INTRODUCTION:

Over the past few decades, plants’ secondary metabolites were used as therapeutic sources, and many advanced medications were developed from them¹. In discovering lead compounds from medicinal plants, traditional medicine involving plants and their extractions play a significant role^2,3.

However, modern studies must scientifically validate ethnomedicinal reports—Cucurbita pepo. (C.pepo), i.e., pumpkin has served as a folk medicine for a variety of diseases. Different varieties of pumpkin are considered edible, but their seeds are discarded⁴. In contrast, according to Ayurvedic medicine, these were used to treat benign prostate hyperplasia (BPH), bladder disorders, gastrointestinal diseases, worm infestation, cancer, and other clinical manifestations.

Neuron degeneration occurs irreversibly in AD and it progresses over time leading to dementia in the elderly, causing impairment in their daily activities⁴. It usually manifests in two stages; it occurs as an insidious onset of impaired cognitive function and altered mood and behavior in the early stages. The later stage progresses to disorientation, severe cortical dysfunction, memory loss, and aphasia. Over 5-10 years of the disease onset, the individual becomes completely disabled, mute, and immobile^5,6.

During neuronal development, essential fatty acids play a vital role⁷. AD's pathophysiology is related to neuron injury and death, initiating memory, and learning in the hippocampus brain region, then atrophy affecting the whole brain. "Positive" lesions such as amyloid plaques, neurofibrillary tangles, and glial responses, as well as "negative" lesions such as loss of neuronal and synapses, are the markers of neuropathology in Alzheimer's disease. Amyloid β-peptide (Aβ-peptide), a 40-42 amino acid peptide generated from a membrane protein, is the predominant protein in neurite plaques, and after enzyme sequential cleavage, the amyloid precursor protein (APP). A gene on chromosome 21 encodes APP. In neuronal culture, APP interacts with the extracellular matrix and stimulates the formation of neuritis, resulting in AD⁸.

Current pharmacological therapy for AD includes NMDA receptor antagonists, Acetyl-cholinesterase inhibitors, and different Cerebro-active medications and are aimed at symptomatic relief by enhancing the concentrations of neurotransmitters at the synapses^9,10. Recently, C.pepo has received a renowned interest in traditional and complementary medicine. C.pepo seeds (pumpkin seeds) contain a very high amount of protein and fat, nutrients, vitamins, minerals, and many bioactive components such as antioxidants, phytosterols, and other phytochemicals that improve brain structure and function^11,12. In addition, there is evidence that micronutrients are present in pumpkin seeds, which can help to improve memory and brain development. Children under the age group 6-8 years old who were tested for memory and brain development with pumpkin supplementation for two months showed improved memory and learning ability compared to the control group¹³. As a result, the current study was proposed to investigate the potential of pumpkin seeds for the treatment and management of AD, which is associated with impaired memory and brain function.

MATERIALS AND METHODS:

The study was conducted after the Institutional Animal Ethics Committee (IAEC) approval was obtained. IAEC /KMC/87/2018. Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal. C. pepo seeds were obtained from Udupi, Karnataka, India, and were authenticated by Professor Mrs. Usharani. S Suvarna, Associate Professor, Head at Department of Botany, Mahatma Gandhi Memorial College Udupi, Karnataka, India (MGMC/2019-20).

Preparation of extract:

Preparation of 80% ethanolic extract of pumpkin seeds by the Soxhlet method¹⁴.

Phytochemical investigation of ethanolic extract of pumpkin seeds:

Alkaloids, tannins, flavonoids, saponins, carbohydrates, and phytosterol were analyzed qualitatively and quantitatively to estimate the total content of phenol, flavonoids, tannins, and saponin^4,15.

Animal Procurement:

Male Sprague Dawley rats weighing 180-220g, aged 12-15 weeks, were housed in cages on standard food and water ad libitum at 23±27°C and 60% humidity with a 14:10-h light/dark cycle

Experimental design:

Thirty male Sprague Dawley rats were categorized into five groups (n=6). The solutions, AlCl_3, and rivastigmine were made fresh every day. AlCl₃ (10 mg/kg) was given intraperitoneally (i.p) to rats to develop Alzheimer's disease. The AlCl₃ dose and rivastigmine dose was chosen based on the previous research^16,17. This dose resulted in substantial memory impairment and a low fatality rate. Rivastigmine (1mg/kg) was given i.p. 30 minutes before AlCl₃ was given for 75 days.

