Nasal Delivery of Asiatic Acid Ameliorates Scopolamine-Induced Memory Dysfunction in Mice

Asiatic acid (AA) has previously shown its neuroprotective effects, but low oral bioavailability limits its penetration into the brain. This study aimed to investigate the effect of intranasal AA administration in mice with memory dysfunction induced by scopolamine. Mice received either intranasal AA (INAA), oral AA (POAA3 or POAA30), or donepezil, followed by scopolamine for 10 days. Morris water maze (MWM) was performed on days 0–5, 30 min after treatment. Locomotor activity was conducted on day 6 followed by brain collection. In MWM, INAA treatment had significantly reduced escape latency on days 2–4, while POAA3 decreased escape latency on day 3 and POAA30 and donepezil decreased escape latency on day 4. INAA inhibited acetylcholinesterase activity, increased catalase protein expression, and decreased malondialdehyde levels in the brain tissue. Therefore, intranasal administration of AA produced a rapid onset in the protection of learning and memory deficits induced by scopolamine through acetylcholinesterase inhibition and antioxidant effect.


Introduction
Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in elderly patients that is often associated with learning defcits and memory loss [1].Aggregated amyloid-beta (Aβ) plaque is one of the main pathological features of AD [2,3].Some evidence suggests that Aβ induces the formation of neurofbrillary tangles (NFTs) by promoting tau hyperphosphorylation of the nerve microtubule [2,4].NFTs, together with Aβ, could lead to neuronal loss in the hippocampus and induce neuroinfammation, oxidative stress, and cortical atrophy, and a decrease in cholinergic transmission [2,5].Current AD therapies such as donepezil and memantine are limited to symptomatic treatment, as they do not exhibit the protective efect to prevent the progression of AD [6].Terefore, fnding alternative treatments that can prevent the progression of AD has become increasingly important.
Asiatic acid (AA) is a bioactive substance that can be extracted from Centella asiatica.AA has been found to exhibit protective activity against neurotoxicity in the neuronal cell line exposed to glutamate and methamphetamine [7,8].In addition, AA prevented memory dysfunction induced by valproate, 5-fuorouracil, quinolinic acid, and AlCl 3 [9][10][11].Te neuroprotective efects of AA involved its antioxidant activity [10,12], anti-infammation [12], and protection of adult neurogenesis [10,11].Moreover, in vitro and in silico studies revealed that AA can inhibit AChE [13].Inclusively, AA is a promising compound in the treatment of AD.
AA has limitations in its pharmacokinetic profle when given orally.Te bioavailability of AA is as low as 16.25% in rodents by oral administration, which could be attributed to the intensive metabolism by cytochrome C450 [14].AA was encapsulated in the solid lipid nanoparticle (SLN) for intranasal delivery to increase AA penetration into the brain.Intranasal administration is seen to increase drug availability in the brain by avoiding frst-pass metabolism and providing rapid onset of action, thus reducing systemic adverse efects [15].Several CNS drugs such as carbamazepine, venlafaxine, olanzapine, and donepezil have been modifed for the nasal delivery system [15][16][17][18].Furthermore, due to the small particle size and lipophilic property, the solid lipid nanoparticles can be delivered to the brain via the olfactory and trigeminal nerves as well as through the blood-brain barrier [19].
Tis study aimed to determine the efect of intranasal AA administration on scopolamine-induced memory impairment in mice.In addition, the protective mechanisms of AA involving AChE activity, antioxidant enzyme, and lipid peroxidation were also examined.

