Effect of Sanguisorba Minor on Scopolamine-induced Memory Loss in Rat: Involvement of Oxidative Stress and Acetylcholinesterase

Sanguisorba minor (S. minor) has neuroprotective and antioxidant activities; nevertheless, its potential benets in ameliorating learning and memory functions have not been explored yet. So, in this study, rats were treated with S. minor hydro-ethanolic extract (50, 100, and 200 mg/kg, intraperitoneal (i.p.)) as well as rivastigmine (0.5 mg/kg, i.p.) for 21 consecutive days. Thereafter, their behavioral performance was assessed using Morris water maze (MWM) and passive avoidance (PA) tasks. Scopolamine was also injected 30 min before conducting the tasks. Finally, oxidative stress biomarkers and acetyl cholinesterase (AChE) activity were determined in the brain. The extract characterization was presented using liquid chromatography-mass spectrometry (LC-MS), which conrmed the presence of quercetin, myricetin, kaempferol, catechin, ellagic acid, and gallic acid derivatives. According to the results, the extract at all doses could signicantly recover the impairment of cognitive performance of the scopolamine-treated rats. In the MWM test, the extract and rivastigmine reduced escape latency and travelled distance, compared to the scopolamine group. Moreover, in the PA test, the latency to enter the dark chamber was signicantly increased by the extract and rivastigmine, compared to the scopolamine group (p<0.05-p<0.001). Similar to rivastigmine, the extract attenuated both AChE activity and oxidative injury in the brain as evidenced by the increased antioxidant enzymes and total thiol content, but it decreased malondialdehyde level (p<0.05-p<0.001). In conclusion, the results suggested the effectiveness of S. minor on preventing cognitive dysfunction induced by scopolamine. Accordingly, these protective effects might be produced through the regulation of cholinergic activity and oxidative stress. The LC-MS analysis was performed in an AB SCIEX QTRAP (Shimadzu) liquid chromatography coupled with a triple-quadrupole mass spectrometer. MS analysis was conducted in both negative and positive modes of ionization, in order to maximize the number of the monitored metabolites ions. Liquid chromatography was performed on a Supelco C18 (15 mm×2.1 mm×3 µm) column at a ow-rate of 0.7 ml/min. The gradient analysis started with 90% of 0.1% aqueous formic acid, isocratic conditions were maintained for 15 min, and then a 20-min linear gradient to 30% methnol along with 0.1% formic acid was applied. From the time of 35 min to 80 min, the acidied methanol was increased to 100%, followed by 10 min of 100% acidied methanol, and nally 10 min of 90% of aqueous formic acid to re-equilibrate the column. Finally, the mass spectra were acquired in a range of 200 to 1200 after the 100-minute scan time. Mass feature’s extraction of the acquired LC-MS data and the maximum detection of peaks were done using MZmine analysis software package, version 2.3.


Introduction
Dementia and neurodegenerative diseases are accompanied with cognitive impairments (Terry et al. 2011). Functional de cits in cholinergic neurotransmission have been found to be directly linked to cognitive impairments. Hence, many studies have recently focused on the cholinergic system (Par tt et al. 2012; Giovannini et al. 2015). The function of acetyl choline (ACh), a major neurotransmitter in the central cholinergic system, is important in the process of perception, attention, learning, and memory. ACh function in the hippocampus and brain cortex is terminated by acetyl cholinesterase (AChE) (Hasselmo 2006).
Acetyl cholinesterase inhibitors (AChEIs), including rivastigmine, galantamine, and donepezil, are currently used as the most effective pharmacotherapeutic agents. Correspondingly, these can ameliorate However, alternative or adjuvant anti-amnestic therapies are required in this regard because these drugs produce some adverse effects (Ng et al. 2006).
The roles of oxidative stress and cholinergic dysfunction in cognitive decline have been explored in some studies . Scopolamine, as a muscarinic cholinergic receptor antagonist, was observed to impair short-term memory and learning ability (Klinkenberg and Blokland 2010). As well, the induction of AChE activity by scopolamine contributes to mitochondria dysfunction and subsequently to oxidative stress (Melo et al. 2003;Leuner et al. 2012).
Sanguisorba minor (S. minor) is a member of the Rosaceae family that has been traditionally used for the treatment of some diseases such as bleeding, eczema, and diarrhea (Zhao et al. 2017). A variety of biological activities, including anti-in ammatory, antibacterial, antiviral, anti-oxidant, neuroprotective, and anticancer effects were proposed for S. minor extracts (Zhao et al. 2017;Cirovic et al. 2020;Finimundy et al. 2020). The ethanol extract of S. minor could also suppress cyclooxygenase-1 and AChE enzymes activity in vitro (Cirovic et al. 2020;Finimundy et al. 2020). Additionally, some in vitro protective properties of S. minor against β-amyloid neurotoxicity were found (Nguyen et

