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[email protected] Current Bioactive Compounds 2017, 13, 000-000
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RESEARCH ARTICLE
Antioxidant Capacity of the Methanol Extract of Dysophylla auricularia Muhammad T. Islam1,2,3,4*, Leonardo R. Sousa3, Ana M.O.F. da Mata3, Marcus V.O.B. de Alencar3,4, Rosália M.T. de Lima3, João M.C. e Sousa5 and Ana A.C. Melo-Cavalcante3,4 Department of Pharmacy, Southern University Bangladesh, Chittagong (Mehedibag)-4000, Bangladesh; 2Laboratory of Research in Experimental Neurochemistry, Federal University of Piauí, Teresina (Paiui)-64009-550, Brazil; 3 Laboratory of Genetic Toxicology, Federal University of Piauí, Teresina (Paiui)-64009-550, Brazil; 4Postgraduate Program in Biotechnology, Northeast Biotechnology Network (RENORBIO), Federal University of Piauí, Teresina (Paiui)-64009-550, Brazil; 5Department of Biological Sciences, Federal University of Piauí, 64.049-550, Picos, Brazil 1
Abstract: Brackground: In our previous study, we have shown that the ethanol, chloroform and nhexane extracts of Dysophylla auricularia revealed antimicrobial, anti-diarrheal, anti-inflammatory and membrane stabilization, alpha-glycosidase inhibitory and antipyretic activities. This study aims to evaluate the antioxidant capacity of methanol extract of D. auricularia (MDA) using some non-clinical test systems. ARTICLE HISTORY Received: August 16, 2016 Revised: November 17, 2016 Accepted: November 18, 2016 DOI: 10.2174/15734072136661611281439 15
Materials and Methods: The methanol hot extract of the herb was undergone for six in vitro and one ex vivo and one in vivo antioxidant assays. An alpha-tocopherol analogue, trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) was taken as a standard for in vitro and ex vivo tests, while hydrogen peroxide (H2O2) as a stressor for in vivo assay. Results: The results suggest MDA within 12.5 to 500 g/ml exhibited prominent radical scavenging, reduction potential, and inhibition of hemolysis capacities. Additionally, MDA dose-dependently exhibited an antioxidative defense against H2O2 induced damage in Saccharomyces cerevisiae (in vivo) test strains. Conclusion: D. auricularia may be one of the potential sources of antioxidant compounds.
Keywords: Dysophylla auricularia, antioxidant, Rattus norvegicus, Saccharomyces cerevisiae. INTRODUCTION In a recent study, we found Dysophylla auricularia has antimicrobial, anti-inflammatory, anti-diarrheal, neuropharmacological and some other important biological activities [1]. About some isolated compounds (Fig. 1) from this plant and other species of Dysophylla were mentioned in the study. Oxidative stress mediated diseases are remarkable [2]. Although, a number of antioxidant defense systems are present in our body, but in severe states we need some external supports to maintain balance between the reactive species and antioxidant systems [3]. Plants are the potential sources of bioactive compounds to treat various human diseases [4]. Our diets, especially plant-based food materials also supply a number of antioxidant compounds. However, in some instances, we need to use synthetic antioxidants, those are already for their unavoidable adverse effects [5, 6]. Thus, to search new, effective and safe antioxidants is a continuous process. *Address correspondence to this author at the Department of Pharmacy, Southern University Bangladesh, Chittagong (Mehedibag)-4000, Bangladesh; E-mail:
[email protected]
1573-4072/17 $58.00+.00
Upon going through the above viewpoint, our present study aims to evaluate the antioxidant capacity of methanolic extract (whole plant) of the D. auricularia (MDA). For this we conducted antiradical activity (DPPH•: 1,1-diphenypicrylhydrazyl radical; ABTS•+: azino-bisethylbenzthiazoline-sulfonic acid radical; •OH: hydroxyl radical and NO•: nitrite oxide radical scavenging), lipid peroxidation (LP), reduction potential (RP), and inhibition of hemolysis (HL) in rat erythrocytes (ex vivo) in comparison with an -tocopherol analogue (Trolox - TRO - positive control). Additionally, to check the oxidative / antioxidative potential of MDA an in vivo test was performed with the wild type (SOD-WT) and deficient (single: SOD1 (cytosolic), SOD2 (mitochondrial), CAT1 (cytosolic); double: SOD1SOD2 and SOD1CAT1) Saccharomyces cerevisiae strains using hydrogen peroxide (H2O2) as a stressor. MATERIALS AND METHODS Plant Collection, Identification and Extraction of Plant Materials The herb D. auricularia was collected from the Chittagong division of Bangladesh and was identified by a taxonomist in Forest Research Institute, Bangladesh (FRIH, © 2017 Bentham Science Publishers
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Islam et al.
