INTRODUCTION:

Alzheimer's disease (AD) is the most important leading cause of dementia in the aging population and the disease is characterized by neurodegeneration affecting the cortex and the limbic system, along with deposition of Aβ and neurofibrillary tangles.There are two major forms of cholinesterases. They are Acetylcholinesterase and butyrylcholinesterase.[1]

Numerous studies have been performed which are in support for the cholinergic hypothesis, which states that the dysfunction of a cholinergic system leads to memory deficit in models of animals which are similar to dementia seen in Alzheimer’s disease. The brains in such models illustrates characteristic degeneration of cholinergic neurons. It also shows a marked decline in cholinergic markets, acetyl cholinesterase and choline acetyltransferase.[2]

The neuropathological features which are seen in AD cannot be completely explained by the cholinergic deficits, but still it shows a significant part of AD pathology and supplementary research in this hypothesis is warranted.[3]

There is a reduction of Ach levels in the hippocampus and cortex of the brain seen in AD patients. Therefore, the current approach to treat AD patients is to inhibit AChE which is responsible for hydrolysis of ACh at Cholinergic synapse.[4]

Reactive oxygen and nitrogen species (ROS & RNS) through various mechanisms are risk factors which causes degeneration of neurons in Idiopathic AD. These are generated extracellularly and intracellularly. Due to brains high oxygen consumption, presence of high lipid content and lesser anti-oxidant compounds when compared with other tissues makes it vulnerable to oxidative stress.[5]

Studies in brain tissues seen in AD patients has been found to contain Advanced Glycation End products, 4HNE (4-Hydroxynoneal), markers of peroxidation, higher levels of oxidized protein. Amyloid β leads to lipoperoxidation of membranes and lipid peroxidation products which causes modification of proteins [6]. The aldehyde product of the peroxidation process is 4-HNE which is generated due to the exposure of Aβ peptide to the neuronal membranes. This leads to the oxidative damage of protein causing an increase in carbonyl groups. [7]

The aim of this study is to investigate the presence of AChE or BuChE inhibitors in Garciniahanburyi. [8] The ethylacetate of the plant is taken because of the presence of Xanthones. The extract is then screened for its AChE, BuChE and anti-oxidant activity to screen them for its efficacy in treating AD.

MATERIALS AND METHODS:

DPPH, Acetylthiocholineidodide (ATCI), AChE, Bovine serum albumin (BSA), DTNB, Physostigmine, Gallic acid, ascorbic acid. All the other reagents are of analytical grade.

PLANT MATERIALS:

The plant materials are procured from Rajesh Chemicals, Mumbai. It is authenticated by Prof. P. Jayaraman, Plant Anatomy Research Center, Tambaram. The registration number of the certificate is PARC/2017/3577.

PREPARATION OF EXTRACT OF GARCINIA HANBURYI:

Extracts were prepared by the maceration method at 60ºc for 72 hours using ethanol 70% and ethylacetate. The extracted samples were evaporated using water bath. The thick extract obtained is made into various concentrations for the study (5,25,100,200 µg/l)

PRELIMINARY PHYTOCHEMICAL ANALYSIS:

The plant extract were used for preliminary screening of active constituents such as Phenols, Alkaloids, steroids, resins, proteins, carbohydrates and saponins. [9] The phytochemical analysis were performed using the standard procedures to investigate the constituents present in the plant. [10,11]

DETERMINATION OF ANTIOXIDANT ACTIVITY BY SCAVENGING EFFECT ON 2,2’-DIPHENYL-1PICRYL HYDRAZYLRADICAL (DPPH):

Anti-oxidant potential of the Ethylacetate extract was estimated using modified DPPH free radical scavenging assay. The stock solution of the Ethylacetate extracts were prepared in 1mg/ml in methanol. Each well was filled in with 200 µl extract in methanol at 1000 µg/ml.

Then, 5µl of the DPPH solution (2.5 mg/ml in methanol) was added to each well. The plate is allowed to develop in the dark for 30 minutes. The optical density of the plate was read using a microplate reader at 517nm. Percentage inhibition was calculated using:

% Inhibition=1 OD (DPPH + Sample)/OD(DPPH)X 100

The percentage inhibition of free radical activity was plotted against concentration of the ethyl acetate extract and IC50 Concentration for 50% inhibition was obtained. The percentage inhibition was compared with ascorbic acid which was the positive control. [12]

DETERMINATION OF ACETYLCHOLINE STERASE AND BUTRYLCHOLINESTERASE INHIBITORY ACTIVITY USING MICROPLATE ASSAY:

AChE activity was measured using a modified 96-well microplate assay which is based on Ellman’s method. [13] In this assay the enzyme hydrolyses the acetylthiocholine or butyrylthiocholine resulting in the formation of thiocholine on its reaction with Ellman’s reagent (DTNB) produces 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which is detected at 412nm. [14]

The reaction volume is made of 220 μl which comprises of 170 μl (0.1 mol/l) sodium phosphate buffer, 10 μl of the extract solution, 10 μl DTNB, 20ul of AChE/BuChE (0.45 U/ml), 10 μl of acetylthiocholine iodide/butyrylthiocholine iodide (Concentration 0.68 mmol/l).

