Anti-Amnesic and Neuroprotective Effects of Ziziphus Jujuba Aqueous Extract on Scopolamine-Induced Cognitive Impairments in Rats

Background: Alzheimer's disease is a neurological condition that affects more than 44 million people worldwide. The available treatments target the symptoms rather than underlying causes. Ziziphus jujuba (Rhamnaceae) is used in traditional Cameroonian medicine to treat many disorders including memory impairments. The study aimed to evaluate the anti-amnesic and neuroprotective effects of Z. jujuba aqueous extract on scopolamine-induced memory disorders in rats. Methods: Learning and memory impairments were induced in rats by scopolamine (1mg/kg, i.p.) for 15 days. Rats that developed cognitive impairments were divided as follows: two positive control groups received piracetam (200 mg/kg, p.o.) or tacrine (1 mg/kg, p.o.); three test groups received the extract (29, 57, and 114 mg/kg, p.o., respectively) daily for 15 days. At the end of treatments, memory impairments were assessed by Morris water maze and Y-maze tests. Thereafter, animals were sacriced and some biochemical parameters (oxidative stress, inammation, and apoptosis) were estimated in the hippocampus and prefrontal cortex. Results: Z. jujuba decreased the time to reach the platform and increased the time in the target quadrant. However, it failed to affect spontaneous alternation in the Y-maze. Furthermore, the extract reversed scopolamine-induced oxidative stress, inammation, and apoptosis. This was conrmed with the prevention of neuronal loss in the hippocampus or prefrontal cortex. Conclusions: These ndings suggest that Z. jujuba extract possesses ant-amnesic and neuroprotective effects. It seems that these effects are mediated in part by antioxidant, anti-inammatory, and antiapoptotic activities. This, therefore, justify its use to treat dementia and psychiatric disorders in Cameroon’s folk medicine. tumor necrosis-alpha; GSH, reduced glutathione; MDA, malondialdehyde; SOD, superoxide dismutase, AD, Alzheimer’s disease.


Introduction
Alzheimer's disease (AD) is the most common form of dementia (60 -70% of cases) [1]. AD is an irreversible and progressive neurodegenerative disorder of the central nervous system, which occurs gradually and leads to memory loss, unusual behavior, and personality changes [1,2]. According to the World Health Organization (WHO), more than 44 million people worldwide are affected by AD with 7.7 million new cases every year [3][4][5]. In Africa, the prevalence of AD is estimated at 5.6% [6]. At the molecular and cellular levels, AD is characterized by extracellular deposits of beta-4-Amyloid (PβA) protein, intracellular entanglements, cholinergic de cit, extensive neuronal loss, and synaptic changes in the cerebral cortex and hippocampus [7]. PβA deposit causes neuronal death via some possible mechanisms including, oxidative stress, in ammation, and apoptosis [2]. At present, there is no curative treatment against AD [8]. Drug therapies are suggested such as acetylcholinesterase inhibitors (Galantamine, Rivastigmine, and Donepezil) and N-methyl-D-aspartic acid (Memantine) receptor antagonists [9]. These treatments are very expensive, di cult to access, have side effects, and are all symptomatic relieves [2,3]. Therefore, medicinal constitute a source for the discovering of effective drugs against AD. Among them, Ziziphus jujuba, a plant from the Rhamnaceae family, is used in Northern Cameroon, Asia, and India for the treatment of many pathologies including typhoid fever, furuncle, sleep disorders, diarrhea, and pain [10,11]. In Cameroon, all parts of the plant are used to treat otitis, in ammation, cancer, anxiety, rickets, typhoid fever, and anorexia. Seeds are used as dewormers [12] and leaves in cases of dementia [12]. In recent years, research on Z. jujuba fruits have shown to possesses anti-in ammatory [13] and neuroprotective activity [14], while the leaves showed to possess antiin ammatory [15], antifungal, anticancer, antifertility, antibacterial, anxiolytic, sedative, and antioxidant properties [16][17][18]. Research undertaken on the anti-amnesic effect of Z. jujuba revealed that the seed possesses a protective effect against spatial memory impairments in rats [19][20][21]. According to other authors, this effect could be mediated by cholinergic blockade [22]. Moreover, the hydroethanolic extract of Z. jujuba was demonstrated to ameliorate cognitive decline and seizures in an experimental model of epilepsy in rats [20]. A toxicity study on Z. jujuba leaves revealed that they are non-toxic [23]. Nonetheless, toxic elements are found in trace amounts in the whole plant [24]. Phytochemical analysis from seeds, leaves, and stem barks revealed the presence of alkaloids, avonoids, tannins, saponins, and polyphenols [25]. The HPLC ngerprint le of Z. jujuba leaves extract identi ed the presence of major constituents such as (-)-catechin, traumatic acid, quercetin-3-O-robinobioside, rutin, and quercetin-3-O-α-L-arabinosyl-(1→2)-α-L-rhamnoside with the total contents of nine avonoids [26]. Furthermore, GC/MS analysis of ethanol extract of Z. jujuba seeds revealed the existence of 20 component, main components were 13-Heptadecyn-1-ol (12.95%), 7-Ethyl-4-decen-6-one (9.73%), Lineoleoyl chloride (8.54%), Linoleic acid (6.37%), 2,5-Octadecadiynoic acid, methyl ester (5.57%) and Palatinol A (4.81%) [27]. To date, there is no scienti c evidence on the anti-amnesic and neuroprotective effects of Z. jujuba leaves aqueous extract. Therefore, this study was undertaken to investigate the anti-amnesic and neuroprotective effects of Z. jujuba leaves aqueous extract on scopolamine-induced cognitive impairments in rats, using Morris water maze and Y-maze paradigms. Possible mechanisms of action have been also explored.

