Department Pharmacology Dr. L.H. Hiranadani College Of Pharmacy , Ulhasnagar
The abstract summarizes research on neurodegenerative disorders, focusing on memory impairment and the potential neuroprotective effects of Hedychium coronarium Linn. Essential oil. Neurological disorders like Alzheimer’s disease involve cognitive decline and are linked to cholinergic system deficits and oxidative stress. Scopolamine-induced amnesia models were used to evaluate memory performance in mice treated with essential oil via inhalation and oral routes, alongside a standard drug (Donepezil). Behavioral tests (Elevated Plus Maze, Pole Climbing Test) and biochemical assays (AChE activity, MDA, GSH levels) demonstrated significant improvements in memory and reduction in oxidative stress markers with essential oil treatment. These findings suggest Hedychium coronarium Linn. essential oil could be a potential therapeutic agent for managing neurodegenerative disorders associated with memory impairment.
Neurodegenerative disorders are disorders of the central and peripheral nervous system. Structural, biochemical or electrical abnormalities in the brain, spinal cord or other nerves can result in symptoms such as paralysis, muscle weakness,poor coordination, loss of sensation ,seizures,confusion, pain .These disorders include epilepsy, cerebrovascular diseases including stroke, migraine, neuroinfections, brain tumors and neurodegenerative diseases like Parkinson’s disease, Alzheimer’s disease and other dementias.[1]Interventions for neurological disorders include preventative measures, lifestyle changes,physiotherapyorotherholisticapproaches, neurorehabilitation, painmanagement, medication, operations performed by neurosurgeons.[2] Amnesia (Memory impairment) is a neurological disorder associated with decline in cognitive abilities; patients also frequently have non-cognitive symptoms, such as depression, apathy and psychosis that impair daily living. [3] Memory is a fundamental mental process, it is an ability of an individual to record event, information and retains them over short or long periods of time and recalls the same whenever needed.[4] Learning is the process of acquisition of information and skills, while subsequent retention of that information is called memory. [5] Learning and memory together are called as cognition. Learning and memory is one of the most intensively studied subjects in the field of neuroscience.[4,6]
Central cholinergic system plays a major role in learning and memory process through various neuronal pathways and neurotransmitters. Deficits occurring in these pathways may result in occurrence of various cognitive disorders like amnesia and dementia. Researches had confirmed that both muscarinic and nicotinic acetylcholine receptors have a role in the encoding of new memories. There are certain agents that are used for memory impairment due to their capability of interfere in memory formation and neuroanatomical structures. According to WHO report, 46.8 million people worldwide are living with dementia in 2015. This number is speculated to double every 20 years and may reach to 74.7 million by 2030 and 131.5 million by 2050. This condition produces a great impact on the healthcare budget and social care. [7] At present, the most appropriate approach for treatment of AD focuses on modulation of acetyl cholinesterase (Ache) activity. Acetylcholine esterase inhibition in the treatment of amnesia is the mainstream pharmacotherapy. Donepezil, tacrine, galantamine, and rivastigmine are cholinesterase inhibitors which are widely used in the treatment of amnesia However, Allopathic drugs are associated with various adverse effects such as diarrhoea, insomnia, nausea, bronchitis, loose stools, muscular cramps etc. [8] This has made their use limited and therefore, it is necessary to develop safer drugs for the treatment of various Neurodegenerative disorders. However their therapeutic effects are not significant. Therefore, other possibilities including herbal medicine sources have been considered and evaluated for memory loss therapy.The use of herbal and natural remedies in the treatment of neurological, psychiatric and neurotoxic logical disorders has been increasing tremendously due to their efficacy and safety benefits. [9,10] Aromatherapy is gaining popularity in a log phase and is currently used worldwide in the management of chronic pain, depression, anxiety, some cognitive disorders, insomnia and stress related disorders. [11-13] The selected essential oil has shown in standard reference book that, this essential oil could be useful for relaxation and stress relief [14]but so far it’s Anti-amnesic activity has not been explored pre-clinically. This study aims at exploring efficacy of Hedychium coronarium Linn. as a neuroprotective agent in Amnesia . From the literature, it was found to possess Anti-microbial[15],Anti-analgesic[16],Anti-inflammatory[17],Anti-bacteria[18],Anti fungal[19],Antiurolithiatic[20],Anti-oxidant[21], Hypoglycaemic activity[22], Mosquito larvicidal [23] and cancer chemoprevention activity[24]. So far it’sAnti-amnesicactivity has not been explored pre-clinically. This study aims at exploring efficacy of essential oil of flowers of Hedychium coronarium linn. as a neuroprotective agent in Amnesia. Most of the studies used scopolamine to induce amnesia; other amnesic agents like diazepam, sodium nitrite are also used as well. Impairments of learning and memory can be induced chemically in experimental animals by scopolamine.[4,25,26] Scopolamine is a blocker of muscarinic acetylcholine receptor ,that serves as a beneficial pharmacological tool in producing a model of amnesia. [4,27,28,29]There is notable evidence that scopolamine causes oxidative stress through the interference with acetylcholine in brain leading to cognitive impairment.[30]
METHODS
1. Procurement of essential oil:
Essential oil of flowers of Hedychium coronarium linn. was procured from JYM Healthcare, Mumbai.(Certificate of analysis was provided) .
