Users Online: 280
Home Print this page Email this page
Home About us Editorial board Search Browse articles Submit article Ahead of Print Instructions Subscribe Contacts Login 


 
Previous article Browse articles Next article 
ORIGINAL ARTICLE
Adv Biomed Res 2015,  4:131

Neuroprotective effects of Rosa damascena extract on learning and memory in a rat model of amyloid-β-induced Alzheimer`s disease


1 Department of Anatomical Sciences and Molecular Biology, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Physiology, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran
3 Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran
4 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
5 Department of Biology, Cells, Molecular Biology and Biochemistry Division, Faculty of Sciences, University of Isfahan, Isfahan, Iran

Date of Submission22-Jan-2014
Date of Acceptance12-May-2014
Date of Web Publication27-Jul-2015

Correspondence Address:
Dr. Mohammad Karimipour
Department of Anatomical Sciences and Molecular Biology, Isfahan University of Medical Sciences, Isfahan
Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2277-9175.161512

Rights and Permissions
  Abstract 

Background: Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease, which is characterized clinically by serious impairment in memory and cognition. Current medications only slow down the dementia progression and the present treatment one-drug one-target paradigm for anti-AD treatment appears to be clinically unsuccessful. Therefore, alternative therapeutic strategies are urgently needed. With respect to multifunctional and multitargeted characteristics of Rosa damascena via its effective flavonoids, we investigated the effects of R. damascena extract on behavioral functions in a rat model of amyloid-β (A-β)-induced Alzheimer's disease.
Materials and Methods: After preparation of the methanolic extract of the R. damascena, HPLC analysis and toxicity studies, median lethal dose (LD50) and dose levels were determined. For evaluation of baseline training behavioral performance, Morris water maze and passive avoidance tests were used. A-β was injected bilaterally into CA1 area of the hippocampus. Twenty-one days after injection of A-β, the first probe trial of the behavioral tests were used to confirm learning and memory impairment. To examine the potential effects of the extract on behavioral tasks, the second probe trials were performed after one month administration of R. damasena extract.
Results: Results showed that the R. damascena extract significantly improved the spatial and long-term memories in the extract- treated groups in a dose-dependent manner, as in the middle and high doses it had significant effect.
Conclusion: According to these results, we concluded that R. damascena can reverse behavioral deficits caused by A-β, and may provide a new potential option for prevention and treatment of the cognitive dysfunction in Alzheimer's disease.

Keywords: Alzheimer`s disease, learning and spatial memory, long-term memory, Rosa damascena


How to cite this article:
Esfandiary E, Karimipour M, Mardani M, Ghanadian M, Alaei HA, Mohammadnejad D, Esmaeili A. Neuroprotective effects of Rosa damascena extract on learning and memory in a rat model of amyloid-β-induced Alzheimer`s disease. Adv Biomed Res 2015;4:131

How to cite this URL:
Esfandiary E, Karimipour M, Mardani M, Ghanadian M, Alaei HA, Mohammadnejad D, Esmaeili A. Neuroprotective effects of Rosa damascena extract on learning and memory in a rat model of amyloid-β-induced Alzheimer`s disease. Adv Biomed Res [serial online] 2015 [cited 2019 Dec 14];4:131. Available from: http://www.advbiores.net/text.asp?2015/4/1/131/161512


  Introduction Top


Alzheimer's disease (AD) is one of the most serious neurodegenerative diseases and the most common cause of dementia in aging population affecting approximately 26 million people worldwide, and whose prevalence has been calculated to triplicate by 2050. [1],[2] AD is characterized clinically by progressive impairment in memory, cognition, and behavioral functions. [3] It is believed that AD initiates with synaptic impairment, neuronal loss, microglial cell proliferation, and is followed by inflammation. [4],[5] The neuropathological feature of AD is complex, including cerebral accumulation of extracellular amyloid-β (A-β) plaques and intraneuronal neurofibrillary tau tangles composed of dystrophic neuritis and hyperphosphorylated tau protein. [6] According to the "amyloid hypothesis," the soluble A-β in the brain, plays an important role in the development of AD. [7] Despite catastrophic increase of dementia patients worldwide, no effective treatment is available yet, although several acetylcholine esterase inhibitors such as donepezil, rivastigmine, and galantamine usually were used, but these medicines can only slow down the progression of dementia, rather than restoring brain function in a real clinical situation. Therefore, alternative and multitargeted therapeutic strategies are urgently needed to prevent or treat Alzheimer's disease. [8],[9] Because AD arises via multiple pathological or neurotoxic pathways, herbal medicines as a result of multifunction, multitarget characteristics have potential of optimum pharmaceutical and nutraceutical effects on AD patients. [10]

Medicinal herbs-derived agents have different mechanisms from conventional drugs, which can be effective in clinical treatment. Herbs contain some of the most powerful natural antioxidants and bioactive secondary metabolites such as flavonoids, phenols, or phenolic components that make them valuable in their antioxidant and antiaging effects. [11] Rosa damascena is a plant that belongs to genus Rosa and family Rosaceae. Rosaceae are well known as ornamental plants and have referred to the king of flowers. Some members of the Rosaceae family have long been used for food and medical purposes. Most of the studies showed that R. damascena as a natural plant with high source of flavonoids such as quercetin, kaemferol, myricetin, gallic acid, and their glycoside derivatives may have curable effect on treatment of diseases. [11],[12] It has been shown that essential oil of R. damascena retards the development of behavioral seizures in amygdale electrical kindling and possesses ability of contract kindling acquisition. [13] There is considerable evidence suggesting that extract of the R. damascena significantly induces the neurite outgrowth and inhibits the A-β fibrilization and deposition in brain. [14] Also it is shown that the R. damascena oil can relieve human stress and depression. [15] Flavonoids perform a multiplicity of neuroprotective functions within the brain, including neural protection against neurotoxin injuries, neuroinflammative suppression and promotion of memory, learning, and cognitive function. These effects could be mediated by flavonoids that inhibit apoptosis, promote neuronal survival and synaptic plasticity. They can also promote cerebrovascular blood flow, angiogenesis, neurogenesis, and neuronal morphology. [16] The aim of the current study was to determine the effects of methanolic extract of R.damascena on behavioral functions in a rat model of amyloid- β (1- 42) induced Alzheimer`s disease.


