Hindawi Publishing Corporation Journal of Tropical Medicine Volume 2014, Article ID 159343, 6 pages http://dx.doi.org/10.1155/2014/159343 Research Article Evaluation of the In Vitro and In Vivo Antioxidant Potentials of Aframomum melegueta Methanolic Seed Extract Samuel Okwudili Onoja,1 Yusuf Ndukaku Omeh,2 Maxwell Ikechukwu Ezeja,1 and Martins Ndubuisi Chukwu2 1 Department of Veterinary Physiology, Pharmacology, Biochemistry and Animal Health and Production, CollegeofVeterinaryMedicine,MichealOkparaUniversityofAgriculture,PMB7267,Umudike,Nigeria 2 Department of Biochemistry, College of Natural Sciences, Micheal Okpara University of Agriculture, PMB 7267, Umudike, Nigeria Correspondence should be addressed to Samuel Okwudili Onoja; [email protected] Received 22 February 2014; Revised 1 May 2014; Accepted 4 May 2014; Published 15 May 2014 Academic Editor: Shyam Sundar Copyright © 2014 Samuel Okwudili Onoja et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aframomum melegueta Schum (Zingiberaceae) is a perennial herb widely cultivated for its valuable seeds in the tropical region of Africa. The present study evaluated the antioxidant effects of methanolic seed extract of A. melegueta. The antioxidant effects were evaluated using in vitro, 2, 2-diphenylpicrylhydrazine photometric assay and in vivo serum catalase, superoxide dismutase and thiobarbituric acid reactive substance assay method. The extract (25–400 g/mL concentration) produced concentration dependent increase in antioxidant activity in 2, 2-diphenylpicrylhydrazine photometric assay. The extract (400 mg/kg) showed a significant ( < 0.05) increase in serum catalase and superoxide dismutase activity when compared with the control group. The extract (400 mg/kg) showed a significant ( < 0.05) decrease in the serum level of thiobarbituric acid reactive substance when compared with the control group. These findings suggest that the seed of A. melegueta has potent antioxidant activity which may be responsible for some of its reported pharmacological activities and can be used as antioxidant supplement. 1. Introduction and glutathione which catalyse neutralization of many types of free radicals [6], while the nonenzymatic antioxidants Antioxidants act as a defence mechanism that protect against include Vitamin C, selenium, vitamin E, carotenoids, and deleterious effects of oxidative reaction produced by reactive polyphenols. There is growing evidence that antioxidants oxygen species (ROS) in a biological system [1]. Reactive play a pivotal role in the prevention of heart disease, cancer, oxygen species not only are produced naturally in cell DNA degeneration, pulmonary disease, and neurological following stress or respiration but also have been reported disorder [7]. Recently, there has been an upsurge of interest to be produced by radiation, bacterial and viral toxin, in the therapeutic potential of plants as antioxidants in smoking, alcohol, and psychological or emotional stress. reducingoxidativetissueinjuries[3]. Plants, herbs, and Overproduction of ROS and/or inadequate antioxidants has spice, rich in phenolic compounds like flavonoids, have been implicated in the pathogenesis and complications of been demonstrated to have anti-inflammatory, antiallergenic, some disease conditions like diabetes, Alzheimer’s disease, antiviral, antiaging, and anticarcinogenic activities which can cancer, atherosclerosis, arthritis, neurodegenerative disease, be attributed to their antioxidant properties [7, 8]. and aging process [2, 3]. Antioxidants have been reported to Aframomum melegueta Schum (Zingiberaceae) also prevent oxidative damage caused by ROS by reacting with known as Guinea pepper, grains of paradise, or alligator free radicals, chelating, and catalytic metals and also by acting pepper (indigenous names include Atare in Yoruba, Ose-oji as oxygen scavengers [4, 5]. The antioxidants in biological in Igbo, and Citta in Hausa) is a perennial herb widely system can be either enzymatic or nonenzymatic. The enzy- cultivated for its valuable seeds in the tropical region of matic antioxidants include catalase, superoxide dismutase, Africa [9, 10].Itgrowsupto1.5minheight,withpurple 2 Journal of Tropical Medicine flower that develop into long pod containing small, reddish with the recommendation of the Guideforthecareanduse brown aromatic and pungent seed. In Nigeria and some other of laboratory animals [13]. The experiment was approved parts of West Africa, the seeds are used as a spicy and have by the University Animal Ethics Committee with reference awiderangeoffolkloricusesintraditionalmedicine.They MOUAU/CVM/EAEC/2013/201. are used as a remedy for treating stomach ache, diarrhoea, and snakebite [9, 10]. Previous studies have established 2.5. Phytochemical Spot Test. The AME was tested for the antiulcer, antimicrobial, anti-inflammatory, and sexual the presence of alkaloids, flavonoids, tannins, glycosides, performance enhancing effects of the seed extract9 [ –12]. The saponins, terpenes/sterols, carbohydrates, and starch using seeds are very rich in the nonvolatile pungent compounds the standard procedures as described by Trease and Evans gingerol,shogaols,paradol,andrelatedcompounds[11]. [14]. The present study aimed at establishing the in vitro and in vivo antioxidant potentials of the methanolic seed extract of Aframomum melegueta. 