Plant collection and extraction
The leaves of Z. jujuba were harvested in Mokolo, in the Far North region of Cameroon, at global position system coordinates 10.7425° North, 13.8042° East. It was identi ed at the National Herbarium of Cameroon (HNC) (database of herbarium index: http://sweetgum.nybg.org/science/ih/herbarium-list) by Mr. Ngansop Eric in comparison to sample N°14446/HNC. The plant name has been checked on http://www.theplantlist.org. The extract of Z. jujuba has been prepared according to the traditional healer's method. Brie y, fresh leaves of Z. jujuba were dried in shade and crushed into powder. The Z. jujuba leaf powder (75 g) was boiled in 1.5 l of distilled water for 20 min. After cooling, the obtained solution was ltered with Whatman No. 3 paper. The ltrate (1.17 l) was dried at 50°C in an oven to a dried extract (10.8 g), yield 14.40%. From the stock solution (5.7 mg/ml), less and most concentrated solutions were respectively made at ratios 1/2, and 2. These solutions were therefore administered in rats at doses 29 and 114 mg/kg, respectively.

Drugs and chemicals
Piracetam tablets were purchased from U.C.B. Pharma SA Braine-l'Alleud (Belgium) and tacrine hydrochloride capsules from Shionogi. Inc (Japan). Scopolamine, trichloroacetic acid, thiobarbituric acid, Ellman reagent, adrenaline, and formalin were purchased from Sigma Chemical Co., St. Louis (United States), while ethyl ether from Cooper Laboratory (Paris). Piracetam, tacrine, and scopolamine were dissolved in distilled water. All solutions were administered per os (p.o.) at a volume of 10 ml/kg except, scopolamine administered intraperitoneally (i.p.).

Animals
Animals were male Wistar rats, 6 to 8 weeks old, weighing between 120 -140 g. These animals were raised in the animal house of the laboratory of Animal Physiology (University of Yaoundé I, Cameroon) under standard light (12-hour day/night cycle) and temperature (24-26°C) with free access to standard animal diet and tap water. Animals procedures were carried out following the guidelines of the Institutional Ethics Committee of the Cameroon Ministry of Scienti c Research and Technological Innovation (Reg. no. FWA-IRD 0001954, 04/09/2006), which adopted the guidelines of the European Union on Animal Care (C.E.E. Council 86/609). Anesthesia of animals has been made according to the American Veterinary Medical Association (AVMA) guidelines for the euthanasia of animals (2020). The ethical procedure (following an institutional guidelines), the study design (group being compared including control group), the sample size (number of animals per group), the experimental procedure (animal model well known and described), the experimental animal (sex, strain and age of animals detailed), housing (Animal facilities of the University of Yaounde I in recommended conditions), allocating animals to experimental group, experimental outcomes (dosage of biomarkers), and statistical methods in this study are detailed in the manuscript and carried out according to the ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357)

