In vitro Acetyl Cholinesterase Inhibitory assay of Acacia catechu Willd Ethanolic Seed Extract

Aim & objective: The aim of this study was to evaluate acetyl cholinesterase inhibitory activity of Acacia catechu ethanolic seed extract to introduce a new source for management of Alzheimer’s disease. Background: Alzheimer’s disease is a complex, multifactorial, progressive, neurodegenerative disease primarily affecting the elderly population and is estimated to account for 50–60% of dementia cases in persons over 65 years of age. It is likely that the inhibition of acetyl cholinesterase by Acacia catechu ethanolic seed extract that is rich in flavonoids and antioxidants may aid in the protection of neurodegenerative disorders and ultimately Alzheimer’s disease. Methods: Anti cholinesterase activity is determined by adopting In vitro standard protocol. Results: According to the obtained results, the inhibitory activity (IC50 values, μg /ml) of extracts was 204.38 ± 2.54 μg/ ml. Conclusion: The results indicated and confirmed the traditional use of Acacia catechu ethanolic seed extract for management of central nervous system disorders. It showed the moderate activity in inhibition of acetyl cholinesterase at various concentrations. However, further investigations on identification of active components in the extracts are needed.


SUMMARY
• Acacia catechu Willd.Belongs to the family fabacaae Commonly known as khadira in Sanskrit and karungali in Tamil is a very potent medicinal plant with diverse pharmacological actions.• Alzheimer's disease (AD) is an neurodegenerative disorder affecting people all over the world.It is associated with loss of cholinergic neurons in the brain and decreased level of Ach.
• Acacia catechu seed is rich in Catechin, Epicatechin and it also exhibits significant antioxidant property.• Acacia catechu seed inhibits Acetyl cholinesterase level hence, recommended for management of Alzheimer's disease.

INTRODUCTION
Alzheimer's disease (AD) is an neurodegenerative disorder associated with loss of neurons in the certain areas of brain which leads to cognitive impairment, neurological disturbances, behavioral abnormalities, even leads to death.The pathophysiology of AD is not clear, but it is believed to be associated with cholinergic pathway impairment, leading to reduction in acetylcholine level in certain regions of the brain. 1 Acetylcholine, a neurotransmitter, which is hydrolyzed by acetyl cholinesterase (AChE) and butyryl cholinesterase (BuChE) is considered to play an important role in the pathology of AD. 2 Neuritic plaques (Neurofibrillary tangles, amyloid plaques) are the major structural abnormality in seen commonly in AD patients.Amino acid Beta-amyloid peptide is an chief protein substance of amyloid in AD individuals. 3The therapy of AD depends on decreasing the progression of the disease by improving the quality of life.Inhibition of acetyl choline level may aid in control of AD.
Cognitive enhancers are used in management of AD, Few of the cognitive enhancers are only approved by FDA in united states. 4few drugs that have received regulatory approval currently includes donepezil, rivastigmine and galantamine, these drugs acts through increasing the concentration of acetylcholine at the neurotransmitter sites or directs by regulating activity at nicotinic receptors. 5rious side effects of medications reported in clinical trials causes nausea, vomiting, diarrhea, syncope and bradycardia. 6Absolutely, there requires a fundamental need for an alternative to anti-cholinesterase compounds with fewer side effects that leads to investigation on plants as a novel source of treatment of AD. 7 Natural sources have been used since antiquity in the treatment of various diseases including cognitive disorders, such as AD.
Considering the importance of plant based compounds in drug discovery, the present study was undertaken to evaluate the anti-cholinesterase activity of a number of selected medicinal plants with various ethno botanical uses, aiming to discover new compounds for anti-cholinesterase activity to be used in management of AD. [8][9][10] Acacia catechu (Family: Fabacaae) is an indigenous tree grown in all the parts of the world.Commonly known as karungali in Tamil and khadira in Sanskrit.Similarly to Neem, Turmeric, Aloe Vera, People in Kerala used this karungali leaves in boiling water, extracted juice for management of digestive disorders. 11It has been used in Ayurveda (Indian Medicinal System) for years extensively as an anti-inflammatory agent.3][14][15][16][17] It also has a role in dye industry the heartwood of khadira is employed for this purpose.Study reported that Acacia catechu Catechin produced promising result in cognitive enhancement in AD patients.Hence, Ethanolic extract of Acacia catechu seed is investigated for its anti-cholinesterase inhibitory activity for the first time.

Plant material
Acacia catechu seeds were collected from Hosur, Tamilnadu and was authenticated by Dr. H.B Singh, NISCAIR, New Delhi and the voucher specimen were stored for further use in Green Chem lab, Bangalore.

Ethanolic Extraction
Seeds were shade dried for a week.Dried seeds were milled to fine powder.Powder was passed through 100 mesh sieve and stored in a sealed polythene bag.

Determination of IC 50
The percentage inhibition was then calculated using the following formula;

% inhibition = [(Control OD -Sample OD)/Control OD] x 100
The experiment was done in triplicate and concentrations of the test extract that inhibit the hydrolysis of the substrate (acetylcholine) by 50% (IC 50 ) were determined by linear regression analysis between the inhibition percentage versus the extract concentration.

RESULT AND DISCUSSION
Neurodegenerative disease is a term applied to a variety of conditions arising from a chronic breakdown and deterioration of the neurons, particularly those of the central nervous system.Alzheimer's disease is associated with loss of cholinergic neurons in the brain and the decreased level of AChE.The major therapeutic target in the AD treatment strategies is the inhibition of brain AChE. 20Cholinesterase inhibitor drugs, inhibiting AChE activity, maintain ACh level by decreasing its breakdown rate. 21Although the underlying patho physiological mechanisms are not clear, AD is firmly associated with impairment in cholinergic pathway, which results in reduced level of acetylcholine (Ach) that is hydrolysed by cholinesterase (ChE) in certain areas of brain.Medications currently approved by regulatory agencies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) to treat the cognitive manifestations of AD and improve life quality of the patients are Donepezil, rivastigmine and galantamine as reversible AChE inhibitors, and memantine as an NMDA receptor antagonist.
][24] Plant based compounds have been used as cognition enhancers herbs like Withania somnifera, Centella asiatica, Curcuma longa, Bacopa monnieri, Convolvulus pluricaulis, Celastrus paniculatus, Nardostachys jatamansi have been reported to be used in management of cognitive impairment in Ayurvedic system of medicine. 25,26cetyl cholinesterase inhibitory activity is dose dependently increasing at a concentration ranging from 50 µg/ml to 300 µg/ml and when compared to standard Galanthamine the inhibitory activity is moderate even though the tested extract shows significant activity.The results are depicted in Table 1 and Figure 1 and can be used for treating neurodegenerative disorders.It was reported that the presence of Catechin, Epicatechin in Acacia catechu is responsible for treating cognitive impairment and the observed anti-cholinesterase activity found in the study could be due to the presence of Catechins, Quercetin and Epicatechin. 27

CONCLUSION
The results concluded that Acacia catechu ethanolic seed extract exhibited potential acetyl cholinesterase inhibitory activity, further In vivo studies are required to prove its cognitive efficacy.

Table 1 :
Anti cholinesterase inhibitory assay of Acacia catechu ethanolic seed