Pharmacological evaluation of Salvadora persica on scopolamine-induced memory disorder

Background: Salvadora persica root has been reported for various pharmacological activities and it can also be used for memory sharpening. Aim and Objective: The aim of the present study is to evaluate the effect of Salvadora persica root extract on scopolamine induced amnesia in rats Materials and Methods: The evaluation of Salvadora persica (S. persica) on scopolamine induced memory impairment was carried out using scopolamine 3mg/kg p.o for 7 days followed by treatment with Donepezil (3mg/kg) and S. persica root extract (250, 500, 1000 mg/kg) for 21 days; assessed by in vivo and in vitro studies. In vivo models Elevated Plus Maze (EPM), Morris Water Maze (MWM) and Passive Avoidance Paradigm (PA) was used for assessment of Transfer Latency (TL), Escape Latency (EL) and Step Through Latency (STL). In vitro studies on rat cerebral cortex and hippocampus AChE enzyme estimation, estimation of reduced glutathione, GSH and histopathological studies. Results: Treated animals shown increased STL whereas decreased TL and EL. In vitro studies of brain and parts of the brain showed decreased AChE enzyme activity in hippocampus and cerebral cortex, increased level of reduced glutathione level and reduced MDA levels. Considerable histopathological changes were seen in treated rat brain, specially in CA1 and CA3 neural intact in hippocampus region. Conclusion: Present study describes attenuation action of S. persica on memory impairment induced by scopolamine may be due to increase in Acetylcholine (ACh) level in brain by inhibiting AChE enzyme and antioxidant property of S. persica. Histopathological changes are seen in scopolamine treated rats and treatment rats.


Introduction
"Memory impairment is de ned as loss of ability to interfere with occupational, social activities, relationship due to the absence of gross clouding of consciousness or motor involvement" (Parle M. et al. 2011). Etiological factors associated with memory impairment such as oxidative stress, hypercholesterolemia, cerebral ischemia, energy failure, calcium overload, glutamate-mediated exitotoxicity and functional changes (Wang J. et al. 2009). AD is the most usual cause of dementia in old population (Sugimoto H. 2008). AD disease is a progressive irreversible neurological disorder occurs gradually and results in memory loss, unusual behavior, personality changes and loss of thinking ability (Parihar M. et al. 2004). AD associate with accumulation of beta-amyloid (Aβ) plaque and intracellular neuro brillary tangles of hyperphosphorylated tau protein (Tiraboschi et al. 2004).
According to world Alzheimer report 2018 50 million people in worldwide suffering from dementia (World Alzheimer report 2018). Treatment of AD involves use of memantine and Acetylcholinesterase (AchE) Inhibitors but the available treatment for AD doesn't treat the disease but reduce the symptoms, hence delays the patient's loss of autonomy. Hence new molecules are required to investigate for treatment of AD.
India is a gold mine for traditional medicine, which are having less side effects and having different phytoconstituents with mechanism of action. Bene cial effects of traditional medicines in the management of infectious and non-infectious diseases is also reported via in silico and in vitro approaches (Khanal et  . Previous report also reported the probable action against memory defect for traditional medicine using multiple approaches reporting the acetylcholinestrase activity of folk medicine (Duyu et al 2020). Similarly. Salvadora persica having tradition claim on memory sharpening (Jivad N. et al. 2014). The aqueous and alcoholic extract of stem and root contain sulfate chlorides, thiocyanate, nitrates, alkaloids, resin and coloring matter, traces of tannins and saponins, uoride, silica, sulfur, vit.C and sterols. The avonoids rutin and quercetin were detected salvadourea has been present in the root of s. persica benzylisothiocynate also present. The meswak shows various pharmacological activities like antibacterial, antimycotic, analgesic, and anti-in ammatory, stimulation of salivary secretion, cytotoxic, locomotor activity, topical medication, antiulcer, anticonvulsant, sedative and antispasmodic. The therapeutic application of meswak are oral hygiene removal of smear layer, root canal, irrigant, plaque control, dental gel, gingival recession, industrial oil production etc (Ahmad H. et al. 2014) and can be used for memory sharpening (Jivad N. et al. 2014). The toxicity doseof S. persica is safe upto 5000mg/kg body weight (Soliman G. et al. 2017).
Scopolamine is a cholinergic antagonist which involved in the transmission of acetylcholine in CNS (El-Sherbiny et al. 2003) and can be used as pharmacological model for AD (Ebert U. et al. 1998).The present study was aimed to evaluate the action of S. persica on scopolamine induced memory impairment in rats. The amnesia can be induced by using scopolamine in concentration 3mg/kg body weight by intraperitoneal route for 7 days (Rahimzadegan M. et al. 2018 The S. persica root was procured from Huma Traders, Miraj (Maharashtra, India) and authenticated by Mr. Ajit Lingayat, authentication expert, KAHER Shri B.M.K. Ayurveda Mahavidyalaya. CRF code: CRF/Auth/2018/122. The roots were shade dried, pulverized into the coarse powder and subjected for extraction process.