Experimental design
To induce cognitive impairments, rats were divided into 2 groups and treated for 15 days: -group of 50 rats treated with scopolamine (1mg/kg, i.p.); -normal control group of 8 rats received distilled water (10 ml/kg, i.p.).
At the end of treatments, learning and memory impairments were assessed on Morris water maze and Ymaze paradigms. Rats that developed learning and memory impairments were selected for the rest of the experiment. These rats were then divided into 5 groups of 8 rats as follows: -two positive control groups received piracetam (200 mg/kg, p.o.) or tacrine (10 mg/kg, p.o.); -three test groups received the aqueous extract of Z. jujuba (29,57, and 114 mg/kg, respectively); Furthermore, the aforementioned normal control group was added as the 6 th group and treated with distilled water (10 ml/kg, p.o.).
All animals were thus treated for 15 additional days. At the end of the experimental period, cognitive declines were again evaluated on the aforementioned paradigms. After completion of behavioral analysis, rats were sacri ced, and the brain was removed for biochemical markers and histological assays ( Figure 1). Spatial long-term learning and memory were studied using the Morris Water Maze [28]. A circular tank (150 cm diameter and 60 cm height) was lled to 40 cm with water (25°C). The pool was virtually divided into four equal quadrants: North, South, East, and West. A white refuge platform (8 cm diameter and 30 cm height) was placed in the center of one of the quadrants, i.e.1 cm below the surface of water. The water was bleached by adding liquid milk so that the platform was invisible on the surface of water. The pool was located in a room with various visual cues. On the 1st day of the test (habituation phase), each rat has been acclimatized for 60 s in the absence of the platform. The following tests (acquisition phase) took place in 4 days with a daily session of 3 sessions per day. The session time for each animal to nd the platform was 120 s. When an animal found the platform, it was left to stay on its top for 10 s. If after 120 seconds an animal was unable to locate the platform, it was taken there and allowed to remain for 10 s. The time interval between sessions was 5 minutes. During each session of the acquisition phase, the latency time to nd the platform was recorded for each animal. The effectiveness of learning was assessed in the retention phase. During this phase, which lasts 120 s, the platform was removed from the tank. The latency time to nd the platform and the time spent in this compartment were recorded.

Y-maze test
The Y-maze test was used to assess working memory in animals by recording spontaneous alternation [29]. The maze used was a wooden device with 3 identical branches (40 cm long x 35 cm high x 12 cm wide) separated by an angle of 120°. The walls of each arm were decorated with a different pattern (A, B, and C) to differentiate them. Rats were individually placed at the end of a branch of the maze, to freely explore the maze for 5 min [30]. The number of entries in each arm of the maze was recorded. After each animal session, the device was cleaned with 10% ethanol. A spontaneous alternation (SA) has been de ned, as three successive entries in the three different arms (example: ABC, CAB, or BCA). The percentage of SA was used as an index of performance of the working memory and calculated according to the following formula: [(Number of AS)/(total number of arms visited -2)] 100.
1.6. Biochemical analysis 1.6.1. Sacri ce and preparation of homogenates At the end of the behavioral assessment, animals were sacri ced by decapitation after ethyl ether anesthesia. The brain of each animal was removed and divided into two hemispheres. Hippocampus and prefrontal cortex from the rst half were isolated, washed in 0.9% NaCl, and wrung out. They were then weighted and homogenized with Tris-HCl buffer (50 mM, pH 7.4) in a ratio of 20%. Following centrifugation at 3000 rpm at 4°C for 25 min, the supernatant was removed and stored at -20°C for neurochemical parameters evaluation. The second half of the brains were xed in 4% formalin for histological analysis.

Reduced tissue glutathione level
The Ellman reagent (1.5 ml) was introduced into tubes containing 100 μl of homogenate (test tubes) or Tris buffer (50 mM HCl, 150 mM KCl, pH 7.4) (blank tube). These tubes were shaken and incubated for 60 min at room temperature. Absorbance was read against the blank at 412 nm. The level of reduced glutathione (GSH) was expressed in mol/g of tissue protein [31].