Liquid chromatography-mass spectrometry (LC-MS) apparatus
The LC-MS analysis was performed in an AB SCIEX QTRAP (Shimadzu) liquid chromatography coupled with a triple-quadrupole mass spectrometer. MS analysis was conducted in both negative and positive modes of ionization, in order to maximize the number of the monitored metabolites ions. Liquid chromatography was performed on a Supelco C18 (15 mm×2.1 mm×3 µm) column at a ow-rate of 0.7 ml/min. The gradient analysis started with 90% of 0.1% aqueous formic acid, isocratic conditions were maintained for 15 min, and then a 20-min linear gradient to 30% methnol along with 0.1% formic acid was applied. From the time of 35 min to 80 min, the acidi ed methanol was increased to 100%, followed by 10 min of 100% acidi ed methanol, and nally 10 min of 90% of aqueous formic acid to re-equilibrate the column. Finally, the mass spectra were acquired in a range of 200 to 1200 after the 100-minute scan time. Mass feature's extraction of the acquired LC-MS data and the maximum detection of peaks were done using MZmine analysis software package, version 2.3.

Animals
Male Wistar rats (aged 6-8 weeks, weighted 200 ± 20 g) were obtained from the animal house of the The animals were housed under the standard conditions (at 22 ± 2°C, 12 h light-dark cycles: light on from 7:00 to 19:00), with free access to food and water ad libitum.

Drug administration and experimental design
After the acclimatization for 1 week, the rats were randomly allocated into the following ve groups (n = 10 per group): Control group (group I) received saline (intraperitoneal (i.p.) injection, 1 mL/kg, daily); scopolamine group (group II) received saline (i.p.) for two weeks and then the treatment with scopolamine was performed (2 mg/kg dissolved in saline, i.

Behavioral assessments
Morris water maze (MWM) test Spatial learning and memory were examined using MWM task (Morris 1984), which is a black circular tank (136 cm in diameter and 60 cm in height) containing water (depth of 30 cm, at 24 ± 1°C). A circular platform (10 cm in diameter) was set 2 cm under the surface of the water in the center of the southwest quadrant of the pool. Swimming behaviors were recorded by a camera that was connected to a computer and the parameters, including latency to nd the hidden platform and traveled distance were calculated.
At the rst day of performing the MWM test, each one of the rats was acclimatized to the maze for 30 sec in the absence of the platform. At each one of the 4-day training period, 4 trials was performed for each animal. For each trial, the rat was separately placed (facing the wall) into each one of four starting points (North (N), East (E), South (S), and West (W)) and then, it was practiced to nd the platform within 60 sec. If the rat found the platform, it was allowed to stay there for 15 sec, otherwise, it was then guided to the platform gently. After each trial, the rat was transferred to its cage and allowed to be dried. By passing 24 hours from the acquisition phase, spatial probe test was performed while removing the platform from the pool and the rat was allowed to seek out the platform in 60 sec (Norouzi et al. 2019).
Passive avoidance (PA) test A passive avoidance (PA) learning test was performed to evaluate memory retention (Norouzi et al. 2019). The apparatus used for PA test was an acrylic shuttle box consisting of a white illuminated compartment and a dark one (30 cm × 20 cm × 20 cm) that were connected to each other via a guillotine door. In a habituation session, the rats were individually put into the light chamber and then allowed to move freely between the two chambers for 5 min. For performing the acquisition trial, an inescapable foot-shock (2 mA for 2 sec) was delivered to the animals through the grid oor by passing 5 sec from entering the dark compartment. The retention trials were done 3h, 24, 48h, and 72h later. In these trails, the rats were placed in the light chamber and an entering delay of the rat to the dark chamber was then measured as the latency time. Additionally, the time spent by the animal in the dark and light compartments was recorded (Bavarsad et al. 2020). Notably, in retention trails, the shock punishment was not applied.