Extraction
The dried coarse powder was extracted by maceration technique using hydro-alcohol as a solvent for seven days with occasional shaking. After 7 days, the extract was ltered & concentrated using evaporator. The obtained sticky extract was dried by using water bath; percentage yield of plant was calculated 2.4. Animals Wistar rats of either sex were procured from CPCSEA approved invivo bioscience Bengaluru, India. They were housed in a clean and transparent polypropylene cage, divided into seven groups; each group contains six animals and was maintained under 12/12hr natural light-dark cycle at room temperature, 45-55 % relative humidity. All the animals were acclimatized one week before the experiment and allowed with free access to standard pellet and water ad libitum. The study protocol was reviewed and approved by the institutional Animal Ethical committee Reg.No.221/Po/Re/S/2000/CPCSEA, KLE College of Pharmacy, Belagavi. Karnataka, India.
2.5. Estimation of Acetylcholinesterase enzyme in Cerebral Cortex and Hippocampus (Ellman G;et al. 1961) AChE enzyme was estimated by method of Ellman et al. The rate of moles of substrate hydrolysed per min per gram of tissue was calculated.
2.6. Lipid peroxidation estimation (Ohkawa, H., et al. 1979) The amount of MDA present in the brain is measured by the method of Ohkawa et al. The MDA level was expressed as nano moles of MDA /mg of protein in brain homogenate. 2.7. Estimation of reduced glutathione (Ellman G. 1959) Glutathione level was measured by the method of Ellman G. L. GSH level was stated in µmoles/mg of tissue.

Histopathological study
Rat brains were collected & kept in formalin. Brains stained by using haematoxylin-eosin. The hippocampal lesions studied using electronic microscope at 40× and 10× magni cations.

Experimental procedure
Animals were divided into seven groups containing six animals in each; Group I received normal saline.
Group VII (Preventive group) S. persica extract (1000mg/kg p.o.) for rst 21 days, followed by Scopolamine (3mg/kg i.p.) along with S. persica extract (1000mg/kg p.o.) for next 7 days. All the animals were subjected for exteroceptive behavioural models of memory using Elevated plus Maze, Passive avoidance paradigm, Morris Water Maze on 0 th , 7 th , 14 th and 21 st day. At the end of the experiment, animals were sacri ced by overdose of anaesthesia, brains were isolated and cerebral cortex, hippocampus Acetylcholinesterase enzyme activity, Lipid Peroxidation, Glutathion level in brain were measured and histopathological study was performed. During the assessment of memory via EPM, rats were individually placed at one end of the open arm facing away from the central platform and the time taken for the rat to move from open to closed arm (Initial Transfer latency, ITL) was recorded using stopwatch. The animals were allowed to explore the maze for 90s. If the animal did not enter the closed arms within 90s, it was guided on the back into one of the closed arm and transfer latency was given as 90s.Later the rat was allowed to explore the maze for 30s to become familiar with the maze and then returned to its cage. A drop in transfer latency time during treatment sessions was taken as an index of memory improvement.

