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LWT - Food Science and Technology 54 (2013) 139e146
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LWT - Food Science and Technology
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Phenolic profile, antioxidant, anti-inflammatory and cytotoxicactivities of small yellow onion (Allium flavum L. subsp. flavum,Alliaceae)
Natasa Simin a,*, Dejan Orcic a, Dragana Cetojevic-Simin b, Neda Mimica-Dukic a,Goran Anackov c, Ivana Beara a, Dragana Mitic-Culafic d, Biljana Bozin e
aUniversity of Novi Sad, Faculty of Sciences, Department of Chemistry, Biochemistry and Environmental Protection, Trg Dositeja Obradovica 3,21000 Novi Sad, SerbiabUniversity of Novi Sad, Faculty of Medicine, Oncology Institute of Vojvodina, Dr Goldmana 4, 21204 Sremska Kamenica, SerbiacUniversity of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Trg Dositeja Obradovica 2, 21000 Novi Sad, SerbiadUniversity of Belgrade, Faculty of Biology, Studentski Trg 16, 11000 Belgrade, SerbiaeUniversity of Novi Sad, Faculty of Medicine, Department of Pharmacy, Hajduk Veljkova 3, 21000 Novi Sad, Serbia
a r t i c l e i n f o
Article history:Received 18 October 2012Received in revised form13 April 2013Accepted 13 May 2013
Keywords:Allium flavumPhenolics LC-MS/MSAntioxidantAnti-inflammatoryCytotoxicity
* Corresponding author. Tel.: þ381 21 4852757; faxE-mail addresses: [email protected] (N. Sim
(D. Orcic), [email protected] (D. Cetojevic-Sdh.uns.ac.rs (N. Mimica-Dukic), [email protected] (D. Mitic-Culafic), [email protected]
0023-6438/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.lwt.2013.05.023
a b s t r a c t
The objectives of this study were to define the phenolic profile, antioxidant, anti-inflammatory andcytotoxic properties of edible Allium flavum subsp. flavum (small yellow onion), which has never beencomprehensively examined before. The presence and content of 44 phenolic compounds in methanolextracts of A. flavum were investigated by LC-MS/MS, where 25 compounds were found, the mostdominant being: ferulic, p-coumaric, caffeic, p-hydroxybenzoic, vanillic, protocatechuic and syringic acid,rutin, quercetin-3-O-glucoside and kaempferol-3-O-glucoside. Antioxidant activity, determined throughseveral assays, was low in comparison to the synthetic antioxidant butylated hydroxytoluene, butcomparable to onion (Allium cepa L.) extract. Anti-inflammatory potential was studied by measuring theinhibitory effect on cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) activity, where A. flavumexpressed high inhibitory potential, especially on 12-LOX activity (IC50 ¼ 0.078 mg mL�1). Treatment offour human cell lines resulted in a considerable inhibition of cell growth, where the extract of A. flavumexpressed selective inhibitory action towards cervix epithelioid carcinoma and colon adenocarcinomacells (IC50 ¼ 71 mg mL�1 and IC50 ¼ 81 mg mL�1, respectively). To conclude, these results support use ofA. flavum as a functional food and indicate that it could be a potent source of health-beneficialphytochemicals.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Allium flavum subsp. flavum is a member of the genus Allium,which is by far the largest genus of the Alliaceae family, comprisingabout 750 species. Some of the species represent the underlyingtaxa for cultivated forms, which are widely used in human dietas spices and vegetables (onion e Allium cepa L., garlic e Alliumsativum L., leek e Allium porrum L.). Considering also the highbiological activity of these well-researched cultivated species,we found it worthwhile to investigate the chemical composition
: þ381 21 454065.in), [email protected]), [email protected] (I. Beara), mdragana@(B. Bozin).
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and biological activities of an unexplored member of the genusAlliume awild-growing A. flavum subsp. flavum. This is a deciduousplant with simple leaves and yellow flowers, known by the com-mon name “small yellow onion”. Leaves and bulbs are edible, are ofa milder taste and smell than onion and are used traditionally inBalkan region as a spice for soups, stews and salads (Grlic, 1986).A. flavum is native to South, Central and Eastern Europe and CentralAsia (Anackov, 2009).
