|Year : 2021 | Volume
| Issue : 1 | Page : 50-59
Potential ameliorative effect of Cynodon dactylon (L.) pers on scopolamine-induced amnesia in rats: Restoration of cholinergic and antioxidant pathways
Laxmi A Pattanashetti1, Basanagouda M Patil1, Harsha V Hegde2, Ranjit P Kangle3
1 Department of Pharmacology and Toxicology, KLE College of Pharmacy, Belagavi (A Constituent Unit of K. L. E Academy of Higher Education and Research), Belagavi, Karnataka, India
2 Department of Ethnomedicine and Medicinal Plants, ICMR- National Institute of Traditional Medicine, Belagavi, Karnataka, India
3 Department of Pathology, Jawaharlal Nehru Medical College, Belagavi, Karnataka, India
|Date of Submission||05-Jun-2020|
|Date of Decision||05-Oct-2020|
|Date of Acceptance||17-Mar-2021|
|Date of Web Publication||28-Apr-2021|
Mrs. Laxmi A Pattanashetti
Department of Pharmacology and Toxicology, KLE College of Pharmacy, KLE Academy of Higher Education and Research, Belagavi - 590 010, Karnataka
Source of Support: None, Conflict of Interest: None
AIM: The present study explored Cynodon dactylon hydro-ethanolic extract (CDE) effect on scopolamine-induced amnesic rats.
MATERIALS AND METHODS: C. dactylon extract was subjected to antioxidant (DPPH and H2O2) and acetylcholinesterase enzyme tests by in vitro methods. Scopolamine (1 mg/kg, i.p) was administered to rats except for normal control. Donepezil (3 mg/kg, p.o), CDE (100, 200, and 400 mg/kg p.o) were administered to treatment groups. Behavioral paradigm: Morris water maze (MWM), elevated plus maze (EPM), and passive avoidance test (PAT) were conducted. Later, rats were sacrificed and brain homogenate was tested for levels of acetylcholinesterase, glutathione, and lipid peroxidase. Histopathology examination of cortex and hippocampus of all the groups was done.
STATISTICAL METHOD: The statistical methods used were ANOVA and Tukey's post hoc test.
RESULTS: CDE antioxidant activity was demonstrated by decreasing DPPH and H2O2 levels confirmed through in vitro analysis. Treatment group rats reversed scopolamine induced amnesia by improvement in spatial memory, decreased transfer latency and increased step through latency significantly (P<0.001) in behavior models such as morris water maze, elevated plus maze and passive avoidance task respectively. CDE modulated acetylcholine transmission by decreased acetylcholinesterase enzyme level (P < 0.001) and scavenging scopolamine-induced oxidative stress by increased reduced glutathione levels and decreased lipid peroxidation levels in the rat brain. CDE and donepezil-treated rats showed mild neurodegeneration in comparison to scopolamine-induced severe neuronal damage on histopathology examination.
CONCLUSION: C. dactylon extract provides evidence of anti-amnesic activity by the mechanism of decreased acetylcholinesterase enzyme level and increased antioxidant levels in scopolamine-induced amnesia in rats.
Keywords: Acetylcholinesterase, antioxidant, Cynodon dactylon, dementia, neuroprotection
|How to cite this article:|
Pattanashetti LA, Patil BM, Hegde HV, Kangle RP. Potential ameliorative effect of Cynodon dactylon (L.) pers on scopolamine-induced amnesia in rats: Restoration of cholinergic and antioxidant pathways. Indian J Pharmacol 2021;53:50-9
|How to cite this URL:|
Pattanashetti LA, Patil BM, Hegde HV, Kangle RP. Potential ameliorative effect of Cynodon dactylon (L.) pers on scopolamine-induced amnesia in rats: Restoration of cholinergic and antioxidant pathways. Indian J Pharmacol [serial online] 2021 [cited 2023 Feb 5];53:50-9. Available from: https://www.ijp-online.com/text.asp?2021/53/1/50/315081
| » Introduction|| |
Dementia is an umbrella term that includes Alzheimer's disease (AD) expressing features of neurodegeneration that leads to memory impairment and behavioral changes that interferes with work, social activities, and impairs a person's ability to perform routine activities. According to Alzheimer's Disease International association, globally over 50 million people are affected by dementia and 152 million people will be living with dementia by the end of 2050.