The experimental groups were as follows:

I – Normal control	distilled water (5 ml/kg orally from day 6- day 75)
II – Disease Control	AlCl₃ (10 mg/kg (i.p) from day 6- day 75)
III - Standard	AlCl₃ (10 mg/kg orally) + rivastigmine (1mg/kg ) from day 48- day 75
IV - Treatment-1	AlCl₃ (10 mg/kg i.p)+ pumpkin seed extract (100mg/kg orally from day 48- day 75)
V - Treatment-2	AlCl₃ (10 mg/kg i.p)+pumpkin seed extract (200mg/kg orally from day 48- day 75)

Dosage:

Previous studies have shown that a Lethal dose (LD) of 2g/kg of pumpkin seed extract is non-toxic to rats⁴.

Behavioral estimations:

To probe the spatial learning and memory involved in AlCl₃-induced Alzheimer’s disease in rats, we performed Behavioral assessment, i.e., morris water maze^18,19 and passive avoidance^20,21.

Dissection and preparation of tissues:

On the last day of the retention trial i.e., the 75^th day, rats were sacrificed, and the brains were collected and preserved at -80°C. The hippocampus and frontal cortex were then extracted from the frozen brain using the method described by Glowinski and Iverson²². A homogenizer was used to homogenize the tissue. Homogenate was made at 8000rpm in a 10% w/v ice-cold phosphate buffer (PBS) pH 7.4 solution. Homogenates were preserved in aliquots at -20°C until they were employed for further analysis.

Estimation of catalase, reduced glutathione, and acetyl-cholinesterase activity:

The supernatant was analyzed for acetyl-cholinesterase (AChE) activity²³, catalase (CAT)²⁴, and reduced glutathione (GSH) levels²⁵ in the hippocampus and the frontal cortex. The Pierce BCA Protein Assay Kit was used to calculate the total protein in all tissue samples as per Thermo Fisher Scientific's experimental protocol (Waltham, USA).

Statistical Analysis:

Graph Pad Prism version 5.0 software was used to do a two-way analysis of variance (ANOVA) followed by a post hoc Bonferroni's test on retention trials completed on days 5, 47, and 75. The data is presented as a mean with a standard error of the mean (SEM). One-way ANOVA was used to examine biochemical estimates, followed by Tukey's multiple comparison tests. It was determined that p < 0.05 was statistically significant.

RESULT:

Table 1: Qualitative analysis of 80% ethanolic extract of pumpkin seed

Phytoconstituents	Result
Flavonoids, Triterpenoids, steroids, tannins, Phytosterol, Saponins	Positive
Alkaloid, Carbohydrate	Negative

Table 2: Quantitative analysis of 80% ethanolic extract of pumpkin seed

Total flavonoid	2.98mg quercetin equivalent/g
Total phenolic content	3.63 mg GAE/g
Total tannin	0.858 mg TAE/g
Saponin	72.8%

Figure 1: Effect of seed extract on brain index in 5 groups. p˂0.05 all other groups and Disease control.

Table 3: Showing the effect of seed extract on frontal cortex tissue homogenate catalase (n=5), reduced glutathione (n=5), and acetylcholinesterase (n=4) in different groups. All values are expressed in Mean ± SEM. (One-way Anova, Tukey’ test)

Biochemical parameters	Group 1 Mean ± SEM	Group 2 Mean ± SEM	Group 3 Mean ± SEM	Group 4 Mean ± SEM	Group 5 Mean ± SEM	p-Value
Catalase unit/ mg of protein	32.27 ± 4.746	14.69 ± 3.48 *	20.24 ± 2.46	11.94 ± 2.03	16.84 ±3.27 ^#	˂ 0.05
GSH µM/mg of protein	9.53 ± 1.11	1.26 ± 0.16 *	5.35 ± 0.48	3.22 ± 0.54	3.77 ± 1.01 ^#	˂ 0.05
AChE unit/ mg of protein	210.64 ± 20.305	28.85 ± 9.19 *	119.82 ± 7.90	94.58 ± 7.021	101.84 ± 4.37 ^#	˂ 0.05

p*, ^# ˂ 0.05= Comparison between disease control Vs treatment 2

Figure 2: Showing the effect of seed extract on frontal cortex tissue homogenate catalase (n=5), reduced glutathione (n=5), and acetylcholinesterase (n=4) in different groups.

