INTRODUCTION:

Alzheimer's disease (AD) is a neurological condition that worsens with time and mostly affects the hippocampus and cortical areas of the brain. It leads to memory loss and behavioral issues, significantly impairing daily tasks, particularly in learning and recall. AD influences between 60-80% overall instances of memory loss, making it the primary factor. This disease involves in general shrinkage, particularly in the frontal cortex, temporal lobe, parietal lobe, and cingulate gyrus. It does this by inducing the degeneration of neurons and synapses within the cerebral cortex and subcortical regions. Key pathological features include intracellular beta amyloid plaques (Aβ) and neurofibrillary tangles^1,2. In 2022, 6.46 million Americans over 65 were diagnosed with AD, with this number projected to rise to 13.85 million by 2060. The prevalence is higher in women and varies across different racial and ethnic groups. In 2020, cognitive impairment was more common in those aged 85+, but by 2022, more cases were reported in the 75–84 age range³.

AD pathophysiology involves several factors, including glutamate excitotoxicity, neuronal degeneration, synapse deprivation, oxidative stress, inflammation, mitochondrial failure, and decreased cholinergic transmission⁴. The amyloid hypothesis of Alzheimer's disease (AD) posits that amyloid plaques, neurofibrillary tangles (NFTs), and neuronal loss are key histopathological features^5,6. Amyloid precursor protein (APP) processing abnormalities lead to an imbalance between amyloid formation and clearance^7,8,9, resulting in toxic amyloid-β (Aβ) plaques^10,11. The most toxic Aβ isoform, 1-42, forms aggregates that contribute to plaque formation. The build-up of extracellular Aβ plaques and intracellular NFTs, composed of hyper phosphorylated tau protein¹², disrupts cellular function and causes neurodegeneration. Oxidative stress, characterized by an imbalance between antioxidant levels and reactive oxygen species (ROS)¹³, exacerbates this process by promoting further amyloid and tau aggregation, leading to cell death¹⁴. This damaging cycle is sustained in AD patients by altered processes involving antioxidant enzymes that include catalase and superoxide dismutase. In AD, cholinergic neurons are significantly reduced, especially the ones that extend throughout the basal forebrain to cortical along with hippocampal areas¹⁵. This reduction in acetylcholine (Ach) is central to the 'cholinergic theory' of AD, which underpins treatments aimed at enhancing cholinergic function using acetylcholinesterase inhibitors like Tacrine, Donepezil, Rivastigmine, and Galantamine^16,17,18. Rather of repairing the damage to brain cells, the current therapies try to stabilize the disease's course and reduce its symptoms. Galantamine and other cholinesterase inhibitors work by inhibiting Ach breakdown, which enhances cognition and memory. The majority of side effects are moderate and include nausea, vomiting, increased bowel movements, and lightheadedness¹⁹.

Recent research addresses the protective effects on neurons of ferulic acid (FA), an active ingredient having a number of advantageous characteristics, such as antioxidant and anti-inflammatory activities^20,21. FA may reduce Aβ plaque deposition in the brain²². Research on rats experiencing scopolamine-induced²³ cognitive decline is being conducted to assess the potential benefits of galantamine and/or FA in improving memory, learning, and cholinergic system function. Preliminary findings suggest that FA could be therapeutic in AD by preventing amyloid plaque formation. This research aims to further assess the neuroprotective effects of FA and its combination with Galantamine against AD-related memory impairment in a Scopolamine-induced model.

MATERIALS AND METHODS:

Materials:

Galantamine was purchased from Sisco Research Laboratories Pvt. Ltd. (SRL), Mumbai. Scopolamine powder (Hbr) and Ferulic acid powder were obtained from Yucca Enterprises, Mumbai. All other chemicals used were of analytical grade.

Animals:

Female Wistar rats, 250 to 300g were housed in rooms with 12/12 h light/dark cycle at 22±3°C and fed with standard dry pellet and water ad libitum. Before being used in experiments, the animals were allowed to adapt and get used to their surroundings lab environment.The experimental protocol was approved by Institutional Animal Ethics Committee and care of the animals was carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA with CCSEA) India (Protocol No. PIPR 984/2023/02/12).

Instrumentation:

Y-maze apparatus was used for screening neuroprotective effect. All weighing was done on electronic balance.

Experimental design:

The animals used in this investigation were distributed among five groups, each with 6 rats.

Group I (NC): Normal control (0.9% saline) i.p for 7 days.

