Effect of Rosa damascena Extract on Rat Model Alzheimer's Disease: A Histopathological, Behavioral, Enzyme Activities, and Oxidative Stress Study

The purpose of the current study is to investigate the effect of aquatic Rosa damascena extract against the oxidative damage induced by aluminum chloride intoxication in Alzheimer's model of Wister rats. Rats were divided randomly into seven groups (n = 10). Control group received no treatment, sham group received distilled water orally, aluminum group (AL) was administered AlCl3 (100 mg/kg) orally, extract 1 and 2 groups were treated with only aqueous R. damascena extract (DRE) (500 and 1000 mg/kg), and treatment 1 and 2 groups received aqueous R. damascena extract (500 and 1000 mg/kg) and AlCl3 (100 mg/kg) orally. The brain tissues were sampled for histopathological examination, and biochemical analysis was conducted for estimating the enzyme activities of acetylcholinesterase and catalase (CAT), the levels of GSH and MDA, and ferric reducing antioxidant power. According to the results of behavioral tests, AL administration showed a reduction in spatial memory and remarkably increased the time needed for reaching the invisible platform. The administration of Al-induced oxidative stress and an increase of the enzyme activity of AChE. Al administration increased AChE level from 1.176 ± 0.173 to 3.62 ± 0.348, which was a significant rise. However, treating with the extract at the dose of 1000 mg/kg downregulated it to 1.56 ± 0.303. Administration of the R. damascene extract caused an increased level of catalase and glutathione levels in treatment groups, attenuated MDA level, and regulated AChE activity. Our results illustrate that administration of R. damascene extract has a protective effect against the oxidative damage induced by AlCl3 intoxication in Alzheimer's model.


Introduction
Aluminum (Al) is an abundant metal in our environment used widely by humans, which makes toxicity particularly relevant to human health.Tis metal is widely identifed as a neurotoxin.A relationship exists between exposure to aluminum and neurodegenerative diseases, such as Parkinsonism dementia, amyotrophic lateral sclerosis, and Alzheimer's disease (AD).Exposure to high levels of Al leads to neurofbrillary degeneration, and higher Al concentration is associated with degeneration, oxidative stress, neuronal apoptosis, and neurotransmission alterations in AD [1,2].
Tis metal is known to speed up extracellular β amyloid generation and aggregation.It acts as a cholinotoxin and causes alterations in the cholinergic activity, a key event in the neurochemistry of AD [3].Also, it is known to speed up extracellular β amyloid generation and aggregation.Moreover, it can act as a cholinotoxin and causes alterations in the cholinergic activity, a key event in the neurochemistry of AD [3].However, many medicines, toothpaste, food additives, and even water contain aluminum [4].Te most common form of human exposure to aluminum is through the gastrointestinal tract with a rate of absorption, of about 0.2% [5].
Alzheimer's disease is a common form of dementia in aged people that is characterized by a gradual loss of neuronal function, memory loss, and a decline in language skills [6,7].Te AD is defned by the following two pathological features: b-amyloid protein (A) deposition and tau hyperphosphorylation. Age, gender, genetic factors, and some environmental factors such as a history of head trauma, diabetes, hypothyroidism, increased cholesterol, and accidental use of aluminum and zinc, as well as lifestyle may contribute to the risk of the AD [8].High levels of Al have been reported in the brain of patients with AD relative to controls [5].In this regard, the correlation between aluminum and AD appears stronger than that of other neurological disorders [9].Oxidative stress poses a signifcant risk to AD.It is believed that redox abnormality is involved in the neurodegenerative process leading to the impairment mediated by reactive oxygen species and reactive nitrogen species in the AD brain.Besides, some studies reported that the levels of oxidative markers of biomolecules including nucleic acids, lipids, proteins, carbohydrates, and antioxidant enzymes changed.It is believed that natural antioxidants are neuron protectants [10].
Numerous traditional and herbal medicinal plants are being employed to treat neurological disorders such as AD, which act as antioxidants, anti-infammatories, and cholinesterase and β-secretase inhibitors.Tese natural compounds also prevent the accumulation of amyloid beta and its fbril formation [11].
Rosa damascena, known as Damask Rose, belongs to the family of Crassulaceae and grows in Northern Asia and the mountains of Central Europe [12].For several centuries, this plant has been used as an advantageous and efective treatment for a wide range of diseases and in folk medicine for treating many diseases such as anti-infammatory treatments [13], menstrual bleeding, digestive problems [14], strengthening the heart and chest [15], and abdominal pain.Moreover, Rosa damascena is widely used in Iran's traditional medicine in various forms, such as extract, powder, and essential oil.In addition, this plant has multiple active ingredients, which are massively investigated in numerous studies for their bioactive efects, such as anticancer and antioxidant properties [16].Tis important ornamental plant has long been applied for industrial, physiological, and medicinal purposes [17].Te physiological functions of Damask Rose may be somewhat related to the plenty of anthocyanidins and favonoids.Anthocyanidins and favonoids possess a broad spectrum of biological operations and play an important role in human health as astringents, antiinfammatory agents, antioxidants, and free radical scavengers.Te treatment properties of this plant may include stimulating the nervous system, reducing depression and anxiety, and improving performance [18][19][20].Tere are essential compounds of favonoid glycoside in this plant, which inhibit the activity of the AChE enzyme.Terefore, due to the presence of such compounds, the plant will be able to control the disturbances caused by AChE [21].
Te present study aims at providing a behavioral, biochemical, and histopathologic evaluation of the efect of aquatic R. damascene extract against the oxidative damage induced by aluminum chloride intoxication in Alzheimer's model of Wistar rats.