Biochemical measurements Tissue preparation
Following conducting the behavioral assessments, the rats were euthanized under deep anesthesia (using i.p. injection of ketamine and xylazine at 100 mg/kg and 15 mg/kg, respectively) and the wholebrain tissues were quickly isolated afterward. A tissue homogenate of cerebral cortex and hippocampus (10 % w/v) was prepared in ice-cold 0.1 M PBS (with pH level of 7.4), which was then stored at -80 o C for biochemical assessments (Norouzi et al. 2019).

Measurement of oxidative stress biomarkers
Assay of lipid peroxidation Lipid peroxidation was measured on the basis of formation of thiobarbituric acid reactive substances (TBARS) following MDA reaction with TBA (Norouzi et al. 2019). In this regard, MDA was regarded as the main product of lipid peroxidation and TBARS (red color) was then expressed as MDA equivalent. In brief, the reaction mixture was prepared by adding TBA/TCA/HCl reagent (2 mL) to the homogenates (1 mL).
Subsequently, the obtained mixture was heated in boiling water and after 40 min, the samples were cooled and the absorbance was spectrophotometrically measured at 412 nm. The following equation was used to calculate the concentration of MDA (Bavarsad et al. 2020).
Assessment of total thiol content Estimation of total thiol content was done based on the reaction of the thiol groups with DTNB. Firstly, the homogenates were centrifuged at 4°C and 1000×g, and the supernatants were also collected.
Thereafter, Tris-EDTA buffer (1 ml, pH 8.6) was mixed with the supernatant obtained from each sample (50 ml) and the absorbance was then recorded at 240 nm using a UV-vis spectrophotometer (A1). After adding the DTNB solution (20 µl, 10 mM in methanol) to each sample, the absorbance was recorded again (A2). The following equation was used to calculate the total thiol concentration (Bavarsad et al. 2020): Where B is the absorbance of the blank.

Assessment of antioxidant enzymes activity
Superoxide dismutase (SOD) activity in the homogenates was assessed using the previously described procedure (M). The amount of SOD inhibiting 50% of pyrogallol auto-oxidation was de ned as one enzyme unit (Bavarsad et al. 2020).
The estimation of catalase (CAT) activity was also done based on its ability in converting hydrogen peroxide to water. The absorbance was read at 240 nm using the UV-vis spectrophotometer. In this method, the amount of hydrogen peroxide consumed per each milligram of the protein sample was considered as one unit of CAT activity. Finally, the obtained results were shown as unit per gram of tissue (Norouzi et al. 2019).

Assessment of AChE activity
The AChE activity was assessed using the method of Ellman (Ellman et al. 1961). The supernatant obtained from tissue homogenate (40 µl) was added to a solution containing PBS (2.55 ml) and DTNB 10 mM (0.1 ml). The absorbance of the resultant mixture was determined after the incubation for 5 min at 37°C. Then, the reaction mixture was further incubated (for 5 min at 37°C) after adding 0.02 ml of acetylthiocholine. The absorbance of the sample was spectrophotometrically recorded at 412 nm and AChE activity was estimated as µmol/g tissue/min.

Statistical analysis
All the obtained results were presented as mean ± SEM (standard error of mean) and then analyzed by GraphPad Prism software 8.0 (GraphPad Software, La Jolla, CA, USA). P < 0.05 was considered as statistically signi cant. For the data of the acquisition part of the MWM, including the time and distance during the 4 days, the experimental design included treatment and day as a repeated measures. Therefore, two-way analysis of the variance (ANOVA) with repeated measures was used, and interactions were also reported (treatment x day). The remained data were analyzed using one-way ANOVA followed by Tukey's post hoc multiple comparison test.