Group II (DC): Disease control (Scopolamine, 1mg/kg) i.p for 7 days.

Group III (GA): Standard (Galantamine, 3mg/kg) p.o for 21 days.

Group IV (FA): Test-1 (Ferulic acid, 100mg/kg) p.o for 21 days.

Group V (GA+FA): Test-2 (Galantamine, 3mg/kg + Ferulic acid, 100mg/kg) p.o for 21 days.

Every animal underwent physical training at the start of the study simultaneous with the administration of Galantamine, Ferulic acid and scopolamine for 28 days. On 29^th day all the physical parameters were to be tested.

Y-maze: The Y-maze test evaluated an animal's functioning location with short-term memory by taking advantage of its innate curiosity to seek out novel surroundings. A wooden apparatus having 3 alike arms (A, B, and C) measuring 120°, 40cm in length, and 35 cm in height was used to carry out the assessment²⁴. In the habituation phase, each rat being randomly assigned to ending of a particular arm and allowed to roam around among the three arms for five minutes. The animal was seen to favor its first arm, whereas its second arm was observed to be discriminated against. Twenty-four hours later, during the acquisition phase, food as a reward were given inside the favored region, whereas an animal's arm of discrimination was shut. After being positioned in the initial arm, the animal was permitted to go to the open arm. Each rat was involved in this exercise for five minutes. Throughout the retention stage, distilled water were employed for the treatment of the NC and DC, galantamine (3mg/kg, po) were used to treat the standard, and (100mg/kg, po) of ferulic acid were given to the FA and a mixture associated with galantamine and ferulic acid being administered to the GA+FA. An animals beingpositioned within the apparatus sequentially over a duration of two minutes following a seven-day course of Scopolamine treatment. To get rid of any smelly residue from the earlier rat as much as possible, alcohol (70% ethanol) was used to wipe the apparatus soon after subsequent transit. Data was collected on two specific factors: the latency time to opt for a favored arm and the spontaneous alternation % (SA): no. of alternation/(total no. of armentries − 2) × 100²⁵.

Biochemical analysis:

Sample preparation:

After the Y-maze activity, 24hours later, all the rats were decapitated. The brain was collected and immediately placed in ice-cold saline. Following brain excision, the hippocampus area separated and maintained around 4°C. Rat hippocampi were fixed in 10% formaldehyde for the purpose of measuring neurochemical characteristics in each group.

Tissue preparation:

After homogenizing 10% (w/v) of brain tissue in 0.1 M phosphate buffer saline (PBS) at pH 7.4, the resulting solution were thencentrifuged for ten min at 4 °C around 4000 rpm. Acetylcholinesterase and other biochemical markers of oxidative stress (SOD) were measured in the supernatant.

Assessment of Acetylcholinesterase (AChE) activity:

An enhanced Ellman approach was utilized to evaluate AChE activity²⁶. Ach iodide served as a substrate to acquire colorimetric evaluation to assess the Ach activity in the hippocampal area of the human brain. The Ach esterase activity was determined by using the DTNB (5' dithionitrobenzoic acid) method. The final 1 ml reaction mixture contained 10mM Ach iodide, 5 mM DTNB, and phosphate buffer (0.1 M pH 7.4). An optical density was detected around 412nm in five minutes.The concentration of AChE was measured via a molar extinction value of 1.36 x 104 M-1 cm-1, and represented as nmolethiocholine produced as µmoles/mg protein.

Estimation of Superoxide dismutase (SOD):

Superoxide dismutase activity is evaluated via spectrophotometry, in accordance with earlier research findings.After quickly stirring the solution, which contained 1 milliliter of brain homogenate and 4 milliliters of n-butanol, it was centrifuged at 4000 revolutions per minute multiplied by the force of gravity for 15 minutes. Using a spectrophotometer set to 560 nm as the reference wavelength, the color intensity was measured. Units/min/mg/protein were used to quantify the super oxide dismutase activities²⁶.

Statistical analysis:

Employing Graph Pad Prism software version 10.2.3, data was assessed via one way analysis of variance (ANOVA) along with Bonferroni multiple comparisons test. The data was expressed as Mean±SEM. P<0.05 was defined as statistical significance.P-values <0.05, 0.01, <0.001, and <0.0001 were represented as *, **, ***, and ****, respectively, whereas P-values ≥ 0.05 are denoted by "ns".