Malondialdehyde level
The MDA assay was carried out according to the method described by Wilbur et al. [32]. Brie y, 250 µl of homogenate was introduced into test tubes and 250 µl of tris buffer (50 mM HCl; 150 mM KCl; pH 7.4) into a blank tube. To each tube was added 125 µl of 20% trichloroacetic acid and 250 µl of 0.67% thiobarbituric acid. Tubes were incubated for 10 min at 90°C. They were then cooled and centrifuged at 3000 rpm for 15 min at room temperature. The supernatant was removed and absorbance was read at 530 nm against the blank. The level of MDA was expressed in mmol/g of tissue proteins.

Superoxide dismutase activity
The activity of SOD was determined according to Misra and Fridovish method [33]. Into blank tube, was introduced 1666 µl of carbonate buffer (50 mM, pH 10.2), and 134 µl of homogenate in test tubes. The reaction was started by adding 200 µl of 0.3 mM adrenaline solution. After fast inversion for homogenization, optical density was read at 480 nm after 20 and 80 s. The speci c activity of SOD was expressed in units of SOD/min/g of organ.

Nitrite level
The determination of nitrite level was carried out according to Grand et al. method [34]. The absorbance was read at 570 nm against the blank. The nitrite level was expressed in mg/ml. 1.6.6. Total protein level The total protein assay was carried out according to Gornall et al. method [35]. The protein concentration was expressed in mg/ml.

Pro-in ammatory markers level
The level of TNF-α, IL1-β, and IL-6 was determined by Enzyme-Linked Immunosorbent Assay (ELISA) using the Quantikine kit (R and D Systems, Inc. Minneapolis, USA).

Apoptosis markers level
The determination of the level of caspases 3 and 9 was carried out by the ELISA technique using the Novus Biologicals kit (R and D Systems, Inc. Minneapolis, USA).

Histopathological analysis of brain tissue
The histological analysis included xing, cutting, dehydration, inclusion, cutting, coloring, mounting, and observation. The stained and mounted slides were observed at 250x magni cations, using Scientico STM-50 optical microscope (HSIDC Industrial Estate, Haryana, India) equipped with a Celestron 44421 digital camera connected to a computer.

Statistics
Statistical analysis was performed using Graphpad Prism software version 7. 1. Difference between groups was analyzed using one-way or two-way analysis of variance (ANOVA) followed by post-hoc test of Turkey. The difference was considered signi cant at p <0.05.

Phytochemical composition of Z. jujuba aqueous extract
Qualitative phytochemical screening of Z. jujuba aqueous extract showed the presence of avonoids, phenols, anthraquinones, coumarins, saponins, tannins, triterpenes, anthocyanins, phenols, and reducing sugars (Table 1). On the 4th day of the acquisition phase, scopolamine signi cantly (p <0.001) induced learning de cit in the negative control group compared to the normal control group (Table 2). the extract of Z. jujuba at all doses increased the latency to reach the platform to 5.2 ± 0.4 s (p <0.001) at dose 29 mg/kg compared to the negative control (Table 2). Piracetam increased (p <0.01) this time to 7.7 ± 0.7 s (Table 2).   (Tables 3 and 4). Piracetam and Tacrine decreased (p <0.001) the MDA level in the hippocampus. In the prefrontal cortex, piracetam (p <0.05) and tacrine (p <0.01) decreased in this level (Tables 3 and 4).
The activity of superoxide dismutase (SOD) increased (p <0.001) in the hippocampus and prefrontal cortex in negative control compared to normal control (Tables 3 and 4). The extract at all doses reduced (p <0.001) the activity of SOD in the hippocampus and the prefrontal cortex (Tables 3 and 4). Piracetam and Tacrine also reduced (p <0.001) this activity in the hippocampus and prefrontal cortex (Tables 3 and  4). The level of nitrite increased (p <0.001) in the hippocampus and the prefrontal cortex of the negative control group compared to the normal control group (Tables 3 and 4). The extract at doses 29 and 114 mg/kg decreased (p <0.001) the level of nitrite in the hippocampus and prefrontal cortex (Tables 3 and 4).
Piracetam and Tacrine also led to a reduction (p <0.001) in the level of nitrite in the hippocampus and prefrontal cortex (Tables 3 and 4). Administration of scopolamine in the negative control group decreased (p <0.001) total protein level in the hippocampus and prefrontal cortex compared to the normal control group (Tables 3 and 4). The extract (29 and 144 mg/kg) increased (p <0.001) total protein level in the hippocampus and prefrontal cortex (Tables 3 and 4). Piracetam and Tacrine increased (p <0.001) total protein level in the hippocampus and prefrontal cortex (Tables 3 and 4).
Administration of scopolamine in the negative control group increased the IL-6 level in the hippocampus and prefrontal cortex to 346.1 ± 8.7 pg/ml (p <0.001) and 3526.3 ± 3.4 pg/ml (p <0.001), respectively compared to the negative control ( Figure 3C). The extract at all doses decreased (p <0.001) the IL-6 level in the hippocampus and prefrontal cortex ( Figure 3C). Piracetam and Tacrine decreased (p <0.001) the IL-6 level in both organs ( Figure 3C).