LC-MS analysis of S. minor hydro-ethanolic extract
In total, 26 compounds were identi ed in the hydro-ethanolic extract of S. minor using LC-MS analysis in its negative mode. These compounds were phenolic compounds, including quercetin, myricetin, kaempferol, kaempferol-3-glucuronide, quercetin − 3-glucuronide ellagic acid, catechin, and various gallic acid derivatives (consisting of galloyl glucoside, galloylquinic acid, methylgallate hexoside, and catechin gallate). Moreover, Ayoub (2003) isolated unique phenolic carboxylic acids, 4, 8-dimethoxy-7-hydroxy-2oxo-2H-1-benzopyran-5,6-dicarboxylic acid, and 2-(4-carboxy-3-methoxystyryl)-2-methoxysuccinic acid in relatively high amounts in the hydro-ethanolic extract of S. minor. This extract also contained a high level of glucogallin, which is a phenolic compound formed from β-D-glucose and gallic acid. The compounds identi ed in the extract characterization are shown in Table 1 and total ion chromatograms obtained in ESI-mode are also shown in Fig. 1. The MS spectral data were compared with those that were available in the literature. The results also show that both the treatment and day affected the distance during the 4-day period of learning (F (5, 936) = 167.09; p < 0.001 for the treatment and F (3, 936) = 25.87; P < 0.001 for the day). As well, an interaction was found between the treatment and the day (F (15,936) = 3.07, p < 0.001). Correspondingly, escape latency time and traveled path were observed to be longer in the scopolamine-injected animals compared to the control group (p < 0.01-p < 0.001). However, the animals treated with both 50 and 100 mg/kg of the extract at the rst day showed shorter latency time compared to the scopolamine group (p < 0.05-p < 0.001). Notably, no signi cant difference was found in terms of the latency time between the 200 mg/kg extract group or rivastigmine-treated group and the scopolamine group at the rst day. Besides, the treatment with the extract (at all doses) and rivastigmine signi cantly reduced the escape latency time compared to that of the scopolamine group, on days 2-4 (p < 0.001). Additionally, it was observed that the administration of the extract (at all doses) and rivastigmine signi cantly decreased the traveled path compared to that of the scopolamine group (p < 0.001). Therefore, it can be stated that the treatment with rivastigmine and the extract signi cantly improved memory performance compared to the scopolamine group (p < 0.001).
According to the results of the probe test (Figs. 3a and b), both the time spent and the distance traveled in the target quadrant signi cantly decreased in the scopolamine group compared to the control group (p < 0.001). Moreover, the animals treated with rivastigmine and the extract (at all doses) exhibited longer time spent and distance traveled in the target quadrant than those of the scopolamine group (p < 0.001). Some signi cant differences were also found in terms of the time spent in the quadrant between the groups treated with 50 and 100 mg/kg of the extract as well as between the groups treated with 100 and 200 mg/kg of the extract (p < 0.001). Similarly, a signi cant difference was found in terms of the distance traveled in the quadrant between the two groups treated with 50 and 100 mg/kg of the extract (p < 0.001).

