from 0.31-0.41 g. Before starting the actual experiment, the zebrafish would be acclimatized for two weeks to get used to the conditions of housing. All fish were acclimatized to the treatment chambers before the test sessions.

A stock solution of all the three test drugs, 7- methoxyflavone, 7,4’-dimethoxyflavone, and 7,2’,4’-trimethoxyflavone, standard Rivastigmine, and Scopolamine, was prepared. From the stock solution, drugs at the desired concentration were prepared by dilution of a desired volume of stock solution in 1 L of system water. Standard drug 1.5 mg/L Rivastigmine and Scopolamine (200 μM) were dissolved in water, while in the control group, system water from the maintenance tank was used.

The controlled behavioral observations will take place between 9:30 AM and 1:00 PM to minimize neuroendocrine fluctuations that may interfere with neurobiological or neurobehavioral outcomes.

Experimental study groups:

Group I: Control (fed by water)

Group II: Scopolamine 200 μM (SCO)

Group III: Scopolamine 200 μM+Rivastigmine 1.5 mg/ kg/L (SCO+RV)

Group IV: Scopolamine 200 μM+7,2’,4’ -trimethoxyflavone 5 mg/L (SCO+TMF-5)

Group V: Scopolamine 200 μM+7,2’,4’-trimethoxyflavone 10 mg/L (SCO+TMF-10)

Group VI: Scopolamine 200 μM +7,2’,4’ -trimethoxyflavone 20 mg/L (SCO+TMF-20)

Group VII: Scopolamine 200 μM+7,4’-dimethoxyflavone 5 mg/L (SCO+DMF-5)

Group VIII: Scopolamine 200 μM+7,4’ -dimethoxyflavone 10 mg/L (SCO+DMF-10)

Group IX: Scopolamine 200 μM+7,4’-dimethoxyflavone 20 mg/L (SCO+DMF-20)

Group X: Scopolamine 200 μM+7-methoxyflavone 5 mg/L (SCO+MF-5)

Group XI: Scopolamine 200 μM+7-methoxyflavone 10 mg/L(SCO+MF-10)

Group XII: Scopolamine 200 μM+7-methoxyflavone 20 mg/L(SCO+MF-20)

Experimental procedures

Colour-biased appetite conditioning T-maze test [15] zebrafish cognitive capacity was assessed using a T-maze test with color-biased hunger conditioning. The acrylic glass T-maze has one long arm (50 cm × 10 cm × 10 cm) and two short arms (20 cm × 10 cm × 10 cm). The zebrafish were given the test drug and Rivastigmine at a dosage of 1.5 mg/L two hours before the experiment. An hour prior to the experiment, a 200 μM scopolamine solution was administered. Every solution, including test extracts and standard drugs, was made fresh on the day of the experiment. Zebrafish was placed at the end of the long arm during the training. The sliding door opened after a minute, allowing the fish to swim. The sliding door at the intersection closes as soon as a fish enters the short arms. Fish in the short arms are watched for four minutes. The fish was rewarded with food if it had entered the green arm. However, if it entered the red arm, the training was repeated for three days in a row without any sustenance. Fish were subjected to the identical methods during the testing sessions, but they were not given food in the green arm or punishment in the red arm. Behavioral metrics of all fish were recorded for further analysis by measuring the time spent in each arm and total number of entries into both the red and green arms.

Light and dark preference test

The light and dark preference test has been a standard model for analysing cognitive and memory functions in zebrafish. The experimental apparatus consisted of a non-reflective rectangular acrylic tank (15 ×10 × 45 cm) that was designed to avoid shoaling behaviour based on reflection. The tank had sliding central doors with the same color as the tank sides. The water level in the tank was kept at a height of 10 cm. Illumination was provided by a 500–600 lux bulb placed 1.8 m above the tank, and trials were recorded with a video camera positioned 50 cm above the setup [16].

