Evaluation of Antioxidant Potential of Melanthera Scandens

J Acupunct Meridian Stud 2010;3(4):267−271 RESEARCH ARTICLE Evaluation of Antioxidant Potential of Melanthera scandens Sunday Adeleke Adesegun1*, Sukurat Olasumbo Alabi1, Patricia Taiwo Olabanji1, Herbert Alexander Babatunde Coker2 1Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos, Lagos, Nigeria 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Lagos, Lagos, Nigeria Received: Jun 22, 2010 Abstract Accepted: Oct 12, 2010 A methanol extract of dried leaves of Melanthera scandens was examined for antioxidant activities using a variety of assays, including 1-diphenyl-2-picrylhydrazyl KEY WORDS: (DPPH) radical scavenging, reducing power, ferrous chelating, and ferric thio- antioxidant activities; cyanate methods with ascorbic acid and EDTA as positive controls. The extract lipid peroxidation; showed noticeable activities in most of these in vitro tests. The amount of phenolic compounds in the extract expressed in gallic acid equivalent was found to be Melanthera scandens; 52.8 mg/g. The extract demonstrated inhibition of linoleic acid lipid peroxidation, radical scavengindg; active reducing power, and DPPH radical scavenging activities which were less than reducing power that of the positive controls. The extract also showed weaker iron chelating effect when compared with the EDTA positive control. The present results showed that M. scandens leaf extract possessed antioxidant properties and this plant is a potential useful source of natural antioxidants. 1. Introduction and reducing chronic diseases and lipid peroxidation [5−7]. There is increasing interest in compounds with Oxidation and reduction reactions are essential pro- antioxidant activity that could be used as supple- cesses in all living organisms, but intermediates known ments in humans and, thus, naturally occurring anti- collectively as reactive oxygen species, such as su- oxidants, especially of plant origin, are a major area . − . peroxide radical ( O2 ), hydroxyl radical ( OH), and of scientific research [8−10]. Melanthera scandens hydrogen peroxide (H2O2) are also generated during (Schum. & Thonn.) Roberty (family Asteraceae) is a some reactions [1,2]. These species can react with scrambling or scandent herb of waste thickets com- biological substrates, such as DNA and proteins, monly dispersed in forest areas and widely distrib- leading to degenerative diseases, ageing, cancer, uted across tropical Africa, providing forage for all arthritis, and atherogenesis [3,4]. Antioxidants are stock in thicket edges around villages and being a compounds that function as free radical scavengers, favorite food of hares in southern Nigeria. In the complexers of prooxidant metals, reducing agents, Ivory Coast, the leaves are used as a purgative and and quenchers of singlet-oxygen formation. They are an antidote against poisoning, and the leaf decoction regarded as protection agents minimizing reactive is used as a soothing cough mixture and for sore oxygen species-related oxidative damage in the body throat. In Nigeria, pulped, decocted, or macerated *Corresponding author. Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos, PMB 12003, Lagos, Nigeria. E-mail: [email protected] ©2010 Korean Pharmacopuncture Institute 268 S.A. Adesegun et al leaves are used as hemostatic preparations, applied 2.4. Determination of total phenolic content to cuts and wounds to promote healing and curb inflammation [11]. Six saponins have been reported The total phenolic content of the M. scandens ex- to have been isolated from M. scandens leaves and tract was determined with Folin-Ciocalteu reagent their structures elucidated by two-dimensional nu- [15]. Gallic acid equivalents (GAE, mg/g) were de- clear magnetic resonance techniques, including termined from a calibration concentration curve. rotating frame nuclear Overhauser effect Spectro- The extract (100 mg/mL, 1.0 mL) was mixed thor- scopy. They are 3-O-beta-D-glucuronopyranosyl- oughly with 5 mL Folin-Ciocalteu reagent (diluted oleanolic acid, 3-O-[beta-D-xylopyranosyl(1−>4) 10-fold) and after 5 minutes, 4.0 mL of sodium car- beta-D-glucuronopyranosyl]-oleanolic acid, 3-O- bonate (0.7 M) was added and the mixture allowed ([beta-D-glucopyranosyl(1−>2)][beta-D-xylopyranosyl- to stand for 1 hour with intermittent shaking. The (1−>4)]beta-D-glucuronopyranosyl)-oleanolic acid, 765 nm absorbance of the resulting blue color was and the corresponding 28-O-beta-D-glucosides [12]. measured in a spectrophotometer, with all determi- In vitro antiplasmodial activity of a M. scandens ex- nations in triplicate and the values for total phenolic tract against a chloroquine-resistant FcB1 strain of content expressed as GAE. Plasmodium falciparum has revealed weak antima- larial activity [13]. Studies in our laboratory revealed 2.5. Determination of DPPH radical the presence of phenolic compounds in the leaf scavenging activity extract. The relationship between phenolic compounds and antioxidant activity is well documented The free radical scavenging activities of