  Materials and methods Top


0Methanolic extraction of R. damascena

Dried fresh flowers of R. damascena Mill was purchased from Isfahan Pharmaceutical Sciences Research Center (Iran). It was ground to a fine powder (2 kg) and then macerated with methanol (10 L). Extraction procedure was performed three times and each time for 4 consecutive days with intermittent stirring (1 h stirring at every 12 h interval) using magnetic stirrer until the extract was light colored. The combined extracts were filtered and concentrated under reduced pressure in a rotary vacuum evaporator (200 mbar) in a water bath at 40°C. Dried gummy extract (380 g) was stored in 4°C until use. [11]

HPLC analysis and extract standardization

High-performance liquid chromatographic (HPLC) analysis was performed on a Waters system, equipped with 515 HPLC pump, UV-Visible detector (2487 dual absorbance) operated at 365 nm, and millennium software for the determination of quercetin in the extract. After acid hydrolysis of 100 mg of the extract (1 h in 2N HCl, at 95°C), the hydrolyzed flavonoids were extracted through ethyl acetate to 5 mL. Then 20 μl of the sample injected into a Nova-Pak C18, 3.9 × 150 mm (Waters, Milford, MA, USA) using H 3 PO 4 10 mM in water (solvent A) and acetonitrile (solvent B) with gradient elution at a flow rate of 0.8 mL/min. A standard calibration curve in the range of 100-1000 μg/mL for quantitative analysis was prepared using different concentrations of quercetin (Sigma Aldrich, USA) as standard material (100, 200, 500, and 1000 μg/mL). The relationship between the concentration and peak-area of standard was measured using the minimum square method (R2 value). [17]

Acute toxicity studies and determination of median lethal dose (LD50)

Toxicity is defined as "the potential of a substance to exert a harmful effect on humans or animals, and a description of the effect and the conditions or concentration under which the effect takes place." [18]

Acute toxicity is involved in the estimation of LD50 (the dose which has proved to be lethal (causing death) to 50% of the tested group of animals). Determination of acute oral toxicity is usually an initial screening step in the assessment and evaluation of the toxic characteristics of all compounds. [19],[20]

Assignment and housing of the animals for toxicity studies

Each animal was assigned a unique identification number. Thirty-six male Wistar rats (200-250 g) were housed six per cage. Six rats in each group at each dose level were chosen. In the present study, rats were gavaged via a gastric tube with increasing six series doses of R. damascena extract to get 50% lethality according to [Table 1]. One dose being used per group. The animals were observed in several times up to 72 h for survival and morbidity. [21],[22] Median lethal dose (LD50) was calculated as given in [Table 1].
Table 1: Determination of (LD50)

Click here to view


Dose levels and dose selection

This is based on the results of (LD50) finding test. At least three dose levels were used, spaced appropriately to produce test groups. For dose selection, 20% of (LD50) 6000 mg/kg was determined as the highest dose (1200 mg/kg) and decreasing spaced doses of 600 and 300 mg/kg were considered as middle and low doses, respectively, in our experimental study. [23]

Animals and stereotaxic surgery

Male Wistar rats (200-250 g) were housed four per cage and maintained on a 12 h light-dark cycle in an air-conditioned constant temperature (23 ± 1°C) room, with food and water made available ad libitum. The Ethics Committee for Animal Experiments at Isfahan University of Medical Sciences approved the study, and all experiments were conducted in accordance with the international guiding principles for biomedical research involving animals, revised in 1985. Initially, all animals were randomly divided into 6 groups (n = 10) for evaluation of baseline training performance in Morris water maze and passive avoidance tests. After spatial acquisition phase of Morris water maze and training (learning) phase of the passive avoidance test, animals were grouped as following: First group = control, Second group = sham, Third group = Alzheimer's disease + Normal saline, Fourth group = Alzheimer's disease + 300 mg of R. damascena extract, Fifth group = Alzheimer's disease + 600 mg of R. damascena extract and sixth group = Alzheimer's disease + 1200 mg of R. damascena extract. The protocol of experimental design is summarized in [Figure 1].
Figure 1: Experimental schedule of the research design during the course of the study

Click here to view


Rats (groups 3 rd to 6 th ) were anesthetized by intraperitoneal injection of chloral hydrate (350 mg/kg) and placed into stereotaxic device (Stoelting, Kiel, WI, USA). A heating pad was used to maintain body temperature at 36.5 ± 0.5°C. An incision was made along the midline, the scalp was retracted, and the related area was cleaned and dried. In addition, lidocaine and epinephrine solution (0.4 mL) were injected in several locations surrounding the incision. In order to make minimum brain trauma during infusion, injections were performed very slowly (1 μL/min) using a microinjection pump. Amyloid-β (1-42) (Sigma) was dissolved in phosphate buffered saline (PBS 0.1 M) and aliquots at a concentration of 1 μg/μL were stored at −20°C until used. For induction of aggregation, aliquots of amyloid-β (1-42) were incubated for 5 days at 37°C before administration. Then 3 μg/3 μL of fibrillar A-β (1-42) or vehicle (PBS 0.1) were injected into the CA1 area of hippocampus at the following coordinates: Incisor bar −3.3 mm, −3.6 mm anterior-posterior to the bregma, 2.4 mm lateral to the sagittal suture, 2.8 mm dorsoventral from top of the skull bilaterally according to the atlas by Paxinos and Watson. [24] The needle was kept in place for 5 min to allow the injected solutions and tissue to equilibrate and avoid the possible reflux through the needle track. Incisions were ligated with silk thread. In sham group, same surgery was performed except that PBS was injected in CA1. No surgery was done in the control group.