Materials and Methods
2.1.Animals.Adult female albino Wistar rats (approximately 8-10 weeks old, 200-250 g) [22,23] were purchased from the Pasteur Institute of Iran, North Research Center (Amol, Iran) [24].Rats were kept in an insulated room with a 12-h light/dark cycle at an ambient temperature of 22 ± 3 °C and relative humidity of 52 ± 5% and were fed adequately and appropriately [25,26].Experimental methods were approved by the ethics committee of Babol University of Medical Sciences with the approval ID Mubabol.rec.1394.24.

Chemicals.
All chemical compounds for biochemical examination were procured from Sigma (Sigma-Aldrich GmbH, Steinheim, Germany).

Plant Extract Preparation.
Fresh petals of R. damascena were collected from Kashan 34.0258 °N, 51.0540 °E, Iran.A voucher specimen of the plant (HUMZ 8115) was deposited in the herbarium center.20 grams of fresh petals were placed in a 200 mL conical fask, and 100 mL of water was added.Te Erlenmeyer fask was covered and kept in a reciprocating shaker for 24 hours for persistent agitation for thorough mixing and complete elucidation of active materials to dissolve in water.Ten, the extract was fltered and the water from the extract was removed by using a rotary vacuum evaporator.Eventually, the remainders were gathered and used [27].

Experimental Design.
Prior to starting the study, rats were divided randomly into the following seven groups (n � 10): control group: received no treatment; sham group: received distilled water orally for ensuring they receive the same administration stress as the treatment groups; group AL: administered AlCl 3 (100 mg/kg daily) (dissolved in distilled water) orally for four weeks [28].Groups DRE 500 and DRE 1000 were treated with aqueous R. damascena extract (500 and 1000 mg/kg) [29,30] daily for eight weeks orally and groups AL + DRE 500 and AL + DRE 1000 received aqueous R. damascena extract (500 and 1000 mg/kg) as gavage daily for eight weeks and AlCl 3 (100 mg/kg) orally daily for the last four consecutive weeks.

Separation and Identifcation of the Constituents of the
Extract.Te chemical content of the extract was analyzed by a gas chromatograph mass spectrophotometer (GC-MS) system (Agilent Technologies 7890A).GC/MS analyses were performed using the Agilent Technologies device with Electric shocks were applied through a metal lattice foor when the rat was in the dark room.All rats were adapted with a device for the frst two days in the dark chamber for 5 min.On the third day, they entered the bright chamber and after staying there for 2 min, the door was opened.Due to the lack of desire for light, the rats entered the dark chamber and the door was closed.Afterward, an electric shock was given to the rats at a rate of 2 s and 1 mA from the foor of the room.After 24 h, each rat was placed again in the bright chamber.Te latency of stepping through the dark part (maximum 600 s) was measured and logged as the index for the passive avoidance behavior [32,33].

