Original Research Article Antioxidant, cytotoxic and DNA protective properties of Achillea eriophora DC. and Achillea biebersteinii Afan. extracts: A comparative study Maryam Varasteh-kojourian1, Parvaneh Abrishamchi1*, Maryam M. Matin1,2, Javad Asili3, Hamid Ejtehadi1, Fatemeh Khosravitabar1 1Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran 2Cell and Molecular Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran 3Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Article history: Abstract Received: Sep 24, 2015 Objective: Achillea is a traditional medicinal herb which contains Received in revised form: Feb 15, 2016 different phenol and flavonoid compounds that are responsible for Accepted: May 08, 2016 Achillea pharmacological effects. We aimed to determine phenol Vol. 7, No. 2, Mar-Apr 2017, and flavonoid contents, besides antioxidant activities of different 157-168. extracts from Achillea eriophoraa (A. eriophora) DC. and Achillea biebersteinii (A. biebersteinii) Afan. (endemic species in Iran) and * Corresponding Author: to investigate their effects on human cells. Tel: +989151246726 Materials and Methods: Achillea extracts, were prepared by Fax: +985138796416 maceration and shaking methods, from different parts (aerial parts, [email protected] stem, leaves and inflorescence) of two species using methanol and ethanol as solvents. Total phenol and flavonoid contents were Keywords: Phenol measured by spectrophotometry, and antioxidant activities of the Flavonoids extracts were determined by DPPH radical scavenging, BCB and DPPH TBARS assays. Cytotoxicity and antioxidant activities of the MTT assay extracts were investigated in Human Foreskin Fibroblast (HFF3) Comet cells using MTT, comet and H2O2 assays. HFF3 cells Results: Methanol extracts of A. biebersteinii prepared from leaves and inflorescence by maceration method exhibited maximum phenol (1657.58 ± 36.45 mg GAE/100 g DW) and flavonoid (264.00 ± 62.16 mg QUE/100 g DW) contents. Leaf methanol extract showed significantly higher antioxidant activity (0.0276 ± 0.003, 0.16 ± 0.016 and 13.96 ± 0.26 mg/ml for DPPH, BCB and TBARS IC50s, respectively) than those of the other extracts. Leaf extract of A. biebersteinii was not cytotoxic even at the highest examined dose (512 µg/ml) and inhibited cell toxicity induced by H2O2 (98% viability for the cells pretreated with plant extract in the presence of H2O2). Comet assay also confirmed high DNA protective activity of leaf extracts. Conclusion: Achillea extracts possess remarkable antioxidant activity, and could be good natural alternatives to synthetic antioxidants in pharmaceutical and food industries. Please cite this paper as: Varasteh-kojourian M, Abrishamchi P, Matin MM, Asili J, Ejtehadi H, Khosravitabar F. Antioxidant, cytotoxic and DNA protective properties of Achillea eriophora DC. and Achillea biebersteinii Afan. extracts: A comparative study. Avicenna J Phytomed, 2016. Epub ahead of print. AJP, Vol. 7, No. 2, Mar-Apr 2017 157 Varasteh-kojourian et al. Introduction flavonoid-enriched extracts from different Achillea, which belongs to the family parts of A. eriophorea and A. biebersteinii. Compositae (Asteraceae), is a genus with The antioxidant activity of the methanol over 100 species all around the world. extracts prepared by maceration from Although these medicinal perennial different parts of the plants, was evaluated rhizomatous plants are native to Europe using different assays. To the extent of our and Western Asia, they are also found in knowledge, this is the first report about the Australia, New Zealand and North antioxidant activity of leaf methanol America (Chevallier, 2000). The two extracts of A. eriophorea and A. studied species Achillea eriophora (A. biebersteinii in human fibroblast cells. eriophora) DC. And Achillea biebersteinii (A. biebersteinii) Afan. are endemic plants in Iran. Materials and Methods Achillea, known as “Bumadaran” in Chemicals Persian, is one of the most widely used Folin-Ciocaltue, sodium carbonate, medicinal plants in Iran. It is used as methanol, ethanol, gallic acid, aluminum hypoglycemic, nerve tonic, anti- chloride, 1,1-diphenyl-2-picrylhydrazyl hemorrhoid, anti-diarrhea, antacid, (DPPH), beta-carotene, linoleic acid, carminative, appetizer, anthelmintic and tween40, chloroform, butylated hydroxyl anti-bacterial remedies (Amiri and toluene (BHT), 2,2’-azobis-(2- Joharchi, 2013; Ghorbani, 2005; Pirbalouti amidinopropane) dihydrochloride (ABAP), and Golparvar, 2007; Zargari, 1993). acetic acid, tiubarbituric acid, sodium These pharmacological properties have dodecyl sulfate (SDS), butanol, 3-(4,5- been mainly attributed to the phenolic and dimethylthiazol-2-yl)-2,5-diphenyl- polyphenolic