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. However, its potential benefits in ameliorating learning and memory functions have been explored in no studies up to now. So, in the current 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. Notably, 30 min before conducting the tasks, scopolamine was injected. Finally, the biochemical assessments were done using the brain tissue. The extract characterization was performed by liquid chromatography-mass spectrometry, which confirmed the presence of quercetin, myricetin, kaempferol, catechin, ellagic acid, and gallic acid derivatives. In the MWM test, the extract reduced both escape latency and the travelled distance, compared to the scopolamine group. Moreover, in the PA test, the latency to enter the dark chamber significantly increased by the extract, compared to the scopolamine group (p < 0.05-p < 0.001). Notably, the beneficial effects of S. minor on cognitive performance of the scopolamine-treated rats appeared to be similar or even better than rivastigmine in behavior performance. Similar to rivastigmine, it was observed that the extract attenuated both AChE activity and oxidative injury in the brain as evidenced by the increased antioxidant enzymes and total thiol content; however, it decreased malondialdehyde level (p < 0.05-p < 0.001). In conclusion, the results suggested the effectiveness of S. minor in preventing cognitive dysfunction induced by scopolamine. Accordingly, these protective effects might be produced by the regulation of cholinergic activity and oxidative stress. S. minor could be considered as a potential alternative therapy in cognition disorders.

Correspondingly, these inhibitors can ameliorate cognitive deficits through the activation of the central cholinergic system (El-Marasy et al. 2012). However, alternative or adjuvant anti-amnestic therapy is required 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, excessive AChE activity mediated by scopolamine was shown to contribute 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). In a number of previous studies, a variety of biological activities, including anti-inflammatory, 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 enzyme activities in vitro (Cirovic et al. 2020;Finimundy et al. 2020). Additionally, anti-Alzheimer properties of methanol extract of S. minor were found in a primary neuron culture (Akbari et al. 2019;Soodi et al. 2017). In this regard, methanol extract of S. minor exhibited the neuroprotective effects on both Aβ-induced toxicity and oxidative stress in the cultured cerebellar granule neurons (Akbari et al. 2019;Soodi et al. 2017). However, memory-enhancing activity of S. minor has not been assessed in animal models of cholinergic dysfunction induced by scopolamine, so far. Thus, the present study aimed to examine the effect of hydro-ethanolic extract of S. minor on learning and memory process, oxidative stress indicators, and cholinergic dysfunction in the rats with scopolamine-induced memory loss.

Preparation of S. minor hydro-ethanolic extract
S. minor was collected from Ghoochan region (Khorasan Razavi province, Iran), and then identified by a botanist (M.R. Joharchi) at Ferdowsi University, Mashhad,Iran (herbarium No. 45489). Thereafter, the aerial parts of the plant were shade-dried, then crushed to a fine powder (50 g), and finally soaked in 200 ml of a hydro-ethanolic solution (50%, v/v) for 48 h at the temperature of 40 °C. Finally, the hydro-alcoholic extract was filtered and then concentrated at 37 °C using a rotary vacuum evaporator (Norouzi et al. 2019;Rajabian et al. 2016).

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

Animals
For the purpose of this study, male Wistar rats (n = 60, aged between 8 and 10 weeks, weighted 200 ± 20 g) were obtained from the animal house of the Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. The needed experimental procedures were then performed in compliance with the National Institutes of Health Guidance for the Care and Use of Laboratory Animals. Additionally, the Animal Ethics Committee of the Mashhad University of Medical Sciences approved the experimental protocols of this study (IR.MUMS.MEDICAL.REC.1398.655). Thereafter, 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 a free access to food and water ad libitum.

Drug administration and experimental design
At this stage, after the acclimatization for 1 week, the rats were randomly allocated into the following six 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.p.) (Marefati et al. 2019;Sun et al. 2019) 30 min before conducting the behavioral tests (Brinza et al. 2021;Hoang et al. 2020;Marefati et al. 2019;Ghasemi et al. 2019); and the treatment groups III-V received 50, 100, and 200 mg/kg of S. minor extract (i.p., Cirvonic et al. 2020;Akbari et al. 2019) and group VI received 0.5 mg/kg rivastigmine (i.p., Budzynska et al. 2015;Gawel et al. 2016), as the standard drug, for a two-week period. Accordingly, scopolamine was not injected during these two weeks. In the third week, the extract or rivastigmine was administered (i.p.) 30 min before administration of scopolamine (i.p.). In groups II-VI, a single dose of scopolamine was administered 30 min before performing the retention trial in the behavioral tests (Ghasemi et al. 2019;Marefati et al. 2019;Brinza et al. 2021;Hoang et al. 2020) (Fig. 1). The same rats were used for both the behavioral tests.

