International Food Research Journal 19 (3): 1125-1130 (2012) Antioxidant proprieties of methanolic and ethanolic extracts of Euphorbia helioscopia, (L.) aerial parts 1Ben Mohamed Maoulainine, L., 2Jelassi, A., 2Hassen, I. and 1Ould Mohamed Salem Ould Boukhari, A. 1 Laboratoire de Biotechnologies, Faculté des Sciences et Techniques, Université de Nouakchott. BP 5026 Nouakchott, Mauritania 2 Unité de Recherche: Etude Physico- chimique des Milieux et Substances Naturels. Institut National de Recherche et d’Analyse Physico- chimique (INRAP), Pôle Technologique Sidi Thabet, 2020. Tunisia Abstract: Sun spurge plants (Euphorbia helioscopia, L.) were collected from the north of Tunisia. Dried plant parts namely flowers, leaves and stem were individually extracted with methanol and ethanol. Extracts were screened for their antioxidant activity using the 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical test. Total phenolics and total flavonoids amounts were also measured. The highest radical scanvenging effect was observed in flowers methanolic extract with IC50 value of 26.66 ± 0.000µg/ml. While, relatively poorer antioxidant activity were observed in the same extracts of leaves and stem with respective IC50 values of 65.25 ± 0.004 and 80.17± 0.012 µg/ml. The IC50 values ranged from 27.55 ± 0.005 to 179.02 ± 0.957µg/ml in ethanol extracts of the above mentioned tested samples. Polyphenols and total flavonoids amounts varied in significant way among tested aerial parts of Euphorbia helioscopia and among two used solvents, the highest phenolics and flavonoids contents were found in methanolic flowers extracts (51.49 ± 0.012 mg GAE/g dry weights, 11.38 ± 0.004 mg QE/dry weight respectively). However, ethanol extract of stem gave the lowest amounts of total phenolics and flavonoids (4.80 ± 0.001 mg GAE/g dry weight and 1.69 ± 0.001 mg QE/dry weight respectively). Keywords: Euphorbiacae, sun spurge, total phenolics, flavonoids, DPPH scavenging. Introduction an antiviral (Ramezani et al., 2008), an antibacterial (Al younes and Abdullah, 2009; Papp, 2004), a Sun spurge (Euphorbia helioscopia, L., nematicidal (Devi and Gupta, 2000), an antifungal Euphorbiacae family), is an annual plant rising (Uzair et al., 2009) and a molluscicidal (AL Zanbagi, 10 to 50 cm high with erected reddish stem, oval 2005) effects. Moreover, Euphorbia helioscopia was alternate leaves and small yellow green flowers. It is reported to have antioxidant proprieties (Uzair et al., an indigenous plant of North Africa and most of the 2009, Nikolova et al., 2011). Nevertheless, in those Europe and Asia. Euphorbia helioscopia has been studies the entire sun spurge plant was investigated considered as medicinal plant and was used in folk but there are no reports about antioxidant activities medicine of various countries around the world (Lai of separated plant parts. Hence, the objective of et al., 2004; Pieroni et al., 2004; Barla et al., 2006; this investigation was to evaluate total phenolics, Qureshi et al., 2007). In traditional Tunisian medicine, flavonoids contents of methanolic and ethanolic sun spurge, locally named ‘Labeena’ (referring to extracts of Euphorbia helioscopia leaves, flowers the latex that plant bleeds when cat) have been used and stem and examine their antioxidant proprieties as treatment of warts through local applications of using the DPPH radical scavenging assay. the latex on the infected area. A large number of secondary metabolites have been reported from sun Materials and Methods spurge plant including diterpenoid, triterpenoid, tannins and steroid (Yamamura et al., 1989; Lee et Chemicals and reagents al., 1990; Wen and Yue-Wei, 2006; Barla et al., 2006, All chemicals used in this study were of analytical Wu et al., 2009) which offered to sun spurge herb a grade and were purchased from Sigma (USA), Fluka wide array of bioactive functions. In fact, Euphorbia Chemie (Buchs, Switzerland) and Merck (Germany). helioscopia plant was reported to have an antitumor Deionized distilled water (ddH2O) was used all through (Cai et al., 1999) a vasodepressor (Barla et al., 2006), the experiment. All absorbance measurements were *Corresponding author. Email: [email protected] © All Rights Reserved 1126 Ben Mohamed Maoulainine, L., Jelassi, A., Hassen, I. and Ould Mohamed Salem Ould Boukhari, A. made using a JSCO V-530 (WITEG Labortechnik., plant extracts (prepared from 1mg of crude extract Gmbh) UV-VIS. spectrophotometer. dissolved in 1ml of methanol) were mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride Plant collection and sample preparation (AlCl3) solution, 0.1 ml of (1M) sodium hydroxide Sun spurge (Euphorbia helioscopia) plants were solution (NaOH) and 2.8 ml of ddH2O. The resulting collected on April 2008 from the area around the mixtures were well mixed and incubated for 30 INRAP institute (National Institute of Research and minutes at obscurity. The absorbance of the reaction Physical and chemical Analysis) at Sidi Thabet region mixture was measured at 430 nm with a UV/visible (latitude 36°55′N, longitude 10004’ altitude 41m) spectrophotometer. Samples