In the present study we investigated antioxidative properties of leaves of different wild (Allium flavum L., Allium sphaerocephalum L., Allium atroviolaceum Bois., Allium vienale L., Allium scorodoprasum L.) and grown (Allium nutans L., Allium fistulosum L., Allium vienale L., Allium pskemense B. Fedtsch, Allium schenoprasum L., Allium cepa L., Allium sativum L.) Allium sorts were investigated. Activities of antioxidant enzymes (superoxide dismutase, catalase, peroxidase, glutathione peroxidase), quantities of malonyldialdehyde superoxide and hydroxyl radicals and reduced glutathione and also the content of total flavonoids, chlorophylls a and b, carotenoids, vitamin C and soluble proteins were determined. Our results indicate that leaves of grown Allium sativum L., Allium cepa L., Allium vineale L., Allium fistulosum L. and Allium nutans L. and wild Allium flavum L. and Allium ursinum L. exibited high antioxidant activites

Stajner, Dubravka

Szőllősi Istvánné Varga, Ilona

English

Stajner Dubravka

Szőllősi Istvánné Varga Ilona

University of Szeged

Volume 47(1-4):103-106, 2003Acta Biologica Szegediensishttp://www.sci.u-szeged.hu/ABSSYMPOSIUM1Faculty of Agriculture, University of Novi Sad, Novi Sad, Yugoslavia, 2Department of Genetics and Molecular Biology,University of Szeged, Szeged, HungaryAn evaluation of the antioxidant abilities of Allium species+Dubravka Stajner1*, Ilona Szöllôsi Varga2ABSTRACT                        In the present study we investigated antioxidative properties of leaves ofdifferent wild (Allium flavum L., Allium sphaerocephalum L., Allium atroviolaceum Bois., Alliumvienale L., Allium scorodoprasum L.) and grown (Allium nutans L., Allium fistulosum L., Alliumvienale L., Allium pskemense B. Fedtsch, Allium schenoprasum L., Allium cepa L., Allium sativumL.) Allium sorts were investigated. Activities of antioxidant enzymes (superoxide dismutase,catalase, peroxidase, glutathione peroxidase), quantities of malonyldialdehyde superoxide andhydroxyl radicals and reduced glutathione and also the content of total flavonoids, chlorophyllsa and b, carotenoids, vitamin C and soluble proteins were determined. Our results indicate thatleaves of grown Allium sativum L., Allium cepa L., Allium vineale L., Allium fistulosum L. andAllium nutans L. and wild Allium flavum L. and Allium ursinum L. exibited high antioxidantactivites. Acta Biol Szeged 47(1-4):103-106 (2003)KEY WORDSantioxidant enzymesflavonoidschlorophyllcarotenoidAllium speciesAccepted April 30, 2003*Corresponding author. E-mail: stajnerd@polj.ns.ac.yu+In memory of Professsor Béla Matkovics103Oxygen free radicals – highly reactive species (O•2-, •OH,HO•2) with one or more unapaired electrones, H2O2 andactivated oxygen species (1∆g, 1Σg) are formed in almostevery cell of the body at an astonishing rate during normaloxdative metabolism (Gey 1994). Environmental factors suchas UV light, ozone, tobaco smoke, different xenobiotics,herbicides, pesticides, ionizing radiation etc. cause theirformation in greater extent (Halliwel and Gutteridge 1984).They react voraciosly with almost every cellular componentand contribute to many types of pathology. Antioxidantdefense mechanism counteract free radicals formation andreactions. In free radicals caused pathology antioxidantsneutralize free radicals and increasing levels of antioxidantsshould decrease pathology. Combinations of different naturalantioxidants which could be find in different medicinal plantswork better that separate antioxidants alone. Many epidemi-ological studies also support the idea that antioxidants areinterdependent (Kery et al. 2001).Onions are widely used in all parts of the world as aflavoring vegetable in various types of food. According totraditional medical literature they are source of many vita-mins and are useful in fever, dropsy, catarrh and chronicbronchitis (Block 1985). Roasted or otherwise they areapplied as a poultice to indolent