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 at 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 connected to a computer and the parameters, including latency to find hidden platform, the traveled distance, and speed were calculated. At the first day of performing the MWM test, each one of the study rats was acclimatized to the maze in the absence of the platform for 30 s. At each one of the 4-day training period, a trial was performed for each animal. For each trial, the rats were separately placed (facing the wall) into each one of the four starting points (North (N), East (E), South (S), and West (W)) and then, they were practiced to find the platform within 60 s. If the rat found the platform, it was allowed to stay there for 15 s, 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 h 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 s. (Norouzi et al. 2019).

Passive avoidance (PA) test
A passive avoidance (PA) learning test was performed to evaluate memory retention (Norouzi et al. 2019). Notably, 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 the habituation session, the rats were individually put into the light chamber and then allowed to move between the two chambers freely for 300 s. To conduct the acquisition trial, an inescapable foot-shock (2 mA for 2 s) was delivered to the animals through the grid floor after 5 s from entering the dark compartment. The retention trials were done 3 h, 24, 48 h, and 72 h later. In these trials, 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. Finally, the times spent by the animal in the dark and light compartments were recorded (Bavarsad et al. 2020). Notably, in the retention trials, the shock punishment was not applied.

Tissue preparation
Immediately after 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 thereafter, the whole-brain tissues were quickly isolated. 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 °C for further biochemical assessments (Norouzi et al. 2019).

Assay of lipid peroxidation
Lipid peroxidation was measured using the formation of thiobarbituric acid reactive substances (TBARS) following the malondialdehyde (MDA) reaction with TBA (Norouzi et al. 2019). In this regard, MDA was considered as the main product of lipid peroxidation and TBARS (red color) was then expressed as the MDA equivalent. In brief, the reaction mixture was prepared by the addition of 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 rate of the supernatants 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
The 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 rate was then recorded at 240 nm using a UV-vis spectrophotometer (A1). After the addition of the DTNB solution (20 μl and 10 mM in methanol) to each one of the samples, the absorbance rate was recorded again (A2). To calculate the total thiol concentration, the following equation was used (Bavarsad et al. 2020): where, B stands for the absorbance of the blank.

Assessment of antioxidant enzyme activities
Superoxide dismutase (SOD) activity was assessed in the homogenates based on the ability of SOD enzyme to inhibit the autoxidation of pyrogallol (Madesh and Balasubramanian 1998). The amount of SOD inhibiting 50% of pyrogallol auto-oxidation was defined as one enzyme unit (Bavarsad et al. 2020).
The catalase (CAT) activity was estimated based on its ability to convert hydrogen peroxide to water. Thereafter, the absorbance rate was read at 240 nm using the UV-vis spectrophotometer. Using 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 proposed by 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 rate of the resultant mixture was determined after incubation for 5 min at 37 °C. Thereafter, the reaction mixture was further incubated (for 5 min at 37 °C) after the addition of 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 data 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 significant. For the data obtained from the acquisition part of the MWM, as the time, distance, and speed during the 4-day period, the experimental design included treatment and day as the repeated measures. Therefore, two-way analysis of the variance (ANOVA) with repeated measures was used, and interactions were also reported (treatment × 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 identified in the hydro-ethanolic extract of S. minor using LC-MS analysis in its negative mode. Accordingly, 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, relatively high amounts of unique phenolic carboxylic acids, 4, 8-dimethoxy-7-hydroxy-2-oxo-2H-1-benzopyran-5,6-dicarboxylic acid, and 2-(4-carboxy-3-methoxystyryl)-2-methoxysuccinic acid were isolated in the hydro-ethanolic extract of S. minor (Ayoub 2003). Furthermore, this extract contained a high level of glucogallin, which is a phenolic compound formed from β-D-glucose and gallic acid. The identified compounds in the extract characterization are shown in Table 1 and total ion chromatogram obtained in ESI-mode is also shown in Fig. 2. The MS spectral data were compared with those that were available in the literature.