Morris Water Maze (MWM).
Morris water maze apparatus was applied for spatial memory testing.Tis apparatus includes a black circular pool (height 50 cm and 160 cm in diameter) flled with 30 cm of water (25 ± 2 °C).
Te pool was divided into four quadrants (1, 2, 3, and 4).An invisible platform (280 mm in height and 100 mm in diameter) was located 20 mm below the water at the center of the third quadrant, which remained in the same quadrant throughout the experiment.Each trial was started with the rat being placed in the pool at one of the four cardinal positions around the Pool environment according to a pseudorandom sequence.Te topmost duration of the trial was 60 s.Rats were assessed in eight trials (twice from each starting place) for each session for 3 days.On the 4th day, a probe test was done through the platform removal.Te rats could swim at will for 60 s.A computer tracking system was used for measuring the time for reaching the platform and spending in the target quadrant [34,35].

Tissue Preparation.
Te animals were decapitated and then the whole brain was rapidly dissected, washed with isotonic saline, dried, and then weighed (n � 10).Next, it was homogenized immediately to give a 10% (w/v) homogenate in an ice-cold medium containing 50 mmol/l Tris-HCl (pH 7.4) and 300 mmol/l sucrose.Te homogenate tissue was centrifuged at 3000 rpm for 10 min.Biochemical analysis (AChE, CAT, GSH, and ferric-reducing antioxidant power) was done after separating the supernatant.All processes were carried out at 4 °C.Supernatants were used to determine AChE activity and the level of the antioxidant parameter using a spectrophotometer (Shimadzu UV-mini 1240).
Histopathological lesions (necrosis and gliosis) were scored as previously described [38].To neuronal count the cortex and the hippocampus, the images were taken from hippocampal regions and cortex.Hence, 45 sections (nine sections from a single animal in each group) were analyzed for each experimental group.Measurement of the area was performed using the capture software (Tucsen, Fuzhou, China).Histological evaluations were performed by pathologists blinded to the experimental groups.

Tissue Cholinesterase Activity Measurement.
For the cholinesterase activity assay, the kinetic photometric method was adopted.Cholinesterase can break down butyrilthiocholine to thiocholine which reacted with 5, 5′-dithiobis-2-nitrobenzoate (DTNB), forming the yellow product of 5thio-2-nitrobenzoate with an absorbance at 412 nm.Te rate of absorbance change was regulated to 1 g tissue and it was compared with the references.Cholinesterase activity was calculated using a molar extinction coefcient value of 13.6 mM expressed as U/mg protein.We used a human blood serum sample for positive control provided by healthy volunteers of our lab personnel [39].Te cholinesterase activity was calculated using an extinction coefcient of thiolate dianion of DTNB as a product at 412, which was produced by the enzymatic hydrolysis of acetylthiocholine iodide.In this method, it is not necessary to draw a calibration curve.

Measurement of Malondialdehyde (MDA)
. Tissue homogenate (0.5 ml) was diluted to 1 ml using Tris-HCl bufer, then incubated at 37 °C for 2 h.Afterward, 1 ml of cold trichloroacetic acid (TCA) was added, vortexed, and centrifuged at 800 × g for 10 min.To 1 ml of supernatant was added 1 ml of thiobarbituric acid (TBA) and the reaction mixture was placed in a boiling water bath for 15 minutes.Te pink-colored complex was formed whose absorbance was read at 532 nm.Te amount of MDA formed (index of lipid peroxidation) was calculated using an extinction coefcient of 1.56 × 10 5 M −1 •cm −1 for MDA-TBA chromophore, and the results are expressed as nmol/mg protein [40].A standard solution of ferrous sulfate (FeV100 to 2000 mM) was prepared in distilled water.Te FRAP value is expressed as mmol of tissue weight [42].