Morris water maze

The circular tank (180 cm in diameter) was filled with water (22 ± 2°C) made opaque and was surrounded by a variety of extramaze cues. The tank was divided into four equal quadrants (northeast, northwest, southwest, and southeast) and four start positions were located at the intersection of the quadrants [Figure 2]a. A platform (12.5 cm in diameter) was placed in the northeast (the target quadrant) and submerged 2.0 cm below the water surface where it remained for all spatial trials. [25],[26],[27] Twenty-four hours before water maze training, all rats were habituated to the water and apparatus. In the spatial acquisition phase, the rats learned to find a submerged platform using extramaze cues. Each rat participated in eight trials that were organized into two blocks of four trials (one trial/start position within a block). Each block was considered a separate test session and blocks were separated by 30 min. For each trial, the rat was given a maximum time of 60 s to locate the platform. After mounting the platform, the animals were allowed to remain there for 30 s, and then were placed in a holding cage for 30 s until the start of next trial. If the rat did not locate the platform within 60 s, it was guided to it by the experimenter. After completion of spatial acquisition phase, the animals were returned to their home cages until the initiation of probe trials in the test days. During the different phases of the maze, the animals were monitored by digital camera fixed 2 m above the maze and different parameters were analyzed using computer-based software (Radiab 1). Swim time (in s), swim distance (in cm), and swim speed (in cm/s) were recorded.
Figure 2: (a) Tank of the Morris water maze and site of the platform. (b) Schematic representation of the passive avoidance test apparatus

Click here to view


The first and second probe trials (spatial retention) 21 days after surgery and following extract treatment were performed, respectively. In the probe trials, the hidden platform was removed and animals were allowed to swim freely for 60 s. During the probe trials, the percentage time in target quadrant was calculated. Cued acquisition task conducted 20 min after completion of the probe trials in order to test their nonmnemonic aspects of water maze performance such as swimming ability, motivation, and visual ability. In this phase, the platform was elevated above the water surface and was placed in different positions, and rats were allowed to swim freely toward visible platform for 60 s. Twenty-one days after operation the 1 st probe trial of Morris water maze and passive avoidance tests were performed and memory impairment was confirmed in groups 3-6.

Then dried R. damascena extract was dissolved in normal saline 0.9% +1% Tween 20, and then was administrated orally at 300, 600, 1200 mg/kg/day for 1 month via gavage in groups 4-6. Sham and 3 rd groups received only normal saline at the same time. After R. damascena extract treatment 2 nd probe trial of Morris water maze and passive avoidance tests were performed to evaluate the effects of extract on learning and spatial and long-term memories.

Passive avoidance test

The apparatus for the step-through passive avoidance test is an automated shuttle-box divided into an illuminated chamber and a dark chamber of the same size (20 × 25 × 30 cm each) by a wall with a guillotine door [28],[29],[30] [Figure 2]b. In the present study, passive avoidance test was used as an index of long-term memory.{Figure 2}

In the adaptation trial, each rat was trained to adapt to the step-through passive avoidance apparatus. The animal was placed into the illuminated chamber, facing away from the dark chamber. After 60 s, the door between these two boxes was opened and the rat was allowed to move into the dark chamber. The training trial was performed 24 h after the adaptation trial. In training trial, each rat was placed individually in the illuminated chamber, and once it entered the dark chamber an electric shock (40 V, 0.3 A, 2 s) was delivered to its feet through the floor grid. The rat was immediately removed and returned to cage. The first probe trial (retention trial) was conducted 21 days after surgery, in which rats were placed again in illuminated chamber and the interval (step-through latency) between placement in illuminated chamber and entry to the dark one was recorded. If the animal did not enter the dark chamber within 5 min, the test was terminated and the step through latency was recorded as 300 s. Memory impairment was confirmed in groups 3-6. Then R. damascena extract was applied as described previously. After R. damascena extract treatment, 2 nd probe trial of passive avoidance was performed to evaluate the effects of extract on long-term memory.

Statistical analysis

Data were expressed as mean ± SEM (standard error of mean) and were analyzed using either SPSS version 19 or GraphPad Prism ® 5.0. software. Statistical analysis was performed using the following tests. The escape latencies, swim distance, and swim speed in the water maze were analyzed by two-way analysis of variance (ANOVA) for between-subject differences between groups and repeated measures (within subjects) for effects across block interval 1-2 ("BLOCK" effect). The probe trials data for percentage of time in target quadrant were analyzed by multivariate ANOVA followed by Tukey's honestly significant difference (HSD) test as a post hoc analysis. One-way ANOVA followed by Tukey's HSD multiple comparison test as a post hoc analysis were performed for evaluation of training trial and retention trials of passive avoidance test. A value of P < 0.05 was considered statistically significant.


  Results Top


0HPLC standardization of the extract

Methanolic extraction of the R. damascena was performed and standardized using quercetin as the bioactive marker for standardization of the extract. Quercetin peak of extract appeared at a retention time of 13.48 min and using calibration curve, the extract was standardized to contain 548.89 ± 20.23 mg/100 g of the total quercetin (0.55% w/w) [Figure 3]a and b. By using of millennium processing software, the calibration curve was determined by linear regression in the range of 100-1000 μg/mL. The regression equation was y = 86419x + 794975, where x was the concentration of total quercetin in the extract (μg/mL) with the correlation co-factor (R2) of 0.998 [Figure 3]c. [14]
Figure 3: (a) HPLC chromatogram of quercetin standard at 365 nm; 20 μL of the quercetin standard (Sigma Aldrich, USA) in the range of 100– 1000 μg/mL was injected into a Nova‑Pak C18, 3.9 × 150 mm (Waters, Milford, MA, USA) using H3PO4 10 mM in water (solvent A) and acetonitrile (solvent B) with gradient elution at a flow rate of 0.8 mL/min. Quercetin peak appeared at a retention time of 13.64 min. (b) HPLC chromatogram of Rosa damascena extract at 365 nm. After acid hydrolysis of 100 mg of the extract (1 h in 2N HCl, at 95°C), the hydrolyzed flavonoids were extracted through ethyl acetate to 5 mL. Then 20 μL of the sample was injected into a Nova-Pak C18, 3.9 × 150 mm (Waters, Milford, MA, USA) using H3PO4 10 mM in water (solvent A) and acetonitrile (solvent B) with gradient elution at a flow rate of 0.8 ml/min.Quercetin peak of the extract appeared at a retention time of 13.48 min. (c): Calibration curve of quercetin using HPLC method and acetonitrile/water as mobile phase with pH adjusted to 2.3 at 365 nm. Using the millennium processing software, the calibration curve was determined by linear regression in the range of 100– 1000 μg/mL. The regression equation was y = 86,419x + 7, 94,975