MWM test
Figures 3a, b, and c illustrate the time spent and the distance traveled to reach the platform during the 4-day training trials in this study, respectively. The results show that both the treatment and day affected the time during the 4-day period of learning (F (5, 936) = 64.11; p < 0.001 for the treatment and F (3, 936) = 63.49; p < 0.001 for the day). As well, an interaction was found between the treatment and the day (F (15, 936) = 2.84; p < 0.001).
The results also show that both the treatment and day affected the distance during the four-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 between the treatment and the day was found (F (15,936) = 3.07, p < 0.001). Correspondingly, escape latency time and the traveled path were observed to be longer in the scopolamine-injected animals compared to those of 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 first day showed shorter latency time compared to the scopolamine group (p < 0.05-p < 0.001). Notably, no significant difference was found in terms of the latency time between the group treated with 200 mg/kg of the extract or rivastigmine-treated group and the scopolamine group at the first day. Besides, Chlorogenic acid 3.5 353.28  the treatment with the extract (at all doses) and rivastigmine significantly reduced the escape latency time compared to that of the scopolamine group, on days 2 to 4 (p < 0.001).
Additionally, it was observed that the administration of both the extract (at all doses) and rivastigmine significantly decreased the traveled path compared to the scopolamine group (p < 0.001). Therefore, it can be stated that the treatment with both rivastigmine and the extract significantly improved memory performance in the treatment groups compared to the scopolamine group (p < 0.001). Furthermore, the time spent and the distance traveled in the groups treated with the extract (at doses of 50 and 200 mg/kg) were similar to the rivastigmine-treated group (p > 0.05). Among the different treatment groups, only the animals treated with 100 mg/kg of the extract exhibited shorter escape latency time and the traveled path than those of both the control and rivastigmine group (p < 0.01).
As shown in Fig. 3c, this treatment had no significant effect on the speed during the 4-day period of learning (F (5, 936) = 2.58; p > 0.05 for the treatment). On the other hand, the day affected the speed during the 4-day period of learning (F (3, 936) = 6.48; p < 0.001 for the day). As well a significant interaction was found between the treatment and the day (F (15, 936) = 5.82; p < 0.001).
According to the results obtained from the probe test ( Fig. 4a and b), both the time spent and the traveled distance in the target quadrant significantly decreased in the scopolamine group compared to the control group (p < 0.001). Moreover, the animals treated with both rivastigmine and the extract (administered at all doses) exhibited longer time spent and the traveled distance in the target quadrant compared to those of the scopolamine group (p < 0.001). Notably, the traveled distance and the time spent in the target quadrant were similar to the rivastigmine-treated group. However, the animals treated with 100 mg/kg of the extract exhibited a shorter time in the target quadrant than those of the rivastigmine group (p < 0.01). Some significant differences were also found regarding 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 significant difference was observed in terms of the distance traveled in the quadrant between the two study groups treated with two 50 and 100 mg/kg doses of the extract (p < 0.001). Compared to the control group, the time spent in the target quadrant significantly increased in the groups treated with 50 and 200 mg/kg of the extract (p < 0.001). Similarly, the animals treated with 50 mg/kg of the extract exhibited longer traveled distance in the target quadrant than those of the control group (p < 0.01).