Each fish was acclimatized for two minutes in the middle chamber of the tank before the session. Following the acclimatization phase, the sliding door was opened, and the fish allowed to explore both the light and dark chambers for 15 minutes. Throughout the training sessions, rewards in the form of food were given upon entry into the light chamber, while entry into the dark chamber was disturbed by a glass rod. Fish selecting the dark chamber continuously were retrained for three consecutive days. There was no food reward in the light chamber, nor any disturbance in the dark chamber during each of the testing sessions. Behavioural parameters-time spent and number of entries in both the light and dark chambers were recorded and analyzed to study learning and memory performance (Figure 1).

Figure 1: Experimental design of this study

Statistical analysis

Sigma Plot (version 12.0) was used to analyse the data. The mean ± SEM was used to express all values. The Mann Whitney U test was used for multiple comparisons, and the Kruskal-Wallis test was used for data analysis. At p<0.05, differences between the groups were deemed statistically significant.

Results

Colour-biased appetite conditioning T-maze test

As seen in Figures 2 and 3, the time spent and the total number of entries in the green/red arms were recorded, and the mean difference (green/red arm) was computed. It was determined that the zebrafishes spent more time in the green arms than in the red arms for groups showing a mean difference larger than 1 in both time spent and total number of entries. This shows that the zebrafish in these groups had high memory. Rivastigmine and test medications (7,4’-dimethoxy flavone 20 mg/L and 7-methoxyflavone at all doses) caused zebrafish to exhibit a mean difference in time spent and total number of entries greater than 1. Zebrafish that received 7-methoxyflavone showed a more extended time spent ratio and a greater total number of entries ratio, which were moderately comparable to those of the positive control (Figure 2).

Figure 2: Effect of Scopalamine (SCO), Rivastigmine (RV), TMF (7,2’,4’-trimethoxyflavone), DMF (7,4’-dimethoxyflavone), MF (7-methoxyflavone) on time spent in the colour-biased appetite conditioning T-maze test

The mean difference in time spent between the green and red arms was expressed as mean ± SEM. Zebrafish that received RV+SCO, DMF20+SCO, MF20+SCO showed mean difference of time spent at more than 1 as 1.95 ± 0.228, 1.041 ± 0.099 and 1.230 ± 0.207, respectively (Figure 3).

Figure 3: Effect of SCO, RV, TMF (7,2’,4’-trimethoxyflavone), DMF (7,4’-dimethoxyflavone), MF (7-methoxyflavone) on number of entries in the colour-biased appetite conditioning T-maze test

The mean difference in the total number of entries into the green and red arms was expressed as mean ± SEM. Zebrafish that received RV+SCO, DMF10+SCO, DMF20+SCO, MF5+SCO, MF10+SCO, MF20+SCO showed mean difference of total number of entries more than 1 as 2.34 ± 1.01, 1.18 ± 0.34, 1.24 ± 0.67,1.16 ± 0.33,1.29 ± 0.49 and 1.42 ± 0.35, respectively.

Zebrafish that received 7-methoxyflavone at doses of 5, 10, and 20 mg/L had significantly increased the time spent in the light chamber, i.e., increased latency time as 151 ± 6.548, 169.1 ± 7.95 and 176.2 ± 6.442 seconds, respectively, compared to the scopolamine group (83.5 ± 13.083) as shown in Figure 4.

Figure 4: Effect of SCO, RV, TMF (7,2’,4’-trimethoxy flavone), DMF (7,4’-dimethoxyflavone), MF (7-methoxyflavone) on the time spent in light chamber–light and dark test

7,2’,4’-trimethoxyflavone and 7,4’-dimethoxyflavone showed moderate results when compared to 7-methoxyflavone. The preference for the light chamber was considered an improvement in memory function. The control and rivastigmine groups showed more preference for the light chamber. The entries to the dark chamber were relatively more in 7,2’,4’-trimethoxyflavone treated zebrafish.

Discussion

This study investigates the potential of methoxyflavones and their derivatives to mitigate scopolamine-induced dementia in zebrafish, a widely used model for studying memory impairment. Scopolamine, a potent muscarinic receptor antagonist, disrupts cholinergic signalling by preventing acetylcholine from binding to its postsynaptic receptors. This is manifested by an increased activity of AChE, enhanced oxidative stress and neuroinflammation, and an increased expression of APP and Tau proteins, which are common hallmarks of neurodegeneration in AD [17].