boils, bruises, wounds, torelieve hot sensations and applied to the navel for dysenteryand fever (Brewster and Rabinowitch 1990).Today Alliums are used for their flavor, aroma and taste,beeing prepared domestically or forming raw material for avariety of food manufacturing processes (dehydration,freezing, canning and pickling). Also, dehydrated onionproduction is widely used, especially in the manufacture ofother processed foods (Brewster and Rabinowitch 1990). Onthe other hand therapeutic and medicinal values of garlic andonions are the subjects of many researches. The differentclinical studies have shown their benefit in the reduction ofcardiovascular disease risk by inducing lowering of serumcholesterol and blood pressure (Steiner and Lin 1994). Theyhave liver protective (Dion and Miler 1996), imune en-hancement and anti-infection (Lau 1989), anti-stress andanti-fatigue (Kawashima 1986), anti-cancer and cancerpreventive effects ( Dion and Milner 1997; Pinto et al. 1997;Balasenthil et al. 2001), brain and neurotrophic (Moriguchi1996) and other pharmacological effects (Yeh 1996). Manyrecent studies showed that Alliums have antioxidant effectwhat could be of the great importance for its use in pre-vention and treatment of different diseases (Lau 1989;Numagami 1996; Geng and Lau 1997) and contribute to itstherapeutic physionomy (Kyo et al. 1998).Our previous studies (Stajner et al. 1998a; 1988b; 1998c,1999) showed that different Allium species posses well-defined antioxidant activity. Therefore the aim of this studywas to perform the screening of different Allium species bydetermination activities of antioxidant enzymes superoxidedismutase (SOD), catalase (C-ase), peroxidase (P-ase),glutathione peroxidase (GP-ase), quantities of malonyl-dialdehide (MDA), superoxide (O•2-) and hydroxyl radicals(•OH), reduced glutathione (GSH) and contents of totalflavonoids, chlorophylls a and b, carotenoids, vitamin C andsoluble proteins. Our results could evaluate their antioxidantvalues and point to easy accessible sources of natural antioxi-104Stajner, Szöllôsidants that could be used as a possible food supplements orin the cosmetic and pharmaceutical industries.Materials and MethodsThe leaves of next wild (Allium flavum L., Allium sphaero-cephalum L., Allium atroviolaceum Bois., Allium vienale L.,Allium scorodoprasum L.) and grown (Allium nutans L.,Allium fistulosum L., Allium vienale L., Allium pskemenseB.Fedtsch, Allium schenoprasum L., Allium cepa L., Alliumsativum L.) Allium sorts were investigated.One g of plant material was ground with quartz sand ina cold mortar. The ground material was suspended in 5 ml 1mol/l K2HPO4 at pH 7.0. After a 10 min centrifugation at 4ºCand 15,000 g, the aliquots of the supernatant were used forSOD activity measurements. 20 µl of Tsuchihashi solution(chloroform:ethanol – 3:5) was added to the supernatant priorto measurement of the enzyme activity. The SOD activity wasdetermined in aliquots by the method of Misra and Fridovich(1972) based on the inhibition of transformation of adrena-line to adrenochrome at pH 10.2 (Matkovics et al. 1977).For the other antioxidant enzymes and biochemicaldeterminations, the plant material was treated in the sameway but the medium was 0.1 mol/l phospthate buffer (pH 7)with a plant material to medium ratio of 1:5, centrifuged for10 min at 15.000 g. After the centrifugation the supernatantwas evaluated for: P-ase activity, using guiacol as substrate(Matkovics et al. 1977); GP-ase activity using cumenehydroperoxide and GSH as substrates (Chiu et al. 1976); C-ase activity spectrophotometrically at 240 nm (Simon et al.1974); lipid peroxidation by the thiobarbituric acid (TBA)method; values were given as equivalent amounts od MDA;the calibration curve was prepared with malonyldialdehydebis-diacetal (Placer et al. 1968); superoxide radical wasdetermined by adrenaline autooxidation (Misra andFridovich 1972); hydroxyl radical