Measurement of Glutathione (GSH)
. GSH measurement was performed by the Ellman procedure.For this purpose, 1 ml of supernatant was taken after sedimentation of 0.5 ml of brain homogenate with 2 ml of 5% TCA.Ten, 0.5 ml of Ellman's reagent (0.0198% DTNB in 1% sodium citrate) and 3 ml of phosphate bufer (1 M, pH 8.0) were added.Te developed color was observed at 412 nm.A standard curve was drawn using known levels of reduced GSH concentration and described as mg/g of tissue [43].

Statistical Analysis.
Data are expressed as the mean-± standard error.Latency data and covered distance during the training days were analyzed using repeated measures within groups, two-way analysis of variance (ANOVA), followed by a Tukey post hoc test to compare between groups [44].Additionally, Kruskal-Wallis and Mann-Whitney U tests were used for histopathological scoring diferences between the groups.Te statistical comparisons were analyzed through SPSS 26 version [25,45].

Morris Water Maze (MWM)
(1) Latency.Tere was no diference between groups in terms of latency in the pretest MWT assessment (P > 0.05).Tere was a statistically signifcant diference between the AL and control groups in escape latency (P < 0.01).Escape latency reduction was observed in animals that received the extract (treatment groups).Group AL + DRE 1000 showed no signifcant escape latency compared to the control group (P > 0.05).According to the results, treatment with R. damascene reduced the escape latency in the treatment groups compared to the Al group (Figure 1).
(2) Probe Trial.Te amount of time spent in the target quadrant during the frst half of the probe trial session was assessed in the post-test phase.Te time spent in the target quarter between the AL and control groups was signifcantly diferent.Te AL + DRE 1000 group showed improved spatial memory and a signifcant increase in the time spent in the target quadrant compared to the Al group (P < 0.01, Figure 1).

Malondialdehyde (MDA).
Te highest level of (MDA) was observed in the AL group.Te MDA level decreased in groups AL + DRE 500 and AL + DRE 1000.Tere was not any signifcant diference in MDA level between AL + DRE 1000 and the control group (P > 0.05) but there was a signifcant diference between the AL group in comparison to the sham and control groups (P < 0.0001, Table 2).

Ferric-Reducing Antioxidant Power (FRAP).
FRAP values in the aluminum group were the lowest while it was increased in groups AL + DRE 500 and AL + DRE 1000.It seems that the negative efects of aluminum are treated with R. damascene extract at a dose of 1000 mg/kg.Also, there was not any signifcant diference in FRAP level in the AL + DRE 1000 group compared to the control group (P > 0.05, Table 2).  2 and 3; Table 3).In addition, exposure to AL signifcantly reduced the number of cells in the cerebral cortex and diferent regions of the hippocampus compared to the control group (Table 4).R. damascene treatment signifcantly increased the cell numbers in diferent regions of the hippocampus and cortex compared to the AL-treated group (Table 4).