Passive avoidance test
As illustrated in Fig. 5a 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 delivery of the shock (p < 0.01, p < 0.001). Besides, no significant difference in terms of the time latency was observed between the scopolamine group and the control group, 72 h post-shock.
In contrast, higher time latencies were observed 3, 24, 48, and 72 h after delivery of the shock in those rats that  (a) Escape latency, (b) travelled distance, and (c) speed of the rats to reach to the platform during the 4 days of training trials were assessed using Morris Water Maze task. Values were expressed as mean ± SEM (n = 10). ** p < 0.01 and *** p < 0.001 Scopolamine group Vs. Control group. ### p < 0.001 Rivastigmin group Vs. Scopolamine group; + p < 0.05 and +++ p < 0.001 S. minor extracttreated group Vs. Scopolamin group. $$ p < 0.01 Rivastigmin group Vs. S. minor extract were treated with rivastigmine compared to the rats in the scopolamine group (p < 0.05 and p < 0.001). Moreover, the administration of the extract at both 100 and 200 mg/kg was found to be associated with a significant increase in the time latency compared to the scopolamine group, 3, 24, 48, and 72 h post-shock (p < 0.001). As well, some significant differences were observed in the time latency between these two groups treated with 50 and 100 mg/kg of the extract as well as between the two study groups treated with 50 and 200 mg/kg of the extract after 3, 24, 48, and 72 h post-shock (p < 0.001).
Interestingly, the administration of the extract at doses of 100 and 200 mg/kg caused the higher time latencies than those of the rivastigmine group, 24 (p < 0.01 and p < 0.05), 48 (p < 0.05 for both 100 and 200 mg/kg), and 72 h postshock (p < 0.001 for both 100 and 200 mg/kg). Moreover, the time latencies in the group treated with 200 mg/kg of the extract was higher than the rivastigmine group, 3 h postshock (p < 0.05). However, the time latencies were shorter in the group treated with 50 mg/kg of the extract compared to the rivstigmine group, 3, 24, and 48 h post-shock (p < 0.001). Compared to the control group, the groups treated with 100 and 200 mg/kg exhibited higher time latencies, 3, 48, and 72 h post-shock (p < 0.01 and p < 0.001).
On other hand, the animals in the scopolamine group were observed to spend longer times in the dark chamber compared to the control group, 3, 24, and 48 h post-shock (p < 0.001). However, no significant difference was 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 compared to the scopolamine group, 3, 24, 48, and 72 h post-shock (p < 0.01 and 0.001). Furthermore, the administration of the extract (at all doses except 50 mg/ kg) caused a significant decrease in the time spent in the dark compared to the scopolamine group, 3, 24, 48, and 72 h post-shock (Fig. 5b, p < 0.05, p < 0.01, and p < 0.001). However, some significant differences were reported in the time spent in the dark 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 3, 24, 48, and 72 h post-shock (p < 0.001).
Notably, the animals treated with 100 and 200 mg/kg of the extract were found to spend shorter times in the dark chamber compared to the rivastigmine group, 24 and 48 h post-shock (p < 0.01 and p < 0.05). However, the spent time in the dark chamber was longer in the animals treated with the low dose of the extract compared to the rivastigmine group, 72 h post-shock (50 mg/kg, p < 0.01).
As illustrated in Fig. 5c, the time spent in the light chamber significantly decreased following the injection of scopolamine in comparison to the control group, 3, 24, 48, and 72 h post-shock (p < 0.001). 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 72 h post-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) compared to the scopolamine group, 3, 24, and 72 h post-shock (p < 0.05p < 0.001). Similarly, the animals treated with 100 and 200 mg/kg of the extract indicated a significant difference in terms of the time spent in the light 48 h after delivery of the shock compared to the scopolamine group. However, some significant differences were indicated in the time spent in the light, 3, 24, 48, and 72 h post-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).
Notably, the animals treated with 100 and 200 mg/kg of the extract were found to spend longer times in the light chamber compared to the rivastigmine group, 24 and 48 h post-shock (p < 0.01 and p < 0.05). However, the spent time in the light chamber was shorter in the animals treated with the low dose of the extract compared to the rivastigmine group, 72 h post-shock (50 mg/kg, p < 0.01). Furthermore, the animals treated with 100 and 200 mg/kg of the extract were found to spend longer times in the light chamber compared to the control group, 72 h post-shock (p < 0.01 and p < 0.05).
According to the results illustrated in Fig. 5d, although the frequencies of the entry to the dark chamber were higher were assessed using passive avoidance test. Values were expressed as mean ± SEM (n = 10). *** p < 0.001 Scopolamine group Vs. Control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 S. minor extract and rivastigmine groups Vs. Scopolamine group. + p < 0.05, ++ p < 0.01, +++ p < 0.001 S. minor extract Vs. rivastigmine group in the scopolamine group compared to that of the control group 3, 24, and 48 h post-shock delivery, the found differences were not significant. The groups that received the extract at 100 and 200 mg/kg demonstrated a significant decrease in the frequencies of dark chamber entry compared to those of the scopolamine group, 24 h (p < 0.05, p < 0.01) and 72 h (p < 0.05, p < 0.001) post-shock. However, no significant 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. Notably, the frequencies of entry to the dark chamber in the groups treated with 100 and 200 mg/kg of the extract, 48 and 72 h post-shock as well as 100 mg/kg, 24 h post-shock were similar to the rivastigmine group (p > 0.05). Furthermore, the frequencies of entry to the dark chamber in the animals treated with 100 and 200 mg/kg of the extract 3 h post-shock (p < 0.01 and p < 0.001) as well as 200 mg/kg of the extract 24 h postshock were found to be less than that of the rivastigmine group (p < 0.05). However, the same value in the animals treated with 50 mg/kg of the extract 24, 48, and 72 h postshock were found to be more than that of the rivastigmine group (p < 0.001).