The methanolic and the ethanolic extracts were tested for the BuChE and AChE inhibitory activity from concentrations 5,25,100,200μg/ml respectively. They are prepared using phosphate buffer. Galanthamine was used a positive control with the same concentrations. The buffer, extract and the enzme were incubated 15 mins at 40c. The reaction is initiated by addition DTNB and the substrate. It is incubated at 25ºc for 30 minutes and absorbance is read at 405nm. The blank consists of buffer, enzyme without the test extracts. The percentage inhibition is calculated using the following formula:

% inhibition = (E S)/E X 100

E= Activity of enzyme without extract

S= Activity of enzyme with extract

IC50 was calculated from the % inhibition values of the plant extract.

STATISTICAL ANALYSIS:

Data were expressed as mean ± standard deviation for separate groups for determinations in triplicates. IC50 values were calculated using GraphPad Prism v 5.0 (GraphPad software Inc., USA).

RESULTS:

Table 1: Determination Of Anti-Oxidant Activity By Dpph Assay

Method	Sample	Concentration μg/ml	Activity (%)	IC50
DPPH	Methanolic Extract	5	20.12	17.604
		25	65.14
		100	81.74
		200	82.11
	Ascorbic Acid	5	18.14	15.36
		25	67.78
		100	83.22
		200	83.91
	Ethylacetate Extract	5	24.9	14.693
		25	75.24
		100	89.14
		200	89.50
	Ascorbic Acid	5	18.14	15.36
		25	67.78
		100	83.22
		200	83.91

Table 2: Determination of Ache and Buche Inhibition by Ellman’s Method

	Concentration μg/ml	% BuChE Inhibition	%AChE Inhibition
Methanolic Extract	5	6.4 ± 0.4	50.7 ± 2.0
	25	12.3 ± 0.3	53.2 ± 2.7
	100	25.4 ± 0.4	60.2 ± 9.2
	200	57.7 ± 0.1	67.4 ± 9.3
Ethylacetate Extract	5	4.0± 0.1	50.1 ± 0.9
	25	15.4 ± 0.5	58.7 ± 3.6
	100	59.6 ± 3.5	61.3 ± 4.1
	200	60.2 ± 4.2	73.5 ± 2.4
Galanthamine	5	14.1	18.4
	25	32.1	39.4
	100	60.3	59.4
	200	60.3	81.4

Figure 1: Determination of Anti-Oxidant Activity by Dpph Assay

Figure 2: Determination of Buche Inhibition by Ellman’s Method

Figure 3: Determination of Ache Inhibition by Ellman’s Method

DISCUSSION:

The deficiency of Acetylcholine is responsible for most of the symptoms seen in Alzheimer's disease such as cognitive decline and memory impairment. Reduction in the levels of Acetylcholine, butyrlcholine is one of the characteristic feature of Alzheimer's disease. There has been various drugs to treat this particular decline such as Rivastigmine, Donepezil, Galanthamine etc. These AChE inhibitors show various side effects such as toxicity, tolerability and a loss of efficiency. The side effects of these drugs as well as their low efficacy in treating Alzheimer’s has posed a serious question on the treatment of the disease. This has led to a search for alternative drugs from natural origin. [15]

In the present study, the ethyl acetate and ethanolic extract of the plant Garciniahanburyi has been selected due to the presence of Xanthones. Xanthones an important phytoconstituent whose pharmacological actions has been studied extensively during recent times. Garciniahanburyi containing xanthones has been proven to have actions such as anti-inflammatory, anti-cancer agent etc. [16,17] Since extracts can contain various number of phytochemical constituents and secondary metabolites they may interact together to produce a biological activity. The activity demonstrated by this extract maybe produced due to the dominant major constituent which in this case was found to be Xanthones.

Acetylcholinesterase and Butrylcholinesterase Inhibitory Activity of the Extracts:

The ethyl acetate fraction has been utilized for AChE inhibitory activity using Ellman’s colorimetric assay using 96 well plate. [18,19] Results are shown in Table 1. Galanthamine was used as the standard AChE inhibitor in this study which showed a % inhibition of AChE at 81.4%.

The percentage inhibition of the Ethanolic extract for AChEwas found to be73.5 % and Ethylacetate extract was found to be 67.4 % respectively.

The percentage inhibition of the Ethanolic extract for BuChE was found to be 57.7 % and Ethylacetate extract was found to be 60.2 % respectively.

Free Radical Scavenging Activity of the Extracts using the DPPH Assay:

The anti-oxidant activity of the ethyl acetate fractions were determined by using free radical 1,1-diphenyl-2- picrylhydrazyl (DPPH) performed by the addition of various concentrations of extracts with DPPH. The DPPH amount which is remaining was determined at 30 minutes based on the absorbance at 517 nm. IC50 values for DPPH scavenging activity is calculated for concentrations of the extract. The IC50 values are shown in Table 2. [20] The IC50 values of theethanolic extract was found to be 17.604 and for Ethyl acetate extract 14.693 It exhibited good activity for DPPH scavenging when compared with that of the standard Ascorbic acid with an IC50 value of 15.36. [21, 22]

CONCLUSION:

The various biological activities which were examined such as anti-oxidant, anti-AChE and BuChE property of the plant Garciniahanburyi warrants further study. The results which have been obtained has indicated that the anti-oxidant activity and potent activity to inhibit acetylcholine esterase and butrylcholinesterase has made them an effective, new therapeutic agents for the treatment of Alzheimer's disease. The extract which are analyzed can be further investigated either as a single isolated compound or as an extract to be used as a therapeutic agent.

ACKNOWLEDGMENTS:

The authors are grateful for SRM College of Pharmacy, SRM University for supporting this study.

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Received on 07.12.2017 Modified on 07.01.2018

Research J. Pharm. and Tech 2018; 11(7): 2918-2921.

DOI: 10.5958/0974-360X.2018.00538.3