2.Route of Administration:
Essential oil (EO) of flowers of Hedychium coronarium linn. was inhaled to the mice in a closed chamber. A constant volume (100?l) of essential oil was placed on filter paper. The soaked filter paper was inserted into electrically heated evaporating machine. Animals were exposed to aroma for 15min. for 7 days. Each filter paper was used only once and renewed each time to maintain concentration of essential oil in the inhalation chamber.
4 .Standard
Donepezil (Acetyl cholinesterase inhibitor):Donepezil was administered at a dose of 4mg/kg orally for 7 days
5. Animal Husbandry /protocol
Swiss albino male mice ((25-30g), 5 weeks of age of 69 animal were procured from Bombay veterinary College, Parel,Mumbai and National institute of bioscience, Pune . Animals were brought to animal house of Dr. L. H. Hiranandani College of pharmacy, Ulhasnagar-03. These animals were acclimatized in animal house under standard husbandry conditions, i.e. room temperature of 24 ±100C, relative humidity 45-55% and 12:12 hr. light/dark cycle. The institutions animal house is registered with Govt. of India, having registration number 879/PO/Re/S/05/CPCSEA and abides by CPCSEA guidelines for the use and care of experimental animal research. The animals were housed in standard propylene cages with wire mesh top and husk as bedding. The animals had free access for food and water supplied ad libitum under strict hygienic condition. Each experimental group had separate set of animals and care was taken to ensure that animals used for one response were not employed elsewhere. The approval of the Institutional animal ethical committee (IAEC) of Dr. L.H. Hiranandani College of pharmacy was taken prior to the start of experiments. All the protocols and the experiments were conducted in strict compliance according to ethical principles and guidelines provided by committee for the purpose of control and supervision of experiments on animals (CPCSEA) and with the protocol no. PCOL 12/IAEC/2017 and PCOL 13/IAEC/2017.
6. Toxicity study
Limit test for acute toxicity was performed according to OECD guideline 423.
7. Model for Anti amnesic activity (Scopolamine Induced Amnesia Model)
Scopolamine, a nonselective muscarinic cholinergic antagonist (anticholinergic drug), causes amnesia in humans and also impairs learning in animals by reducing cholinergic activity. Scopolamine have been used for decades in experimental animals to induce impairment in their performance of a variety of tasks requiring intact working and reference memory. Indeed, blockade of central muscarinic receptors could induce a pattern of cognitive impairment. Therefore, scopolamine is considered as reliable tool to study neuroprotective effects of candidate molecules.
