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929 Advances in Environmental Biology, 5(5): 929-932, 2011 ISSN 1995-0756 This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Antioxidant Activity, Total Phenolic and Flavonoids Contents in Methanolic and Aqueous Extract of Achillea millefolium L. 1 Anoosh Eghdami, 2Vahid Fateh, 2Davood Eradatmand Asli and 2Alireza Houshmandfar 1 Department of Biology, Islamic Azad University, Saveh Branch, Saveh, Iran Department of Agronomy and Plant Breeding, Islamic Azad University, Saveh Branch, Saveh, Iran 2 Anoosh Eghdami, Vahid Fateh, Davood Eradatmand Asli and Alireza Houshmandfar; Antioxidant Activity, Total Phenolic and Flavonoids Contents in Methanolic and Aqueous Extract of Achillea millefolium L. ABSTRACT Achillea millefolium L. sensu lato (yarrow) is the best-known species of the genus Achillea due to numerous medicinal applications both in folk and conventional medicine. Phenolic compounds such as flavonoids and phenol carbonic acids which present in yarrow are constitute one of the most important groups of pharmacologically active substances. In the present study, Achillea millefolium L. flowers gathered from native populations in different locations of Saveh region of Iran were analyzed for composition of phenolic compounds. The extract was analyzed by aluminum chloride (AlCl3) method using the Folin-Ciocalteu reagent. The antioxidant activity was further tested by the DPPH (2,2'-diphenyl-1-picrylhydrazyl) free radical scavenging method. High-performance liquid chromatography (HPLC) was used for chemical analyze. The extract of Achillea millefolium L showed scavenging effects against the DPPH radical. Furthermore, the antioxidant and total phenolic content levels were positively and significantly correlated. Key words: Phenolic compounds, DPPH, Antioxidants, Folin-Ciocalteou Introduction Phenolic compounds are secondary metabolites that are derivatives of the pentose phosphate, shikimate, and phenylpropanoid pathways in plants [18,9,5]. Phenolics are antioxidants with redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers [12]. Antioxidants are important because they have the ability of protecting organisms from damage caused by free radical-induced oxidative stress [2]. Besides well known and traditionally used natural antioxidants from tea, fruits, vegetables and spices, some natural antioxidant are already exploited commercially either as antioxidant additives or a nutritional supplements [16]. In this regard, although many of plant species have been investigated in the search for novel antioxidants [3], there is still a demand to find more information concerning the antioxidant potential of plant species. Flavonoids are a group of poly phenolic compounds with known properties included free radical scavenging, inhibition of hydrolytic and oxidative enzymes, and antiinflammatory action. Some evidence suggests that the biological actions of these compounds are related to their antioxidant activity [8,13]. According to physiological properties of different flavonoids, the flavonols having orto or para hydroxyl group in the 2- phenyl ring are known to have strong antioxidant properties, while free hydroxyl at the 5,7- position proved to have a pro-oxidant effect (Table 1). Achillea millefolium L. has a long history of use as traditional herb medicine even in veterinary medicine, preparations in the form of infusions, decoctions or fresh juices have been applied against anorexia, stomach cramps, flatulence, gastritis, enteritis, internal Corresponding Author Anoosh Eghdami, Department of Biology, Islamic Azad University, Saveh Branch, Saveh, Iran E-mail: [email protected] 930 Adv. Environ. Biol., 5(5): 929-932, 2011 and external bleeding (coughing blood, nosebleed, hemorrhoidal and menstrual bleeding, bloody urine), wounds, sores, skin rash as well as dog and snake bites. In the present study, Achillea millefolium L. flowers were analyzed for composition of phenolic compounds. followed by 5% NaNO2 (0.03 ml). Then, AlCl3 (0.03 ml, 10%) was added, after 5 min and at 25°C. After further 5 min, the reaction mixture was treated with 0.2 ml of 1 mM NaOH. Finally, the reaction mixture was diluted to 1 ml with water and the absorbance was measured at 510 nm. The results were expressed as mg quercetin (QE) g-1 of Achillea millefolium L. Materials and methods Antioxidant Activity: Plant Collection: The plants were collected in July 2010 from Saveh region (35°15´ N, 49°45´ E; 1680 m above mean sea level), located in Markazi state of Iran. Chemicals: 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy4H-chromen-4-one) were purchased from Sigma Chemical Co. (St., Louis, USA). Gallic acid, FolinCiocalteu reagent and methanol were purchased from Merck Co. (Germany). Sample Preparation: The samples were first ground to fine powder. For water extraction, 0.5 g of the fine powder was extracted with 10 ml of ultra-filtered water at 100 °C for 30 min in a water bath. For methanol extraction, 0.5 g of the powder was extracted