In vitro Anti-Alzheimer and Anti Oxidant activity of the Peels of Citrus maxima fruits

P. Vijayalakshmi*, R. Radha

Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai – 600003

*Corresponding Author E-mail: vijayapharma7@gmail.com

ABSTRACT:

Alzheimer’s disease is an age-associated, irreversible, progressive neurodegenerative disease which is characterized by severe memory loss, unusual behavior, personality changes, and a decline in cognitive function. Even today Alzheimer disease is spreading in very fast pace though large number of researchers and manufacturers are engaged to find effective medication but result is not satisfactory. Most of the drugs or medicines have high tendency of side effect. This fact made me inclined towards plants to execute the research work. Normally it is observed that side effects in plant derivative are very less. Most potential target for the treatment and slowing down of Alzheimer disease progression is cholinesterase inhibitors from plants. The anti oxidant and anti cholinesterase activity of various extracts of Citrus maxima fruit peels were studied by DPPH assay and Hydrogen Peroxide radical scavenging effect for in vitro analysis; Whereas Ellman’s colorimetric methods were used for anti cholinesterase activity estimation. Ethanolic extract shown the potent anti oxidant (DPPH; IC₅₀= 25 µg/ml , HPSA; IC₅₀= 54 µg/ml) and anti cholinesterase activity with IC₅₀value of 115µg/ml.

These plants were found to be potent enough to be studied further for its in vivo Anti Alzheimer disease and to perform formulations.

KEYWORDS: Alzheimer disease, Citrus maxima, anti oxidant, anti cholinesterase activity.

INTRODUCTION:

Alzhiemer’s or Alzhiemer disease accounts for about 60 to 70% of cases of dementia. It is a chronic neurodegenerative disease that usually starts slowly and gets worse over time^1,2.

It is a peculiar disease of cerebral cortex identified by Dr. Alios Alzheimer in 1906 and was later named as Alzheimer’s disease³. It is described as thin cortex speckled with usual brown clumps and irregular knots that was found to be growing inside the brain cells and are now known as amyloid plaques and neurofibrillary tangles. The brains of people with AD have been found to have abundant of two abnormal structures. Amyloid plagues and neurofibrillary tangles which are made of misfolded proteins. Diagnosed in both the gender over 65 years of age⁴.

In 2006 26.6 million people of age more than 65 years was found to be affected by AD (Alzheimer’s disease). It was predicted to affect 1 in 85 people globally by 2050⁵. The symptoms of Alzheimer’s disease is unique for every individual⁶. AD is diagnosis confirmed with behavioral assessments, cognitive tests and followed by brain scan⁷. Recently researchers have found that activity-dependent neuroprotective protein (ADNP) in elderly individuals can assess the patients at risk for Alzheimer's disease. ANDP levels in blood and amyloid plaques (a hallmark Alzheimer's) in the brain," said lead researcher Illana Gozes from Tel Aviv University in Israel.

Several biochemical and pathological mechanism contribute to AD. These include (i). Imbalance in production of amyloid β (Aβ) vs its clearance resulting in accumulation of Aβ in the form of senile plaques (Amyloid hypothesis)⁸. These plaques can induce neuroinflammatory process and trigger the production reactive oxygen species⁹. ( ii) Cholinergic deficits resulting due to enhanced activity of acetylcholinesterase and Butyl cholinesterase, contributing to the cognitive impairments seen in AD (Cholinergic hypothesis)¹⁰. Based on those hypotheses several anti amyloid¹¹ and cholinesterase inhibitor¹² are investigated for their disease modifying stratergies

Plant have been a major source of medicine. About 80% of the population in developing countries yet relies on plant based medicines to obtain primary health care WHO 1978¹³.

Citrus maxima (Linn) of family Rutacea commonly known as Pamelo, Pomelo, Sadhaphal etc. It was traditionally used in Epilepsy, cholera, Cough, Asthma etc¹⁴. It has been evaluated for its anti diabetic¹⁵ , CNS activity¹⁶, Hypocholestremic activity¹⁷, Analgesic, anti inflammatory¹⁸, anti depressant¹⁹, anti tumor²⁰,anti oxidant activity of leaves²¹, using various parts of plant. The present study is on the in vitro anti oxidant and in vitro anti alzheimer activity of the dried peels of Citrus maxima.