AD is a progressive disorder and has a major risk impact on the aging process. The pathological hallmarks for AD are hypothesized such as interruption in the cholinergic neuronal system, abnormal amyloid pathway, hyperphosphorylation of protein “tau,” and its accumulation in the brain. Hence, multiple cellular and biochemical changes diversify pathological abnormalities. The well-recognized pathology in AD is the cholinergic neuronal loss in the basal forebrain; which is essential to regulate cognition, sleep-wake cycle, and learning new tasks. Modulation of cholinergic transmission is necessary in the treatment of AD; hence, acetylcholinesterase enzyme inhibitors (AChEI's) are considered as major targets since these decrease the breakdown of acetylcholine. Currently, FDA-approved drugs such as donepezil, rivastigmine, galantamine (AChEI's), and memantine (NMDA antagonist) slow down the progression and show symptomatic relief but do not cure AD. The donepezil is used as the first choice of treatment which prevents hydrolysis of residual acetylcholine in the brain along with the anti-inflammatory pathway. However, its beneficial effects are up to 36 weeks and can cause central and peripheral cholinergic side effects for long-term therapy.
Many medicinal therapies have been used in the treatment of neurological diseases since ancient times. Cynodon dactylon (L.) Pers is included in the family Poaceae known by Bermuda grass (English), traditionally used to treat epilepsy, dropsy, wound infections, piles, and snakebite.
The preclinical studies of different types of C. dactylon extracts possess central nervous, cardiovascular, antidiabetic, gastrointestinal, antioxidant, immunological, anti-allergic, anti-inflammatory, antipyretic, analgesic, anticancer, dermatological, diuretic, protective, antimicrobial, and antiparasitic activities.
As per the Unani system of medicine, C. dactylon was reported as the heart and brain tonic. However, scientifically, limited literature is available regarding research of C. dactylon effects for its potential to treat dementia so far. Hence, the present study was planned to test C. dactylon hydro-ethanolic extract (CDE) effect on scopolamine caused amnesia in rats and may explore the potential traditional claim for the modern treatment of dementia.
| » Materials and Methods|| |
Collection plant and preparation of extract
The C. dactylon whole grass was collected from the local regions of Belagavi, Karnataka, India. The plant was authenticated by a botanist and voucher number RMRC-1391 was deposited at ICMR-NITM, Belagavi. Collected plants were washed carefully and dried in shade for 15 days. The powdered material was macerated using ethanol: water (70:30) for 7 days; the residue was refluxed by Soxhlet apparatus up to 8–12 h. The hydro-ethanolic extract was cooled and concentrated by evaporation and stored under refrigerated conditions, until further studies.
The CDE was screened for the presence of ﬂavonoids, tannins, phenols, saponins, glycosides, and alkaloids qualitatively according to protocols described by Oshadie et al. Ash values, water, and alcohol-soluble extractives were tested as described by Kokate.
The quantitative estimations of total flavonoid and total phenolic contents were determined by methods explained by McDonald et al. Results were expressed for flavonoid as quercetin equivalent and phenolic content as gallic acid equivalent.
Drugs and chemicals
All the reagents used were of analytical grade. Quercetin, acetylthiocholine iodide, donepezil hydrochloride, DPPH, acetylcholinesterase enzyme (Source: Electrophorus electricus), Tris HCl, DTNB, and H2O2 were procured from Sigma-Aldrich (USA). Gallic acid, ethanol (Hi-media, India), and scopolamine hydrobromide (Vital Laboratories; Gujarat, India) were used.
In vitro assays
Estimation of DPPH and H2O2
DPPH and H2O2 scavenging activity of C. dactylon extract was tested as described by Teh et al. and Ruch et al., respectively. Three independent assays were carried out using ascorbic acid as a standard control to ensure precision.
Acetylcholinesterase enzyme inhibition assay
Acetylcholinesterase activity of CDE by in vitro was measured by 96-well microplate assay using Ellman's method described by Dhanasekaran et al. The AchE enzyme inhibition was recorded in triplicates to determine inhibition concentration50 (IC50) values by linear regression analysis.
In vivo studies
The Wistar albino rats 18 months old (280–300 g; either sex) were procured from In vivo Biosciences; Bangalore, India. Animal ethical approval was obtained from IAEC-KLECOP/CPCSEA and Reg. No-221/Po/Re/S/2000/CPCSEA. These rats were acclimatized and housed in stainless steel cages in room temperature 25°C ± 2°C, 12 h light-dark cycle with access to water ad libitum.