There have been very few published data on either its chemicalconstituents or biological potential. Besides a taxonomic study ofsulfoxide compounds in this species (Kusterer, 2010), there are noother data on the chemical profile of this species. Furthermore,there are only a few biochemical reports confirming that A. flavumextracts possess a high antioxidant (Curcic et al., 2012; Stajner, Igic,Popovic, & Malencic, 2008), antibacterial and antiproliferativeactivity (Curcic et al., 2012), as well as anti-Aspergillus properties
N. Simin et al. / LWT - Food Science and Technology 54 (2013) 139e146140
(Yin & Tsao, 1999). However, there have been no available reportson anti-inflammatory activity of this species. Considering thatA. flavum is traditionally used as a food, we considered it importantto examine whether this species could be regarded as functionalfood possessing both nutritional and healthy properties.
Therefore, the aim of the present study was to explore thephenolic profile and anti-inflammatory potential of A. flavumsubsp. flavum, as well as to expand knowledge of their antioxidantand antiproliferative properties and to compare these biologicalactivities with the activities of A. cepa, the official drug (WHO,1999).
2. Materials and methods
2.1. Plant material and extract preparation
The whole plants of wild-growing A. flavum L. 1753 subsp. fla-vum var. flavum f. flavum were collected in July 2009 from threedifferent locations in Serbia (Vrsacki breg, Dimitrovgrad, Babusn-ica). Samples of the grown onion (A. cepa L.) were collected also inJuly 2009 in the village of Neradin, the Fru�ska Gora Mountain,Serbia. The voucher specimens (A. flavum subsp. flavum, Vrsackibreg e no. 2-1769, Dimitrovgrad e no. 2-1765, Babusnica e no. 2-1767; A. cepa, no. 2-1762) were prepared, identified and depositedat the Herbarium of the Department of Biology and Ecology (BUNSHerbarium), University of Novi Sad, Faculty of Sciences.
30 g of air-dried and ground plant material (whole plants, aerialparts, bulbs) were macerated with 70% aqueous methanol (8 mLper 1 g of dw) during 72 h at 30 �C. After filtration, the solvent wasevaporated to dryness under vacuum at 45 �C and dry residueswere re-dissolved in 70% aqueous methanol to the final concen-tration of 300 mg mL�1 (for antioxidant assays) or in DMSO toobtain 300 mg mL�1 stock solutions (for evaluation of anti-inflammatory and cytotoxic activity). Prepared extracts(300 mg mL�1 in 70% aqueous methanol) were diluted with amixture of 0.5% aqueous formic acid and methanol (in ratio of 7:3)to obtain 2 mg mL�1 stock solutions for LC-MSeMS analysis of thephenolic profile.
2.2. Quantitative LC-MS/MS analysis of the selected phenolics
The content of quinic acid and 44 selected phenolic compounds(14 phenolic acids, 25 flavonoids, 3 coumarins and 2 lignans) wasinvestigated by LC-MS/MS according to the previously reportedmethod (Beara et al., 2012). Standards of the compounds werepurchased from SigmaeAldrich Chem (Steinheim, Germany), FlukaChemie GmbH (Buchs, Switzerland) or from ChromaDex (SantaAna, USA).
The Agilent 1200 series liquid chromatograph, coupled withAgilent series 6410B electrospray ionization triple-quadrupolemass spectrometer and controlled by MassHunter ver. B.03.01.software, was used for analysis. Analytes were separated using aZorbax Eclipse XDB-C18 4.6 mm � 50 mm � 1.8 mm (AgilentTechnologies) reversed-phase column. Compound-specific, opti-mized MS/MS parameters are given in Table 1.
