Evaluating the antioxidant and acetylcholinesterase inhibitory activities of some plants from Kohgiluyeh va Boyerahmad province, Iran

Research Journal of Pharmacognosy (RJP) 3(4), 2016: 1-7 Received: June 2016 Accepted: Aug 2016 Original article Evaluating the antioxidant and acetylcholinesterase inhibitory activities of some plants from Kohgiluyeh va Boyerahmad province, Iran H. Hajimehdipoor1, L. Ara2, H. Moazzeni3, S. Esmaeili1* 1 Traditional Medicine and Materia Medica Research Center and Department of Traditional Pharmacy, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2 Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3 Department of Botany, Research Center for Plant Sciences, Ferdowsi University of Mashhad, Mashhad, Iran. Abstract Background and objectives: Alzheimer’s disease (AD) is a neurodegenerative disorder. Nowadays, many investigations are performed to find new drugs for AD and medicinal plants are considered as one of the most important sources for developing new drugs. According to the role of oxidant agents and acetylcholinesterase enzyme (AChE) in AD, plants with antioxidant and AChE inhibition properties could be good candidates for AD studies. In the present investigation, acetylcholinesterase inhibition (AChEI) and antioxidant effects of some plants from Kohgiluye va Boyerahmad province of Iran have been determined. Methods: The plants collected from Kohgiluyeh va Boyerahmad province (56 species) were extracted with methanol by using maceration method. AChEI activity of the extracts was determined using Ellman method in 96-well microplates. Antioxidant activity was determined using DPPH and FRAP methods. Results: The results showed that aerial parts of Amygdalus scoparia had the highest AChEI effect (50% inhibition in concentration of 300 µg/mL). The plant also demonstrated suitable antioxidant effects. Epilobium minutiflorum found to be the most potent species for DPPH inhibition and reduction of ferric-TPTZ complex (IC50 3.6 µg/mL and FRAP value 335.0 mmol FeSO4.7H2O/100g Extract). Conclusion: Our results confirmed that almost all species with AChEI activity showed to be effective as potent antioxidant agents. Keywords: acetylcholinesterase, antioxidant, DPPH, FRAP, Kohgiluyeh va Boyerahmad Introduction Alzheimer's disease (AD) is one of the most widespread degenerative illnesses of the human brain which involves multiple neuronal systems. In this case, a specific pattern of lesions evolves slowly over time and remains remarkably consistent across cases [1]. The pathogenesis of AD has not yet been elucidated. It is widely accepted that a combination of genetic susceptibility factors and environmental triggers are responsible for AD. It is proposed that beta amyloid protein, abnormal tau protein, oxidative damages, cholinergic deficit and slow inflammatory processes are possible mechanisms involved. No product with proven disease modifying properties is available yet, and current treatments offer symptomatic benefit Available at: http://rjpharmacognosy.ir Copy right© 2014 by the Iranian Society of Pharmacognosy * Corresponding author: , Tel/Fax: +9821-88776027 Hajimehdipoor H. et al. only [2-6]. One of the ways to control AD is using acetylcholinesterase inhibitors (AChEIs) [7]. Because of low efficacy and side effects of the existing drugs, many researches being performed in order to find new drugs and plants are usually considered as good sources for drug discovery [8-12]. One of the causes of AD is oxidative destruction of brain neurons [3]. Free radicals are usually produced in human body during biochemical reactions and they are harmful for body cells; but there are several endogenic mechanisms to eliminate these radicals. If free radicals are produced more than toleration of body defense system, they cause damage to body. It has been established that free radicals can induce many diseases in human body such as AD, Parkinson’s disease, inflammations, cardiovascular disorders, cancers, diabetes, etc; therefore, using antioxidant agents may prevent mentioned disorders. Many medicinal plants have antioxidant activity and could be used in AD [13-16]. Kohgiluyeh va Boyerahmad is a mountainous province in south-west of Iran. Lots of endemic plants grow in this area are used for prevention and treatment of disorders as folklore medicine [17]. Despite many medicinal herbs grow in this area, a few researches have been carried out about their properties. In the present investigation, in order to find medicinal plants for further AD studies, antioxidant and AChEI activities of 56 species of this area have been evaluated. Experimental Plant material Fifty six species were collected from Kohgiluyeh va Boyerahmad province, Iran during 2009-2011. They were identified by Dr. H. Moazzeni and Dr. A. Pirani. A voucher specimen from each plant was kept at the Herbarium of TMRC, SBMU, Tehran, Iran. Chemicals Acetylthiocholine iodide (ATCI) was obtained from Fluka (Germany). Acetylcholinesterase enzyme (AChE) from bovine erythrocytes and 2,2-diphenyl-1-picryl hydrazyl (DPPH) were 2 prepared from Sigma (Germany). 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) and TPTZ (2,4,6tri-(2-pyridyl)-1,3,5-triazine) were purchased from Merck (Germany). Methanol and all other solvents were provided from Merck (Germany). Plants extraction Fifty g of each plant powder was macerated with methanol for 24 h. The mixture was filtered and concentrated using rotary evaporator and dried. It was kept in refrigerator before test. Acetylcholinesterase inhibitory assay AChEI activity was determined using a microplate reader based on Ellman’s method [8,18,19]. Briefly, 125 μL of 3 Mm DTNB, 25 μL of 15 mM ATCI and 50 μL of phosphate buffer pH 8, and 25 μL of sample (were dissolved in methanol 3 mg/mL) were added to 96-well plates. The absorbance was measured at 405 nm every 13 sec for 65 sec. 25 μL of 0.22 U/mL of AChE enzyme was then added and the absorbance was recorded every 13 sec for 104 sec. The absorbance was plotted against time and the enzyme activity was calculated. Any increase in the absorbance due to the nonenzymatic hydrolysis of substrate was corrected by subtracting the rate of reaction before addition of the enzyme from the rate after addition of the enzyme. Percentage of enzyme inhibition was calculated by comparing the rates for the sample to the blank (using methanol without extract). Donepezil was used in the experiment as the positive control and its IC50 was calculated. 2,2-Diphenyl-1-picryl hydrazyl (DPPH) radical scavenging assay This technique is one of the most widely used methods for evaluating antioxidant activity of samples with natural sources. The method is based on scavenging of DPPH free radicals by antioxidants which induce a decrease in absorbance at about 520 nm. When a DPPH solution is mixed with a hydrogen donating substance, the reduced form of DPPH radical is generated accompanied by loss of color [20]. In the current experiment, in order to determine DPPH r (...truncated)