Acute oral toxicity studies
Organization for Economic Co-operation and Development guidelines, 423 (OECD 2001) was used to determine observable adverse effects and safety of C. dactylon extract for the purpose to find out a safe dose for further studies.
The hydro-alcoholic extract of C. dactylon was dispersed in CMC (0.5%) and rats were administered as single dose of 2000 mg/kg by gastric gavages using an oral feeding needle to rats (n = 3). Control group rats were administered water as a vehicle. After the treatment of CDE, the number of rats survived was considered. Symptoms of toxicity were observed and reported 24 h; further housed up to 14 days with daily observations. Mobility, skin changes, sensitivity to pain, sensitivity to sound, respiratory movements, and aggressiveness were the visual observations in this study. Toxic effects of the extract were analyzed based on mortality and were expressed as LD50.
Preparation of drug solutions
Based on previous reports, the dried hydro-ethanolic C. dactylon extract was reconstituted in carboxymethyl cellulose (0.5%) and administered for 15 days, and as per the previous report, the present study used scopolamine 1 mg/kg, i.p route as inducing agent for 30 days as because it provokes cognitive impairment in animal models. Scopolamine and donepezil were dissolved in a normal saline solution; fresh drug solutions were prepared on each day of experimentation.
Rats used for the experiment were selected randomly, (n = 6) into six groups as mentioned: Group I: received normal saline + 0.5% CMC (normal control), Group II: scopolamine (SCO) 1 mg/kg, through i.p route (disease control), Group III: SCO + donepezil 3 mg/kg p.o (standard control), Group IV: SCO + CDE 100 mg/kg p.o; Group V: SCO + CDE 200 mg/kg p.o, Group VI: SCO + CDE 400 mg/kg, p.o. The scopolamine 1 mg/kg was administered intraperitoneally to groups II-VI for 30 day to provoke cognitive impairment. Meanwhile, donepezil and treatment control C. dactylon extract with CMC (0.5%) were administered from 16 to 30th day to treatment groups to intervene scopolamine-induced amnesia.
Morris water maze test
The water-filled circular pool with 10 cm × 10 cm immersed 2 cm under the water surface. The individual rat was subjected to 4 times test/day for 4 days and retention of memory was tested on 30th day. During each training day, the starting place was randomized but kept the same for all the rats in each experiment; in each trial, rats' head pointed at the sidewall when allowed to swim for 90 s to check missing platform. Before the test, rats were habituated for training trials and were allowed to swim. If it is not habituated, then the rat was placed manually for 30 s suppose if rats do not reach the platform in 120 s. On day 30, the platform was taken away from position, then time spent to the search of the missing platform is considered as probe trial referred as retrieval of memory.
Elevated plus maze
This apparatus is composed of four arms (two closed and two open arms). Day 1, rats were individually placed near the either ends of open arm. Transfer latency was considered as acquisition trial if rat moves towards closed arm. Retention of memory of rats was recorded on day 30 using TL and percentage time spends in the open arm.
The apparatus has a light and dark compartment connected by an opaque guillotine door. The floors of both compartments were made of stainless steel rods (3 mm diameter) spaced 1 cm apart. The test was carried out according to the method followed as per Pitchaimani et al. Step-through latency was considered on day 1 as acquisition test and day 30 as a retention test.
Collection of brain sample
The cervical dislocation was performed to sacrifice rats immediately after cognitive tests. From the skull, the brain was gently taken out then homogenized brain and centrifuged; the supernatant was collected then used for the biochemical assays.
Acetylcholinesterase enzyme test
Brain tissue 20 mg (hippocampus)/mL of phosphate buffer (0.1 M; pH 8) was homogenized and 0.4 mL aliquot of brain homogenate was added to a cuvette containing 2.6 mL of 0.1 M phosphate buffer, 100 µL of DTNB reagent. The substrate acetylthiocholine iodide 20 µL was added. The change in optical absorbance was measured every 2 min for 10 min at 412 nm to provide a measure of enzyme activity.
Estimation of lipid peroxidase
Lipid peroxidation (LPO) is measured by the amount of TBARS present in the hippocampus of a rat brain. Ohkawa et al. described the method was followed to test LPO. The TBARS value is expressed as nanomoles of MDA/mg of protein.