by the inhibition ofdeoxyribose degradation (Cheesman et al. 1988).The amount of GSH was determined with Ellman (Sedlakand Lindsay 1968) and protein by Folin reagents (Lowry etal. 1951).Total flavonoids were estimated according toMarckam (1989). Pigments were extracted with acetone anddetermined spectrophotometrically using molar extinctioncoefficients according to Wettstein (1957). The content ofvitamin C was determined according to AOAC OfficialMethods of Analysis (1984).Table 1. Enzyme activities in leaves of different Alliums.Allium sort SOD C-ase P-ase GP-aseU/mg proteinAllium sativum L. 52.47±7.11 8.78±0.43 40.48±4,01 0.038±0.011Allium vineale L. (grown) 39.95±2.34 23.77±15.92 8.54±0.40 0.035±0,010Allium vinaele L. (wild) 3.63±0.52 7.32±4.54 22.51±2.26 0.020±0.006Allium sphearocephalum L. 8.91±2.52 6.42±1.10 105.83±8.00 0.050±0.014Allium scorodoprasum L. 45.54±23.88 7.32±11.96 130.10±10.00 0.977±0.052Allium atroviolaceum Boiss. 7.46±1.51 8.81±6.63 17.06±1.18 0.048±0.004Allium cepa L. 2.69±0.76 5.51±3.13 10.23±0.91 0.051±0,011Allium pskemenese L. 4.08±0.84 3.16±1.25 4.87±0.04 0.065±0,049Allium fistulosum L. 62.20±3.85 20.59±11.89 23.28±0.88 0.537±0.043Allium schenoprasum L. 77.46±5.98 5.91±0.62 63.26±4.21 0.083±0.024Allium nutans L. 20.27±3.27 10.01±5.11 10.46±0.45 0.018±0.002Allium ursinum L. 2.51±1.21 11.48±6.90 8.85±0.19 0.052±0.007Allium flavum L. 10.62±2.02 13.59±8.28 21.03±1.17 0.022±0.005Table 2. Quantities of (O•2-), .OH and MDA in leaves of different Alliums.Allium sort (O•2-) .OH MDA(nmol/mg protein)Allium sativum L. 508.8±218.1 2.10±0.29 24.55±1-02Allium vineale L. (grown) 155.9±78.0 2.32±0.05 13.19±0.51Allium vineale L. (wild) 608.8±189.5 2.56±0.03 30.67±0.47Allium sphearocephalum L. 1341.8±372.1 1.29±0.11 50.83±0.73Allium scorodoprasum L. 1587.6±357.8 25.52±1.209 130.19±1.24Allium atroviolaceum Boiss. 1376.2±315.3 3.21±0.20 67.09±1.02Allium cepa L. 473.5±147.4 0.82±0.16 7.61±0.36Allium pskemenese L. 129.4±86.2 0.07±0.02 7.12±0.77Allium fistulosum L. 153.4±124.3 0.20±0.02 4.98±0.34Allium schenoprasum L. 623.4±150.4 4.06±0.24 36.85±0.60Allium nutans L. 1611.9±619.6 0.30±0.04 12.40±0.16Allium ursinum L. 108.0±70.7 0.38±0.03 7.27±0.21Allium flavum L. 639.6±81.5 0.43±0.13 7.74±0.40105Antioxidant abilities of Allium speciesAll measurements were made in triplicate. The values areexpressed as mean ± standard error.Results and discussionThe results obtained from the study are presented in Tables1, 2, 3. and 4. The SOD activity was detected in leaves of allinvestigated Allium sorts. It had values between 2.51 U/mgpotein in Allium ursinum L. and 77.46 U/mg protein inAllium schenoprasum L.. C-ase activity ranged from 3.16 U/mg protein (Allium pskemense L.) to 23.77 U/mg protein(Allium vineale L. grown), P-ase activity from 4.87 U/mgprotein (Allium pskemense L.) to 130.10 U/mg protein(Allium scorodoprasum L.) and GP-ase activity from 0.018U/mg protein (Allium nutans L.) to 0.977 U/mg protein(Allium scorodoprasum L.; Table 1). Our results showed thatleaves do to various enzyme activities had different suscepti-bility to the action of toxic oxygen species.The results obtained from the study of oxygen radicalsand MDA are presented in Table 2. The highest O•2- quantitywas obserwed in leaves of Allium schorodoprasuu L. (1587.6nmol/mg protein) and lowest in leaves of Allium ursinum L.(108.0 nmol/mg protein). Quantity of •OH, was also highestin leaves of Allium schorodoprasum L. (25.52 nmol/mgprotein) where the MDA quantity was the highest (130.19nmol/mg protein) due to toxic oxygen radicals action. Thequantity of O•2- was lowest in leaves of Allium psekemenseL. (0.07 nmol/mg protein) and MDA quantity in leaves ofAllium fistulosum L. (4.98 nmol/mg protein).The results obtained from the study of the nonenzymicantioxidants and proteins are presented in Table 3. Quantityof GSH ranged from 0.108 nmol/mg protein (Allium vinealeL. wild) to 2.182 nmol/mg protein (Allium