The outcomes indicated that cognitive disturbances in zebrafish, such as passive avoidance and learning response impairment, are a reliable simulation of the symptoms produced in AD conditions. Rivastigmine, an already known acetylcholinesterase inhibitor and clinically used medication for AD, was used as a positive control to validate the ability and neurotransmitter ACh. The organ responsible for memory of this type, which involves learning, is the hippocampus. Against this background, destruction of the hippocampal cells leads to disturbances in memory functions and loss of short-term memory, which is reflected in the inability of animals to learn and perform appropriate behavioral patterns. In the T-maze test, four colors (blue, green, red, and yellow) were used to train the zebrafish.They showed a higher response to red and green than to the remaining colors. Consequently, these colors were used in the experiments, and feeding was done in the green arm to enhance learning and memory through a phenomenon called synesthesia. The T-maze test measured the time spent in the green and red arms to evaluate zebrafish learning and memory [18-20]. The study found that in the colour-biased appetite conditioning T-maze test, the time spent ratio and the total number of entries in the green/red arm ratio greater than 1 indicated good memory performance. Zebrafish treated with Rivastigmine exhibited the highest time spent ratio and total number of entries ratio, suggesting that Rivastigmine significantly improved spatial learning and recognition. This improvement is attributed to Rivastigmine’s inhibition of Acetylcholinesterase (AChE) activity [21]. In this study, 7-methoxyflavone doses (5, 10, 20 mg/L) were tested, and the time spent ratio and the total number of entries in the green arm were significantly higher than in the red arm and compared to the positive control. 7,4’-dimethoxyflavone in a dose of 20 mg/L indicated a cognitive improvement when compared to its other doses of 5 and 10 mg/L and 7,2’,4’-trimethoxyflavone improved memory performance compared to the negative control. The light-dark preference test is a widely used behavioral model for assessing cognitive and memory functions in zebrafish, particularly in response to pharmacological treatments. In this test, a preference for the light chamber signifies improved memory function. Both the control group and the group treated with Rivastigmine showed a marked preference for the light chamber, indicating enhanced memory. Conversely, zebrafish treated with 7,2’,4’-trimethoxyflavone and 7,4’-dimethoxyflavone exhibited a higher number of entries into the dark chamber, suggesting a lesser degree of memory improvement compared to the control and rivastigmine groups. In contrast, 7-methoxyflavone doses showed increased latency time and significant time spent in the light chamber, indicating increased memory function. Thus proving it to be a potential compound for improving cognition in Alzheimer’s disease. This study supports the findings of the survey by Mhalhel, et al. on the effects of flavonoids in neurodegenerative disorders such as Alzheimer’s disease [14,21].

Conclusion

The effects of 7-methoxyflavone, 7,4’-dimethoxyflavone and 7,2’,4’-trimethoxyflavone on memory deficit induced by scopolamine were investigated. Results proved that methoxyflavone and its derivatives showed significant improvement in memory, and 7-methoxyflavones in all doses (5, 10 and 20 mg/L) had significant cognitive improvement in comparison with Rivastigmine. 7-methoxyflavone has the potential to be developed as a health product to help alleviate memory impairment or treat Alzheimer’s disease

Study Limitations

This study was conducted as a preliminary investigation to identify behavioural parameters of methoxyflavone and its derivatives. Our findings can be helpful for further research on methoxyflavone in biochemical estimation of Acetylcholinesterase activity (AchE) and Glutathione S-transferase (GST) and histopathological detection of degenerative changes in brain tissues of zebrafish, to prove it as one of the promising compounds in the management of dementia in Alzheimer’s disease.

Ethical Approval

The study was approved by the Institutional Animal Ethics Committee, Registration No: 863/PO/Re/S/04/CPCSEA in meeting dated 18/12/2021. The authors have no conflict of interest regarding this study

Financial Disclosure

The authors declare that no funds, grants, or other support were received for study

Author Contributions

Conceptualisation, methodology, software, validation formal analysis: PK, NP; Investigation resources, data curation, writing-original draft,writing-review and editing visualisation: PK,NP, HKS; Supervision: PK.

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Copyright: © 2025 Nithya Panneerselvam, et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.