were analyzed in which is located at 20 Km North- Ouest of Tunis. triplicates. The calibration curve was prepared by This region belongs to the semi arid area of Tunisia quercetin solutions at concentrations of 12.5 µg/ml characterized by a rainfall range higher than 400mm/ to100 µg/ml (standard curve equation: y = 0.0078x – year. Flowers stem and leaves of collected sun spurge 0.0133, R2= 0.999). plants were separated and dried in the shade at an airy place. Plant materials (flowers, stem and leaves) were DPPH radical scavenging activity assay then grounded in fine powders and stored in hermetic The antioxidant activity was investigated using glass bottles at dark until extraction processing. the stable free radical 1,1 diphenyl-2-picrylhydrazyl (DPPH) assay, which estimated the hydrogen donating Preparation of plant extracts or the radical scavenging ability of the examined Powders of stem, flowers and leaves ofEuphorbia extract. When reacting with an antioxidant compound, helioscopia plants (100 gram of each plant part) the DPPH solution, initially purple in color, changed were primarily extracted in Soxhlet extractor by to yellow. The degree of discoloration denotes the hexane for 6 hours at 65°C in order to remove fatty scavenging potency of the tested compound which materials. 25 grams of defatted powders of each can be measured spectrophotometrically at 517nm. plant part material were then individually reextracted The antioxidant activity of Euphorbia helioscopia in Soxhlet apparatus for 12 hours at 60°C by 250ml flowers, leaves and stem extracts was monitored of solvent (methanol or ethanol). Following the according to the method described by Brand-William extractions, solvents were removed using rotary et al., (1995) with modifications, briefly 1ml of each vacuum evaporator at 40°C. Resulting crude extracts sample extract of varying concentrations (20, 50, 100, were collected into small dark sterile vials and stored 200 and 500µg/ml) was mixed with 2ml of DPPH at 4°C. methanol solution (10-4M). The mixture was then allowed to stand at room temperature in the dark for Total phenolics content determination one hour. Absorbance of the mixture was measured at The total phenolic contents were determined 517nm by an UV-Visible spectrophotometer. Trolox using the Folin-Ciocalteu reagent as described by (6-hydroxyl-2578 tertramethylchrome-2-carboxylic (Mc Donalds, 2001), 0.5ml of each diluted extract acid) was used as positive control and measurements (1:10 mg/ml) was thoroughly mixed with 5 ml of were carried out in triplicate. Folin Ciocalteu reagent (1:10 diluted with ddH2O) Inhibition free radicial DPPH in percentage terms and 4ml of (1M) solution of sodium carbonate was calculated using the following equation: % of Na2CO3 and allowed to stand for 15 minutes. The inhibited DPPH= ((AC517-AE517)/ AC517) x100), absorbance of all samples was measured at 765nm Where AC517 is the absorbance of the control and with a UV-visible spectrophotometer. Total phenolics AE517 is the absorbance of the plant extract after 1 were quantified based on standard curve prepared hour incubation. from various concentrations (0-250 mg/ml) of gallic Extract concentration that provide 50% inhibition acid (prepared in 80% methanol) and expressed as (IC50) and expressed in µg extract /ml was calculated mg of gallic acid equivalent per gram of dry weight from the graph plotted inhibition percentages against (mg GAE/g dry weight, standard curve equation: tested samples extracts. The higher the IC50, the lower y= 0.005x+0.0173, R2= 0.998). All samples were is the antioxidant activity of the examined sample. analyzed in triplicates. Statistical analysis Total flavonoids contents determination Data were analyzed using SPSS statistics 17.0 The total flavonoids contents were determined by (release 2008) and expressed as mean ± standard colorimetric method and expressed as mg quercetin deviation of triplicate determinations. Duncan’s test equivalent per g of dry weight, using the method was carried out to determine statistical significance described by Chang et al., (2002). 0.5 mL of each International Food Research Journal 19(3): 1125-1130 Antioxidant proprieties of methanolic and ethanolic extracts of Euphorbia helioscopia, (L.) aerial parts 1127 between the means. Differences were considered Table 1. Total phenolics and flavonoids contents of different extracts of statistically significant at the p≤ 0.05 level. Euphorbia helioscopia. Results and Discussion Extraction yields The percentage yields of prepared extracts from the sun spurge plant parts are summarized in table 1. Percentage yields ranged from 4.24% to 19.2%. Furthermore, our results showed that methanol extracts of all three tested parts were found to be higher than those obtained by the ethanol extraction, which is in agreement with several studies reporting the efficiency of methanol in polyphenols recovery compared to ethanol and other solvents of different polarity degrees (Sun et al., 2005; Dongmei et al., 2007; Anokwuru et al., 2011; Chinedu et al., 2011; Ghasemzadeh et al., 2011).