S. minor restored MDA and thiol concentration in the brain
According to Fig. 6a, 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 significantly 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 significantly 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. However, MDA level was found to be greater in cortex of the animals treated with 50 and 100 mg/kg as well as in hippocampus of the animals treated with 200 mg/kg of the extract, compared to the control group (p < 0.01 and p < 0.001).
By determining the thiol content in both 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 significantly 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). It was found that all doses of the extract significantly restored the decreases in the concentrations of thiol in the hippocampus and cortex caused by scopolamine compared to the scopolamine group (p < 0.01 and p < 0.001) (Fig. 6). Notably, MDA and thiol levels in both the hippocampus and cortex of the groups treated with the extract (at all doses) were comparable to the rivastigmine group (p > 0.05). However, thiol level was found to be lower in the cortex of the animals treated with 50 mg/kg (p < 0.001) as well as in the hippocampus of the animals treated with 50 and 100 mg/kg of the extract, compared to the control group (p < 0.01).

S. minor enhanced the antioxidant defense in the brain
As shown in Fig. 7a and b, the antioxidant enzyme (SOD and CAT) activities were significantly 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 significant increase in the activities of the enzymes in the hippocampus and cortex (p < 0.001). However, SOD and CAT activities were lower in the hippocampus and cortex of the rats treated with the low dose (50 mg/kg) of the extract compared to the control group (p < 0.001). More importantly, 200 mg/kg of the extract was observed to bring the most marked effects on restoring the scopolamine-induced suppression of antioxidant enzymes (p < 0.001). Notably, SOD and CAT activities in the hippocampus and cortex of the rats treated with the extract (at all doses) were similar to rivastigmine group (p > 0.05).

S. minor suppressed the activity of AChE in the brain
As shown in Fig. 8, AChE activity significantly increased in the hippocampal and cortical tissues of the scopolamineinjected rats compared to the control group (p < 0.005). However, in the current study, the administration of rivastigmine significantly 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 was found in the hippocampus of in the animals treated with different doses (50, 100, and 200 mg/ kg, p < 0.001) of the extract in this study. Meanwhile, only the highest dose of the extract (i.e. 200 mg/kg) was observed to reduce the AChE activity in the cortex significantly compared to the scopolamine group (p < 0.05). Moreover, among the groups treated with different doses of the extract, only the group treated with the lowest dose (50 mg/kg) exhibited a greater AChE activity in the cortex and hippocampus compared to the control group (p > 0.05). Notably, the AChE activities in the hippocampal and cortical tissues of the rats treated with the extract (at all doses) were similar to the rivastigmine group (p > 0.05).