H H3CO H
C C
C C
C C
OH
H3CO
OCH3 C
CH2
C
HC
CH3
CH
C C
C
OH
O
A. Phlorobytyrophenone
H3CO
OCH3 C
CH
CH
CH3
R2 R3
HC
CH C
C C
OCH3 C
CH
CH
CH3
OCH3 O
O
B. Acyl phloroglucinol
H3C
C
C. Methyl acyl phloroglucinol CH2
H R1O 2C
H CH3
D. Auricularic acid: R1=H; R2=CH2; R3=H E. OH-derivative: R1=H; R2=CH2; R3=OH F. Ac-derivative: R1=CH3; R2=CH2; R3=OAc
Fig. (1). Isolated and synthesized chemical compounds reported in Dysophylla genus.
BD). A voucher specimen was deposited with the accession number BFRIH-4688.
concentration range 12.5-100 g/ml, while in in vivo test it was 64.5-500 g/ml.
After collection, D. auricularia (whole plant) was dried (temperature not exceeding 50°C), and the dried materials were then subjected to course grinding. Methanol hot extraction (16 h) was carried out by using the Soxhlet apparatus. The extract was then filtered through a cotton plug followed by Whatman filter paper (No. 1). For a rapid evaporation of solvent, rotary evaporator (temperature not exceeding 50°C) was used. The yield was 12.93%.
Screening for Antioxidant Activity
Reagents and Chemicals Tween-80 (0.05%) dissolved in 0.9% saline was used as a vehicle for the extract and TRO. Hydrogen peroxide (H2O2) (reconstituted with sterile distilled water) was used as a stressor (STR) for S. cerevisiae test. All the other necessary reagents and chemicals were purchased from (SigmaAldrich, St. Louis, MO; USA.). Rattus norvegicus and Saccharomyces cerevisiae For hemolysis (ex vivo) activity test, a male Wister albino rat (Rattus norvegicus) of 220 g body weight (2 months old) was collected from Central Animal House of the Federal University of Piaui. The animal was allowed to free access to water and food (Purinas pellets) ad libitum and was kept under controlled lighting (12 h dark/light cycle) and temperature (24 ± 2°C). For in vivo assay, the above mentioned proficient and deficient S. cerevisiae strains were collected from the Genetic Toxicology of Federal University of Rio Grande do Sul (UFRGS), Brazil. Sample Preparation For in vitro and ex vivo antioxidant assays, MDA and TRO were dissolved in above mentioned vehicle to attain a
In vitro Assays DPPH (1,1-diphenyl-2-picrylhydrazyl) Scavenging Activity The method described by Manzocco et al. [7] was used in this case. Both MDA and TRO were tested at concentrations 12.5, 25, 50 and 100 g/ml. Briefly, 0.3 ml of sample (MDA/TRO) was added to 2.7 ml of ethanolic solution of DPPH (0.5 mM). After 30 min, the absorbance was measured by using a spectrophotometer at 517 nm. A negative control (NC) group was performed with 0.3 ml of vehicle. The blank contains no sample. The DPPH radical scavenging potential was calculated using the equation as given below: % inhibition of DPPH radical = [(Abr – Aar)/Abr]100 where, Abr and Aar are the absorbance of DPPH free radicals before and after the reaction, respectively. ABTS+ (2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic Acid)) Radical Cation Decolorization Assay This test was done by slight modification of the method of Seeram et al. [8]. Briefly, ABTS cationic radicals were produced by adding the solid manganese dioxide (80 mg) to a 5 mM aqueous stock solution of ABTS (in 75 mM Na+/K + buffer of pH 7.0). Then 2.8 ml sample was added to 0.2 ml of ABTS+ solution. The absorbance was read at 750 nm after 5 min of sample application. The percentage of scavenging capacity was calculated as follows: % scavenged ABTS+ = [(Abr – Aar)/Abr]100 where, Abr and Aar are the absorbance of ABTS•+ free radicals before and after reaction with test samples, respectively.
Antioxidant capacity of Dysophylla auricularia
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Hydroxyl Radical ( OH) Scavenging Activity
Reduction Potential (RP) Test
This test was carried out according to the method of Kunchandy and Rao [9]. Briefly, 0.5 ml of the test sample (MDA/TRO) was added to 0.1 ml of 2-deoxy-ribose (28 mM in 20 mM KH2PO4-KOH buffer, pH 7.4). Then 0.2 ml EDTA (1.04 mM) and 0.2 ml FeCl3 (200 M) was added to the mixture and followed by the addition of 0.1 ml of H2O2 (1.0 mM). After incubating at 37°C for 1 h, 1 ml thiobarbituric acid (1%) and 1 ml of trichloroacetic acid (2.8%) were added to the reaction vessel. Incubation was done again at 100°C for 20 min and after cooling, the absorbance was measured at 532 nm, against a blank (except sample). As NC group, 0.5 ml of vehicle was used. The percentage of OH scavenging capacity was calculated as follows:
The RP test was conducted by the modified method described by Oyaizu [12]. Briefly, 0.2 ml of sample was added to 0.5 ml of 0.2 M phosphate buffer (pH 6.6) and 0.5 ml of K3Fe(CN)6 (1% w/v) and the reaction mixture was heated at 50°C for 20 min. Then 0.5 ml of trichloroacetic acid (10% w/v) was added with constant shaking, followed by the addition of 1.175 ml distilled water and 0.125 ml of FeCl3 (0.1%, w/v) after 5 min. The absorbance was taken at 700 nm. To NC tube, only 0.2 ml vehicle was added. The activity as reducing power capacity is calculated as follows: -
% scavenged OH = [(Abs – Ats)/Abs]100 where, Abs is the absorbance of blank and Ats is the absorbance of the test sample.