M. scandens [14]; thus it was considered rational to determine extract was measured using DPPH and the method the antioxidant potential of the extract. of Yen and Chen [16] with modifications. A 1.0 mL volume of extract (0.2−5.0 mg/mL) was diluted to 20% of the original concentration with methanol, 2. Materials and Methods 1.0 mL of 1 mM methanolic DPPH added, and the mixture shaken vigorously and allowed to stand in dark 2.1. Chemicals for 30 minutes. The 517 nm absorbance was then measured by spectrophotometer, with ascorbic Folin-Ciocalteu phenol reagent, linoleic acid, ammo- acid as a positive control and deionized water as a nium thiocyanate, [4,4Ј-[3-(2-pyridinyl-1,2,4-triazine- blank. Lower absorbance values of the reaction mix- 5,6-diyl]bisbenzenesulfonic acid] (ferrozine), 90% ture indicated higher free radical scavenging activity. 1,1-diphenyl-2-picrylhydrazyl (DPPH), FeCl2 tetrahy- drate, gallic acid, anhydrous sodium carbonate, an- 2.6. Determination of iron chelating ability hydrous ferric chloride, potassium ferricyanide, and trichloroacetic acid were purchased from Sigma The extract’s ferrous ion chelating activity was es- Chemical Company (St. Louis, MO, USA). Ascorbic timated by a ferrozine assay [17], in which 1.0 mL acid and all other chemicals were of analytical grade of extract (0.2−5.0 mg/mL) was mixed with 3.7 mL of from BDH Chemical Laboratory (England, UK). deionized water and added to 1.0 mL of 1 mM FeCl2. The reaction was initiated by the addition 0.2 mL of 2.2. Plant material 5 mM ferrozine and then the mixture shaken vigorously and left to stand for 20 minutes. The 562 nm The aerial part of M. scandens was collected at absorbance was measured by spectrometer, with Ibadan, in Oyo, Nigeria and authenticated by Mr. K.I. EDTA as a positive control. The results were expressed Odewo of the Forest Research Institute of Nigeria, as percentage inhibition of ferrozine-Fe2+ complex Ibadan, Nigeria after comparison with the voucher formation, with the ability to chelate ferrous ion specimen with number FHI 94215. The leaves were determined using the formula given below: plugged out and dried at 30ºC and milled to produce fine powder. Chelating effect (%) = [(Acontrol − A sample)/Acontrol] × 100 2+ 2.3. Extraction where Acontrol is the absorbance of the ferrozine-Fe complex and Asample the absorbance in the presence About 500 g of powdered plant material was ex- of extract or standard. tracted with 3 L of methanol using a Soxhlet appara- tus for 48 hours and the resulting extract con centrated 2.7. Determination of reducing ability under reduced pressure using a rotary evaporator until a semisolid sticky mass was obtained. The yield The reducing ability of the extract was determined was 12.63% weight in weight. by the method of Lai et al with modifications [6]. Antioxidant potential of Melanthera scandens 269 Different concentrations of extract (0.1−5.0 mg/mL) radical chain reaction [19,20]. The high phenolic in 1 mL of distilled water were mixed with 2.5 mL of content of this leaf extract suggested that antioxi- 0.2 M phosphate buffer, pH 6.6, and 2.5 mL of 1% po- dant activity of the extract may be related to its tassium ferricyanide. The mixture was incubated at phenolics. 50ºC for 20 minutes, 2.5 mL of 10% trichloroacetic The DPPH method is commonly used to determine acid added, and then the mixture centrifuged at the antioxidant activity of phenolic compounds and 1000 g for 10 minutes. The upper 2.5 mL layer of the plant extracts and was used here to determine the solution was mixed with 2.5 mL of distilled water hydrogen atom or electron donating ability of and 0.5 mL of 0.1% FeCl3, and the 700 nm absorbance M. scandens extract and the standard antioxidant measured against a blank by spectrophotometer, with ascorbic acid. A significant decrease (p < 0.05) in ascorbic acid as a positive control. Increased absor- DPPH· concentration was observed due to the bance of the reaction mixture indicated increased scavenging power of the extract and ascorbic acid reduction capability. (Figure 1). The radical scavenging effect of the extract, ascorbic acid, and butylated hydroxytoluene 2.8. Determination of antioxidant activity on DPPH· concentration decreased in the order: ascorbic acid > extract with EC50 values of 0.05 and Antioxidant activity of the extract was determined 0.43 mg/mL, respectively, which suggested that the according to a ferric thiocyanate method carried out extract’s radical scavenging ability played a role in in a linoleic acid emulsion [18]. Different concen- its antioxidant property but the effect was signifi- trations of extract (0.5−8.0 mg/mL) were mixed with cantly less (p < 0.05) than that of synthetic drugs a 0.56% w/v linoleic acid emulsion, pH 7.0, and 2 mL used as positive controls. DPPH· is a stable free of 0.2 M phosphate buffer, pH 7.0, and incubated radical with an 517 nm absorption band due to its at 60ºC in the dark for 12 hours to accelerate oxida- odd electron and it loses this absorption when re- tion.

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