Elevated plus-maze test (EPM):
EPM serves as the exteroceptive behavioral model (wherein the stimulus existed outside the body) to evaluate short-term learning and memory in mice. The maze is composed of wood, consisting of two open arms and two close arms. Mice were placed at the end of open arm,facing away from central platform. Mice learned to move and spend more time in either of the closed arm because they felt safe there without any fear of falling down. Transfer latency is time taken by a mice to move into either of the closed arms (entry with all four legs) was recorded. Retention of this learned task was examined on 8th day. EPM consists of two opposite open arms (50 x 10 cm), crossed with two closed arms of same dimensions with 10 cm high walls. The arms are connected with Central Square (10 x10 cm). Mouse was placed at the end of open arm, facing away from central square (10cm×10cm) platform and maze kept elevated to a height of 50 cm from the floor. Mouse learned to move and spend more time in either of the closed arm because it felt safe there without any fear of falling down. Transfer latency (TL) is time taken by a mice to move into either of the closed arms (entry with all four legs) was recorded. Reduction in transfer latency indicates improved memory .Retention of this learned task was examined on 8th day. All the animals received scopolamine at the dose of 0.5mg/kg by intra-peritoneal route for induction of amnesia. In addition, the standard treatment group received Donepezil at the dose of 4mg/kg for 7 days. Test groups received essential oil of flowers of Hedychium coronarium linn. by oral (200mg/kg & 400mg/kg) and inhalation route.On the first day of study, mice were allowed to move freely to explore the apparatus for at least 10 min. TL recorded on 1st day was acquisition (learning) and 7th and 14th day TL reflects the retention / consolidation (memory) for learning.[32]
Pole Climbing Test
Pole climbing apparatus is used to study cognitive function, mainly a response to conditioned stimuli i.e. buzzer signal, during learning and its retention. In experiment, mice were placed in the pole-climb apparatus and were subjected to a neutral conditioned stimulus such as sound, followed immediately by an aversive unconditioned stimulus, such as foot-shock. Each mouse was allowed to acclimatize for 2 min and then exposed to a buzzer noise. After 10 secondsof the buzzer, mild electric shock was given through the grid floor. The magnitude of the voltageis adequate (0.6/0.8mA) to stimulate the mice to escape from the floor and climb the pole. Assoon as the mice climb the pole, both the buzzer and foot-shock were switched off.All the animals received scopolamine at the dose of 0.2mg/kg by intra-peritoneal route for induction of amnesia. In addition, the standard treatment group received donepezil at the dose of 4mg/kg for 7 days. Test groups received essential oil of flowers of Hedychiumcoronarium linn. by oral (200mg/kg & 400mg/kg) and inhalation route. On 7th day after treatment animals were subjected to Pole Climbing test.[33]
Biochemical Estimation
Collection of Brain Sample.
Animals were sacrificed using carbon-di-oxide chamber (CO2). The brain tissue was isolated and placed immediately in chilled phosphate buffer (PH 7.4) for bio-chemical estimation. Brain tissue samples were then homogenized with 10 times (w/v) ice cold 0.1M phosphate buffer (ph 7.4). The homogenate was centrifuged at 10000 x g for 15min, supernatant was separated and aliquots were used for biochemical estimations.
Brain Acetylcholinesterase Activity.
The method of AChE activity estimation is popularly known as Ellman’s method developed by George Ellman et al [34] in 1961. Acetylthiocholine iodide was used as a synthetic substrate for the assay of AChE, replacing the natural substrate acetylcholine (Ach). This enzyme hydrolyses the substrate to yield acetate and thiocholine. The free thiol group of thiocholine reacts with DTNB (Ellman's reagent) included in the assay mixture, producing the yellow 4- nitrothiolate anion. The cholinergic marker, acetylcholinesterase was estimated in the whole brain. according to the method of Ellman. Ellman's reagent is 5, 5’-dithiobis (2-nitrobenzoate) DTNB. 0.1ml of brain tissue homogenate was incubated for 5 min with 2.7 ml of phosphate buffer (pH7.2) and 0.1 ml of DTNB (0.5mM). Then, 0.1 ml of freshly prepared acetylthiocholine iodide (pH 8) was added and the absorbance will be taken at 412 nm.[34]
Thiobarbituric Acid Reactive Substance (TBARS) Assay
For the estimation of brain TBARS levels, an index of lipid peroxidation, the method of okhawa et al.[35] was used. Malondialdehyde (MDA) is a secondary product of lipid peroxidation. It is most sensitive marker of oxidative lipids. Lipid peroxidation is a process in which oxidative degradation of lipids occurs. In this process, free radicals steal electrons from the lipids in cell membranes resulting neuronal cell damage. Scopolamine increases brain MDA, a compound that results from the decomposition of polyunsaturated fatty acid lipid peroxides. Reduction in brain MDA level is an indication of neuroprotective activity. The tissue homogenate (0.5ml) was supplemented with 0.5ml of phosphate buffer (pH 7.2) and then with 1ml of 10% trichloroacetic acid. The mixture was centrifuge at 3000rpm at 4°c for 10 min. The supernatants of the tissue homogenates were incubated with 1 ml of 0.8% w/v of the thiobarbituric acid at 100°c for 15 min. After a cooling period, Malondialdehyde formed from the breakdown of polyunsaturated fatty acid reacts with Thiobarbituric acid to form thiobarbituric acid reactive substance (TBARs), the end product of lipid peroxidation, a pink to red color tri-methionine complex exhibiting absorption maximum at 530 to 535nm.
