The potential role of pomegranate and its nano-formulations on cerebral neurons in aluminum chloride induced Alzheimer rat model

Abstract

The oxidative stress leading to degenerative changes in the brain of Alzheimer's disease (AD) is evident. Our aim was to evaluate the therapeutic and protective effects of pomegranate extract (PE) and pomegranate extract-loaded nanoparticles (PE nano) in an AlCl 3-induced AD rat model. Nanoparticles were synthesized with a PE load of 0.68% w/w, and 70 male Wistar rats were divided into 7 groups: Group I was the control, Group II received PE., Group III received PE nano for 2 weeks, Group IV received AlCl 3 (50 mg/kg) daily orally for 4 weeks, Group V received PE for 2 weeks, Group VI received PE nano for 2 weeks, and Groups V and VI were started after AlCl 3 administration was stopped. Group VII received PE for 2 weeks and was stopped before AlCl 3 was administered. The Results revealed that the discrimination index in the novel object recognition test was the least in AD rat model but increased in cases protected with PE treated with PE nano. Similar results were shown based on calculating the brain weight/body weight percent. The biomarkers of antioxidant activity (catalase, glutathione and total antioxidant activity) in brain homogenate were significantly increased in groups treated with either PE or PE nano. The thiobarbituric acid reactive substance measured to estimate lipid peroxidation was significantly increased in AD rat model and decreased in groups protected with PE or treated with PE nano. Histopathological studies using hematoxylin and eosin, cresyl violet, and silver stains revealed hyaline degeneration, chromatolysis, and hallmarks of AD; neurofibrillary tangles and the senile plaques in brains of AD rat model. Restoration of the histological architecture, Nissl granules, and minimal appearance of hallmarks of AD characterized brains treated with PE or PE nano. In conclusion, PE was more effective as a protectant than a therapeutic measure in alleviating the antioxidant, lipid peroxidative effects and histopathological hallmarks in AD brains. But, the therapeutic PE-loaded nanoparticles increased the efficacy of active components and produced similar results as the protective PE.

Keywords: Alzheimer's; Antioxidant markers; Histopathology; Lipid peroxidation; Nanoformulations; Novel object recognition test; Pomegranate extract.

Fig. 1

A diagram showing Novel object recognition test.

Fig. 2

Size measurement of PE-SLNPs using Dynamic Light Scattering (DLS). Average particle size is around 297 nm in diameter; PDI = 0.212. The entrapment efficiency was found to be around 45%.

Fig. 3

Bar graph showing DI calculated in NORT in all groups. The one-way analysis of variance (ANOVA) test was used. When equal variance could be assumed, the Fisher’s least significant difference (LSD) t-test was applied. Data are presented as means ± standard deviation (SD). (a) Significantly different from the control, PE, PE Nano at P ≤ 0.05. (b) Significantly different AD group at P ≤ 0.05.

Fig. 4

Bar graph showing brain weight /body weight percent in all groups. The one-way analysis of variance (ANOVA) test was used. When equal variance could be assumed, the Fisher’s least significant difference (LSD) t-test was applied. Data are presented as means ± standard deviation (SD). (a) Significantly different from the control, PE, PE Nano at P ≤ 0.05. (b) Significantly different AD group at P ≤ 0.05.

Fig. 5

Bar graph showing the mean concentration of TBARS µM in brain homogenate of rats of all groups. The one-way analysis of variance (ANOVA) test was used. When equal variance could be assumed, the Fisher’s least significant difference (LSD) t-test was applied. Data are presented as means ± standard deviation (SD). (a) Significantly different from the control, PE, PE Nano at P ≤ 0.05. (b) Significantly different AD group at P ≤ 0.05.

Fig. 6

Photomicrograph of sections of cerebral cortex of rats. H&E reveals well organized structure with neurons having rounded pale nuclei and basophilic cytoplasm in Con, PE and PE Nano. Disturbed structure, shrunk neurons with condensed pyknotic nuclei (arrow) and areas of hyaline degeneration (head arrow) noted in AD. Sections of AD treated with PE and PE Nano formulation or PE + AD reveals restoration of the well-organized structure of the cortex and the neurons. The cresyl violet stain shows prominent basophilic Nissl’s granules in the cytoplasm of neurons (arrows) present in Con, PE and PE Nano formulation. In AD, chromatolysis is evident (dashed arrows). Nissle granules is restored in sections of PE Nano formulation or PE + AD but partly in AD + PE (arrows). Silver stain shows numerous NFT (arrows) and SP (circles) in AD sections. NFT (arrows) appeared to decrease in AD + PE or PE nano but disappeared in PE + AD. (H&E X200, Cresyl violet and silver X600).