Table 4: Showing the effect of seed extract on hippocampus tissue homogenate catalase, reduced glutathione, and acetylcholinesterase in different groups (n=6)

Biochemical parameters	Group 1 Mean ± SEM	Group 2 Mean ± SEM	Group 3 Mean ± SEM	Group 4 Mean ± SEM	Group 5 Mean ± SEM	p-Value
Catalase unit/ mg of protein	65.33 ± 18.18	43.32 ± 12.59	60.05 ± 8.29	50.77 ± 4.81	53.30 ± 16.57	ns
GSH µM/mg of protein	6.38 ± 1.08	1.372 ± 0.503	4.906 ± 1.01	2.84 ± 0.55	3.0 ± 0.73	˂ 0.05
AChE unit/ mg of protein	163.66 ± 6.33	48.24 ± 3.35	64.63 ± 11.09	50.39 ± 11.42	54.57 ± 11.09	ns

Figure 3: Showing the effect of seed extract on hippocampus tissue homogenate catalase (n=5), reduced glutathione (n=5), and acetylcholinesterase (n=4) in different groups.

Table 5: Effect of seed extract on time spent in the dark chamber, latency time, and the number of entries into the dark chamber aftershock in different groups (n=6). All values are expressed in Mean ± SEM

Behavioral parameters	Group 1 Mean± SEM	Group 2 Mean± SEM	Group 3 Mean± SEM	Group 4 Mean± SEM	Group 5 Mean± SEM	p-Value
Time spent in the dark chamber	3.76 ±0.65	57.13±7.54**	25.06 ± 6.72	30.93± 3.03	29.84 ±0.52^##	˂ 0.005
Latency time	6.16±1.3	65.33± 11.5**	9.66±1.52	18.066± 7.46	13.58±3.68^##	˂ 0.005
No. of entries into dark Chamber	0.19±0.06	1.46±0.06**	0.66±0.27	0.86±0.16	0.73 ±0.13^##	˂ 0.005

p**, ^## ˂ 0.005= Comparison between disease control Vs treatment 2

Figure 4: Effect of seed extract on time spent in the dark chamber, latency time, and the number of entries into the dark chamber aftershock in different groups (n=6). All values are expressed in Mean ± SEM.

Figure 5: Graph showing Latency to reach the Island zone on 1s, 2nd, 3rd and 4th day during Spatial acquisition trial, n = 6 rats in each group. (One-way Anova, Tukey’ test)

Figure 6: Graph showing time spent in the target quadrant during the probe trial. n=6 rats in each group. (One-way Anova, Tukey' test)

Figure 7: GC-MS chromatogram for 80% ethanolic extract of pumpkin seed

DISCUSSION:

Alzheimer’s disease (AD) is a progressive, irreversible, and the most common cause of dementia in older people because of the impairment of general activities^26,27. More remarkable than 36.5 million individuals over 60 years worldwide have either AD or any of the various sorts of dementia. Approximately 115 million will have AD by 2050 and trusted that 1 of every 85 individuals would develop the symptoms and factors for AD. Age is also one of the critical risk factors for AD, the occurrence of AD is 3% for people 65 to 74 years of age, 19% for 75 to 84 years, and 47% for 85 years or more²⁸. Life is dependent on plants as a source of medicine, and many modern medications are produced from secondary plant compounds²⁹.

Phytochemical analysis of the extract showed the presence of flavonoids, tannin, saponins, and, phenolic contents (Table: 1 and 2); the secondary plant metabolites may resist oxidative stress-induced AD as they have the potential to function as antioxidants by scavenging the free radicals and increasing the acetylcholinesterase activity^30,31. In addition, the weight of the brain in AD rats was significantly decreased compared to the control (Fig: 1). Typically, in AD patients, 100-200gms of brain weight will be reduced due to atrophy³².