MATERIAL AND METHODS:

Plant Material

Citrus maxima fruits were collected from Cuddalore (Tamil Nadu) in June and July 2015. The plant was authenticated for its identity by Scientist F and Head of office Dr. G.V.S. Muruthy Botanical Survey of India, Coimbatore with survey no: BSI/SR/5/23/2016/Tech/33. It was identified as Citrus maxima (Burm.) Merr. (= Aurantium maximum Burm.)-Rutaceae

Preparation of samples

The Peels of fruit Citrus maxima were shade dried, cut into pieces, powdered and was stored in the air tight container and a portion of it is used for the Pharmacognostical and Phytochemical studies and remaining it was extracted.

Extraction

Plant material was dried in shadow and ground to powder for extraction. The extraction was done using Soxhlet apparatus and extracted with the solvents of increasing polarity like Hexane, Ethyl acetate, ethanol and was macerated with water at the end of soxhlet extraction.

In Vitro Anti oxidant

In vitro antioxidant activities for the extracts were carried out using the following methods.

1. DPPH assay

2. Hydrogen peroxide scavenging assay.

DPPH Assay: 1, 2-Diphenyl-2-picryl hydrazyl Radical (DPPH)²²

Initial volume 0.1 mL of various concentrations of samples was mixed with 0.4 mL of 0.3M DPPH reagent prepared in ethanol. The mixture was shaken thoroughly and incubated in the darkness at room temperature for 30 min. The absorbance of the reaction was measured spectrophotometrically at 517nm, immediately after mixing and then after incubation as well. The scavenging effect of DPPH free radical was calculated by using the following equation.

% Scavenging activity =

Abs (control) - Abs (standard) / Abs (control) × 100

Where control is the absorbance of the blank (a reaction with all the reagents except the test extract), and absorbance of sample is the absorbance of the test extract. Tests were carried out in triplicates to obtain 50% inhibition (IC50). Using Butylated hydroxy Toulene.

Hydrogen Peroxide Scavenging Assay (HPSA)²³

The ability of the extracts to scavenge hydrogen peroxide was determined according to the method of Ruch. A solution of Hydrogen peroxide (2mmol/l\lt) was prepared in phosphate buffer (PH 7.4). Various concentrations of extracts (10-100µg/ml) were added to hydrogen peroxide solution (0.6ml). Absorbance at 230nm was determined after 10min against a blank solution containing phosphate buffer without hydrogen peroxide.

% Scavenging activity =

Abs (control) - Abs (standard) / Abs (control) × 100

Compared with the ascorbic acid standard.

Anti-Acetylcholinesterase Assay²⁴

The enzymatic activity was determined by Ellman’s methods developed in year 1961. It is used to measure extract for its Ache inhibition. Electric cell was considered as the enzyme source, acetyl thiocholine iodide was used as substrate and 5, 5, bis dithionitrobenzene, a dye was used for the measurement of cholinesterase activity.

In this assay, 150 µl of 0.1M sodium phosphate buffer (pH 8), 10 µl of test compound solution and 20µl of enzyme solution (0.09 units/ml) were mixed and incubated at 25⁰c for 15 min. 10 µl of the DTNB solution, 10 µl of acetyl thiocholine iodide were added to the resultant and allowed to withstand for 10 min. The intensity of the colour produced were studied using spectrophotometric observation at 410 nm. Physostigmine dissolved in ethanol is used as a standard. Percentage of enzyme inhibition was determined using the formula below

% Enzyme inhibition =

[(Optical density of test-blank) X 100]

Optical density of standard-blank

Statistical analysis:

All determinations were done for three times and results reported as mean ± Standard deviation. IC₅₀ values were calculated and reported.