with 10 ml of 80% methanol at 40 °C for 24 h. The samples were then cooled down to room temperature and centrifuged at 4500 rpm for 15 min. The supernatant was recovered and used for the DPPH assay and total phenolic analysis. Determination of Total Phenolic Content: The total phenolic content of the Achillea millefolium L. extracts was determined using the Folin-Ciocalteu reagent [19]. The reaction mixture were contained 200 μl of diluted rice bran extract, 800 μl of freshly prepared diluted Folin-Ciocalteu reagent and 2 ml of 7.5% sodium carbonate. The final mixture was diluted to 7 ml with deionized water. Mixtures were kept in dark at ambient conditions for 2 h to complete the reaction. The absorbance at 765 nm was measured. Gallic acid was used as standard and the results were expressed as mg of Gallic acid equivalents (GAE) g-1 of Achillea millefolium L. Determination of Total Flavonoid Content: Total flavonoid content was determined using aluminium chloride (AlCl3), and quercetin (standard) as described by Ordon Ez et al. [11]. The plant extract of 0.1 ml was added to 0.3 ml distilled water The antioxidant activity was determined using DPPH free radical scavenging method. A dilution of 1 M DPPH was prepared and the absorbance of a mixture of 1 ml of the extract and 1 ml of the DPPH solution was measured at 517 nm. The radical scavenging activity was calculated from the following equation [1]: HPLC Analysis: The analysis was performed with a flow rate of 0.75 mL min-1, using 0.2% trifluoroacetic acid (TFA) as solvent A and Methanol as solvent B, with a linear gradient from 5 to 80% methanol in 50 min (Table 2). All solvent used were filtered on Acetate Plus (0.22) before analysis. The selected flavonoid standards required a greater concentration of Methanol (80%) and a longer HPLC run for their proper elution than phenolic acids. Each standard was first injected individually to determine the exact retention time and chromatographic characteristics (.max, absorbance ratio) followed by the analysis of the standard mixture. The methanolic extract of Achillea millefolium L was filtered on qualitative circle, Whatman filter paper No. 1 (Whatman International Ltd., UK) under vacuum and subsequently on Acetate Plus filters (0.22 μm) to remove the undesirable contaminants. HPLC analysis was performed using a liquid chromatographic Agilent 1200 equipped with UV detector G1314B. Separations were carried out using an analytical column (150 × 4.6 mm, 5 μm) and with G1311A quaternary pump. All the analysis was performed in triplicate. Results and discussion Table 3 indicated the total phenolic and flavonoids contents and antioxidant activities of methanol and aqueous extract of Achillea millefolium L. Furthermore, HPLC chromatograms for some of polyphenol from methanolic exteract of Achillea millefolium L. and for some of polypehnol standard were presented in Figure 1 and 2 respectively. The 931 Adv. Environ. Biol., 5(5): 929-932, 2011 Table 1:. Some of the flavonoids compounds name and structure Table 2:. Gradient elution method performed with binary solvent system using 0-5 5-8 8-11 11-14 14-17 17-20 20-23 23-26 26-29 29-32 A 5 10 15 20 25 30 35 40 45 50 B 95 90 85 80 75 70 65 60 55 50 as mobile phase 32-35 35-38 55 60 45 40 38-41 65 35 41-44 70 30 44-47 75 25 47-50 80 20 Table 3; Total phenolic and flavonoids contents and antioxidant activities of methanol and aqueous extract of Achillea millefolium L. Variety Phenolic content Flavonoids content Antioxidant activity by (QE g-1) DPPH (Inhibition %) (mg GAL g-1) Methanolic extract 123.9 ± 2.6 41.2 ± 1.7 75% Aqueous extract 48.4 ± 2.7 13.15 ± 1.8 50.8% Fig. 1: HPLC chromatogram for some of polypehnol standard (gallic acid, vanillic acid, coumaric acid and benzoic acid respectively). Fig. 2: HPLC chromatogram for some of polyphenol from methanolic exteract of Achillea millefolium L. 932 Adv. Environ. Biol., 5(5): 929-932, 2011 analysis of the extract of Achillea millefolium L. showed scavenging effect against DPPH radical. The hierarchy for antioxidant capacity with respect to their EC50 values was methanolic extract > aqueous extract. Correlation coefficient showed that total phenolic content was responsible for antiradical efficiency in the extract of Achillea millefolium L. The antioxidant and total phenolic content levels are also positively and significantly correlated. It has been recognized that flavonoids show antioxidant activity and their effects on human nutrition and health are considerable [13]. The mechanisms of action of flavonoids are through scavenging or chelating process [7,3]. Phenolic compounds are a class of antioxidant agents which act as free radical terminators [17]. The results strongly suggested that the extract of Achillea millefolium L. can be a promising source of potential antioxidants. The antioxidant activities observed can be ascribed both to mechanisms exerted by phenolic compounds and also to synergistic effects of different phytocompounds. 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