RESULTS:

Effect on latency time using Y-maze:

The willingness that allows the animal to investigate the concealed surface within its designated zone gradually improves after a week of Y-maze activity retraining. Throughout courses of study, the rats receiving scopolamine (102.7 ± 4.5, P < 0.0001) showed greater latency (the time it takes to discover surface) than NC (72.67 ± 3.1, P < 0.0001), suggesting trouble in remembering.The NC animals figured out the platform's position rather quickly. In comparison to the disease control (scopolamine-induced) groups (102.7 ± 4.5, P < 0.0001), the effects of scopolamine on the duration of time to reach the favorable arm were notably diminished in the group receiving galantamine (41.0 ± 3.2, P < 0.0001), Ferulic acid (50.0 ± 1.39, P < 0.0001), and galantamine with Ferulic acid (37.0 ± 1.5, P < 0.0001) treated groups represented in (Figure. 1).

Figure 1: The Effect of Ferulic acid individual and along with Galantamine on Latency time (s) in scopolamine induced cognitive impairment. Values stated as Mean ± SEM for n = 6, in comparison with scopolamine, a statistically significant variance is defined as ****p < 0.0001.

Effect of spontaneous alteration during study period:

These study trial data of rat exposed to scopolamine (16.67±2.1, P<0.0001) shows significant reduced number of successive three arm visits in comparison NC (30.00±2.5, P<0.01). Oral administration of Galantamine with Ferulic acid (50.00 ± 3.6, P<0.0001) diminished the disease induced memory disruption while elevated mental ability in terms of working memory in the apparatus by animals comparing with disease (50.00 ±3.6, P<0.0001), Galantamine (43.33± 4.2, P<0.0001) and Ferulic acid treated group (40.00± 3.6, P<0.001) represented in (Figure 2).

Figure 2: The Effect of Ferulic acid individual and along with Galantamine on Spontaneous Alteration (%) in scopolamine induced cognitive impairment. Values stated as Mean ± SEM for n = 6, Group II was significant in comparison to Group I, while Group II DC significant in comparison to Group III, IV, and V.

Effect of drugs on the activity of Acetylcholinesterase activity:

When rats were exposed to scopolamine, their hippocampal AChE activity increased significantly (41.8 ±1.72, p<0.0001) in comparison to the NC (25.50±1.25, p<0.0001). AChE is an enzyme that's involved in the breakdown and utilization associated with acetylcholine. Furthermore, the scopolamine group, animals in the Galantamine, Ferulic acid and Galantamine+Ferulic acid groups exhibited a substantial decrease in acetylcholinesterase activity (14.6±0.66, p<0.0001, 17.3±1.22, p< 0.0001, 12.6±0.84, p <0.0001) depicted in (Figure 3).

Effect of drugs on the activity of superoxide dismutase:

When rats were exposed to scopolamine, their hippocampal radical was significantly lower (22.17± 0.6, P<0.0001) than when rats were given in NC (60.83 ±2.1, p<0.0001). Furthermore, the scopolamine group, animals in the Galantamine, Ferulic acid and Galantamine+Ferulic acid groups exhibited a substantial decrease in acetylcholinesterase activity (42.83±2.0, p < 0.0001, 69.67±2.3, p<0.0001, 84.67±1.33, p <0.0001) depicted in (Figure 4).

Figure 3: Graph represents the effect of Ferulic acid individual and along with Galantamine on AchE Activity in scopolamine induced cognitive impairment. Values stated as Mean ± SEM for n = 6, Group II was significant in comparison to Group I, while Group II DC significant in comparison to Group III, IV, and V.

Figure 4: Graph represents the effect of Ferulic acid individual and along with Galantamine on SOD Levels in scopolamine induced cognitive impairment. Values stated as Mean ± SEM for n = 6, Group II was significant in comparison to Group I, while Group II DC significant in comparison to Group III, IV, and V.