Catalase and GSH are endogenous enzymes with antioxidant activity; they govern the intracellular free radical levels. Reactive Oxygen Species (ROS), like hydroxyl ions in the cells, are responsible for damaging the beta-amyloid peptides and other biological compounds around them, thus producing the plaques. Therefore, estimating these enzymes' levels in the homogenate of the brain’s frontal cortex and hippocampus is essential as they play an important role in the formation of beta-amyloid plaques and AD pathogenesis^33,34,35. In the catalase estimation, the disease control group showed a significant (p<0.05) decrease when compared to the control group and treatment groups (Table 3). Similarly, GSH neutralizes the free radicals by donating their electrons, thereby converting them to an oxidized form. Due to increased free radicals in the disease control group, reduced GSH was observed, which was statistically significant (p<0.05) compared to the standard and treatment groups. Conversely, the control group showed significantly high GSH levels (Table 3). A previous study showed similar results in brain tissue lysates as this enzyme is crucial for maintaining a proper balance between oxidative and antioxidant forms in cells³⁶.

Acetylcholine is a functional and important neurotransmitter that is degraded by the enzyme acetyl-cholinesterase. Decreased acetylcholine release at the synapse is responsible for impaired cognition. Therefore, increasing the activity of AChE is considered a potential target for the treatment of AD. A significant (p<0.005) decrease in the acetyl-cholinesterase levels in the disease control group compared with the control. Whereas in the treatment groups, there is a significant (p<0.05) increase in the AChE activity (Table 3). The literature suggests and shows corresponding results similar to the current study results¹⁶.

During the passive avoidance assay, an increase in the time spent in the dark chamber was observed in the disease control group and showed significance (p<0.005) with standard drug Rivastigmine and treatment groups. In addition, treatment group-2, before and after the induction of memory impairment, showed a positive effect. Similarly, an increase in the latency of the first entrance and the number of entries from one chamber was observed in disease control which also showed a significance (p<0.005) with the standard drug rivastigmine and treatment groups. A time period of 3 minutes was used to check the animals' fear-motivated avoidance learning and memory activity.

Morris water maze includes a spatial acquisition trial for four days and a probe trial for one day. In the acquisition trial, various parameters were assessed, like escape latency, D time, and D entries. In latency to reach the island zone or escape latency, all the animals in the treatment groups gradually learned and took less time to reach the island zone from day one to day four (Spatial Acquisition Trial) than the disease control group took more time. Compared to disease control, treatment groups showed a remarkable decrease in escape latency as detected in the Morris water maze test. The disease control group showed a memory deficit when compared to the treatment groups. A significant difference (p ≤ 0.05) between the disease control group and treatment models from day 1 to day 4 of the spatial acquisition trial. These results show similar to the previous studies^37,38.

An induction of AlCl₃ in rats for 75 days, in the disease control group, showed significant spatial memory impairment during the probe trial performed on the 75^th day in the Morris water maze. However, on the 5th day, i.e., probe trial, animals in the drug treatment group significantly spent more time in the target quadrant than the disease control group. In addition, there was a statistically significant difference (p ≤ 0.05) among drug treatment groups compared to the probe trial's disease control group.

A total of 35 compounds from the ethanolic extract of Cucurbita pepo were identified. Major Compounds by GC-MS, which accounted for over 60% of the overall content were, 9,12-Octadecadienoic acid (21.52%), linoleic acid ethyl ester (19.24%), 9,12-Octadecadienoic acid (Z,Z)-, 2,3-dihydroxy propyl ester (12.33%) and ethyl alpha-d-glucopyranoside (7.99%). Polyunsaturated fatty acids have a significant role in neurite outgrowth activity; thus, the presence of these compounds in pumpkin seed extracts resulted in improved memory and behavior changes in the animal model^39,40. To conclude 80% ethanolic extract of C.pepo seed administration to the AlCl₃-induced memory impairment group animals showed remarkably enhanced spatial memory function, shown in the Morris water maze, and enhanced fear-motivated avoidance learning and memory showed in the Passive avoidance test. The activity may be due to phytochemical constituents and PUFA present in the ethanolic extract. 80% ethanolic extract of C.pepo (pumpkin) seeds has cholinergic, antioxidant, and anti-inflammatory levels and prevents memory impairment.

FUNDING:

PG research grant, Manipal academy of higher education, Manipal

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We thank Kasturba Medical College, Manipal, Manipal Academy of Higher Education for providing the facility to conduct the study and also providing a Postgraduate research grant.

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Received on 02.12.2021 Modified on 11.07.2022

Research J. Pharm. and Tech 2023; 16(3):1065-1071.

DOI: 10.52711/0974-360X.2023.00178

In vivo Acetylcholinesterase activity and Antioxidant property of Cucurbita pepo ethanolic extract in Alzheimer’s disease induced by Aluminium chloride in Sprague Dawley rat model