Chemicals and Reagents.
Asiatic acid (Sigma-Aldrich, St. Louis, MO, USA) was prepared in the SLN formulation for intranasal delivery.AA concentration in the SLN formulation was 2.26 mg/mL.Te particle size, polydispersity index, and zeta potential of AA in SLN were 189.27 ± 4.22 nm, 0.321 ± 0.047, and −18.33 ± 0.45 mV, respectively [20].AA at a concentration of 3 and 30 mg/mL was prepared in 0.5% carboxymethylcellulose (CMC) for oral administration.Scopolamine, donepezil, CMC, pyridine, 1,1,3,3-tetraethoxypropane (TEP), sodium dodecyl sulfate (SDS), thiobarbituric acid (TBA), and butanol were obtained from Sigma-Aldrich (St. Louis, MO, USA).[22,23], while controls were injected with normal saline solution (NSS) (10 mL/kg, i.p).All treatments were given 30 min before the experiments for 10 consecutive days (days 1-10).All mice were performed MWM on days 0-5 and locomotor activity on day 6.On day 10, mice were euthanized by CO 2 .Mice' brains were then quickly removed and dissected for the hippocampus.Te brains were snapfreezed in liquid nitrogen and kept in a −80 °C freezer for later analysis.Te hippocampus was determined by AChE activity and CAT and SOD protein expression.Te rest of the brain except the cerebellum was kept for thiobarbituric acid reactive substance (TBARS) assay.

Morris Water Maze.
MWM apparatus consisted of a circular water pool (130 cm in diameter and 50 cm in depth) with four visual cues and a platform (8 cm in diameter and 18 cm in height) placed in the center of the northeast quadrant.Te water pool was flled with water at a temperature of 22 ± 2 °C.On day 0, the platform was visible at 1 cm above the water level, while on days 1-4, the platform was hidden at 1 cm below the water level.On days 0-4, mice were allowed to fnd the platform for 60 s.Tree consecutive trials were performed each day, while mice were started from three quadrants.Te escape latency was recorded using a video tracking system (VideoMot2, TSE Systems, Germany), and the average escape latency from the three trials was presented.Mice that failed to reach the platform within 60 s were placed on the platform and allowed to stay for 20 s.On day 5, the probe trial was performed by removing the platform from the pool.A mouse was allowed to fnd the platform location for 60 s.Te time spent in the platform quadrant was recorded.

Open-Field Test.
Locomotor activity was performed in a square box (50 × 50 × 40 cm).Mice were placed in the open-feld chamber for 30 min following the treatment.Te locomotion time was detected using the VideoMot2 system (VideoMot2, TSE Systems, Germany) for 5 min.

Results and Discussion
3.1.Intranasal AA Administration Prevents Scopolamine-Induced Learning and Memory Impairment.MWM was conducted to evaluate the learning and memory behavior in mice.On day 0, mice received no treatment and performed MWM with a visible platform.It was noted that the mean escape latency at baseline was not diferent between the groups (p > 0.05, one-way ANOVA).On days 1-4, mice received treatment 30 min before the MWM test with a hidden platform.Scopolamine-treated mice had signifcantly higher escape latency than that of control on days 1, 3, and 4 (p < 0.01, p < 0.01, and p < 0.001, respectively).Donepezil signifcantly reduced escape latency on day 4 compared to scopolamine treatment alone (p < 0.05).Intranasal administration of AA signifcantly decreased escape latency on days 2, 3, and 4 compared to scopolamine treatment alone (p < 0.05, p < 0.05, and p < 0.001, respectively).POAA3 treatments signifcantly reduced escape latency on day 3 compared to scopolamine treatment alone (p < 0.05).Te escape latency of POAA30 mice was not diferent from that of the control group and the scopolamine group (Figure 1(a)).
Te probe trial on day 5 showed that mice in the INAA group spent signifcantly higher time in the target quadrant than scopolamine-treated mice (p < 0.05) (Figure 1(b)).Alternatively, POAA3, POAA30, and donepezil treatment failed to increase the time spent in the target quadrant in the probe trial.
Locomotor activity was conducted on day 6 to determine the efect of treatment on mice' motor performance.As per the results, no diference was observed in terms of locomotion time among groups (Figure 2), indicating that scopolamine, donepezil, INAA, and POAA do not afect locomotor activity.
AA in solid lipid nanoparticle formulation has been developed for intranasal administration to enhance brain penetration.Our previous study demonstrated successful delivery of AA to the olfactory bulb and hippocampus after a single intranasal administration within 30 minutes [20].Te present study showed that the intranasal administration of AA in SLN prevented scopolamine-induced learning and memory impairment.Acetylcholine plays a vital role in the learning and memory processes in the hippocampus [24].Learning and memory defcits in patients with AD can be alleviated by acetylcholinesterase inhibitors [6].In this study, scopolamine, a muscarinic receptor antagonist, induced memory impairments in the MWM test.Intranasal and oral AA treatment as well as donepezil, an AChE inhibitor, can reverse the memory impairment efect of scopolamine.Interestingly, AA given intranasally was noted to produce faster efects than donepezil and oral administration of AA.Tis result could be due to that intranasal route accelerated drug delivery of AA to achieve the target site of action in the brain.In line with our previous study, nasal administration of AA improved cognitive impairment induced by Aβ 1−42 in ICR mice [20].
It is noticeable that a low dose of oral AA (3 mg/kg) prevented memory impairment induced by scopolamine, while a high dose of oral AA (30 mg/kg) had no efect.A Advances in Pharmacological and Pharmaceutical Sciences previous study showed that AA dose-dependently increased the expression of the P-glycoprotein (P-gp) efux transporter in a concentration-dependent manner [25].It is likely that low-dose AA did not induce P-gp, thus, AA could reach the efective concentration in the brain and produced an acute efect.