Click here to view


Evaluation of the spatial acquisition phase of the Morris water maze before animal model induction

There were no significant differences in the mean time latency (F (5, 54) =0.041, P = 1 >0.05), mean swimming distance (F (5, 54) =0.128, P = 0.9 >0.05), and mean swimming speed (F (5, 54) =0.426, P = 0.8 >0.05) in blocks 1 and 2 of the spatial acquisition among all groups before application of A-β [Figure 4]a-c, blocks 1 and 2], but repeated measure test showed a significant difference in escape latency, swimming distance, and swimming speed (F (1, 54) = 74.96, P < 0.001); (F (1, 54) = 397.22, P < 0.001); (F (1, 54) = 20.14, P < 0.001 respectively) between blocks 1 and block 2 were observed [Figure 4]a-c, blocks 1 and 2] to locate the platform across blocks of trials, indicating spatial acquisition performance in all groups improved significantly during the training days and were able to learn the task.
Figure 4: Behavioral assessment. In spatial acquisition phase there were no significant differences in block 1 and 2 among the six groups before application of amyloidβ, but Repeated measure test showed a significant reduction in (a) escape latency (P < 0.001), (b) swimming distance (P < 0.001), and (c) swimming speed (P < 0.001) in 2nd block compared with 1st block to locate the platform across blocks of trials

Click here to view


Evaluation of the training trial of the passive avoidance test before animal model induction

One-way ANOVA showed that the step-through latency did not differ in the training trial among the six groups before operation (F (5, 54) =0.069, P = 0.9 > 0.05) [Figure 5].
Figure 5: In training trial of the Passive avoidance test, there were no significant differences in step-through latency among the six groups (P = 0.9)

Click here to view


R. damascena extract improved the spatial learning and memory parameters in a rat model of Alzheimer's disease

Multivariate ANOVA analysis showed significant differences in 1 st probe trial among groups (P < 0.001) [Figure 6]. Post hoc Tukey's HSD analysis showed that, the mean percentage of time spent in target quadrant in A-β-injected groups (3 rd to 6 th ) after 21 days surgery was significantly decreased in comparison to control and sham groups (P < 0.001), but no significant differences were demonstrated in mean percentage of time spent in target quadrant between control and sham groups, (P = 0.9 > 0.05). These results showed that A-β-induced learning and memory impairment in the rats. Multivariate ANOVA analysis showed that mean percentage of time spent in target quadrant in 2 nd probe trial after one month R. damascena extract treatment was significantly increased among groups (P < 0.001) [Figure 6]. Post hoc Tukey's HSD analysis showed that, mean percentage of time spent in target quadrant was significantly increased in extract-treated groups (5 th and 6 th groups) relative to 3 rd group, which had received normal saline (P = 0.006 < 0.05, P < 0.001, respectively), but mean percentage of time in target quadrant in 4 th group did not significantly increase relative to 3 rd group (P = 0.3 > 0.05). Also mean percentage of time in target quadrant in 6 th group was significantly increased in comparison to 4 th (P = 0.002 < 0.05). Taken together, these results showed that R. damascena extract improved spatial learning and memory in a dose-dependent manner.
Figure 6: Effects of the amyloid-β and Rosa damascena extract on spatial memory during the first (A, white color) and second (A, black color) probe trials, respectively, as measured by mean percentage time in target quadrant.**P < 0.01; ***P < 0.001 different from the control and sham groups. ##P < 0.01, ###P < 0.001 different from the Aβ-injected group (Alz + NS), @P < 0.05, @@@P < 0.001 different from the Aβ-injected group (Alz + 300)

Click here to view


R. damascena extract improved the long-term memory parameters in a rat model of Alzheimer's disease

One-way ANOVA showed that significant differences in mean step-through latency among groups were observed following surgery in the 1 st probe trial (F (5, 54) =275.94, P < 0.001) [Figure 7]. Post hoc Tukey's HSD analysis showed that mean step-through latency reduced in Aβ-treated groups (3 rd to 6 th ) compared with control and sham groups (F (5, 54)=119.24, P < 0.001). One-way ANOVA demonstrated that following extract treatment, step-through latency significantly increased among groups (P < 0.001) [Figure 7]. The mean step-through latency in groups 5 and 6 was significantly increased in comparison to 3 rd group (P < 0.001). Mean step-through latency in 4 th group did not significantly increase relative to 3 rd group (P = 0.07). This parameter in 5 th and 6 th groups significantly increased when compared with 4 th group (P < 0.001). All together these data show that R. damascena extract in medium (600 mg/kg) and high dose (1200 mg/kg) improved long-term memory in a dose-dependent manner.
Figure 7: Effects of the amyloid-β and Rosa damascena extract on long‑term memory during the first (B, white color) and second (B, black color) probe trials, respectively, as measured by mean time step-through latency. **P < 0.01; ***P < 0.001 different from the control and sham groups. ##P < 0.01, ###P < 0.001 different from the Aβ-injected group (Alz + NS), @P < 0.05, @@@P < 0.001 different from the Aβ-injected group (Alz + 300)

Click here to view



  Discussion Top


The goodness of usual anti-AD drugs, is just a temporary relief of clinical symptoms and signs of dementia, but the efficacy of these cholinesterase inhibitors in AD treatment is not supported by any high-quality and long-lasting clinical trials. [9] These medications cannot cure Alzheimer's dementia or stop the disease progression. [31],[32] Because AD is a multifactorial disease, using multifunctions and multitargeted agents have been considered extensively. [33],[34] Enormous medical combination therapies have been proposed to cure Alzheimer's disease. [35] Herbal medicines as a result of multifunction, multitarget characteristics have potential of optimum pharmaceuticals and nutraceuticals effects on AD patients. [10] Preclinical and epidemiological studies suggest that herbal drugs might be effective at reversing neurodegenerative pathology and age-related declines in neurocognitive performance. [36] Herbal drugs may act to protect the brain in a number of ways, including by protection of vulnerable neurons, promotion of peripheral and cerebral vascular system, enhancement of existing neuronal function or by stimulating neuronal regeneration and inhibition of neuroinflammation. [37] Moreover, medicinal herbs-derived drugs are natural and hence safer than synthetic drugs, and that a complex mixture of herbs can effectively treat complex diseases. These advantages may account for the sudden increase in herbal use in the last decade. [38] In addition, some of the experimental reports suggest that some herbs may have neuroprotective effects against amyloid-β. [39],[40]

