J Basic Clin Physiol Pharmacol 2016; aop

Ganiyu Oboh*, Veronica O. Odubanjo, Fatai Bello, Ayokunle O. Ademosun, Sunday I. Oyeleye, Emem E. Nwanna and Adedayo O. Ademiluyi

Aqueous extracts of avocado pear (Persea americana Mill.) leaves and seeds exhibit anticholinesterases and antioxidant activities in vitro DOI 10.1515/jbcpp-2015-0049 Received May 13, 2015; accepted August 21, 2015

Abstract Background: Avocado pear (Persea americana Mill.) leaves and seeds are used in traditional medicine for the t­reatment/management of Alzheimer disease (AD); however, information on the mechanism of actions is limited. This study sought to investigate the effect of P. americana leaf and seed aqueous extracts on some enzymes linked with AD (acetylcholinesterase [AChE] and butyrylcholinesterase [BChE] activities) and their antioxidant potentials in vitro. Methods: The inhibitory effects of extracts on AChE and BChE activities and antioxidant potentials (inhibition of Fe2+- and sodium nitroprusside-induced thiobarbiturate reactive species [TBARS] production in rat brain homogenates, radicals [1,1-diphenyl-2-picrylhydrazyl, hydroxyl, and nitric oxide] scavenging and iron [Fe] chelation abilities) were investigated. Phenolic content and phytochemical screening were carried out. Alkaloid profile was also determined using gas chromatography coupled with flame ionization detector (GC-FID). Results: The extracts inhibited AChE and BChE activities and prooxidant-induced TBARS production in a dose-dependent manner, with the seed extract having the highest inhibitory effect and the leaf extract exhibiting higher phenolic content and radical scavenging

*Corresponding author: Ganiyu Oboh, Department of Biochemistry, Federal University of Technology, Akure, Nigeria PMB 704, Akure 340001, Nigeria, Phone: +234 7031388644, Fax: +234 7098721306, E-mail: [email protected] Veronica O. Odubanjo: Department of Biochemistry, Federal University of Technology, Akure, Nigeria; and Department of Biochemistry, Adekunle Ajasin University, Akungba Akoko, Ondo State, Nigeria Fatai Bello, Ayokunle O. Ademosun, Sunday I. Oyeleye, Emem E. Nwanna and Adedayo O. Ademiluyi: Department of Biochemistry, Federal University of Technology, Akure, Nigeria

abilities, but lower Fe chelation ability compared with that of the seed. The phytochemical screening revealed the presence of saponins, alkaloids, and terpenoids in both extracts, whereas the total alkaloid profile was higher in the seed extract than in the leaf extract, as revealed by GC-FID. Conclusions: The anti-cholinesterase and antioxidant activities of avocado leaf and seed could be linked to their phytoconstituents and might be the possible mechanisms underlying their use as a cheap and natural treatment/ management of AD. However, these extracts should be further investigated in vivo. Keywords: alkaloids; antioxidant; ­cholinesterases; phytochemicals.

avocado

pear;

Introduction Persea americana Mill. (Lauraceae) is one of the most widely cultivated varieties of avocado pear in tropical and subtropical areas [1]. The plant is widely used in traditional medicine as a treatment for toothache, intestinal parasites, diarrhea, dysentery, skin treatment, menorrhagia, stomachache, and bronchitis [2]. The oil from the seed is reportedly used [3, 4] for weight loss in obese people. The aqueous extract of the P. americana seed has been found to have glycemic and anti-hypertensive properties, with effects on some biochemical indices [5]. The leaf possesses anti-inflammatory and analgesic activities [6]. In traditional medicine in southwestern Nigeria, the leafy part of the avocado pear is usually used to prepare infusions for older people in order to enhance their memory, but there is a dearth of information on the biochemical rationale underlying its use in the management and/or treatment of Alzheimer disease (AD). AD is an age-related dementia characterized by progressive degeneration of the nervous system, which results in cognitive impairments. AD is a multi-­factorial disease, among which is “cholinergic hypothesis”,

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2      Oboh et al.: Anti-cholinesterases and antioxidant abilities of avocado pear leaves and seeds which involves the hydrolysis of acetylcholine (ACh) to the choline and acetate groups by acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme activities. The decline in ACh level could in turn cause the blockage in signaling in the brain of AD patients [7]. Therefore, inhibition of these enzymes has been accepted as a modern therapeutic approach to AD management, as this restores the Ach level in AD patient [8, 9]. Recent research on AD management focuses on medicinal plants in order to discover safe and active natural cholinesterase (ChE) inhibitor because currently available synthetic drugs used in the treatment and management of AD such as donepezil, rivastigmine, and galantamine are known to have several side effects (e.g. nausea, vomiting, hepatotoxicity, dyspepsia, myalgia, anorexia, dizziness) [10, 11]. Free radicals and oxidative stress are the other implicated factors that trigger and induce AD, as evidenced by the high incidence of lipid oxidation in specific areas of the brain of an AD patient [12]. This incidence might be the result of higher polyunsaturated fatty acid (PUFA) content, higher level of oxidation-induced metals such as iron (Fe) in certain regions, low antioxidant defense, and high and constant use of oxygen in the mammalian brain [13]. These characteristics make the brain more susceptible to peroxidation and oxidative modification compared with other organs in the body [13, 14]. Free radicals, especially reactive oxygen species (ROS), such as superoxide anion (O2−), hydroxyl (OH) radical, nitric oxide (NO) radical, are active oxygen compounds generated by normal aerobic cellular metabolism [14]. These radicals are capable of stealing electrons from surrounding molecules and oxidizing the vital components of cellular organelles such as proteins, lipids, and DNA, thereby inducing cellular damage and subsequent cell death [15, 16]. However, natural antioxidants such as polyphenols have been suggested to be effective in the management of neurodegenerative conditions owing to their ability to block the actions of free radicals via O2 removal, to scavenge and/or inhibit ROS or their precursors (e.g. H2O2), to chelate metal ions (e.g. Fe, Cu) involved in the catalysis of ROS production, and upregulate endogenous antioxidant defenses [14]. This study sought to investigate the effect(s) of aqueous extracts from P. americana leaves and seeds on AchE and BChE activities as well as antioxidant potentials in order to establish their therapeutic use and mechanism of action in the treatment and/or management of AD in the context of finding cheap and active therapeutic agents with little or no side effect from plant materials.

Materials and methods Sample collection Avocado pear (P. americana Philip Miller) fruits and leaves were collected from a botanical garden in Akure metropolis, Nigeria. Identification and authentication were carried out by Bernard Omomoh at the Botany Department of Obafemi Awolowo University, “” Nigeria. The sample was deposited at the university herbarium with voucher number IFE 17380.

Chemicals Chemicals and reagents used such as acetylthiocholine and butrylthiocholine iodide, gallic acid, and Folin-Ciocalteau reagent were procured from Sigma-Aldrich (St Louis, MO, USA). Thiobarbiturate (TBA), trichloroacetic acid (TCA), quercetin, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2-dioxyribose were sourced from Sigma-Aldrich Chemie (Steinheim, Germany). Sodium carbonate, ferric chloride, aluminum chloride, potassium acetate, potassium ferricyanide, sodium nitroprusside (SNP), Tris buffer, ferric sulfate, and all other reagents were of analytical grade, and the water used was glass distilled.

Preparation of the aqueous extracts The leaf and fruit were washed with distilled water to remove dirt. The seed of the fruit was separated from the edible portion, chopped into small pieces by a table knife, and dried alongside the leaf to constant weight. The samples were pulverized, and an aqueous extract was subsequently prepared by soaking 1 g each of the pulverized studied samples in 100 mL of distilled water for about 24 h. Thereafter, the extracts were filtered and centrifuged to obtain a clear supernatant that was stored in the refrigerator for subsequent analysis.

