الفهرس 
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Abstract
Alzheimer’s disease (AD) is a neurodegenerative condition that most often influences elderly people and manifests as memory and cognitive loss. By its very nature, the condition progresses, meaning that over time, the severity of the symptoms worsens and the quality of life decreases. The severity of Alzheimer’s disease can range from mild to severe. In humans, neurofibrillary tangles (NFTs), senile plaques (SPs), and cholinergic neuron loss in the basal forebrain are the three main pathogenic features of AD (Guerriero et al., 2017).
Prior research has demonstrated that the use of D-gal and AlCl3 together can cause neurodegenerative alterations in rodents that are similar to those seen in AD, including in the cholinergic system, expression of elevated levels of amyloidogenic proteins, oxidative stress, memory problems, and cell loss in the cerebral cortex and hippocampus (Wei et al., 2017).
Lately, the utilization of phytonutrients and medicinal plants has attracted particular attention for the treatment of neurological illnesses like AD (Shah et al., 2017). Rosemary and saffron were included in the current study as examples of these medicinal herbs for their capacity to improve cognition and memory (Ahmed and Babakir‐Mina, 2020).
This study aimed to evaluate the protective effect of aqueous extracts of rosemary and saffron on AlCl3 & D-gal induced Alzheimer rat model to mitigate the symptoms of Alzheimer’s disease from one side and to avoid the serious side effects of synthetic drugs from the other side.
In the present study, the optimum dose of both rosemary and saffron was determined according to OECD criteria (Oecd, 2000), and the selected dose was 500 mg/kg for each. After that, we conducted phytochemical analysis and screening for our investigated herbal extracts using high performance liquid chromatography (HPLC) analysis and different tests.
The study included sixty male Wistar rats which were divided into 6 groups, 10 rats per groups as follows:
G1, Control: rats were orally administered 0.9 % saline and treated as a normal control. G2, AD model: rats were intraperitoneally administered AlCl3 and D-gal at doses of 150 & 300 mg/kg for one week to induce Alzheimer’s disease. G3, Donepezil: AD rat models were orally treated with the conventional medicine (Donepezil®) at a dose of 1.5 mg/kg for 15 days. G4, Rosemary: AD rat models were orally treated with rosemary aqueous extract at a dose of 500 mg/kg for 15 days. G5, Saffron: AD rat models were orally treated with saffron aqueous extract at a dose of 500 mg/kg for 15 days. G6, Combined (Rosemary and Saffron): AD rat models rats were orally treated with combined extracts of both rosemary and saffron for 15 days.
At the end of the experiment blood samples were collected from the retro-orbital plexus of the rats 24 hours under light ether anesthesia and then centrifuged at 3000 rpm for 10 min for serum separation. The obtained serum samples were then stored at -80 °C for biochemical analysis. To obtain the brain samples, the rats were then decapitated. After that, the brain tissue was divided into two halves. The first half was utilized for the estimation of LC3 (an autophagy marker) by flow cytometry. The second half was preserved in 10% formaldehyde for further histopathological examination.
The behavioral test (Y-maze test) was performed to assess the spatial memory. The biochemical analysis included the measurements of Malondialdehyde (MDA), Glutathione peroxidase (GPx), Catalase (CAT), Superoxide dismutase (SOD), Nrf2, tau, β-amyloid, Acetylcholine (Ach), Acetylcholinesterase (AchE), Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), and LC3 (Microtubule-associated protein 1A/1B-light chain 3).
The behavioral results of the current study showed a significant improvement in total arm entries and spontaneous alternation percent (SAP%), which reflect the potential capacity of both extracts in repairing spatial memory in particular in the combined treated group P < 0.0001.
The AD model exhibited a significant rise in MDA and SOD level accompanied by a decline in other oxidative stress markers in comparison to the control one at P < 0.0001. However, both saffron and rosemary individually and in combination exhibited a significant improvement in these markers as compared to the AD model at P < 0.05.
In comparison to the control group, the AD model showed a prominent elevation in tau, β-amyloid, and AchE levels with a marked decline was seen in Ach level P < 0.0001. However, these markers showed a marked improvement when treated with both extracts (P < 0.0001) with special attention to the combined treated group as it showed the most improvement (P < 0.0001).
Further, consideration was given to some selected proinflammatory markers, TNF-α and IL-6 in this study. where these parameters significantly showed obvious improvements in all treated groups as their levels downregulated after a sharp elevation in AD model P < 0.0001.
Autophagy (self-eating) is a lysosome-mediated elimination process. Lysosomal degradation routes trigger autophagy, a catabolic process for unwanted or defective cytoplasmic components. Several cellular conditions, namely food deprivation, hypoxia, oxidative stress, microbial infection, and endoplasmic reticulum (ER) stress, trigger autophagy activation (Khandia et al., 2019).
Microtubule associated protein light chain 3 (LC3) is considered one of the markers of autophagy induction (Huang and Liu, 2015). In this study LC3 was precisely measured by flowcytometry. The results revealed an obvious disturbance in the LC3 autophagy marker percentage as compared to control. This situation was modulated when treating with both extracts and there combinations as well P < 0.0001.
At the end of our study, it was clearly noticed that our biochemical examinations were all well confirmed by the histopathological investigations, as they corrected the tissue changes in cortex and hippocampus and reflected the potential role of each of rosemary and saffron in overcoming the AD symptoms.
In conclusion, the two herbal extracts revealed their pivotal role in Alzheimer’s symptoms amelioration, especially when used in combination because of their active components that can efficiently bind with some target proteins that are crucial to the development of the disease. Based on the aforementioned results, we advocate conducting additional research on the use of both plant extracts individually or in combination to assess their therapeutic potential in Alzheimer’s disease management. Finally, we recommend using both rosemary and saffron in food preparation in which they help enhance memory, combat oxidative stress and offer protection against Alzheimer’s disease. Moreover, we advise limiting our daily exposure to aluminum to prevent serious conditions like Alzheimer’s disease.