The present study examined the effects of a methanolic extract of R. damascene on behavioral deficits in a rat model of amyloid-β (Aβ1-42)-induced Alzheimer`s disease. Morris water maze and passive avoidance tests were used for examination of behavioral changes caused by amyloid-β and possible effect of R. damascena extract on learning, spatial memory, and long-term memories. [29],[30],[41],[42] The memory impairment induced by intra-CA1 injection of Aβ in our study was in agreement with Yamada et al.[43] One month after R. damascena extract treatment, spatial memory and long-term memory were improved, whereas cognitive decline was reversed in a dose-dependent manner. The neuroprotective effect of R. damascena against cytotoxicity of A-β was investigated by Awale et al. in vitro. They showed that R. damascena extract significantly causes neurite outgrowth and suppresses the Aβ - induced atrophy and cell death. Moreover, the length of dendrite in the cells treated with R. damascena extract was similar to those of nerve growth factor (NGF)-treated cells. [14] Significant effect of R. damascena on Aβ-injected groups in our study could be because of its multifactorial effects of bioactive flavonoids, polyphenols, and secondary active metabolites. Kumar et al. had detected and quantified some of polyphenols including gallic acid, rutin, quercitrin, myricetin, quercetin, and kaempferol, in methanolic extract of R. damascena by using improved HPLC analysis method. [17] In agreement with Kumar et al., our HPLC analysis showed that quercetin as bioactive compound exists in the R. damascena extract. Flavonoids comprise the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants. [44],[45] The neuroprotective effects of R. damascena were mediated by flavonoids that induce memory and cognitive improvement function via scavenging free radicals, and releasing neurotransmitter (acetylcholine). Other multifunctional effects of flavonoids include antioxidant, antiapoptotic, anti-inflammatory, anticholinesterase, antidepressant, and modulation of signaling pathway cascade [44],[45],[46],[47],[48] and cerebrovascular blood flow improvement. [49] Cellular and molecular mechanisms of flavonoids effects may carry out via protein kinase activation and phosphatase inhibition that could lead to activation of cyclic AMP-response element-binding protein (CREB). CREB could increase blood flow, angiogenesis, neurogenesis, dendritic spine growth, neuronal communication, and synaptic plasticity via production and activation of neurotrophic factors such as brain-derived growth factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VGEF), and transform growth factor-β (TGF-β). Flavonoids prevent neurodegeneration and brain aging via reduction of induced nitric oxide synthase (iNOS), nitric oxide releasing, suppression inflammatory cytokines, such as TNF-α, IL-1β. [50],[51] Quercetin as a flavonoid which is present in R. damascena attenuates microglia- and/or astrocyte-mediated neuroinflammation. [52] Many neurodegenerative diseases pathogenesis including Alzheimer's disease correlate to oxidative stress enhancement in brain. [16] Brain biological functions of flavonoids attribute to their antioxidant functions. [53] Phenolic compounds are effective hydrogen donors, and because of this they are known as good antioxidants. [54] Their high antioxidant potential is attributed to their capacity of scavenging reactive oxygen species (ROS), inhibiting lipid peroxidation, chelating metal ions, and other free radicals, which are originated from various cellular activities and lead to oxidative stress. [55] The cholinergic system is involved in many physiological processes, including synaptic plasticity and learning and memory. [56],[57] Quercetin and rutin, natural compounds widely found in R. damascena and diet have anticholinesterase inhibition effects without severe side effects. [58] Recent studies have focused on amyloid precursor protein (APP) proteolysis and Aβ-generation as potential targets for AD therapy. [59] Aβ is generated from the APP by beta-site APP cleaving enzyme-1 (BACE-1, β- secretase) and g-secretases through pro-amyloidogenic processing pathway, but α-secretase via nonamyloidogenic processing pathway inhibits Aβ production and promote Aβ degradation. [60] Myricetin, one of the natural flavonoids has been found in R. damascena is a strong antioxidant and has a dual neuroprotective effect by activation of α-secretase and direct inhibition of β-secretase (BACE-1). As myricetin-bound BACE-1 does not hydrate APP at the β-cleavage site, Aβ production would be reduced. It has three hydrogen-binding groups, which stabilize BACE-1 binding for concerning AD drug therapy. [60],[61] Myricetin binds also to Aβ through its hydroxyl arms and traps Aβ hydrogen bond, which is necessary to form β-structures or oligomers. [62] Myricetin via this mechanism prevents Aβ structural alteration from a random coil to β-sheet and inhibits Aβ fibril generation. The β-structure of Aβ forms soluble oligomers that are considered to be AD cytotoxic component. [7],[63] It is demonstrated that quercetin also disrupts the β-sheet structure to form random coils. [61],[64] Therefore R. damascena with high concentration of quercetin, myricetin, and other flavonoids decreases level of Aβ (1-42) in brain and finally improves learning and memory.


  Conclusion Top


In summary, R. damascena reversed the Aβ-associated memory abnormalities in a rat model of amyloid-β-induced Alzheimer`s disease. Taken together, the diverse actions of individual natural components of the R. damascena on learning and memory represent a new promising way for generation of memory-enhancing drugs, although complementary experiments are still to be down for better characterizing this valuable effect and its underlying mechanisms, in order to provide a basis for proper clinical trials.


  Acknowledgments Top


This work was supported by Isfahan University of Medical Sciences. The authors thank Dr. Parham Reisi of Department of Physiology for his excellent technical assistance. None of the authors claim any conflict of interest.