Glutathione assay
For the estimation of brain glutathione levels, an index of lipid peroxidation, the method of Beutler et al. was used. Glutathione (GSH) is an important anti-oxidant. GSH having capability of preventing cellular damage due to free radicals or reactive oxygen species. Scopolamine decreases the brain GSH level causes oxidative stress. Increase in brain GSH level is an indication of neuroprotective activity.[36] The method for assessment of GSH (mg/g wet tissue) in the brain homogenates was based on that of Beutler et al. Homogenates were deproteinized with 5-sulfuosalicylic acid (10%) for 30 min at 40C then centrifuge at 3000g for 15min at 40C. An aliquots of the acid soluble supernatant were diluted with phosphate buffer (0.3 M, pH-7.7) and 5, 5’-dithiobis-2- nitrobenzoic acid (1mM) was added to the samples. The optical density of the produced colored product was determined at 412nm.
9. Statistical analysis of data:
The statistical analysis was carried out by one way ANOVA followed by Dunnet’s multiple “t” test using software graph Pad Prism 5. P values < 0>
RESULT
Limit test for acute toxicity was conducted according to OECD guidelines 423. The animals were observed for 14 days for any signs of toxicity.
Clinical observation was noted:
Observed rating N = Normal O = Absent
There were no changes in clinical signs and symptoms viz. respiratory, circulatory, autonomic,and central nervous system and somatomotor activity and behavioral pattern throughout the period of 14 days. None of the animals demonstrated any signs viz. tremors, convulsion,salivation, diarrhea, leathery or comma. (Cage side observation)
No mortality was observed.
The essential oil of flowers of Hedychium coronarium linn. was found to be safe at the maximum dose of 2000mg/kg bodyweight.
2 Model 1: Anti-amnesic activity (Scopolamine induced amnesia model)
1 Activity 1: Elevated Plus Maze (EPM)
Animals were subjected to Elevated Plus maze test to record ITL and Retention TL on 7th and 8th day respectively. [Table no -1.1] Values are Mean ±SEM, n=6. Significant difference by Dunnets ‘t’ test, *p?0.05 when compared with Disease Control Group, **p?0.01 when compared with Disease Control Group. $ p?0.05 when compared with Vehicle Control Group, $ $ p?0.01 when compared with Vehicle Control Group. Animals were subjected to EPM test on 7th and 8th day to record ITL and Retention TL, respectively.The results of EPM conducted are illustrated above in table no. 1.1. Administration of Scopolamine (0.5mg/kg, i.p) showed significant (p<0>
Activity 1: Pole Climbing Test:
Animals were subjected to Pole Climbing test on 7th day to record escape latency.[table no-1.2]
Values are Mean ±SEM, n=6. Significant difference by Dunnets t test,*p?0.05 when compared with Disease Control Group, **p?0.01 when compared with Disease Control Group. $ p?0.05 when compared with Vehicle Control Group, $ $ p?0.01 when compared with Vehicle Control Group. Animals were subjected to Pole Climbing test on 7th day to record escape latency.