Effect of Z. jujuba extract on the neuronal alteration in the hippocampusand prefrontal cortex
The micro-architecture of the hippocampus of normal control rats shows intact neurons in CA1 and CA3 layers ( Figures 5A and B) and regular thickness of dentate gyrus ( Figure 5C). The normal density of neurons is also observed in the prefrontal cortex ( Figure 5D). In the negative control, the thickness of layers (CA1 and CA3) and the density of neural cell bodies are reduced ( Figures 5E and F). We also observe the presence of spongiosis (S), granulovacuolar degeneration (GVD) as well as chromatolysis (CH) in the CA3 layer ( Figure 5F). Moreover, the dentate gyrus shows the presence of perivascular edema (PE) ( Figure 5G). The prefrontal cortex shows a low density of neurons ( Figure 5H). In rats treated with the extract (29 and 57 mg/kg), CA1 and CA3 layers showed a thickness comparable to the normal control group ( Figures 5Q, R, U, and V). In the dentate gyrus, there is a decrease in perivascular edema ( Figures 5S  and W), while in the prefrontal cortex there is a normal density of neurons ( Figure 5T and X).
2.9. Effect of Z. jujuba extract on the CA1 and CA3 neurons density of the hippocampus Table 5 presents the effect of treatments on the density of CA1 and CA3 neurons. In Administration of scopolamine in distilled treated rats decreased the number of neurons in CA1 and CA3 to 61.1 ± 1.7 (44.51%) and 37.5 ± 1.5 (19.01%), respectively compared to the normal control group. The extract (29 mg/kg) prevented a decrease in the number of CA1 and CA3 neurons (Table 5).