Discussion
In this study, it was observed that aluminum is a neurotoxicant and accumulates in diferent parts of the brain.Al induces oxidative stress and increases MDA and reduces antioxidative enzymes such as SOD, CAT enzyme activities, and TAC levels in the rat brain [46][47][48].Our results showed the highest level of MDA was observed in the AL group and the levels of catalase, FRAP, and GSH were reduced in the group AL, our fndings are in agreement with previous studies.
Overall, Al-induced oxidative stress in brain tissue and can cause the death of neuronal cells by shifting ROS production.It should be noted that reactive oxygen types (ROS) resulting from a normal aerobic metabolism are potentially harmful.Tese free radicals are usually removed or disabled by antioxidant groups in vivo.However, since R. damascene is a promising source of antioxidants, it was likely to see reduced lesions in the treatment groups after it reduced the free radicals in the tissue.Also, the potential of treatment was investigated by employing DRE at 2 diferent doses [29].
Our results show that the administration of R. damascene extract increased catalase and glutathione levels in the treatment groups.Hence, regarding the protective and antioxidant efects of R. damascene extract, the best form of efcacy was exhibited in a group of (AL + DRE 1000).We also found the highest level of MDA in the AL group and the enzyme activities of CAT, FRAP, and GSH reduced in the group AL, confrming earlier studies.
Acetylcholine is an important neurotransmitter in the memory and learning process.Te behavior and performance defcits in AD are due to the inability the transmission of nerve impulses in the cholinergic synapses [66].AChE plays a role in cholinergic transmission.In addition, cholinesterases may also play a role during morphogenesis and in the onset of neurodegenerative diseases [67].Te observed elevated activity of AChE is described as the direct efect of aluminum [68].Aluminum is a strong cholinotoxin that has a biphasic efect on acetylcholinesterase activity with a primary increase in the activity of this enzyme.Tis biphasic efect is ascribed to the gradual agglomeration of Al in the brain and this is the reason for increasing AChE in the rat [69].
Chlorogenic acids are a family of polyphenolic compounds, which is esters such as quinic acid that are found in R. damascene extract.Chlorogenic acids are potent antioxidants [70] and inhibited acetylcholinesterase activity in the frontal cortex and hippocampus [71].Also, quinic acid has been characterized as a prometabolite that leads to the induction of efcacious levels of tryptophan and nicotinamide as antioxidants [72].Te results of our study showed aluminum chloride administration increased AChE activity and R. Damascene extract attenuated AChE activity.
Because of the presence of this material and the antioxidant characteristics of the plant, it can be used as a treatment and prevention of brain illnesses such as AD.According to the results of behavioral tests, it was observed that the aluminum group had a signifcant diference from other groups.Te results of this study are consistent with the fndings of the study by Burimau, who concluded that aluminum reduced the behavior scores of the Wistar rats [73].In another study, Azui found gavage administration of aluminum in mice reduced their vigilance [74].Moreover, Goat and Caorah also found that the intake of AlCl 3 in male mice causes neuronal damage and behavioral changes [75].
Al administration showed a decrease in spatial memory and notably increased the time needed to overtake the hidden platform.Abulfadl et al. [48] investigated the exposure to aluminum signifcantly reduced memory in the MWM test and AlCl 3 -induced neurotoxicity in rats.An earlier study reported aluminum administration had     Evidence-Based Complementary and Alternative Medicine a neurodegeneration efect resulting in learning defcits [1,76].Te results of these studies are consistent with our study.In this study, R. damascene extract improved signifcantly the latency periods of a passive avoidance response in the treatment groups.Also, R. damascene treatment during aluminum exposure reduced the time to reach the hidden platform, which this reduction was more in Al + DRE 1000 group.On the probe day, the time spent on the target quadrant was notably reduced in the AL group compared to other groups.It seemed that the negative efects of aluminum-receiving groups were downregulated with R. damascene extract, and both treatment groups, which received 500 and 1000 DRE, respectively, did not show signifcant diferences compared to the control group.Our results are in agreement with the previous study [77] that reported R. damascene extract administration improved learning and memory by using Morris Water Maze (MWM) performance and the passive avoidance test in treated rats.
It was indicated that the toxic efects of Al on mice's brain confrm the damage in the hippocampus and cortex, including necrosis, gliosis, and neurofbrillary degeneration due to the accumulation of Al in these regions [78].Tese results are in agreement with our study.Similarly, histopathology fndings of the brain and hippocampus confrmed the protective activity of R. damascene extract against aluminum-induced brain and hippocampus damage, as is evident by the reduction of brain and hippocampus lesions such as gliosis and necrosis in treatment groups, especially in AL + DRE 1000 group.
Overall, it seemed that the extract was quite successful at reducing the Al-induced lesions in brain tissue.Tese fndings are in line with each other, which suggest that can be used as a potent natural substance for fghting with AD.DRE showed than has wide efects on multiple pathways, and can efectively upregulate the functions of the brain such as remembering.

Conclusion
In summary, this study demonstrated that Al results in toxic efects, inducing oxidative stress, increasing AChE, reduced behavior scores, and histopathological changes in brain tissue.Also, R. damascene extract in a dose of 1000 mg/kg could improve signifcantly the levels of acetylcholinesterase enzymes and the rate of stress indicators, improving the level of consciousness and brain function.Terefore, R. damascene extract can be used as an efective treatment agent for preventing complications from Al intoxication.