In the given treatment, the CAR (time taken by mice to climb the pole to avoiding the foot shock) time was recorded.[table no-1.2]Administration of Scopolamine (0.5mg/kg, i.p) showed significant (p<0>
Biochemical Estimations
1.Acetylcholinesterase (AChE) assay
The result mentioned in Mean ± SEM in table no.1.3 Administration of Scopolamine (0.5mg/kg, i.p) showed significant (p<0>
2.Malondialdehyde (MDA) level:
The result mentioned in Mean ± SEM in [table no.1.4] Administration of Scopolamine (0.5mg/kg, i.p) showed significant (p<0>
3.Glutathione (GSH) level:[ Table No. 1.5]
The level of anti-oxidant enzyme glutathione (GSH) was evaluated for their free radicals scavenging activity showed in table no.1.5 Administration of Scopolamine (0.5mg/kg, i.p) showed significant (p<0>
DISCUSSION
Neurological disorders are disorders of the central and peripheral nervous system. In other words, injury to the brain, spinal cord, cranial nerves, and peripheral nerves or in the neuromuscular junction and muscles leading to or from them can result in symptoms such as muscle weakness, poor coordination, loss of sensation, seizures, confusion, pain and altered levels of consciousness. These disorders include epilepsy, cerebrovascular diseases including stroke, migraine, neuroinfections, brain tumors and neurodegenerative diseases like Parkinson’s disease, Alzheimer’s disease and other dementias. According to WHO report, worldwide, around 50 million people have dementia, [2] and there are nearly 10 million new cases every year. This condition produces a great impact on the healthcare budget and social care.[7] Central cholinergic system plays a major role in learning and memory process through various neuronal pathways and neurotransmitters. Deficits occurring in these pathways may result in occurrence of various cognitive disorders like amnesia and dementia. Researches had confirmed tha both muscarinic and nicotinic acetylcholine receptors have a role in the encoding of new memories. There are certain agents that are used for memory impairment due to their capability of interfere in memory formation and neuroanatomical structures. Impairments of learning and memory can be induced chemically in experimental animals by Scopolamine. Scopolamine is a blocker of muscarinic acetylcholine receptor that serves as a beneficial pharmacological tool in producing a model of amnesia. There is notable evidence that scopolamine causes oxidative stress through the interference with acetylcholine in brain leading to cognitive impairment. In the present study, Scopolamine administration causes Significant increase in retention latency and escape latency in Elevated plus maze test[32] and Pole Climbing tests[33], respectively, in comparison to vehicle control group.[table no-1.1,1.2]. Administration of essential Oil of Hedychium coronarium linn. reversed the scopolamine induced memory impairment in mice. The results obtained in the present investigation demonstrated increase in brain AChE Activity in scopolamine induced amnesic mice as compared to vehicle control group. Treatment with Donepezil and EO of Hedychium coronarium linn. showed significant reduction of Scopolamine induced upsurge in AChE activity.[34]Along with cholinergic disturbances, oxidative stress can also induce impairment in learning and cognitive function. Scopolamine-induces neuronal damage by indusing oxidative stress in brain. Which is evident of increase lipid peroxidation and increase in malondialdehyde reduction in glutathione in scopolamine treated animals. Reduction in malondialdehyde and GSH level in brain after treatment with donepezil and essential Oil of Hedychium coronarium linn. indicates neuroprotective activity against scopolamine induced oxidative neuronal damage. The observed neuroprotective effect of essential oil of Hedychium coronarium linn. and Donepezil in the Present study was paralleled by decreased brain lipid peroxidation and increased GSH stores.[31]
The present study demonstrated that essential oil from flowers of Hedychium coronarium linn. has shown promising antiamnesic activity.[37] The observed neuroprotective effect of essential oil of Hedychium coronarium linn. in the present study was paralleled by decreased brain lipid peroxidation and increased GSH stores and reduction of scopolamine induced upsurge in AChE activity in mice. Hence essential oil of Hedychium coronarium linn. can be used for the management of Alzheimer’s disease and other neurodegenerative disorders.
|
Sr No. |
Abbreviation |
Full Form |
|
1 |
AD |
Alzheimer’s disease |
|
2 |
WHO |
World Health Organization |
|
3 |
AchE |
Acetylcholinesterase |
|
4 |
EO |
Essential Oil |
|
5 |
EOoralI |
Essential Oil oral I |
|
6 |
EOOralII |
Essential Oil oral II |
|
7 |
EOinhl |
Essential oil inhalation |
|
9 |
EPM |
Elevated Plus Maze |
|
10 |
SEM |
Standard Error Mean |
|
11 |
ITL |
Initial Transfer latency |
|
12 |
TL |
Transfer Latency |
|
13 |
CAR |
Conditioned Avoidance Response |
|
14 |
MDA |
Malondialdehyde |
|
15 |
GSH |
Glutathione |
|
16 |
DTNB |
5,5’- Di-thio-bis-(2-nitrobenzoate) |
|
17 |
DMAE |
Dimethylaminomethanol |
|
18 |
cAMP |
Cyclic Adenosine Monophosphate |
|
19 |
CPCSEA
|
Committee for the purpose of control and supervision ofexperiments on animals |
|
20 |
IAEC |
Institutional Animal Ethics Committee |
|
21 |
OECD |
Organisation for Economic Co-operation and Development |
REFERENCES
Sonali Pradeep Gopale , Screening Of Neuropharmacological Activity Of Herbal Extract, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 9, 1060-1075. https://doi.org/10.5281/zenodo.13824794
10.5281/zenodo.13824794