Passive avoidance test
As illustrated in Figs. 4a and b, in comparison to the control group, the scopolamine-injected animals exhibited lower time latency to enter the dark chamber 3, 24, and 48 h after the shock (p < 0.01, p < 0.001).
Besides, no signi cant difference was observed in terms of the time latency 72 h after the shock between the scopolamine group and the control group.
In contrast, higher time latencies were observed 3, 24, 48, and 72h after the shock in those rats that were treated with rivastigmine compared to the rats in the scopolamine group (p < 0.05, p < 0.001). Moreover, the administration of the extract at both 100 and 200 mg/kg was found to be associated with a signi cant increase in the time latency 3, 24, 48, and 72h after the shock compared to the scopolamine group (p < 0.001). As well, some signi cant differences were observed in the time latency 3, 24, 48, and 72h after the shock between the two groups treated with 50 and 100 mg/kg of the extract as well as between the two groups treated with 50 and 200 mg/kg of the extract (p < 0.001).
On other hand, the animals in the scopolamine group were observed to spend longer times in the dark chamber than the control group at 3, 24, and 48 h time points (p < 0.001). However, no signi cant differences were observed in this regard between the scopolamine and control groups 72 h after the shock. Besides, the animals treated with rivastigmine were found to spend shorter times in the dark compartment 3, 24, 48, and 72 h after the shock compared to the scopolamine group (p < 0.01, 0.001). Furthermore, the administration of the extract (at all doses) caused a signi cant increase in the time spent in the dark at 3, 24, 48, and 72 h after the shock compared to the scopolamine group (p < 0.05 to p < 0.001). Moreover, the animals treated with the extract (50 mg/kg) indicated no signi cant difference in the time spent in the dark, 48h after the shock compared to the scopolamine group. However, some signi cant differences were reported in the time spent in the dark 3, 24, 48, and 72 h after the shock between the two groups treated with 50 and 100 mg/kg of the extract as well as between the two groups treated with 100 and 200 mg/kg of the extract (p < 0.001).
As illustrated in Fig. 4c, the time spent in the light chamber signi cantly decreased following the injection of scopolamine (p < 0.001) in comparison to the control group 3, 24, and 48h after the shock (p < 0.001).
However, no signi cant difference was observed in this regard between the scopolamine and the control groups 72 h after the shock. However, a longer time was spent in the light room by the rats that received rivastigmine compared to the scopolamine group 3, 24, 48, and 72h after the shock (p < 0.01, p < 0.001).
Notably, the spent time in the light chamber increased after the treatment with the extract (at all doses) 3, 24, 48, and 72 h after the shock compared to the scopolamine group (p < 0.05-p < 0.001). The animals treated with 50 mg/kg of the extract indicated no signi cant difference in terms of the time spent in the light 48 h after the shock compared to the scopolamine group. However, there were some signi cant differences in the time spent in the light 3, 24, 48, and 72 h after the shock between the two groups treated with 50 and 100 mg/kg of the extract and also between the two groups treated with 100 and 200 mg/kg of the extract (p < 0.001).
According to the results illustrated in Fig. 4d, although the frequencies of entry to the dark chamber were higher in the scopolamine group compared to that of the control group 3, 24, and 48h post-shock delivery, the differences were not signi cant. The groups that received the extract at 100 and 200 mg/kg demonstrated a signi cant decrease in the frequencies of dark chamber entry compared to those of the scopolamine group 24 (p < 0.05, p < 0.01) and 72 h (p < 0.05, p < 0.001) post-shock delivery. However, no signi cant decrease was found in the frequency of entry to the dark chamber in the rivastigmine group at either time point compared to the scopolamine group.
Biochemical estimation S. minor restored MDA and thiol concentration in the brain According to Fig. 5a, scopolamine-injected rats exhibited the elevated levels of MDA in their hippocampal and cortical tissues in comparison to the control group (p < 0.001). However, the rivastigmine administration signi cantly restored the elevated levels of MDA induced by scopolamine in the rats' hippocampus and cortex, compared to the rats of the scopolamine group (p < 0.001). It is notable that the treatment with all doses of the extract signi cantly counteracted scopolamine-induced increase in MDA level in both the hippocampus (p < 0.001) and cortex (p < 0.01, p < 0.001) compared to the scopolamine group.
By determining thiol content in the hippocampal and cortical tissues, the non-enzymatic defense potential of the extract against the oxidative stress was indicated. Accordingly, the thiol concentrations in both the hippocampus and cortex of scopolamine-injected rats were signi cantly lower than those of the control group (p < 0.001). As well, the administration of rivastigmine effectively restored the scopolamine-induced decreases in thiol contents in both tissues compared to the scopolamine group (p < 0.001). All doses of the extract signi cantly restored the decreases in the concentrations of thiol caused by scopolamine in the hippocampus and cortex compared to the scopolamine group (p < 0.01, p < 0.001) (Fig. 4b).
S. minor enhanced the antioxidant defense in the brain As shown in Figs. 5a and b, the antioxidant enzymes (SOD and CAT) activities were signi cantly attenuated in the hippocampus and cortex of the rats of the scopolamine group in comparison to those of the controls (p < 0.001). The treatment of these animals with rivastigmine and all doses of the administered extract resulted in a signi cant increase in the activities of the enzymes in the hippocampus and cortex (p < 0.001). More importantly, 200 mg/kg of the extract had the most marked effects on restoring the scopolamine-induced suppression of antioxidant enzymes (p < 0.001).
S. minor suppressed the activity of AChE in the brain As shown in Fig. 7, AChE activity signi cantly increased in the hippocampal and cortical tissues of the scopolamine-injected rats compared to the control group (p < 0.005). However, the rivastigmine administration signi cantly reduced the AChE activity in the hippocampal and cortical tissues compared to the scopolamine group (p < 0.05, p < 0.001). An ameliorative effect on the AChE activity in the hippocampus was found in the animals treated with different doses (50, 100, and 200 mg/kg, p < 0.001) of the extract. Meanwhile, only the highest dose of the extract (i.e. 200 mg/kg) was observed to reduce the AChE activity in the cortex signi cantly compared to the scopolamine group (p < 0.05).