Morris Water Maze (MWM):
It consists of a large circular water tank having size of 110cm diameter and 60cm height. It is made of black opaque polyvinyl chloride or hard board coated with berglass or resin with a white surface and lled with water (26 ± 2 0 C) to a depth of 30cm. the oor of the circular tank is marked off into 4 equal quadrants and were designed as North, East, West and South. In all trials, 2cm below the surface of the water, a black round platform of diameter 10cm was placed in a constant position. The cut off time given to individual rat is 60s. The time taken for the rat to locate the escape platform is noted. If the rat is unable to locate the platform within cut off time, it was gently guided to the platform and was allowed to stay on it for 10s. Escape Latency Time (Time taken for the rat to locate hidden platform in the water maze) was taken as an index of learning.

Passive Avoidance Test (PA):
Each test consists of two distinct trials such as acquisition trial and retention trial. For acquisition trial, individual rat is placed in the light compartment, as soon as the rat entered into the dark compartment, an electrical shock of 0.4mA was applied for 3s, the latency time once the rat had entered the dark compartment was recorded as Escape Latency (EL). After 24h of acquisition trial, retention trial was conducted, in which no shock was applied when the rat entered into the dark compartment and retention latency is recorded by time taken the rat to re-enter into the dark chamber. Animals which didn't enter dark compartment even after 180s were removed from the apparatus.

Statistical Analysis:
Outcomes were represented as Mean ± SEM. The difference among mean was determined using one way ANOVA followed by Tukey's Multiple comparison Test, using GraphPad Prism software version 5.0.
Variations between sets readings was deemed signi cant (probability level) at the degree of freedom at 0.05.

Effect of S. Persica on transfer latency (TL) using EPM
The scopolamine brought mental disturbance assessed by behavioral models. The rats were exposed to transfer latency on EPM. The transfer latency was signi cant increased (p<0.001) from acquisition period to 0 th day after the treatment of scopolamine (26.83±0.600). The PC, SP250 and SP1000 showed signi cantly decreased TL on 7 th , 14th and 21 st (p<0.001) on comparing with NC. SP500 showed signi cant decreased activity on 14 th and 21 st day were as preventive group showed signi cant decreased TL from 0 th to 21 st day (p<0.001). The TL on 7 th , 14 th , and 21 st of all groups showed dose dependent decrease in TL. The effect of S. persica TL is shown in Figure 1. 3.2. Effect of S. persica on ELT using MWM Scopolamine treated group i.e. negative control (NC) showed signi cant increased ELT (p<0.001) on 0 th day on compared with acquisition period (21.25±1.499). SP250, SP500 and SP1000 on 7 th 14 th and 21 st day shows signi cant decreased ELT when correlated with NC (p<0.001). The Donepezil treated group or positive control (PC) showed signi cantly decreased ELT on 14 th and 21 st day (p<0.001) but not notable on 7 th day on compared with NC. The preventive group showed signi cant activity from day 0 th to 21 st day which is compared with NC (P<0.001). The ELT on 7 th 14 th and 21 st day decrease exponentially due to all treatment groups showed dose dependent decrease in ELT. The effect of S. persica ELT is shown in Figure 2. 3.3. Effect of S. persica on STL using PA All scopolamine treated groups showed signi cantly decreased in STL on 0 th day as compared with acquisition period (p<0.01). The PC, SP250, SP500, SP1000 and preventive groups showed signi cant increased STL on 21 st day, compared against NC (p<0.001). The effect of S. persica STL is shown in Figure 3.

Effect of S. Persica on AChE level in rat hippocampus
The scopolamine treated group or NC animals showed signi cantly rised AChE (P<0.001) enzyme action when contrast with control group (12.00 ± 1.471). PC group treated with donepezil 3mg/kg compared with NC showed signi cant decreased AChE enzyme activity (P<0.001) (5.613 ± 0.414), SP250 showed signi cant decreased (P<0.01) AChE enzyme activity on compared with NC (6.718± 0.9868), were as SP500, SP1000 and preventive group showed signi cantly decreased AChE enzyme activity in contrast with NC and when compared with SP250 & PC showed signi cant decreased AChE enzyme activity (p<0.01, 0.05) respectively. Action of S. persica on AChE level in rat hippocampus is shown in gure 4.
3.5 Effect of Persica on AChE level in rat cerebral cortex NC group treated with scopolamine showed signi cant increased AChE enzyme activity compared along with cerebral cortex from control group animals (11.75 ± 0.573). Whereas PC (P<0.001), SP250 (P<0.01), SP500 (P<0.01), SP1000 (P<0.001) and Preventive (P<0.01) showed signi cant decreased cerebral cortex AChE enzyme level. Effect of s. Persica on ache level in rat cerebral cortex is shown in gure 5.