Figure 1 :
Figure1: Efect of DRE on escape latency, and the mean percentage of time spent in the target quadrant.Al increased escape latency and R. damascene administration signifcantly reduced the escape latency parameter.* P < 0.05, * * P < 0.01, and * * * P < 0.001 of Al group compared to the control group; # P < 0.05 and ### P < 0.001 compared to Al + extract 1000 mg/kg experimental animal group.During the probe trial, which is an index of spatial memory, it was shown that animals treated with Al + extract 1000 mg/kg had a signifcant preference for the target quadrant in contrast to the Al group.P < 0.01 Al group compared to the control group; P < 0.01 Al group compared to the Al + extract 1000 mg/kg group.

Figure 2 :
Figure 2: Hippocampus tissue: Rosa damascena extracts reduced necrosis and gliosis induced by AlCl 3 in diferent regions of the hippocampus.Necrosis and gliosis were not observed in the control, DRE 500 and DRE 1000 groups in the hippocampus, AlCl 3 group with necrosis (black arrow), and gliosis (star) in the diferent regions of the hippocampus.In AL + DRE 500, AL + DRE 1000 groups showed less necrosis and gliosis compared to the AlCl 3 group, ×40 magnifcations, H&E, and the Nissl staining.Scale bar � 100 μm.

Figure 3 :
Figure 3: Cortex tissue: Rosa damascena extracts (DRE) reduced necrosis and gliosis induced by AL in the cortex.Necrosis and gliosis were not observed in the control, DRE 500 and DRE 1000 groups in the cortex, AL group with necrosis (black arrow), and gliosis (star) in the cortex.In AL + DRE 500, AL + DRE 1000 groups showed less necrosis and gliosis compared to the AL group, ×40 magnifcations, and H & E staining.Scale bar � 100 μm.
[41]vity.CAT activity was measured based on its capability to decompose hydrogen peroxide (H 2 O 2 ) in brain tissue,[41].As a rule, H 2 O 2 decomposition can be assessed by a decrease in absorbance at Evidence-Based Complementary and Alternative Medicine 3 240 nm.Tus, hydrogen peroxide at a fnal concentration of 19 mM and 50 mM phosphate bufer (pH 7) was used as a substrate and an alternative substrate in the blank solution, respectively.Te reaction was initiated by the addition of H 2 O 2 and the decrease in the absorbance was evaluated by a spectrophotometer (Pharmacia, Novaspec II, and Biochrom, England) at 240 nm for 30 s. Te values were expressed as U/mg protein.To calculate u/mg, the enzyme activity was calculated in U/ml based on changes in the adsorption mixture per minute, then the protein concentration was measured in mg/ml and the activity was divided by the protein concentration of enzyme activity in U/mg.Also, it is not necessary to draw a standard diagram to obtain the activity, and the activity is obtained by using the extinction coefcient related to hydrogen peroxide[41].
2.11.Measurement of Ferric-Reducing Antioxidant Power(FRAP) Assay Test.Te FRAP value was measured by comparing the change in absorbance at 593 nm in a test reaction mixture containing the mixture (tissue samples with 2,4,6-tri-(2-pyridyl)-s-triazine) with a defned ferrous ion concentration.When reduced to the ferrous form (FeV) in lower pH values, the FeШ-2,4,6-tri-(2-pyridyl)-s-triazine complex produces an intense blue color product with absorption at 593 nm.Practically, conditions that are favorable for complex development are provided in the presence of reductants 0 (antioxidants), which allow color development.

Table 1 :
Compounds that were identifed in the aquatic extract of Rosa damascena by GC-MS.
* * * P < 0.001 of Al group compared to the control group; # P < 0.05 and ### P < 0.001 compared to Al + extract 1000 mg/kg experimental animal group.

Table 2 :
Efect of R. damascene extract (DRE) on aluminum-induced oxidative stress parameters and AChE activity in rat brain.