% reducing potential = [(Ats – Abs)/Ats] 100 where, Ats is the absorbance of the test sample and Abs is the absorbance of blank sample. Ex vivo Assay
Nitric Oxide (NO ) Scavenging Activity
Inhibition of Hemolysis (HL) Induced by H2O2 in Rat Erythrocytes
In this test, the reaction mixture contains 0.375 ml test sample, 1.5 ml of sodium nitroprusside (10 mM) and 0.375 ml phosphate buffer saline (pH 7.4). The absorbance (Abr) was taken at 546 nm. After incubating the reaction mixture at 37°C for 1 h, 1 ml of supernatant was mixed with 1 ml of Griess reagent [(1.0 ml sulfanilic acid reagent (0.33% in 20% glacial acetic acid at room temperature for 5 min with 2 ml of naphthylethylenediamine dichloride (0.1% w/v)]. Then the reaction mixture was kept at room temperature for 30 min and the final absorbance (Aar) was measured at the same wavelength. For NC, 0.375 ml vehicle was used [10]. The amount of NO inhibition was calculated by the following this equation:
In this test, blood collected from the rat’s retro-orbital plexus was mixed with equal volume of Alsevier’s solution and centrifuged at 4,000 g for 5 min. The RBCs were then reconstituted as 10% suspension in PBS (pH 7.4). To 0.5 ml of RBC suspension, 0.15 ml of H2O2 (200 mM in PBS, pH 7.4) and 0.2 ml of sample solution were added. The reaction mixture was then incubated at 37°C for 30 min and centrifuged (2,500 g for 3 min) immediately, followed by withdrawing 0.2 ml supernatant and subsequently mixing with 2.8 ml PBS (pH 7.4). The absorbance was taken at 475 nm [13]. To NC tube, 0.2 ml of the vehicle was added. The percentage of HL was calculated taking into account the 100% HL induced by H2O2 (blank) using the following formula: -
% inhibition of NO = [(Abr – Aar)/Abr]100
% inhibition of HL = [(Absblank – Abssample) x 100] / Absblank
where, Abr and Aar are the absorbance of NO free radicals before and after reaction with Griess reagent, respectively.
where, Absblank and Abssample are the absorbance of aliquots containing blank and sample, respectively.
Evaluation of the Antioxidant Potential Against the Formation of TBARS
In vivo Assay
The TBARS (thiobarbituric acid substrates) assay was adopted to measure the quantity of lipid peroxidation capacity. Briefly, 0.1 ml of sample was added to the test tube containing 1 ml of 1% w/v homogenized egg yolk (in 20 mM phosphate buffer at pH 7.4). Lipid peroxidation was induced by the addition of 0.1 ml of 2,2'-azobis(2methylpropionamidine) dihydrochloride solution (AAPH; 0.12 M). The reaction mixture was then incubated at 37°C for 15 min. After cooling, 0.5 ml of supernatant mixed with 0.5 ml of trichloroacetic acid (15%) was centrifuged at 1,200 g for 10 min. An aliquot of 0.5 ml of supernatant was mixed with 0.5 ml TBA (0.67%) and heated at 95°C for 30 min. After cooling, absorbance was measured by using a spectrophotometer at 532 nm. The results were expressed as percentage of inhibition of TBARS formed by AAPH alone (induced blank) [11]. The antioxidant activity by TBA method was calculated as follows: % inhibition of lipid peroxidation = [1- {(Ats – Abs)/Ats}] 100
where, Ats is the absorbance of the test sample and Abs is the absorbance of blank sample.
Oxidant/Antioxidant Capacity Test in S. cerevisiae Cells This test was performed according to the earlier described method of Fragoso et al. [14]. Briefly, previously sub-cultured strains were linearly swabbed to the sterile YEPD media (0.5% yeast extract, 2% peptone, 2% dextrose and 2% bacteriological agar). Then 10 l of test sample (MDA/STR/NC; MDA: 64.5, 125, 250 and 500 g/ml; H2O2: 10 mM; NC: 10 l/disc) was applied on the sterile paper disc. The disc was then immediately applied at the center of the previously (strain) seeded dishes. After a 72 h incubation at 32°C the length of inhibition (both sides) in each strain line was measured in millimeters (mm) with a range from 0 mm (full growth) to 40 mm (no growth), these values being the size of the petridish procured. All the treatments were performed in duplicate. Statistical Analysis Values are mean ± standard deviation (SD). The data are analyzed by means of analysis of variance (ANOVA followed by t-Student–Newman–Keuls's as post-hoc (one-way) and Tukey's test (two-way) using the GraphPad Prism soft-
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ware (version 6.0) considering p
