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Antioxidant and Acetylcholinesterase Inhibitory Activity of Selected Southern African Medicinal Plants ⁎ E.A

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Antioxidant and Acetylcholinesterase Inhibitory Activity of Selected Southern African Medicinal Plants ⁎ E.A Available online at www.sciencedirect.com South African Journal of Botany 77 (2011) 638–644 www.elsevier.com/locate/sajb Antioxidant and acetylcholinesterase inhibitory activity of selected southern African medicinal plants ⁎ E.A. Adewusi a, N. Moodley b, V. Steenkamp a, a Department of Pharmacology, School of Medicine, Faculty of Health Sciences, PO Box 2034, University of Pretoria, Pretoria 0001, South Africa b Biosciences, Council for Scientific and Industrial Research, Pretoria, South Africa Received 30 September 2010; received in revised form 23 November 2010; accepted 23 December 2010 Abstract Alzheimer's disease (AD) is the most common type of dementia in the aging population. Enhancement of acetylcholine levels in the brain is one means of treating the disease. However, the drugs presently used in the management of the disease have various drawbacks. New treatments are required and in this study, extracts of Salvia tiliifolia Vahl. (whole plant), Chamaecrista mimosoides L. Greene (roots), Buddleja salviifolia (L.) Lam. (whole plant) and Schotia brachypetala Sond. (root and bark) were evaluated to determine their polyphenolic content, antioxidant and acetylcholinesterase inhibitory (AChEI) activity. The DPPH and ABTS assays were used to determine antioxidant activity and Ellman colorimetric method to quantify AChEI activity. Although all four plants showed activity in both assays, the organic extracts of C. mimosoides root was found to contain the highest AChEI activity (IC50 =0.03±0.08 mg/ml) and B. salviifolia whole plant had the highest antioxidant activity (ABTS; IC50 =0.14±0.08 mg/ml and DPPH; IC50 =0.23±0.01 mg/ml). The results suggest that the tested plant species may provide a substantial source of secondary metabolites, which act as natural antioxidants and acetylcholinesterase inhibitors, and may be beneficial in the treatment of AD. © 2011 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Acetylcholinesterase; Alzheimer's disease; Antioxidant; Medicinal plants; Neurodegeneration 1. Introduction deficient cholinergic neurotransmission (Giacobini, 1998; Krall et al., 1999). Dementia is characterized by the gradual onset and con- Selective cholinesterase inhibitors, free of dose-limiting side tinuing decline of higher cognitive functioning (Dhingra et al., effects, are not currently available, and current compounds may 2005). Alzheimer's disease (AD), the most common form of not allow sufficient modulation of acetylcholine levels to elicit the dementia (Nie et al., 2009), is a progressive age-related full therapeutic response (Felder et al., 2000). In addition, some of disorder that is characterized by the degeneration of neuro- the synthetic medicines used e.g. tacrine, donepezil and logical function. The latter is due to the reduction in levels rivastigmine have been reported to cause gastrointestinal dis- of the neurotransmitter acetylcholine, in the brains of the turbances and problems associated with bioavailability (Melzer, elderly as the disease progresses, resulting in loss of cog- 1998; Schulz, 2003). Therefore, the search for new AChEIs, nitive ability (Felder et al., 2000). Acetylcholinesterase in- particularly from natural products, with higher efficacy continues. hibitors (AChEIs) have been shown to function by increasing Oxidative stress, caused by reactive oxygen species (ROS), acetylcholine within the synaptic region, thereby restoring is known to result in the oxidation of biomolecules, thereby leading to cellular damage and it plays a key pathogenic role in the aging process (Zhu et al., 2004). In recent years, there has ⁎ Corresponding author. Tel.: +27 12 3192457; fax: +27 12 3192411. been growing interest in finding natural antioxidants in plants E-mail address: [email protected] (V. Steenkamp). because they inhibit oxidative damage and may consequently 0254-6299/$ - see front matter © 2011 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2010.12.009 E.A. Adewusi et al. / South African Journal of Botany 77 (2011) 638–644 639 prevent aging and neurodegenerative diseases (Fusco et al., the wells and the absorbance measured five times consecutively 2007). every 45 s. Galanthamine served as the positive control. Any In an effort to discover new sources which can potentially be increase in absorbance due to the spontaneous hydrolysis of the used in the treatment of AD, four plants — Salvia tiliifolia Vahl. substrate was corrected by subtracting the absorbance before (Lamiaceae), Chamaecrista mimosoides L. Greene (Caesalpi- adding the enzyme from the absorbance after adding the niaceae), Buddleja salviifolia (L.) Lam. (Buddlejaceae) and enzyme. The percentage inhibition was calculated using the Schotia brachypetala Sond. (Fabaceae), traditionally used in the equation: treatment of neurodegenerative diseases (Orhan et al., 2007; Stafford et al., 2008), were evaluated for their AChEI and ÀÁ InhibitionðÞ % =1− A = A × 100 antioxidant capacity. sample control 2. Material and methods where Asample is the absorbance of the sample extracts and Acontrol is the absorbance of the blank [methanol in Buffer A 2.1. Chemicals (50 mM Tris–HCl, pH 8)]. Extract concentration providing 50% inhibition (IC50) was obtained by plotting the percentage Acetylthiocholine iodide (ATCI), acetylcholinesterase inhibition against extract concentration. (AChE) type VI-S, from electric eel, 5,5′-dithiobis[2-nitroben- zoic acid] (DTNB), galanthamine, 1,1-Diphenyl-2-picrylhydra- zyl (DPPH), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic 2.4. Determination of total phenolics acid (ABTS) and trolox were purchased from Sigma. Methanol and all other organic solvents (analytical grade) were purchased Total phenolic content in the extracts were determined by the from Merck. modified Folin–Ciocalteu method of Wolfe et al. (2003). The extract (1 mg/ml) was mixed with 5 ml Folin–Ciocalteu reagent 2.2. Plant collection and extract preparation (diluted with water 1:10 v/v) and 4 ml (75 g/l) sodium carbonate. The mixture was vortexed for 15 s and allowed to The plant species; S. tiliifolia (whole plant; P03649), stand for 30 min at 40 °C for color development. Absorbance C. mimosoides (root, P08814), B. salviifolia (whole plant, was measured at 765 nm using the Hewlett Packard UV–VIS P01281), S. brachypetala (bark, P08514) and S. brachypetala spectrophotometer. Total phenolic content is expressed as mg/g (root, P06300) were collected in Gauteng Province, South tannic acid equivalent using the following equation based on the Africa. Identities of the specimens were confirmed by the South calibration curve: y=0.1216x, where x is the absorbance and y African National Biodiversity Institute (SANBI), Tshwane and is the tannic acid equivalent (mg/g). voucher specimens are deposited at this institution. The plant samples were cut into small pieces and dried in an oven at 30–60 °C for 48 h. Dried material was ground to a coarse 2.5. Determination of total flavonoids powder using a hammer mill and stored at ambient temperature prior to extraction. Six grams of the powdered plant material Total flavonoid content was determined using the method of was extracted with 60 ml of either dichloromethane/methanol Ordonez et al. (2006). A volume of 0.5 ml of 2% AlCl3 ethanol (1:1) or distilled water for 24 h. Organic extracts were solution was added to 0.5 ml of sample (1 mg/ml). After one concentrated using a rotary vacuum evaporator and then further hour at room temperature, the absorbance was measured at dried in vacuo at ambient temperature for 24 h. The aqueous 420 nm. A yellow color is indicative of the presence of extracts were concentrated by freeze-drying. All extracts were flavonoids. Total flavonoid content was calculated as quercetin stored at −20 °C prior to analysis. The residues were equivalent (mg/g), using the following equation based on the redissolved in DCM:MeOH or distilled water, respectively to calibration curve: y=0.025x, where x is the absorbance and y is the desired test concentrations. the quercetin equivalent (mg/g). 2.3. Micro-plate assay for inhibition of acetylcholinesterase 2.6. Determination of total proanthocyanidins Inhibition of acetylcholinesterase activity was determined using Ellman's colorimetric method as modified by Eldeen et al. The procedure reported by Sun et al. (1998) was used to (2005). Into a 96-well plate was placed: 25 μl of 15 mM ATCI determine the total proanthocyanidin content. A volume of in water, 125 μl of 3 mM DTNB in Buffer C (50 mM Tris–HCl, 0.5 ml of 1 mg/ml extract solution was mixed with 3 ml of a 4% pH 8, containing 0.1 M NaCl and 0.02 M MgCl2.6H2O), 50 μl vanillin–methanol solution and 1.5 ml hydrochloric acid. The of Buffer B (50 mM, pH 8, containing 0.1% bovine serum mixture was allowed to stand for 15 min after which the albumin) and 25 μl of plant extract (0.25, 0.5, 1 or 2 mg/ml). absorbance was measured at 500 nm. Total proanthocyanidin Absorbance was measured spectrophotometrically (Labsystems content is expressed as catechin equivalents (mg/g) using the Multiscan EX type 355 plate reader) at 405 nm every 45 s, three following equation based on the calibration curve: y=0.5825x, times consecutively. Thereafter, AChE (0.2 U/ml) was added to where x is the absorbance and y is the catechin equivalent (mg/g). 640 E.A. Adewusi et al. / South African Journal of Botany 77 (2011) 638–644 2.7. Antioxidant activity 3. Results and discussion 2.7.1. DPPH radical scavenging activity Four plants — S. tiliifolia Vahl. (Lamiaceae), C. mimosoides The effect of the extracts on DPPH radical was estimated L. Greene (Caesalpiniaceae), B. salviifolia (L.) Lam. (Bud- using the method of Liyana-Pathiranan and Shahidi (2005), dlejaceae) and S. brachypetala Sond. (Fabaceae), traditionally with minor modifications. A solution of 0.135 mM DPPH in used in the treatment of neurodegenerative diseases (Orhan methanol was prepared and 185 μl of this solution was mixed et al., 2007; Stafford et al., 2008) were the focus of the current with 15 μl of varying concentrations of the extract (0.25, 0.5, 1 study.
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