Histological investigation:

Under a microscope, hippocampal histopathology microstructure was ordinary as there were no histopathology alterations within portions of the specific region from the NC (Figure 5a). The brain sections of the AD-induced group's images revealed substantial synaptic collapse, along with the production of neurofibrillary tangles and amyloid plaques (Aβ)(Figure 5 b). The Galantamine treated group showed few shrunken cells and many normal cells (Figure 5c), images demonstrate a portion of the AD-induced rats' brain received Ferulic acid aloneshowed minute shrinking of cells(Figure 5d) and theimages shows the region of rats' brain received combination of Galantamine + Ferulic acid showing in the hippocampus area, there were primarily normal cells and relatively few dark, shrunken cells (Figure 5e).

a) Group I (NC): Normal histological structure was observed in hippocampal region.

b) Group II (DC): AD induced rat showing shrunken nerve cells in hippocampal regionA) Amyloid plaques (Aβ) formation B) Neurofibrillary tangles.

c) Group III (GA): In the hippocampus area, there were several normal cells and a small number of shrunken cells.

d) Group IV (FA): In the hippocampal area cells were seen to be somewhat diminishing.

e) Group V (GA+FA): Showing in the hippocampus area, there were primarily normal cells and relatively few dark, shrunken cells.

Figure 5: The effect of Ferulic acid individual and along with Galantamine in histopathological images of scopolamine induced cognitive impairment.

DISCUSSION:

Alzheimer's disease (AD) is a neurological illness which impacts the cortex of the brain along with hippocampal. It progresses over time AD is a disease of agingwhich can make it challenging for a person to interact with others or react to their surroundings. The first is a mild cognitive impairment. The intricate process of aging modifies a person's capacity to function regularly throughout time. This change is causing a decline in biological functioning, particularly those related to the brain and cognition. It might begin as mild memory loss and advance to the point where daily duties become impossible^1,16.

To find out how various chemicals affected the way neurons interacted in the brain, both traditional drugs and experimental treatments were used. Although these treatments could offer short-term symptom alleviation or stability, it is doubtful that they would reverse the brain cell damage brought on by dementia. It is recommended to treat problems affecting logic, memory, language, judgment, and other mental functions with drugs called antagonists of cholinesterase. These kinds of drugs stop acetylcholine, a neurotransmitter vital to memory and thought processes, from breaking down. These drugs facilitate communication between nerve cells. During mild-to-moderate phases of AD, the drug galantamine is licensed for usage¹⁹.

Studies have suggested that Ferulic acid may aid in reducing the amount of Aβ accumulated in the cortex of the brain. It could be helpful for AD as it stops amyloid plaques from developing and it also helps to improve antioxidants. In the present study, the pharmacological effect of Ferulic acid and effect of combination of Galantamine with Ferulic acid were assessed thoroughly on experimentally induced scopolamine in rats.

The animals were divided in five groups and among them, the control group was the normal one without Alzheimer’s induction to which each group were compared. Group II to V were administered scopolamine 1 mg/kg (i.p.) one time a day throughout weeks in order to cause memory impairment as it is a muscarinic receptor antagonist, it causes the muscarinic acetylcholine receptor to switch from active to inactive, resulting in temporary forgetfulness and electrophysiological abnormalities that are similar to those seen in Alzheimer’s disease. On contrast, Galantamine which was administered as a standard drug for treatment to group III caused a significant delays in the breakdown of ACh, allowing it to build up and utilize its full efficacy when compared with disease control (group II) confirming recovered state.

As per the treatment of this study, our combination with Galantamine (GA) and Ferulic acid (FA) reversed the condition of memory impairment exhibiting positive readings that were even greater than those of the standard medication. Treatment with Ferulic acid was administered to group IV of animals which was effective but not much as compared to Galantamine.

In scopolamine model, after the training sessions of 7 days in the Y-maze, the administration of scopolamine resulted in an increased time required to locate the submerged platform in rats. Both of these outcomes were consistent with earlier studies showing scopolamine impairs rat performance. GA+FA treated, could easily locate the platform when compared to the scopolamine group. Scopolamine is known to induce neuronal loss and cause amyloid-β build-up in the rat brain. In accordance with previous studies, our findings indicated that scopolamine-induced impairments in learning and memory were accompanied by an increase oxidative stress in the rat brain, and a decrease in SOD enzymes GA+FA treated rats reduced the AchE levels and increased the levels of SOD enzyme, indicating a diminished impact of scopolamine in rats. And the amyloid-β plaque formation and neurofibrillary tangles hyper phosphorylation gets decreased.

The present study concludes that the combination of Galantamine and Ferulic acid could be a promising therapeutic target for AD treatment.

ACKNOWLEDGEMENTS:

For granting access to the facilities needed to conduct the research, the authors are extremely grateful to Parul Institute of Pharmacy and Research, Parul University.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 19.06.2024 Revised on 09.10.2024

Accepted on 10.12.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2207-2212.

DOI: 10.52711/0974-360X.2025.00316

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