 
  References Top

1.
Selkoe DJ. The molecular pathology of Alzheimer's disease. Neuron 1991;6:487.  Back to cited text no. 1
    
2.
Lee JK, Jin HK, Endo S, Schuchman EH, Carter JE, Bae JS. Intracerebral transplantation of bone marrow-derived mesenchymal stem cells reduces amyloid-beta deposition and rescues memory deficits in Alzheimer's disease mice by modulation of immune responses. Stem Cells 2010;28:329-43.  Back to cited text no. 2
    
3.
Selkoe DJ. Alzheimer's disease: Genes, proteins, and therapy. Physiol Rev 2001;81:741-66.  Back to cited text no. 3
    
4.
Rogers J, Webster S, Lue LF, Brachova L, Civin WH, Emmerling M, et al. Inflammation and Alzheimer's disease pathogenesis. Neurobiol Aging 1996;17:681-6.  Back to cited text no. 4
    
5.
Scheff SW, Price DA, Schmitt FA, DeKosky ST, Mufson EJ. Synaptic alterations in CA1 in mild Alzheimer disease and mild cognitive impairment. Neurology 2007;68:1501-8.  Back to cited text no. 5
    
6.
Huang HC, Jiang ZF. Accumulated amyloid-beta peptide and hyperphosphorylated tau protein: Relationship and links in Alzheimer's disease. J Alzheimers Dis 2009;16:15-27.  Back to cited text no. 6
    
7.
Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: Progress and problems on the road to therapeutics. Science 2002;297:353-6.  Back to cited text no. 7
    
8.
Querfurth HW, LaFerla FM. Alzheimer's disease. N Engl J Med 2010;362:329-44.  Back to cited text no. 8
    
9.
Salloway S, Ferris S, Kluger A, Goldman R, Griesing T, Kumar D, et al. Efficacy of donepezil in mild cognitive impairment: A randomized placebo-controlled trial. Neurology 2004;63:651-7.  Back to cited text no. 9
    
10.
Kim HG, Oh MS. Herbal medicines for the prevention and treatment of Alzheimer's disease. Curr Pharm Des 2012;18:57-75.  Back to cited text no. 10
    
11.
Kalim MD, Bhattacharyya D, Banerjee A, Chattopadhyay S. Oxidative DNA damage preventive activity and antioxidant potential of plants used in Unani system of medicine. BMC Complement Altern Med 2010;10:77.  Back to cited text no. 11
    
12.
Schiber A, Mihalev K, Berardini N, Mollov P, Carle R. Flavonol glycosides from distilled petals of Rosa damascena Mill. Z Naturforsch C 2005;60:379-84.  Back to cited text no. 12
    
13.
Ramezani R, Moghimi A, Rakhshandeh H, Ejtehadi H, Kheirabadi M. The effect of Rosa damascena essential oil on the amygdala electrical kindling seizures in rat. Pak J Biol Sci 2008;11:746-51.  Back to cited text no. 13
    
14.
Awale S, Tohda C, Tezuka Y, Miyazaki M, Kadota S. Protective Effects of Rosa damascena and its Active Constituent on A {beta}(25-35)-induced Neuritic Atrophy. Evid Based Complement Alternat Med 2011;2011:131042.  Back to cited text no. 14
    
15.
Hongratanaworakit T. Relaxing effect of rose oil on humans. Nat Prod Commun 2009;4:291-6.  Back to cited text no. 15
    
16.
Spencer JP. The impact of fruit flavonoids on memory and cognition. Br J Nutr 2010;104:S40-7.  Back to cited text no. 16
    
17.
Kumar N, Bhandari P, Singh B, Gupta AP, Kaul VK. Reversed phase-HPLC for rapid determination of polyphenols in flowers of rose species. J Sep Sci 2008;31:262-7.  Back to cited text no. 17
    
18.
Yu KH, Nation RL, Dooley MJ. Multiplicity of medication safety terms, definitions and functional meanings: When is enough enough? Qual Saf Health Care 2005;14:358-63.  Back to cited text no. 18
    
19.
Akhila J. Shetty, Deepa Shyamjith, Alwar M. C. Acute toxicity studies and determination of median lethal dose. Curr Sci 2007;93:917-20.  Back to cited text no. 19
    
20.
Poole AF, Leslie GB. A practical approach to toxicological investigations. Cambridge, England: Cambridge University Press;1989.  Back to cited text no. 20
    
21.
Javan M, Ahmadiani A, Semnanian S, Kamalinejad M. Antinociceptive effects of Trigonella foenum-graecum leaves extract. J Ethnopharmacol 1997;58:125-9.  Back to cited text no. 21
    
22.
Pant J, Deshpande SB. Acute toxicity of Bisphenol A in rats. Indian J Exp Biol 2012;50:425-9.  Back to cited text no. 22
    
23.
Jagetia G, Baliga M, Malagi K, Sethukumar Kamath M. The evaluation of the radioprotective effect of Triphala (an ayurvedic rejuvenating drug) in the mice exposed to g-radiation. Phytomedicine 2002;9:99-108.  Back to cited text no. 23
    
24.
Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates: Hard Cover Edition. United States: Academic press; 2007.  Back to cited text no. 24
    
25.
Morris RG, Garrud P, Rawlins JN, O'Keefe J. Place navigation impaired in rats with hippocampal lesions. Nature 1982;297:681-3.  Back to cited text no. 25
    
26.
de Quervain DJ, Roozendaal B, McGaugh JL. Stress and glucocorticoids impair retrieval of long-term spatial memory. Nature 1998;394:787-90.  Back to cited text no. 26
    
27.
Frick KM, Kim JJ, Baxter MG. Effects of complete immunotoxin lesions of the cholinergic basal forebrain on fear conditioning and spatial learning. Hippocampus 2004;14:244-54.  Back to cited text no. 27
    
28.
Venault P, Chapouthier G, de Carvalho LP, Simiand J, Morre M, Dodd RH, et al. Benzodiazepine impairs and beta-carboline enhances performance in learning and memory tasks. Nature 1986;321:864-6.  Back to cited text no. 28
    
29.
Mazzola C, Micale V, Drago F. Amnesia induced by beta-amyloid fragments is counteracted by cannabinoid CB1 receptor blockade. Eur J Pharmacol 2003;477:219-25.  Back to cited text no. 29
    
30.
Abdel-Aal RA, Assi AA, Kostandy BB. Rivastigmine reverses aluminum-induced behavioral changes in rats. Eur J Pharmacol 2011;659:169-76.  Back to cited text no. 30
    