Glutathione (reduced) level estimation
The procedure described by Ellman et al. was used to estimate the level of reduced glutathione (GSH) in brain homogenate and values were expressed in µmoles/mg of tissue.
Histopathology of rat brain
The brain tissue of the rat was collected and fixation was done using 10% neutral buffer formalin. These were processed to obtain 4 µm paraffin-embedded sections. These different specimens were stained using Hematoxylin and Eosin and observed under a microscope.
Mean ± standard error of mean was indicated for all test values. Tukey's post hoc multiple comparison test followed by ANOVA was used to analyze statistical differences of various groups. Mean values were considered statistically significant if P < 0.05, P < 0.01, or P < 0.001.
| » Results|| |
Phytochemical screening of Cynodon dactylon hydro-ethanolic extract
The CDE % yield was 9.47% W/W and showed the presence of phytoconstituents such as phenols, flavonoids, tannins, saponins, and steroids. The total ash value (9.5%), acid insoluble ash (6.0%), water-soluble extract (10.2%), and alcohol-soluble extract (4.2%) W/W were reported. The quantitative estimation of CDE such as total phenolic and total flavonoid content was found to be 71.07ug/ml (GAE/g) and 106 ug/ml (QE/g), respectively.
Antioxidant activities of Cynodon dactylon
The C. dactylon extract inhibits DPPH at IC50 value at 255.6 ± 17.54 µg/ml as compared to ascorbic acid IC50 at 35.73 ± 0.85 µg/ml. H2O2 was inhibited at IC50 at 56.94 ± 0.594 µg/ml as compared to ascorbic acid 47.81 ± 0.6639 µg/ml indicated antioxidant potency.
Effect of Cynodon dactylon extract on acetylcholinesterase enzyme inhibition (in vitro analysis)
C. dactylon extract inhibited the AChE enzyme significantly with IC50 value (38.47 ± 2.56 µg/ml) as compared to donepezil (2.23 ± 0.141 µg/ml). This showed that the possibility of C. dactylon extract may act on the cholinergic pathway. Hence, in vivo studies were further tested.
Acute oral toxicity studies
Observations from normal rats and CDE administered groups showed the absence of mortality. The extract was found to be safe at 2000 mg/kg; therapeutic doses 100, 200, and 400 mg/kg were selected depending on oral toxicity studies for further studies.
Effect of Cynodon dactylon extract on spatial memory in Morris water maze test
[Figure 1] explains escape latency in seconds using Morris water maze (MWM) as task to evaluate the restoration of spatial memory. [Figure 1]a shows training trials for 7 days before commencing treatment to rats; which expressed significant decreased escape latency on day 7 as compared to day 1 of the training trail. [Figure 1]b shows the donepezil and C. dactylon extract treatment to respective groups intervened and showed a decrease in escape latency significantly compared to scopolamine-induced rats. [Figure 1]c shows that the scopolamine induction elevates escape latency significantly (P < 0.001) as compared to normal control, donepezil (3 mg/kg), and C. dactylon extract at 100, 200, and 400 mg/kg treatment expressed significant decreased escape latency (P < 0.001) as compared to scopolamine-induced rats. [Figure 1]d represents a probe trial in which scopolamine-induced rats showed decreased time spent in the target quadrant as compared to normal control rats; while donepezil and CDE-treated rats demonstrated increased time spent in the target quadrant (P < 0.001) compared to scopolamine-induced rats significantly. These observations indicate improvement in spatial memory.
|Figure 1: Data were represented as mean ± SEM (n = 6), (a) escape latency (s) of rats during training trials, (b) escape latency expressed by rats for every 7 days of treatment, (c) Probe trial (time spent to reach target quadrant) compared to acquisition trial, (d) time spent in target quadrant by rats on probe trial. *As compared to normal control group, ***Compared to scopolamine control group. P < 0.001 was considered as statistically significant. SEM = Standard error of mean|
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Effect of Cynodon dactylon extract on transfer latency in elevated plus maze task
[Figure 2]a shows the effect of CDE on the TL and [Figure 2]b expresses the percentage time spent in open arms following 5 min of exploration in an elevated plus-maze. The scopolamine-induced rats showed increased TL as compared to normal group rats. The donepezil (3 mg/kg) and C. dactylon extract treatment at 100, 200, and 400 mg/kg doses showed a significant decrease in TL as compared to scopolamine-induced rats.