RESULT AND DISCUSSION:

Antioxidant activities

A number of studies shown that oxidative stress is involved in age related neurodegenerative diseases. Likewise, there are numerous studies which have examined the positive benefits of antioxidants to reduce or block neuronal death occurring in the pathophysiology of these disorder. In addition, the antioxidant potential of a compound can be attributed to its radical scavenging ability and total antioxidant activity. In order to determine the ability of the plant extracts to serve as antioxidants, two activities were measured; ability to scavenge DPPH and Hydrogen Peroxide Scavenging assay.

DPPH radical scavenging activity :

Various extracts were studied for its anti oxidant activity using 1, 2-diphenyl-2-picryl hydrazyl radical (DPPH). Results are discussed in Table 1 and Fig 1.

Table 1: DPPH Radical Scavenging activity of various extract

S.No	Concentration (µg/ml)	% Inhibition
S.No	Concentration (µg/ml)	Standard (Butylated hydroxyl Toulene )	Hexane	Ethyl acetate	Ethanol	Aqueous
1.	20	53.95	19.43	23.61	45.94	19.34
2.	40	64.81	29.32	31.28	59.86	29.34
3.	60	76.62	30.54	33.59	68.18	30.34
4.	80	85.21	31.34	43.16	76.95	34.32
5	100	91.45	37.34	52.23	88.87	37.45
IC₅₀(µg/ml)		10	152	97	25	147

Fig 1: DPPH Radical Scavenging activity of various extract

Table 2: Hydrogen peroxide Radical Scavenging activity of various extract

S no	Concentration	% inhibition
S no	Concentration	Standard (Ascorbic acid)	Hexane	Ethyl acetate	Ethanol	Aqueous
1	20 µg/ml	32.23	16.43	36.51	40.43	12.34
2	40 µg/ml	38.13	20.32	42.23	45.65	24.54
3	60 µg/ml	56.34	23.34	46.43	50.81	28.45
4	80 µg/ml	73.32	29.34	52.34	57.65	32.54
5	100 µg/ml	88.34	43.23	53.43	65.56	38.56
IC ₅₀(µg/ml)		47	127	77	54	131

Fig 2: Hydrogen peroxide Radical Scavenging activity of various extract

The acetyl cholinesterase enzyme is a biomarker in amnesia. In this assay the percentage enzyme inhibition has been recorded for all the extracts. The ethanolic extract shown IC₅₀ value of 115 µg/ml were the hexane, ethyl acetate, aqueous extract shown the IC₅₀(µg/ml)value of 215, 159 and 950 respectively. It was observed that all the extract posses inhibitory activity against AChE, but ethanolic extract of Citrus maxima had good AChE inhibitory activity. Ethanol used as a solvent and it produced better activity as compared to aqueous is described in various reports²⁵. The higher activity of the ethanolic extract may be due the organic solvents which are able to extract compounds with higher AChE inhibitory activity²⁶. Thus the ethanolic extract can be used for the further studies.

CONCLUSION:

The study included in vitro anti oxidant and in vitro AChE inhibitory activity of the various extracts of Citrus maxima. Ethanolic extract of Citrus maxima fruit peels were found to show good radical scavenging activity in dose dependent manner. In vitro acetyl cholinesterase enzyme inhibition study by modified Ellman’s method confirms that the ethanolic extract of Citrus maxima maximum inhibition while comparing the IC₅₀Values of all extracts. Thus these extract have good anti oxidant potency and AchE inhibitory activity. It has thus paved a new path for an alternative anti oxidant and AChE inhibitor and futher in vivo studies could be carried out based on these reports.

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Received on 17.02.2016 Modified on 05.03.2016

Res. J. Pharmacology & P’dynamics. 8(1): Jan.-Mar., 2016; Page 17-22

DOI: 10.5958/2321-5836.2016.00005.7

Extract	IC₅₀ value
Hexane extract	215 µg/ml
Ethyl acetate extract	159 µg/ml
Ethanol extract	115 µg/ml
Aqueous extract	950µg/ml