31.
Scarpini E, Scheltens P, Feldman H. Treatment of Alzheimer's disease: Current status and new perspectives. Lancet Neurol 2003;2:539-47.  Back to cited text no. 31
    
32.
Cummings JL. Alzheimer's disease. N Engl J Med 2004;351:56-67.  Back to cited text no. 32
    
33.
Youdim MB, Buccafusco JJ. Multi-functional drugs for various CNS targets in the treatment of neurodegenerative disorders. Trends Pharmacol Sci 2005;26:27-35.  Back to cited text no. 33
    
34.
Pimplikar SW. Reassessing the amyloid cascade hypothesis of Alzheimer's disease. Int J Biochem Cell Biol 2009;41:1261-8.  Back to cited text no. 34
    
35.
Klafki HW, Staufenbiel M, Kornhuber J, Wiltfang J. Therapeutic approaches to Alzheimer's disease. Brain 2006;129:2840-55.  Back to cited text no. 35
    
36.
Manach C, Scalbert A, Morand C, Remesy C, Jimenez L. Polyphenols: Food sources and bioavailability. Am J Clin Nutr 2004;79:727-47.  Back to cited text no. 36
    
37.
Youdim KA, Joseph JA. A possible emerging role of phytochemicals in improving age-related neurological dysfunctions: A multiplicity of effects. Free Radic Biol Med 2001;30:583-94.  Back to cited text no. 37
    
38.
Raskin I, Ribnicky DM, Komarnytsky S, Ilic N, Poulev A, Borisjuk N, et al. Plants and human health in the twenty-first century. Trends Biotechnol 2002;20:522-31.  Back to cited text no. 38
    
39.
Kim DS, Kim JY, Han YS. Alzheimer's disease drug discovery from herbs: Neuroprotectivity from beta-amyloid (1-42) insult. J Altern Complement Med 2007;13:333-40.  Back to cited text no. 39
    
40.
Park SY, Kim DS. Discovery of natural products from Curcuma longa that protect cells from beta-amyloid insult: A drug discovery effort against Alzheimer's disease. J Nat Prod 2002;65:1227-31.  Back to cited text no. 40
    
41.
Jarrard LE. On the role of the hippocampus in learning and memory in the rat. Behav Neural Biol 1993;60:9-26.  Back to cited text no. 41
    
42.
Jhoo JH, Kim HC, Nabeshima T, Yamada K, Shin EJ, Jhoo WK, et al. Beta-amyloid (1-42)-induced learning and memory deficits in mice: Involvement of oxidative burdens in the hippocampus and cerebral cortex. Behav Brain Res 2004;155:185-96.  Back to cited text no. 42
    
43.
Yamada K, Takayanagi M, Kamei H, Nagai T, Dohniwa M, Kobayashi K, et al. Effects of memantine and donepezil on amyloid beta-induced memory impairment in a delayed-matching to position task in rats. Behav Brain Res 2005;162:191-9.  Back to cited text no. 43
    
44.
Joseph JA, Shukitt-Hale B, Denisova NA, Prior RL, Cao G, Martin A, et al. Long-term dietary strawberry, spinach, or vitamin E supplementation retards the onset of age-related neuronal signal-transduction and cognitive behavioral deficits. J Neurosci 1998;18:8047-55.  Back to cited text no. 44
    
45.
Joseph JA, Shukitt-Hale B, Denisova NA, Bielinski D, Martin A, McEwen JJ, et al. Reversals of age-related declines in neuronal signal transduction, cognitive, and motor behavioral deficits with blueberry, spinach, or strawberry dietary supplementation. J Neurosci 1999;19:8114-21.  Back to cited text no. 45
    
46.
Casadesus G, Shukitt-Hale B, Stellwagen HM, Zhu X, Lee HG, Smith MA, et al. Modulation of hippocampal plasticity and cognitive behavior by short-term blueberry supplementation in aged rats. Nutr Neurosci 2004;7:309-16.  Back to cited text no. 46
    
47.
Goyarzu P, Malin DH, Lau FC, Taglialatela G, Moon WD, Jennings R, et al. Blueberry supplemented diet: Effects on object recognition memory and nuclear factor-kappa B levels in aged rats. Nutr Neurosci 2004;7:75-83.  Back to cited text no. 47
    
48.
Joseph JA, Shukitt-Hale B, Casadesus G. Reversing the deleterious effects of aging on neuronal communication and behavior: Beneficial properties of fruit polyphenolic compounds. Am J Clin Nutr 2005;81:313S-6.  Back to cited text no. 48
    
49.
Schroeter H, Heiss C, Balzer J, Kleinbongard P, Keen CL, Hollenberg NK, et al. (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans. Proc Natl Acad Sci U S A 2006;103:1024-9.  Back to cited text no. 49
    
50.
Spencer JP. The interactions of flavonoids within neuronal signalling pathways. Genes Nutr 2007;2:257-73.  Back to cited text no. 50
    
51.
Spencer JP. The impact of flavonoids on memory: Physiological and molecular considerations. Chem Soc Rev 2009;38:1152-61.  Back to cited text no. 51
    
52.
Chen JC, Ho FM, Pei-Dawn Lee C, Chen CP, Jeng KC, Hsu HB, et al. Inhibition of iNOS gene expression by quercetin is mediated by the inhibition of IkappaB kinase, nuclear factor-kappa B and STAT1, and depends on heme oxygenase-1 induction in mouse BV-2 microglia. Eur J Pharmacol 2005;521:9-20.  Back to cited text no. 52
    
53.
Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 1996;20:933-56.  Back to cited text no. 53
    
54.
Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB. The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic Res 1995;22:375-83.  Back to cited text no. 54
    
55.
Bors W, Heller W, Michel C, Saran M. Flavonoids as antioxidants: Determination of radical-scavenging efficiencies. Methods Enzymol 1990;186:343-55.  Back to cited text no. 55
    
56.
Flood JF, Landry DW, Jarvik ME. Cholinergic receptor interactions and their effects on long-term memory processing. Brain Res 1981;215:177-85.  Back to cited text no. 56
    
57.
Power AE, Vazdarjanova A, McGaugh JL. Muscarinic cholinergic influences in memory consolidation. Neurobiol Learn Mem 2003;80:178-93.  Back to cited text no. 57
    