Effeect of S. Persica extract on reduced GSH in rat brain
The NC animal treated with scopolamine showed signi cant reduce in reduced GSH level (P<0.001) as contrasted with control group (0.56 ± 0.083). PC group treated with donepezil 3mg/kg showed increased in reduced GSH level signi cantly (P<0.001) compared along with NC (1.35 ±0.065), were as animals treated with S. Persica extract with 500, 1000mg/kg denoted signi cant rise in the reduced GSH concentration when compared with NC (P<0.001) but not by SP250. The preventive group showed signi cant increase in reduced GSH level (P<0.01) when contrasted with NC. Effeect of s. Persica on reduced GSH in rat brain is shown in gure 6. 3.7. Action of S. persica extract on brain MDA level NC group in respect of control group showed signi cantly increased in MDA level (44.87±4.533), were as PC, SP250, SP500, SP1000 and PREV groups resulted in signi cant reduced MDA level in brain when compared with NC (p<0.001) (Figure 7).

Histopathological study of rat brain
The NC group when compared with control group histogram indicated that severe cerebral congestion, cerebral odema, moderate meningeal congestion, neuronal eosinophilia and mild neuronal micro vacuolization, neuronal nuclear pyknosis, neuronal karyorrhexis, neutrophilic in ltration, RBC extravasation, macrophage in ux, vascular proliferation, and reactive gliosis. PC and treatment groups did not show any severe change in anatomy; were as mild changes observed in cerebral congestion, meningeal congestion, neuronal eosinophilia and neuronal nuclear pyknosis compared with NC. However, the 10X magni cation of hippocampus shows integrity of the neurons in the region of CA1 and CA3 of all the treated groups are maintained as compared with NC and PC. These neurons are involved in memory and cognitive functions. Histopathology of rat brain is shown in gure 8.