Intranasal AA Administration Inhibits Acetylcholinesterase (AChE)
Activity in the Hippocampus.On day 10, mice were terminated 30 min after treatment.Te brain AChE activity was then determined to elucidate the mechanism of action of AA.INAA, POAA3, and POAA30 had signifcantly reduced AChE activity compared to scopolamine treatment alone in the hippocampus (p < 0.05) (Figure 3).
Our study demonstrated that AA prevented memory impairments by AChE inhibition.A previous study showed that AChE activity was inhibited by AA using the thin-layer chromatography (TLC) bioautographic method [26].In addition, raw extract of Centella asiatica inhibited AChE activity in SH-SY5Y and AW 264.7 cells, and in the animal model [27].Previously, the IC 50 value for AA was determined to be 15.05 μg/mL by in vitro analysis, while the binding energy value was found to be −10.27Kcal•mol −1 by in silico analysis [13].Terefore, AA can increase acetylcholine levels through interactions with the active sites of AChE and consequently prevent memory defcits induced by scopolamine.Several studies showed that scopolamine induces memory decline by increasing AChE activity in the mouse brain [28,29].Scopolamine inhibited cholinergic   4 Advances in Pharmacological and Pharmaceutical Sciences transmission by blocking the interaction of acetylcholine (ACh) with muscarinic receptors, leading to an increase in ACh levels in the synaptic cleft [28].Te upregulation of AChE activities might be due to a feedback mechanism to degrade the excess ACh neurotransmitter.