scorodoprasumL.). Content of flavonoids ranged from 37.87 mg/g (Allimflavum L.) to 496.53 mg/g (Allium cepa L.), vitamin C from0.005 mg/g (Allim cepa L.) to 0.811 mg/g (Allium sphaero-cephalum L.) and soluble proteins from 0.25 mg/g (Allimscorodoprasum L.) to 4.78 mg/g (Allium vineale L. grownand Allium cepa L.).The content of pigments is presented in Table 4. In leavesof Allium pskemense L. the contents of all investigatedpigments were the lowest: 0.39 mg/g for chlorophyll a, 0.11mg/g for chlorophyll b and 0.66 mg/g for carotenoids. Thehighest content of investigated pigments was obserwed inleaves of Allium ursinum L.: 2.87 mg/g for chlorophyll a,1.35 m/g for chlorophyll b and 9.99 mg/g for carotenoids(Table 4).Table 3. Quantity of reduced glutathione and contents of flavonoids, vitamin C and soluble proteins in leaves of different AlliumsAllium sort GSH Flavonoids Vitamin C Soluble proteinsnmol/mg mg/g mg/g mg/gAllium sativum L. 0.215±0.009 333.87±12.22 0.036±0,003 2.22±0.09Allium vineale L. (grown) 0.200±0,003 208.53±4.62 0.253±0.016 4.78±0.23Allium vineale L. (wild) 0.108±0.001 259.20±0.00 0.066±0.010 4.72±0.12Allium sphearocephalum L. 0.453±0.004 216.53±4.62 0.811±0.000 1.81±0.05Allium scorodoprasum L. 2.182±0.016 344.53±9.24 0.142±0.000 0.25±0.03Allium atroviolaceum Boiss. 0.399±0.011 59.20±13.86 0.157±0.005 3.42±0.05Allium cepa L 0.127±0.001 496.53±12.22 0.005±0.000 4.78±0.15Allium pskemenese L. 0.177±0.004 168.53±4.62 0.019±0.003 3.61±0.15Allium fistulosum L. 0.297±0.008 365.87±9.24 0.061±0.000 7.30±0.15Allium schenoprasum L. 0.669±0.009 432.53±4.62 0.122±0.000 1.86±0.12Allium nutans L. 0.125±0.004 53.87±9.24 0.011±0.000 3.68±0.15Allium ursinum L. 0.148±0.003 171.20±13.86 0.020±0.002 3.97±0.09Allium flavum L. 0.146±0.002 37.87±9.26 0.344±0.000 4.51±0.04Table 4. Pigments content in leaves of different Alliums.Allium sort Chlorophyll  a Chlorophyll  b Carotenoids(mg/g)Allium sativum L. 1.64±0.00 0.58±0.00 2.57±0.00Allium vineale L. (grown) 1.91±0.013 0.94±0.05 3.04±0.01Allium vineale L. (wild) 0.97±0.017 0.41±0.06 1.30±0.03Allium sphearocephalum L. 1.08±0.003 0.57±0.06 1.91±0.02Allium scorodoprasum L. 1.28±0.00 0.70±0.00 0.25±0.01Allium atroviolaceum Boiss. 0.41±0.01 0.12±0.00 0.71±0.01Allium cepa L. 1.23±0.05 0.67±0.02 1.92±0.01Allium pskemanese L. 0.39±0.03 0.11±0.03 0.66±0.01Allium fistulosum L. 1.14±0.03 0.58±0.03 0.87±0.03Allium schenoprasum L. 1.48±0.01 0.44±0.03 2.14±0.01Allium nutans L. 1.65±0.02 0.80±0.07 2.24±0.01Allium ursinum L. 2.87±0.03 1.35±0.01 9.99±0.01Allium flavum L. 0.70±0.03 0.33±0.06 1.10±0.01106Stajner, SzöllôsiOur results indicated that some of grown Alliums such asAllium sativum L., Allium vineale L., Allium cepa L.,Allium fistulosum L. and Allium nutans L. possessed highantioxidant activities. They had small quantities of •OH andMDA. At the some time SOD, or P-ase activity were high andthe content of flavonoids and carotenoids was also high whatcontribute to their antioxidant abilities.Among the wild Alliums, Allium flavum L. and particu-lary Allium ursinum L. exibited high antioxidant activities.Allium ursinum L. had extraordinary antioxidant abilitiesinspite lowe SOD activity due to high C-ase activity highcontent of chlorophylls a and b and carotenoids. In leaves ofAllium ursinum L. small quantities of O•2- and •OH weredetected what provoced small lipid peroxidation.Our results indicates that leaves of menshioned grownand wild Alliums could be used as a source of natural untoxicantioxidants in food, cosmetic and pharmaceutical industries.ReferencesAOAC (1984) Vitamins and other nutrient Official methods of analysis.Association of Official Analytical Chemists. Inc. 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An evaluation of the antioxidant abilities of Allium species

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An evaluation of the antioxidant abilities of Allium species

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