58.
Ahmed T, Gilani AH. Inhibitory effect of curcuminoids on acetylcholinesterase activity and attenuation of scopolamine-induced amnesia may explain medicinal use of turmeric in Alzheimer's disease. Pharmacol Biochem Behav 2009;91:554-9.  Back to cited text no. 58
    
59.
Haass C. Take five-BACE and the gamma-secretase quartet conduct Alzheimer's amyloid beta-peptide generation. EMBO J 2004;23:483-8.  Back to cited text no. 59
    
60.
Guo T, Hobbs DW. Development of BACE1 inhibitors for Alzheimer's disease. Curr Med Chem 2006;13:1811-29.  Back to cited text no. 60
    
61.
Shimmyo Y, Kihara T, Akaike A, Niidome T, Sugimoto H. Multifunction of myricetin on A beta: Neuroprotection via a conformational change of A beta and reduction of A beta via the interference of secretases. J Neurosci Res 2008;86:368-77.  Back to cited text no. 61
    
62.
McLaurin J, Kierstead ME, Brown ME, Hawkes CA, Lambermon MH, Phinney AL, et al. Cyclohexanehexol inhibitors of Abeta aggregation prevent and reverse Alzheimer phenotype in a mouse model. Nat Med 2006;12:801-8.  Back to cited text no. 62
    
63.
Mattson MP. Pathways towards and away from Alzheimer's disease. Nature 2004;430:631-9.  Back to cited text no. 63
    
64.
Shimmyo Y, Kihara T, Akaike A, Niidome T, Sugimoto H. Flavonols and flavones as BACE-1 inhibitors: Structure-activity relationship in cell-free, cell-based and in silico studies reveal novel pharmacophore features. Biochim Biophys Acta 2008;1780:819-25.  Back to cited text no. 64
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1]


This article has been cited by
1 Rosa canina L. methanolic extract prevents heat stress-induced memory dysfunction in rats
Marjan Erfani,Zohreh Ghazi Tabatabaei,Saeed Sadigh-Eteghad,Fatemeh Farokhi-Sisakht,Fereshteh Farajdokht,Javad Mahmoudi,Pouran Karimi,Ava Nasrolahi
Experimental Physiology. 2019;
[Pubmed] | [DOI]
2 Age related neurodegenerative Alzheimer’s disease: Usage of traditional herbs in therapeutics
Abhishek Singh,Sindhu Agarwal,Sarika Singh
Neuroscience Letters. 2019; : 134679
[Pubmed] | [DOI]
3 Aerobics or Pilates: Which is More Effective in the Performance of Wechsler Acid Profile Among Children with Learning Disabilities? A Randomized Comparison Trial
Ali Seghatoleslamy,Maryam Masoudi,Marzieh Saghebjoo,Morteza Taheri
International Journal of School Health. 2019; 6(3)
[Pubmed] | [DOI]
4 Unraveling the novel effects of aroma from small molecules in preventing hen egg white lysozyme amyloid fibril formation
Zahra Seraj,Arefeh Seyedarabi,Ali Akbar Saboury,Mehran Habibi-Rezaei,Shahin Ahmadian,Atiyeh Ghasemi,Human Rezaei
PLOS ONE. 2018; 13(1): e0189754
[Pubmed] | [DOI]
5 Exercise as a Positive Modulator of Brain Function
Karim A. Alkadhi
Molecular Neurobiology. 2017;
[Pubmed] | [DOI]
6 Therapeutic Applications of Rose Hips from Different Rosa Species
Inés Mármol,Cristina Sánchez-de-Diego,Nerea Jiménez-Moreno,Carmen Ancín-Azpilicueta,María Rodríguez-Yoldi
International Journal of Molecular Sciences. 2017; 18(6): 1137
[Pubmed] | [DOI]
7 Effects of the hydroalcoholic extract of Rosa damascena on learning and memory in male rats consuming a high-fat diet
Arezoo Rezvani-Kamran,Iraj Salehi,Siamak Shahidi,Mohammad Zarei,Shirin Moradkhani,Alireza Komaki
Pharmaceutical Biology. 2017; 55(1): 2065
[Pubmed] | [DOI]
8 Efficacy of topical Rose ( Rosa damascena Mill.) oil for migraine headache: A randomized double-blinded placebo-controlled cross-over trial
Maria Niazi,Mohammad Hashem Hashempur,Mohsen Taghizadeh,Mojtaba Heydari,Abdolhamid Shariat
Complementary Therapies in Medicine. 2017; 34: 35
[Pubmed] | [DOI]
9 Peripheral and central administration of T3 improved the histological changes, memory and the dentate gyrus electrophysiological activity in an animal model of Alzheimer’s disease
Yaghoob Farbood,Sahreh Shabani,Alireza Sarkaki,Seyyed Ali Mard,Akram Ahangarpour,Layasadat Khorsandi
Metabolic Brain Disease. 2017;
[Pubmed] | [DOI]
10 Comparison of the glycopattern alterations of mitochondrial proteins in cerebral cortex between rat Alzheimer’s disease and the cerebral ischemia model
Houyou Yu,Changwei Yang,Shi Chen,Yang Huang,Chuanming Liu,Jian Liu,Wen Yin
Scientific Reports. 2017; 7: 39948
[Pubmed] | [DOI]
11 Central and peripheral administrations of levothyroxine improved memory performance and amplified brain electrical activity in the rat model of Alzheimeræs disease
Sahreh Shabani,Alireza Sarkakia,Seyyed Ali Mard,Akram Ahamgarpoor,Layasadat Khorsandi,Yaghoob Farbood
Neuropeptides. 2016;
[Pubmed] | [DOI]
12 A review on the management of migraine in the Avicenna’s Canon of Medicine
Arman Zargaran,Afshin Borhani-Haghighi,Pouya Faridi,Saeid Daneshamouz,Abdolali Mohagheghzadeh
Neurological Sciences. 2016;
[Pubmed] | [DOI]



 

Top
Previous article  Next article
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and me...
Results
Discussion
Conclusion
Acknowledgments
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed1955    
    Printed13    
    Emailed0    
    PDF Downloaded414    
    Comments [Add]    
    Cited by others 12    

Recommend this journal