Discussion
The present study was conducted on a scopolamine-induced amnesia rat model and demonstrated the bene cial effects of S. minor on cognitive decline. As shown by performing the passive avoidance and MWM tasks, scopolamine induced a noticeable decline in both learning and memory of the experimental rats compared to the controls. During 4 days of training in the current study, scopolamine prolonged the escape latency and distance to reach the platform. Additionally, the rats of the scopolamine group did not remember the location of the platform in probe trial and the time spent in the target quadrant decreased following the scopolamine injection. In PA task, scopolamine reduced the escape latency in the dark chamber as well as the time in the light chamber, while it prolonged the time in the dark chamber.
Consistent with our ndings, some previous investigations have also reported the cognition-impairing effects of scopolamine using behavioral tests (Marefati et al. 2019;Brinza et al. 2021). Accordingly, Hoang showed that the mean latencies and swimming distances increased by the administration of scopolamine (Hoang et al. 2020). Additionally, scopolamine reduced the latency to enter the darkness after shock (Ishola et al. 2020).
On the other hand, it was observed that the extract of S. minor effectively reversed the behavioral changes induced by scopolamine. By performing the MWM test, the ability of the rats to recall the platform location increased following the treatment with the extract, especially at the dose of 200 mg/kg. Since the MWM task generally indicated spatial memory ability of rodents (Crawley 2008; D'Hooge and De Deyn 2001), so these ndings suggested that S. minor extract boosted the rats' learning and spatial memory abilities in this study. Furthermore, in PA test, the administration of the extract improved the indices of memory in the scopolamine-treated animals. Similar results were also observed in the animals that received rivastigmine, which is a standard anti-amnesia drug considered as an AChE inhibitor. In the current research, the extract restored oxidative stress parameters levels disturbed by the administered scopolamine. The extract and rivastigmine signi cantly reduced lipid peroxidation while, they enhanced total thiol content, SOD, and CAT in the cortex and hippocampus. Additionally, AChE activity was attenuated after the treatment with both the extract and rivastigmine. Accordingly, rivastigmine also exhibited antioxidant activities in an animal model of Alzheimer's disease (Mahdy et al. 2012). Moreover, it was previously shown that rivastigmine causes memory consolidation and acquisition. The compounds with AChE inhibiting properties such as rivastigmine were found to reduce cognitive impairments induced by scopolamine (Howes and Houghton 2003).
Our ndings suggested that the inhibition of oxidative stress may contribute to memory enhancing effects of the extract in the rats, which was also reported in previous studies (Soodi et

Conclusions
The present study provided some evidence that the hydro-ethanolic extract of S. minor could ameliorate the scopolamine-induced memory de cits in rats as shown by the data obtained from the MWM and PA tasks data. In addition, the neuroprotective potential of S. minor extract may be partly linked to its anti-AChE and antioxidant activities in the brain tissue. Therefore, S. minor may be a candidate for the prevention or treatment of the cognitive disorders. However, further in vivo and clinical studies should be done before application of this herbal extract for treatment of human amnesia. (Bunse et al. 2020 Figure 1 The total ion chromatogram of S. minor hydro-ethanolic extract using LC-MS in the positive mode.    Effect of S. minor hydro-ethanolic extract on SOD (a, b) and catalase activities (c, d) in hipocampus and cortex of scopolamine-treated rats. Values were expressed as mean ± SEM (n = 10). ***p<0.001