compounds, which are well tetrazolium bromide (MTT), H2O2, known as antioxidant agents (Evans, 2009; ethylenediaminetetraacetic acid (EDTA), Harborne and Williams, 2000; Weber et dimethyl sulfoxide (DMSO) and all al., 2006). Clinical evidences have solvents were purchased from Merck revealed that antioxidants are effective in (Germany). Ascorbic acid and potassium the treatment of various diseases, including acetate were purchased from Sigma– atherosclerosis, arthritis, ischemia and Aldrich (St. Louis, MO, USA). Dulbecco's reperfusion injury of many tissues, central Modified Eagle's Medium (DMEM) and nervous system injury, gastritis, and fetal bovine serum (FBS) were obtained cancer, and are beneficial to the wound from Gibco Life Technologies (Grand healing process (Cook and Samman, 1996; Island, NY, USA). Kumpulainen and Salonen, 1999). These activities are in accordance with reported Sample collection and extract pharmaceutical properties for Achillea. preparation Antioxidant activity of different species Plants were collected at the flowering of Achillea including Achillea millefolium stage in May 2011 from pastures of L. (Candan et al., 2003), Achillea ligustica Khorassan Razavi (N 36.291886, E All. (Conforti et al., 2005), Achillea 58.583121; 1618 m above sea level) and wilhelmsii (Ozgen et al. 2004) and A. South Khorasan provinces (N 36.34020, E biebersteinii (Tawaha et al., 2007) have 40.710158; 1591 m above sea level), Iran. been previously investigated. However, the The voucher specimens (No. 30348 and protective effects of A. eriophorea and A. 22004 for A. eriophora and A. biebersteinii against oxidative stress in biebersteinii, respectively) were deposited HFF3 cells have not yet been reported. In in Herbarium of Ferdowsi University, this study, two extraction methods and Mashhad, Iran. solvents were used to obtain phenol and AJP, Vol. 7, No. 2, Mar-Apr 2017 158 Antioxidant and cytotoxic properties of Achillea Grinded dry materials from different Tween 40 (200 mg) were added to 1 ml of parts of plants (stem, leaves, aerial parts this mixture. After removing chloroform and inflorescence), were extracted with using a rotary evaporator at 40ºC, the methanol and ethanol (1:20 w/v) using remaining where solved in oxygenated maceration and shaking. Extracts were distilled water (50 ml) and vigorously filtered through the regular filter paper and shacked. An aliquot of 250 µl of the evaporated under vacuum. above-prepared beta carotene–linoleic acid emulsion was applied to each well of a 96- Total phenol and flavonoid assay well plate. Next, 30 µl of different The total phenolic content of all extracts concentrations of the extracts (0.5-7 was determined spectrophotometrically mg/ml) or BHT as a standard (1-100 according to Folin-Ciocalteu method µg/ml) were added to each well in (Pattanayak et al., 2012), and total triplicate. An equal amount of the extracts flavonoid content was determined using or BHT was used as blank. The aluminum chloride colorimetric method microplates were incubated at 55ºC and (Chang et al., 2002). The results were their optical densities were determined at expressed in terms of gallic acid equivalent 492 nm using an ELISA reader. Reading (GAE mg/g of dry weight) for phenolic the absorbance of all samples was carried content and quercetin equivalent (QE mg/g out at the start (t=0) and after 105 min of of dry weight) for flavonoid content, incubation. The antioxidant activity which are the two common reference coefficient (AAC) was estimated compounds. according to the following formula: ACC = [(AT105 - AB105) / (AB0– Antioxidant assays AB105)] DPPH radical scavenging microplate Where AT105 and AB105 are absorbance assay of sample and blank after 105 min, The antioxidant properties of methanol respectively. AB0 is the absorbance of extracts (0.5-7 mg/ml) or gallic acid as a blank at t=0. standard, were investigated by reducing the stable DPPH radical (Yang et al., TBARS assay 2011). The absorbance was then measured A modified TBARS assay (Bazzaz et at 492 nm using an ELISA reader al., 2011), using egg yolk homogenates as (Awareness Technology, USA). The lipid rich media, was applied to measure antioxidant index (AI %) was calculated as the antioxidant capacity of the extracts. [(1 – A1 – A2/ A0) × 100]. Where A0 is the Briefly, 500 µl of yolk homogenate (10% absorbance of the control reaction (without w/v in distilled water) and 100 µl of sample), A1 is the absorbance of sample solution (5-50 mg/ml sample/gallic acid, and A2 is the concentrations of the extracts or standard), absorbance of sample without DPPH. were added to a test tube and made up to 1 Analyses were run in triplicate, and IC50 ml with distilled