2.6. Determination of the In Vitro Antioxidant Activities of AME Using 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) Photomet- ric Assay. The free radical scavenging activity of the extract 2. Materials and Methods was analyzed by DPPH assay using spectrophotometer [15]. Each of the test extracts (2 mL) at different concentrations 2.1. Plant. The freshly harvested fruit of Aframomum (25,50,100,200,and400g/mL) was mixed with 0.5 mM melegueta (Alligator Pepper) were bought from Ndoro mar- DPPH(in1mLofmethanol)inacuvette.Theabsorbanceat ket, Oboro in Ikwuano LGA of Abia State in the month of 517 nm was taken after 30 minutes of incubation in the dark July 2013, and were authenticated by Dr. I. C. Okwulehie of at room temperature. The concentrations were prepared in the Department of Plant Science and Biotechnology, Michael triplicates and the percentage antioxidant activity calculated Okpara University of Agriculture, Umudike, and the voucher as follows: specimen catalogued MOUAU/CVM/VPP/2013/03 was kept for reference purpose in the departmental herbarium. % antioxidant activity (AA) 2.2. Preparation of the Extract. Dried and pulverized seeds = 100 − [ {(absorbance of sample − absorbance of blank) of Aframomum melegueta were extracted by cold maceration −1 method for 48 hours at room temperature using absolute × 100} × (absorbance of control) ]. methanol in a Winchester bottle. The Aframomum melegueta (2) extract (AME) was filtered with Whatman No. 1 filter paper. The filtrate was concentrated in vacuo using vacuum rotary One milliliter of methanol plus 2.0 mL of the extract was ∘ evaporator at 40 C and was later concentrated to dryness in a used as the blank, while 1.0 mL of the 0.5 mM DPPH solution ∘ hot-air oven at 40 C. The extract was stored in a refrigerator plus 2.0 mL of methanol was used as the negative control. ∘ at 4 C throughout the duration of this study. Ascorbic acid (vitamin C) was used as reference standard [16]. 2.3.DeterminationoftheYieldoftheAME. An empty clean 2.7. Determination of the In Vivo Antioxidant Effect of AME. and dry beaker was weighed and later the extract was poured Twenty male albino Wistar rats were randomly divided into into it. The beaker was weighed after the extract has been fourgroupsoffiveanimalseach.Group1servedasthecontrol concentrated to constant weight. The weight of the extract and received 0.4 mL of distilled water. Group 2 received was calculated as follows: 100 mg/kg of the AME. Group 3 received 200 m/kg of the AME,andgroup4received400mg/kgoftheAME.The The percentage yield of extract (%) w/w animalsweredoseddailyfor21daysandwereobserved − daily for changes and other signs of toxicity and death = weight of beaker and extract wieght of empty beaker weight of plant material throughout the period of study. Twenty-four hours after the last treatment, blood obtained through direct cardiac 100 × . puncture was used to assay for in vivo antioxidant activity of 1 AME. (1) 2.8. Analytical Methods 2.4. Animals. Twenty male Wistar albino rats weighing between 120 and 170 g were obtained from Department of 2.8.1. Serum Preparation. The blood used for serum prepa- Zoology, University of Nigeria, Nsukka, and kept in the ration was collected via direct heart puncture with 21 G Animal House of the Biochemistry Department. The animals needle attached to 5 mL syringe, following mild chloroform were allowed access to feed and water ad libitum and were anaesthesia of the rats. The serum was prepared using allowed two weeks to acclimatize before the commencement standard method as described by Yesufu et al. [17]. Briefly, of the experiment. The animals were kept in well-ventilated the method used is as follows. Blood was allowed to clot for aluminium cages at room temperature and under natural 30 minutes and then centrifuged at 2500rpm for 15 minutes light/darkness cycles. They were maintained in accordance and serum was harvested. Journal of Tropical Medicine 3 2.8.2. Determination of the Lipid Peroxidation (LPO) in 120 Serum. The level of thiobarbituric