Discussion
The present study was conducted on a scopolamine-induced amnesia rat model and as a result, demonstrated the beneficial 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 the 4-day 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 has been shortened 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. In consistent with our findings in this study, some previous investigations have also reported the cognition-impairing effects of scopolamine using a number of behavioral tests (Marefati et al. 2019;Brinza et al. 2021). Accordingly, Hoang et al. (2020) in their study showed that the mean latencies and swimming distances increased by the administration of scopolamine. 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), these findings suggest that in this study, the administration of S. minor extract boosted the rats' learning and spatial memory abilities. According to the results of speed, no significant difference was found between the study groups. Hence, the effect of the extract on the animal's performance does not seem is related to its effect on Fig. 6 Effect of S. minor hydroethanolic extract on MDA (a, b) and thiol concentration (c, d) in hipocampus and cortex of scopolamine-treated rats. Values were expressed as mean ± SEM (n = 10). *** p < 0.001 Scopolamine group Vs. Control group; ## p < 0.01 and ### p < 0.001 S. minor extract and rivastigmine groups Vs. Scopolamine group locomotor activity in MWM task. Furthermore, in PA test, the administration of the extract improved the indices of memory in the scopolamine-treated animals. Notably, the results of MWM and PA tests exhibited a significant difference between the control and the treatment groups in some time points that can be linked to the intrinsic properties of S. minor extract. However, for evaluating the effect of S. minor alone, 3 additional groups were needed to be considered in the control group, this can be mentioned as a limiting factor of this research. Therefore, further examinations should be done to confirm the possible cognitive improving effects of the extract in comparison to the control rats.
Interestingly, similar results were also observed in the animals that received rivastigmine, which is known as a standard anti-amnesia drug considered as an AChE inhibitor. The results showed that the effects of the extract on performance of scopolamine treated rats was comparable to the effect of rivastigmine. Accordingly, this drug can elevate the availability of ACh in the central cholinergic synapses and consequently ameliorate cholinergic functions (Mahdy et al. 2012). As well, the scopolamine administration to the rodents has been well-documented to induce memory loss and cognitive deficits through the inhibition of cholinergic transmission (Ishola et al. 2013;Marefati et al. 2019;Ogunsuyi et al. 2018). Notably, scopolamine also triggers oxidative stress by inducing an imbalance in brain oxidative status (Budzynska et al. 2015;Haider et al. 2016). Oxidative stress and cholinergic dysfunction have been previously found to have close associations with the cognitive decline in Alzheimer's disease (Liguori et al. 2018;Dos Santos Picanco et al. 2018). Our data revealed that scopolamine-induced memory loss was accompanied with increase in AChE activity and oxidative stress in the brain tissue. These results are in line with those of the previous studies (Marefati et al. 2019;Brinza et al. 2021;Boiangiu et al. 2020). In this regard, Brinza et al. (2021) reported a significant reduction in the total antioxidant content along with the increased AChE activity. Notably, the impairment of endogenous antioxidant defense systems is known as a key factor in the scopolamine-dependent cognitive impairment (Haider et al. 2016;Muhammad et al. 2019). On the other hand, Fig. 7 Effect of S. minor hydroethanolic 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 Scopolamine group Vs. Control group; ## p < 0.01 and ### p < 0.001 S. minor extract and rivastigmine groups Vs. Scopolamine group some compounds with antioxidant properties like ascorbic acid, has also been observed to cause positive effect on scopolamine-induced cognitive deficits via inhibiting AChE activity and attenuating oxidative injury (Ishola et al. 2013;Harrison et al. 2009).
In the current research, the administered extract restored oxidative stress parameters levels disturbed by the administered scopolamine. The extract, in a similar manner with rivastigmine, significantly reduced lipid peroxidation while, they increased total thiol content, SOD, and CAT activities in the cortex and hippocampus of the studied rats. These results suggested that S. minor was as effective as rivastigmine. 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 both 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 findings suggested that the inhibition of oxidative stress may contribute to memory enhancing effects of the extract on the rats, which was also reported in previous studies (Soodi et al. 2017;Nguyen et al. 2008;Akbari et al. 2019). In this regard, Ferreira et al. (2006) claimed the anti-oxidant and AChE inhibitory potentials of the ethanol extract and essential oil of S. minor. Another previous study also showed neuroprotective effects of S. minor on oxidative injury induced by amyloid β in the cultured cerebellar granule neurons (Soodi et al. 2017). Furthermore, the AChE inhibitory effect has been reported to be involved in the neuroprotective effects of S. minor on amyloid β toxicity in the primary neural cell culture (Akbari et al. 2019).
As previously reported, the phenolic and flavonoid compounds present in S. minor extract could contribute to its antioxidant and neuroprotective functions (Akbari et al. 2019;Cirovic et al. 2020). In this regard, various phenolic compounds, including quercetin and ellagic acid, were observed to alleviate cognitive deficits in the experimental models (Dornelles et al. 2020;Molaei et al. 2020). Different bioactive components were also identified in S. mninor extract, including quercetin, myricetin, kaempferol, ellagic acid, catechin, gallic acid, and their derivatives. Hence, the alleviation of cholinergic dysfunction and the subsequent memory-enhancing effect of S. minor extract may be attributed to the presence of the components with antioxidant activities.

Conclusions
The present study provided some evidences that the hydroethanolic extract of S. minor could ameliorate the scopolamine-induced memory deficits in rats as shown by the data obtained from the MWM and PA tasks data. In addition, the neuroprotective potential of S. minor may be partly attributed to its suppressive effect on AChE activity as well as antioxidant activities in the brain tissue. The anti-amnesic and neuroprotective effects of the extract are also comparable to those of rivastigmine. Therefore, S. minor can be Fig. 8 Effect of S. minor hydro-ethanolic extract on AChE in hipocampus (a) and cortex (b) of scopolamine-treated rats. Values were expressed as mean ± SEM (n = 10). ** p < 0.01 and *** p < 0.001 Scopolamine group Vs. Control group; # P < 0.05 and ### p < 0.001 S. minor extract and rivastigmine groups Vs. Scopolamine group a considered as a candidate either for the prevention or the treatment of the cognitive disorders. However, further in vivo and clinical studies should be done before applying this herbal extract for the treatment of human amnesia.