Discussion
The purpose of this study was to assess the effect of Z. jujuba extract on cognitive impairments induced by scopolamine in rats, using Morris water maze and Y-maze paradigms. The morris water maze is used to assess long term spatial learning and memory in rodents [36]. During acquisition (4th day) and retention phases, injection of scopolamine in distilled water treated-rats increased the latency to nd the platform. It also reduced the time spent in the target quadrant in the Morris water maze. Indeed, scopolamine induces amnesia by antagonizing muscarinic receptors of acetylcholine, which is the main neurotransmitter involved in the learning and memory process [37]]. These ndings corroborate those of Li et al. [38] with increased time to reach the platform and decreased time spent in the target quadrant after scopolamine administration. However, during both phases, the extract of Z. jujuba and piracetam reversed these effects and suggesting an anti-amnesic effect. These results are in agreement with a study demonstrating that Z. jujuba ethanolic extract ameliorates cognitive impairments in rats [22]. The fact that the extract restores learning and memory impairments like piracetam, these ndings might also suggest cholinergic blockade and emphasize its bene cial effect on long-term spatial memory dysfunctions [7,22].
To determine the effect of the extract on short-term spatial memory, the Y-maze test was assessed. The Y-maze paradigm is used to assess short-term spatial memory in rodents [39]. In rats that received scopolamine and distilled water, the percentage of spontaneous alternation decreased. Surprisingly, the extract did not affect the percentage of spontaneous alternation. These results indicate that the extract does not interfere with short-term memory process dysfunctions, because Y-maze is sensitive to drugs that act on the working memory process [29,40,41]. This study gives an insight into the complex mechanisms underlying short term memory dysfunction and reveals that this type of memory would not be affected in AD patients.
In AD, the increased deposition of beta-4-amyloid proteins (PβA) in the brain, induces the generation of reactive oxygen species (ROS) [42,43]. These ROS generations are associated with cognitive impairments. In this study, administration of scopolamine-induced oxidative stress in the hippocampus and the prefrontal cortex. This event is characterized by an increase in the level of MDA, SOD, nitrite, and a decrease in GSH and total protein. Similar results were obtained by Isola et al. [44] and Ghasemi et al. [45]. Administration of the extract as well as piracetam or tacrine decreased SOD, nitrite, and MDA level, and increased those of GSH and protein total in both tissues. These data indicate that the extract possesses antioxidant activity [31]. This activity might be due to the presence of tannins, coumarins, phenols, triterpenes, and avonoids evidenced in this study. Indeed, recent studies showed that the cytoprotective and neuroprotective effects of these molecules are strongly correlated to their antioxidant potential [46,47]. These observations are in agreement with those of Yoo et al. [48], who showed that the neuroprotective effects of the methanolic extract of Z. jujuba fruit against ischemic damage in rodents result from the antioxidant properties of this essence. Taken together, these ndings reveal therefore that the extract possesses a neuroprotective effect.
Neuroin ammation is not generally seen as a cause of AD, but rather as a result of AD [49]. Neuroin ammatory processes are known to be exacerbated by PβA and phosphorylation of tau protein deposit [50]. Therefore, drugs with anti-in ammatory activity could contribute to improving memory in patients with AD. Administration of scopolamine led to an increase in the level of TNF-α, IL-1β, and IL-6 in the hippocampus and prefrontal cortex of rats treated with distilled water [51]. The treatment of animals with the extract signi cantly decreased the level of these cytokines. These data suggest that the extract has anti-in ammatory properties [52]. The identi ed group of avonoids in this plant might underlay this activity [53]. These results also suggest a neuroprotective effect and could explain in part its anti-amnesic effect.
During the pathogenic process in the brain of AD patients, it is well known that oxidative stress, as well as in ammation, could trigger apoptosis mechanisms [54]. Thus, inhibiting these mechanisms may hamper their impact on cognitive functions. In this study, scopolamine increased the level of caspases 3 and 9 in the hippocampus and the prefrontal cortex of distilled water-treated rats. These data are consistent with those of Demirci et al. [55]. In fact, administration of scopolamine induces the formation of PβA in the hippocampus and the prefrontal cortex [56]. PβA via the caspases cascade (mainly caspases 8, 9, and 3) leads to apoptosis of neuronal cells and contributes to the pathophysiology of AD [57]. Treatment of animals with the extract resulted in a signi cant decrease in caspases levels in the hippocampus and prefrontal cortex. These results suggest that the extract possesses anti-apoptotic properties and strongly support its aforementioned neuroprotective effect [58,59]. These properties might involve some major secondary metabolites with anti-apoptotic activity such as avonoids, triterpens and polyphenols [60,61].
Analysis of the histological sections of scopolamine-treated rats showed a reduction in the number of neuronal cells in the CA1 and CA3 regions of the hippocampus. These results support those of Sayyahi et al. [62]. The extract protected the hippocampus from neuronal degeneration in the CA1 and CA3 regions of the hippocampus. This protection was re ected by a preserved density of neurons in these regions.
Given that CA3 and CA1 regions are involved in the learning and memory process [63], these results con rm its neuroprotective effect and subsequently explain its anti-amnesic effect. Consequently, this extract could be used to prevent only long-term memory dysfunctions or neurodegenerative processes in patients with AD, epilepsy, and Parkinson's disease. However, the lack of effect on short-term memory, despite evident antioxidant, anti-in ammatory, and anti-apoptotic in the prefrontal cortex highlights the complexity of short-term memory physiology. Further studies on isolated molecules need to be achieved to establish the exact mechanisms involved in the ant-amnesic and neuroprotective effects of the extract.

Conclusions
In sum, this study aimed to assess the anti-amnesic and neuroprotective effects of Z. jujuba leaves aqueous extract on the scopolamine model of AD in rats. Treatment with the aqueous extract protected animals from cognitive impairments. Analysis of possible mechanisms of action demonstrated that these effects are mediated in part by anti-oxidant, anti-in ammatory, and anti-apoptotic activities. These ndings suggest that the extract possesses anti-amnesic and neuroprotective effects. This justi es its empirical use in the treatment of dementia in Cameroonian's folk medicine. Furthermore, this extract could be used as an adjunct to treat diseases associated with spatial long-term memory impairments such as epilepsy, Alzheimer's, and Parkinson's diseases.

Not applicable
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing of interest
There is no con ict of interest