|Figure 2: Data were represented as mean ± SEM (n = 6), (a) transfer latency (s) of rats on elevated plus maze, (b) percentage time spent by rats in the open arm of EPM, (c) step-through latency of rats in the passive avoidant test: * As compared to the normal control group, ***Compared to the scopolamine control group. P < 0.001 was considered as statistically significant. SEM = Standard error of mean, EPM = Elevated plus maze|
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Scopolamine-induced rats reduced percentage time spent (P < 0.001) at open arms as compared to normal control rats significantly. However, donepezil, CDE at selected therapeutic doses showed increased time spent percentage in open arms, respectively, as compared to scopolamine-induced rats. This indicates that C. dactylon extract shows retention of short-term memory.
Effect of Cynodon dactylon extract on passive avoidance task
On day 30 (Retention trial) SCO administered rats showed significantly decreased (P<0.001) step through latency and this could indicate an increased fear aggravated behaviour. However, treatment groups, namely donepezil (3 mg/kg), CDE-100, 200, and 400 mg/kg doses showed that increased step-through latency indicates the improved nondeclarative memory as shown in [Figure 2]c.
Effect of Cynodon dactylon extracts on acetylcholinesterase enzyme activity
Rats administered with scopolamine increased AChE enzyme activity expressed as micromoles hydrolyzed/min/g (24.71 ± 0.9231; P < 0.001) compared to normal group rats (7.263 ± 0.4508) significantly. The donepezil treated group was considered as a standard treatment showed decreased AChE level as 8.099 ± 0.75; P < 0.001 as compared to SCO induced rats. The rats treated with C. dactylon at 100 mg/kg, 200 mg/kg, and 400 mg/kg significantly inhibited AChE levels by (P < 0.001; 14.04 ± 1.77, 11.34 ± 0.49, 9.326 ± 0.33), respectively, when compared to scopolamine-induced animals. Hence, the C. dactylon extract could improve cholinergic transmission by decreasing the acetylcholinesterase enzyme level as shown in [Figure 3].
|Figure 3: Modulation of acetylcholinesterase enzyme level in rat brain: Data were represented as mean ± SEM (n = 6), * As compared to the normal control group, ***Compared to the scopolamine control group. P < 0.001 was considered as statistically significant|
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Antioxidant activity of Cynodon dactylon extract on reduced glutathione levels
Scopolamine administered rats showed significantly decreased glutathione level (P < 0.001; 0.53 ± 0.05) compared with normal control rats (1.39 ± 0.05). The rats treated with donepezil (1.23 ± 0.115; P < 0.001), C. dactylon extract significantly increased (0.87 ± 0.15; 1.04 ± 0.06; 1.18 ± 0.036: P < 0.001) GSH levels as compared to SCO-induced rats as shown in [Table 1].
|Table 1: Antioxidant activity of Cynodon dactylon hydro-ethanolic extract in rat brain homogenate. Mean±SEM, P<0.001 was considered as statistically signifcant, *As compared to normal control, #As compared to scopolamine control group. MDA=Malondialdehyde, CDE=Cynodon dactylon hydro ethanolic, GSH=Reduced glutathione, LPO=Lipid peroxidation, SCO=Scopolamine, SEM=Standard error.|
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Effect of Cynodon dactylon extract on lipid peroxidation levels
The animals induced with scopolamine reported a significant increase in (P < 0.001; 39.53 ± 3.05) in MDA levels in comparison with normal rats (12.82 ± 2.86). Interestingly, C. dactylon extract administered at therapeutic doses to rats (P < 0.001; 19.23 ± 4.05,17.09 ± 3.16,16.03 ± 2.75) and the donepezil (P < 0.001; 11.75 ± 1.97) significantly reduced MDA level which indicates decreased LPO in the brain in contrast to scopolamine-induced rats as shown in [Table 1].