Discussion
Scopolamine is the muscarinic receptor antagonist causes inhibition of cholinergic signaling (Vogel, H. 1998, Ebert, U. et al. 1998. The mechanism of scopolamine is steel not known but the various studies shows that it act on various neurotransmitters either by stimulatory or blocking action on glutamatergic (Gutierres, J. et al. 2019, Mahmoodi, G. et al. 2010), adrenergic (Azami N. et al. 2010), dopaminergic (Alto S. et al. 2005, Dash P. et al. 2007), serotonergic (Dash P. et al. 2007, Bames N. et al. 2007, Da silva costa-Aze V. et al. 2012) and histaminergic (Zong C. et al. 2000) system could all together scopolamine induced memory impairment suggesting that these neurotransmitters may be involved in induction of memory impairment (Rahemzadegan M. 2018). It also induces oxidative stress (Jang Y et al. 2013), apoptosis (Jahanshahi M. et al. 2013) and in ammatory responses ). The present study explains effect of S. persica on scopolamine induced amnesia in rats was studied using MWM, EPM, & Passive Avoidance (PA) and biochemical estimations. However, the NC group treated with scopolamine signi cantly increase ELT and TL in MWM and EPM respectively, decreased STL in PA. This recommended that scopolamine interfere with acquisition, retention and consolidation of learning task. Treatment with donepezil 3mg/kg, SP 250, 500, 1000mg/kg and preventive group (SP1000mg/kg and scopolamine 3mg/kg) produced signi cant decreased ELT, TL and increased STL. This suggested that donepezil and S. persica root extract prevented rats from impaired learning and memory induced by scopolamine with dose dependent action and showed treatment and preventive learning and memory activity is sharped.
The degradation of ACh by AChE causes decrease in neurotransmission that leads to decreased binding of ACh with M1 receptors which further leads to decrease in generation of secondary messenger system that causes the decreased neurotransmission and convert tau protein to hyperphophorylated tau protein (Francis P. et al. 1999). ACh degraded by AChE to maintain a constant concentration in brain, were as excess action of AChE consequences in decrease in ACh causes memory impairments. The learning and memory can be improved by increasing the level ACh in brain (Zhang Z. et al. 2004). AChE inhibitors rises level of ACh in the brain which is required for nerve cells communication. Examples: Donepezil, and Rivastigmine. Donepezil is powerful, discriminating, noncompetitive & reversible antagonist of AChE used especially in the treatment of AD. Donepezil improve cognition of patient with mild to moderate AD and its effectiveness can be maintained up to 50 weeks (Jelic V. et al. 2010). In present study estimation of AChE enzyme activity in rat hippocampus and cerebral cortex were performed. However Donepezile and S. persica root extract showed signi cant decreased AChE enzyme activity compared with NC.
Oxidative stress performs key role in amnesia & one of the main cause for memory loss in AD (Parihar M. et al. 2004). Free radicals are responsible for oxidative stress and it is also known as reactive oxygen species (ROS) generated by oxygen and nitrogen depend on unpaired electron. The oxidative stress alters cells and its mechanism causing alteration of cellular properties like " uidity, ion transport, enzyme activity & protein cross linking". Excess oxidation leads to cell death normal body handles radicals by antioxidant and antioxidant enzymes (Floyd R. et al. 2002). In this study in vitro antioxidant activity such as reduced glutathione level and lipid peroxidation in rat brain was performed. In NC group treated with scopolamine did not showed increase in reduced glutathione, were as signi cant increased in reduced glutathione was seen in the PC and preventive group but not in Sp250, 500, 1000mg/kg, were as signi cantly increased in MDA in dose dependent manner was observed in S. persica root extract treated rats. Hence,the phytochemical contituens of S. persica like Flavanoids, alkaloids, traces of tannins and saponins, vit.C and sterols, showing Antioxidants and Antiin ammatory property to overcome oxidative stress and neuronal in ammation which damage the neurons and degeneration of neurons, Beta amyloid and Tau protein formation in hippocampus region which are involved in memory pathway.
Histopathological examination of hippocampus and cerebral cortex of rat brain were performed. The changes induced by scopolamine in NC group like cerebral congestion, cerebral odema, moderate meningeal congestion, neuronal eosinophilia and mild neuronal micro vacuolisation, neuronal nuclear pyknosis, neuronal karyorrhexis, neutrophilic in ltration, RBC extravasation, macrophage in ux, vascular proliferation, and reactive gliosis was observed. PC and S. persica root extract treatment groups reversed the damage induced by scopolamine. However, the 10X magni cation of hippocampus shows integrity of the neurons in the region of CA1 and CA3 of all the treated groups are maintained as compared with NC and PC.

Conclusion
Present study describe that S. persica improve scopolamine induced amnesia in rats studied via behavioral, biochemical and histopathological studies. However persistent administration of S. persica improves scopolamine induced amnesia may be due to rising ACh concentration, antioxidant and antiin ammatory activity in hippocampus and cerebral cortex by inhibiting AChE and lipid peroxidation,tau protein and beta amyloids formation which helps in improving the memory. Histopathological ndings provide in uential impact of S. persica on scopolamine induced amnesia. Effect of S. persicain scopolamine induced raise in microtubule associated tau protein which needs to be further con rmation, also further approaches are made to identify the potential phytoconstituents for the inhibition of AChE and lipid peroxidation. Figure 1 The effect of S. persica on amnesia using Elevated plus Maze: results: mean SD: (n=6): analysed by one way ANOVA followed by Tukey's Multiple comparison test whereas *** p< 0.001 compared to control, ### p<0.001 compared to NC, @@@p< 0.001 compared to PC, %p<0.05, %%p<0.01, %%%p<0.001 compared to SP250 <<< p< 0.001 compared to SP500, >>>p<0.001 compared with SP1000 on 0th, 7th 14th and 21st day.

Figure 7
The effect of S. persica on MDA level in rat brain: mean SEM: (n=6): analysed by one way ANOVA followed by Tukey's multiple comparison test whereas *** p< 0.001 compared to control,##p<0.01, ### p<0.001 compared to NC Figure 8 Histopathological study of rat brain (Hippocampus and cerebral cortex 40X magni cation)