AChE and BChE inhibitory activity assay The AChE activity was determined in a reaction mixture containing 200 μL of 0.415 U/mL AChE solution (EC 3.1.1.7 in 0.1  M phosphate buffer, pH 8.0), 100 μL of 5,5′-dithio-bis(2-nitrobenzoic) acid (3.3  mM in 0.1  M phosphate-buffered solution, pH 7.0, containing 6 mM NaHCO3), 0–100 μL of extracts, and 500 μL of phosphate buffer (pH 8.0). The mixture was incubated for 20 min at 25 °C. Thereafter, 100  μL of 0.05  mM acetylthiocholine iodide was added as the substrate, and AChE activity was measured in an ultraviolet spectrophotometer by monitoring the changes in absorbance at 412 nm for 3 min. BChE enzyme activity was determined using butyrylthiocholine iodide (100 μL) as substrate, whereas all other reagents and conditions were the same [17].

Lipid peroxidation and thiobarbituric acid reactions assay Mature male rats (weighing 155–160 g) were decapitated under mild diethyl ether anesthesia, and the brain tissue was rapidly i­solated,

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Oboh et al.: Anti-cholinesterases and antioxidant abilities of avocado pear leaves and seeds      3 placed on ice, and weighed. This tissue was subsequently homo­ genized in cold saline (1:10 w/v) with about 10 up-and-down strokes at approximately 1200  rpm in a Teflon glass homogenizer. The homogenate was centrifuged for 10  min at 3000  g to yield a clear supernatant fraction [18]. Thereafter, 100 μL of the supernatant fraction was mixed with a reaction mixture containing 30 μL of 0.1  M ­Tris-HCl buffer (pH  7.4), 0–100 μL of extracts, and 30 μL of freshly prepared prooxidant (250 μM FeSO4 and 7 mM SNP). The volume was increased to 300  μL by adding distilled water before incubation at 37 °C for 60 min. The color reaction was developed by adding 300 μL of 8.1% sodium dodecyl sulfate to the reaction mixture, followed by 600 μL of acetic acid/HCl (pH 3.4) mixture and 600 μL of 0.8% TBA. This ­mixture was incubated at 100 °C for 60 min. The thiobarbiturate reactive species (TBARS) produced were measured in the UV-visible spectrophotometer (Jenway model 6305, Jenway Barloworld Scientific, ­Dunmow, UK) at 532  nm [19], and absorbance was compared with that of a standard curve using malondialdehyde (MDA). The handling and use of the experimental animals was in accordance with the NIH guide for the use and handling of experimental animals.

liner and an Rtx-5MS (5% diphenyl-95% dimethyl polysiloxane) capil­ lary column (30 m length, 0.25 μm film thickness), and detected with an FID. The following conditions were employed: injector temperature, 23 °C; temperature ramp, 80 °C for 5 min then ramped to 250 °C at 30 °C/min; detector temperature, 320 °C.

Free radical scavenging ability The radical scavenging ability of the extracts against DPPH free ­radical was evaluated as described by Gyamfi et  al. [24]. In brief, appropriate dilution of the extracts (1 mL) was mixed with 1  mL of 0.4 mM methanolic solution containing DPPH*; the mixture was left in the dark for 30 min, and the absorbance of the remaining DPPH* was measured at 516 nm. The percentage DPPH* scavenging ability of the extracts was subsequently calculated.

Inhibition of degradation of deoxyribose assay

The method of Halliwell and Gutteridge [25] was used to determine the  preventive ability of the extracts against Fe2+/H2O2-induced decomposition of deoxyribose. The extracts (0–100 μL) were added The total phenol content of the extracts was determined by mix- to the reaction mixture containing 120 μL of 20  mM deoxyribose, ing appropriate dilutions of the extracts with 2.5  mL 10% Folin-­ 400  μL of 0.1  M phosphate buffer, 40 μL of 500 μM of FeSO4, and Ciocalteau reagent (v/v), and 2.0 mL of 7.5% sodium carbonate was 240  μL of distilled water. The reaction mixture was incubated at subsequently added. The reaction mixture was incubated for 40 min 37 °C for 30 min. Then, the reaction was stopped with the addition at 45 °C, and absorbance was measured at 765 nm in the spectropho- of 0.5  mL of 2.8% TCA, ­followed by 0.4  mL of 0.6% thiobarbituric tometer. The total phenol content was subsequently calculated and acid solution for color development. The reaction mixture was sub­ expressed as the gallic acid equivalent (GAE) [20]. sequently incubated in boiling water for 20 min. Absorbance was measured at 532 nm, and the percentage OH radical scavenging ­ability was ­sub­sequently calculated.