Impact of Cynodon dactylon extract on rat brain histology
H and E-stained brain tissue sections showed normal healthy neurons in the cortex part of normal control rats. The scopolamine-induced rats showed severe neuronal damage which was noted by the presence of gliosis and neuronophagia. C. dactylon extract treated rats showed mild damage to neurons, with vacuolated degeneration as shown in [Figure 4]. The standard control donepezil treated group showed mild neuronal damage and vacuolated degeneration.
|Figure 4: Histopathology of rat brain (cerebral cortex) section) with H and E x400 (a) Normal control group showing normal histological finding. (Black arrow- Normal Neuron). (b)SCO (1mg/kg) (Black arrow- severe neuronal degeneration, neuronophagia and blue arrow pyknotic nucleus) (c) CDE (100mg/kg) + SCO group showing moderate neuronal degeneration with neurophagia with congestion of blood vessels. (Black arrow Moderate edema and Blue arrow shows moderate neurodegeneration) (d) CDE (200mg/kg) + SCO showing neuronal degeneration. (Black arrow-mild neuronal degeneration) e) CDE (400mg/kg) + SCO group (Black arrow show mild congestion and blue arrow shows mild neurodegeneration) (f) Donepezil (3mg/kg) + SCO group showing minimal neuronal degeneration. (Black arrow - Mild congestion, Blue arrow minimal neurodegeneration). *CDE: Cynodon dactylon hydroethanolic extract; SCO: Scopolamine|
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The hippocampus region was histologically normal in normal control rats and scopolamine induction showed severe neuronal damage with perivascular edema. The donepezil-treated rats showed moderate pyramidal cell damage with perivascular edema, whereas C. dactylon extract-treated rats showed mild damage to neurons with few pyknotic cells and mild edema compared to scopolamine-induced rats' brain histology as shown in [Figure 5].
|Figure 5: Histopathology of rat brain (hippocampus)section) with H and E x400 (a) Normal control group showing normal histological finding. (Black arrow- Normal Neuron). (b)SCO (1mg/kg) (Black arrow- severe neuronal degeneration, neuronophagia and blue arrow pyknotic nucleus) (c) CDE (100mg/kg) + SCO group showing moderate neuronal degeneration with neurophagia with congestion of blood vessels. (Black arrow Moderate edema and Blue arrow shows moderate neurodegeneration) (d) CDE (200mg/kg) + SCO showing neuronal degeneration. (Black arrow-mild neuronal degeneration) e) CDE (400mg/kg) + SCO group (Black arrow show mild congestion and blue arrow shows mild neurodegeneration) (f) Donepezil (3mg/kg) + SCO group showing minimal neuronal degeneration. (Black arrow - Mild congestion, Blue arrow minimal neurodegeneration). *CDE: Cynodon dactylon hydroethanolic extract; SCO: Scopolamine|
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| » Discussion|| |
The current study explored the CDE effect on scopolamine-induced amnesic rats. Briefly, C. dactylon extract was subjected to preliminary phytochemical investigation further in vitro, in vivo, and biochemical estimations were carried out to find the ability of the C. dactylon extract to show anti-amnesic effect by the cognitive modulation, inhibition of AchE, and antioxidant activity in rats.
The CDE phytochemical investigation complies with earlier reports of Madan Kumar et al. who observed that the C. dactylon extract showed the active constituents such as flavonoids, tannins, alkaloids, phytosterols, saponins, glycosides, phenols, proteins, and carbohydrates.
The in vitro study revealed CDE scavenges activity of free radical by inhibition of DPPH and this was supported by a previous report and phenolic phytoconstituents present in CDE may be responsible for DPPH scavenging action.
C. dactylon extract scavenges hydrogen peroxide and confirmed promising antioxidant property. The hydrogen peroxide is a neutral molecule formed during the dismutation of superoxide by superoxide dismutase which freely diffuses out of the lipid bilayer, thereby targeting other biomolecules. Polyphenols of C. dactylon may have scavenging property.
Memory loss is a common feature of the elder population; to depict this condition, aged animals can be served as natural model of dementia which has been shown to develop neuropathology, oxidative stress followed by memory impairment similarly observed in patients affected with AD.
Along with this, the current study used scopolamine hydrobromide as to induce amnesia in rats, since earlier reports suggests that SCO binds to cholinergic receptor non- selectively and blocks the transmission of Ach which leads to memory loss. SCO increases oxidative stress which disturbs antioxidant defense system.
Results of in vivo studies include rats administered by scopolamine showed increased step-through latency and escape latency in passive avoidance test and MWM, respectively, along with reduced TL in elevated plus maze (EPM) tests, whereas rats treated with donepezil and C. dactylon extracts showed decreased step-through latency, escape latency, and increased TL in the aforementioned behavioral tasks. These findings emphasize traditional claim, especially in the cognitive-enhancing properties of C. dactylon.