Intranasal AA Administration Elevates Hippocampal Catalase (CAT) Protein Expression and Decreases Brain Lipid
Peroxidation.We investigated the efects of repeated AA administration on the expression of antioxidant enzymes in the hippocampus (Figure 4).It was shown that nasal delivery of AA signifcantly increased CAT expression levels compared to those of control and scopolamine treatment alone (p < 0.05 and p < 0.05, respectively) (Figure 4(a)).However, SOD expression was unaltered (Figure 4(b)).
Malondialdehyde (MDA), which is a lipid peroxidation product, was used as a parameter of oxidative stress in brain tissue.Scopolamine and POAA3 treatment signifcantly increased MDA levels compared to control (p < 0.01 and p < 0.05, respectively), while INAA and POAA30 administration signifcantly decreased MDA levels compared to scopolamine treatment alone (p < 0.01 and p < 0.05, respectively) (Figure 5).
Oxidative stress is one of the main factors involved in the pathological processes in AD [30].Antioxidants are one of the therapeutic approaches to prevent neurodegeneration in AD [31].Catalase and SOD are the two main factors of frstline defence antioxidant enzymes in the living tissue [32].Previous studies indicated that scopolamine-induced oxidative stress in the brains of rodents by decreasing SOD and catalase expression, leading to a decline in MDA levels Advances in Pharmacological and Pharmaceutical Sciences [33,34].A study showed that the antioxidant enzymes were controlled by some transcription factors such as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and peroxisome proliferator-activated receptor-gamma (PPAR-c) [35].Scopolamine injection reduced the expression of Nrf2 in the mouse brain [36].A previous study showed that AA reduced lipid peroxidation by increasing Nrf2 protein expression in chemotherapy-induced neurotoxicity in rats [37] Moreover, AA increased PPAR-c activation in human keloid fbroblasts exposed to TGF-β1 [38].Previous research has demonstrated the antioxidant properties of AA in a rodent model of memory loss produced by quinolinic acid and aluminium chloride (AlCl 3 ) by increasing the expression of superoxide dismutase (SOD), catalase, and glutathione and decreasing lipid peroxidation [10,39].In line with our study, repeated intranasal delivery of AA increased the expression of catalase in the hippocampus and subsequently reduced lipid peroxidation, suggesting the beneft of AA to increase antioxidants in the normal brain.

Conclusions
In summary, our study showed that intranasal AA administration protected against scopolamine-induced learning and memory impairment in mice.Te efect of intranasal administration of AA was faster than AA oral administration.Repeated intranasal administration of AA inhibited acetylcholinesterase activity, elevated antioxidant enzyme expression, and decreased lipid peroxidation in the brain (Figure 6).Tis study suggests that AA can be a potential treatment for AD.

Figure 1 :
Figure 1: (a) Efects of INAA, POAA3, and POAA30 on the escape latency during the acquisition phase (days 1-4) in the MWM test.Data were provided as mean ± S.E.M. (n � 7).* * p < 0.01, * * * p < 0.001 compared with the CON group, # p < 0.05, ### p < 0.001 compared with the SCO group on the same day.(b) Efects of INAA, POAA3, and POAA30 on the time spent in the target quadrant during the probe trial in the MWM test.Data were provided as mean ± S.E.M. (n � 7).* * p < 0.01 compared with the CON group.# p < 0.05 compared with the SCO group.

.
Brain acetylcholinesterase activity was measured using a colorimetric AChE assay kit (Abcam, Cambridge, MA, USA).Brain tissues were homogenized in a lysis bufer on ice and centrifuged at 4 °C.Te supernatants were then incubated (Millipore, Billerica, MA, USA) antibodies at 4 °C overnight.After that, the membrane was incubated with horseradish peroxide (HRP)-conjugated goat anti-rabbit IgG antibodies (1 : 1000) (Millipore, Billerica, MA, USA) or anti-mouse IgG antibodies (1 : 1000) (Santa Cruz Biotechnology, CA, USA) at room temperature for 2 h.Te membrane was prepared with a chemiluminescence solution and analysed with a detector of a luminescent image (ImageQuant LAS 4000, GE Healthcare Biosciences, Japan).USA).Two-way ANOVA with treatment and time as the main factors was used to analyse the escape latency during the acquisition phase in the Morris water test.For other experiments, the comparison between groups was determined via one-way ANOVA, followed by Fisher's (LSD) post hoc test.Te p values <0.05 were defned as statistically signifcant.
* * p < 0.01, * * * p < 0.001 compared with the CON group, # p < 0.05, ### p < 0.001 compared with the SCO group on the same day.(b) Efects of INAA, POAA3, and POAA30 on the time spent in the target quadrant during the probe trial in the MWM test.Data were provided as mean ± S.E.M. (n � 7).* * p < 0.01 compared with the CON group.# p < 0.05 compared with the SCO group.