Determination of total phenol content

Determination of total flavonoid content

Briefly, 0.5 mL of appropriately diluted extracts was mixed with 0.5 mL of methanol, 50 μL of 10% AlCl3, 50 μL of 1 M potassium acetate, and 1.4 mL of distilled water. The mixture was incubated at 25 °C for 30 min. The absorbance of the reaction mixture was subsequently measured at 415 nm, and the total flavonoid content was subsequently calculated and expressed as the quercetin equivalent (QE) [21].

Phytochemical screening of the extracts Qualitative phytochemical screening for saponins, alkaloids, tannins, anthraquinones, phlobatanins, and terpenoids was carried out according to the methods described by Trease and Evans [22].

Determination of NO radical scavenging activity The NO radical scavenging ability was determined using the Griess reagent method. Briefly, 0.3 mL of 5 mM SNP was added to 1 mL each of various extract concentrations. The test tubes were then incubated at 25 °C for 150 min. After 150 min, 0.5 mL of the Griess reagent (equal volume of 1% sulfanilamide on 5% ortho-phosphoric acid and 0.01% naphtyl ethylenediamine in distilled water, used after 12 h of preparation) was added. Absorbance was measured at 546 nm, and the percentage NO radical scavenging ability of the extracts was subsequently calculated [26].

Determination of Fe (II) chelating ability Quantification of alkaloid compounds by gas ­chromatography coupled with flame ionization ­detector (GC-FID) Alkaloid compound quantification was carried out using the modified method of Ngounou et  al. [23]. For the GC-FID detection, a ­Hewlett-Packard 6890 gas chromatograph (Hewlett-Packard, Palo Alto, CA, USA) was used, along with HP Chem station Rev. A09.01 [1206] software equipped with a derivatized, non-packed injection

The method of Minotti and Aust [27], with a slight modification by Puntel et al. [28], was used to determined the Fe (II) chelating ability of the extracts. In brief, freshly prepared 150 μL of 500 μM FeSO4 was added to a mixture containing 168 μL of 0.1 M Tris-HCl (pH 7.4) buffer, 218 μL of saline solution, and 0–25 μL of extracts. The reaction mixture was incubated for 5 min, before the addition of 13 μL of 0.25% 1,10-phenanthroline (w/v). Absorbance was subsequently measured at 510 nm, and the Fe (II) chelating ability was subsequently calculated and expressed as percentage.

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4      Oboh et al.: Anti-cholinesterases and antioxidant abilities of avocado pear leaves and seeds Data analysis The triplicate results were pooled and expressed as mean±standard deviation (SD); the means were analyzed using one-way analysis of variance, and the least significant difference was determined. The IC50 (concentration of the extract causing 50% inhibition/radical scavenging ability) values were calculated by non-linear regression analyses, and significance was accepted at p  ≤  0.05.

Results Figure 1A and B shows the inhibitory effects of the extracts on AChE and BChE activities, respectively. The  result showed a linear increase in the inhibition of the enzymes activities with an increase in extract concentration. ­Nevertheless, there was a significant (p  0.05).

The effects of the studied extracts on TBARS production induced by FeSO4 and SNP are presented in Figure 2A and B, respectively. The result revealed that incubation of brain homogenates with 250 μM FeSO4 caused an increase in TBARS production. A similar trend was also observed when the SNP (7 mM) solution was incubated with brain homogenates. However, there was a significant (p 

Aqueous extracts of avocado pear (Persea americana Mill.) leaves and seeds exhibit anti-cholinesterases and antioxidant activities in vitro.

Avocado pear (Persea americana Mill.) leaves and seeds are used in traditional medicine for the treatment/management of Alzheimer disease (AD); howeve...
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