This study confirmed that scopolamine-induced group showed the decline in memory by increased levels of AChE enzyme, thus indicating signs of dementia. Interestingly, the effect of CDE was administered at doses (100, 200, and 400 mg/kg) showed a decrease in AChE levels in the hippocampus which confirmed the protection of cholinergic neurons affected by scopolamine-induced amnesia in rats. These current observations were pertinent as per the previous reports of Poojary et al. and Rai et al., Another research showed promising reports of C. dactylon aqueous extract improving cognition in amnesia induced by scopolamine in the zebrafish model.
The important biomarkers, GSH (reduced) and LPO of the rat brain, show the antioxidant defense to regulate reactive oxygen species generated in neurons. Aging is an unavoidable part of the living system, considered as a risk factor for chronic diseases including AD and oxidative stress is a natural factor associated with the aging population. In the brain, GSH an antioxidant plays to scavenge damage of neurons against oxidative stress, alongside neurotoxicity occurs due to free radicals mediation possessed by the activity of LPO as per many earlier researcher's suggestions. The current study explained that rats induced by scopolamine imbalanced antioxidants and show decreased GSH (reduced) and increased LPO (MDA levels). In contrast to the response of scopolamine-induced oxidative stress, CDE protected neuronal damage by increased GSH (reduced) and decreased LPO activity. These outcomes were apparently relevant with the report of Rai et al. Moreover, other recent reports that the aqueous extract of C. dactylon potentiated antioxidant status by decreasing LPO levels along with the increased activity of Na+/K+ ATPase and Mg2+ ATPase enzymes and protected brain regions from neurotoxicity induced by aluminum trichloride.
H and E-stained sections are the most commonly used dye-based technique for histopathological examination of the rat brain. Majority of the neuronal changes in the cerebral cortex and hippocampus region can be demonstrated using these stains.
The reports of histopathological interventions show severe neuronal damage induced by scopolamine and administration of donepezil and CDE showed mild neuronal damage in dose-dependent manner as compared with scopolamine-induced rats. These reports were confirmed by earlier reports of Barai et al.
Essential assessments may typically be performed in H and E-stained neuronal tissues; however, to characterize unique cell responses as well as to discern modifications, special histological stains may be required. Congo red, thioflavin S stains bind to characteristic β-pleated sheet conformation of amyloid; hence, they can be used to detect different forms of AD. Routine staining cannot differentiate between astrocytes and glial cells, and antibody-based immunohistochemical stain like glial fibrillary acidic protein (GFAP) has been used to demonstrate the glial scar in a large number of studies. Immunohistochemical detection of GFAP typically marks most parts of the brain because it identifies both resting and active astrocytes, but it is helpful in analyzing changing astrocyte activity patterns. Hence, dye-based techniques and antibody-based techniques coherently are useful in detecting AD.
Drugs such as Galantamine is a natural alkaloid extracted from Galanthus nivalis L. Rivastigmine is a synthetic alkaloid, the structure is similarly modelled on that of natural physostigmine. Hence, FDA approved drugs show anticholinergic effect similar to natural origin compounds including flavanoids and phenolic acid.
For developing effective and safe anti-AD drugs through modulating enzyme-based targets, flavonoids play attractive lead compounds. C. dactylon phytoconstituents play important role in preventing various neurological disorders. This is attributed due to active constituents such as alkaloids, carotenoids, saponins, flavonoids, phytosterols, and proteins in plant. Several flavonoids have been investigated effectively to treat AD. According to Muthukrishnan et al., HPLC analysis of C. dactylon revealed the identification of different flavonoids. Among these, quercetin was found as major flavonoid, followed by apigenin kaempferol, rutin, and catechin. Predominantly, β-carotene, luteolin, and zeaxanthin were also present. Near future, there is scope for bioactivity-guided fraction which may help to identify the lead compound (s) responsible for this activity, which eventually may be a target for AD treatment.
| » Conclusion|| |
The current study showed that scopolamine administration confirmed signs related to amnesia by increased levels of acetylcholinesterase enzyme and oxidative stress; eventually damaging cholinergic pathways which are essential for learning and memory in rats. Meanwhile; the C. dactylon extract provides evidence of anti-amnesic activity by the mechanism of decreased AChE enzyme levels and increased antioxidant levels in amnesia induced by scopolamine in rats; which may be attributed due to the presence of phytoconstituents singly or in their combinations in the extract.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
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