2.3. Antioxidant activity
Antioxidant potential was determined using several assays:assays related to free radical (DPPH�, ABTS�þ), reactive oxygen(HO�) and reactive nitrogen species (NO�) scavenging activity, andability to inhibit lipid peroxidation (LP). The synthetic antioxidantbutylated hydroxyanisole (BHA) was used as a positive control.ABTS�þ scavenging activity was examined by using the Total Anti-oxidant Status kit (Biorex Diagnostics Limited, Antrim, UK), inline
with the specified recommendations. The total antioxidant status(TAS) of the extract is expressed in mmol of Trolox equivalents pergram of dry weight. DPPH radical scavenging activity wasmeasuredaccording to the method described in Beara et al. (2012). NOscavenging capacity and LP inhibition ability were determined bymethods published by Orcic, Mimica-Dukic, Franciskovic, Petrovic,and Jovin (2011). HO� scavenging activity was measured using theESR DMPO spin trapmethod (Babovic et al., 2010). ESR spectrawererecorded after 2.5 min, with the following spectrometer settings:field modulation 100 kHz, modulation amplitude 0.226 G, receivergain 5 � 105, time constant 80.72 ms, conversion time 327.68 ms,center field 3440.00 G, sweep width 100.00 G, x-band frequency9.64 GHz, power 20 mW, and temperature 23 �C.
2.4. COX-1 and 12-LOX inhibition assays
Anti-inflammatory potential was studied by ex vivo COX-1 and12-LOX assay previously described by Beara et al. (2010) andmodified by Lesjak et al. (2013). Estimated IC50 values werecompared to IC50 values of standards e aspirin (acetylsalicylic acid)(Sigma Aldrich, Germany), a well-known COX-1 inhibitor, andquercetin (Sigma Aldrich, Germany), a 12-LOX inhibitor.
2.5. Effect on cell growth
Antiproliferative activity was evaluated in vitro by the estima-tion of cell growth effects in four human cell lines: HeLa (cervixepithelioid carcinoma; ECACC No. 93021013), MCF7 (breastadenocarcinoma; ECACC No. 86012803), HT-29 (colon adenocarci-noma; ECACC No. 91072201) and MRC-5 (human fetal lung; ECACCNo. 84101801). Cell lines were grown in DMEM (PAA LaboratoriesGmbH, Pashing, Austria) with 45 mg mL�1 glucose, supplementedwith 100 mL mL�1 heat inactivated FCS (PAA Laboratories GmbH,Pashing, Austria), 100 IU mL�1 of penicillin and 100 mg mL�1 ofstreptomycin. They were cultured in 25 cm2
flasks at 37 �C in at-mosphere of 5% CO2 and high humidity, sub-cultured twice a weekand a single cell suspension was obtained using 1 mg mL�1 trypsinwith 0.4 mg mL�1 EDTA.
Extracts were diluted in 9 mg mL�1 NaCl and sterilized byfiltration through 0.22 mmmicro filters (Sartorius, Germany). Serialdilutions of extracts (20 mL per well) were added in 180 mL of me-dium to achieve the required final concentrations. Serial dilutionsof standards in DMSO (1 mL) were added in 199 mL of medium. Equalvolumes of solvents were added in control wells. Concentration ofDMSO in cell culture was �5 mL mL�1.
Cell growth was evaluated by Sulforhodamine B (SRB) assaypreviously published by Cetojevic-Simin et al. (2012).
3. Results and discussion
3.1. LC-MS/MS analysis of the selected flavonoids
The phenolic profile of A. flavum extracts has not been investi-gated so far. In this study, 44 plant phenolics and quinic acid (anintermediate in plant phenolics biosynthesis) were quantified inaerial parts and bulb extracts of A. flavum from the three locations inSerbia, by using the LC-MS/MS technique. The MRM mode wasapplied as the preferred acquisition method for the accurate quan-tification. This type of analysis provides the high sensitivity andspecificity, due to the fact that only ions specific to targeted analytesare monitored. Representative chromatograms are shown in Fig. 1,while the overall data concerning the content of the phenolic com-pounds are presented in Table 2. The results of the analysis showedthat the aerial parts extracts of A. flavum are rich in phenolic acidsand flavonoids (25 compounds were detected), while the bulb
Table 1LC-MS/MS parameters for standard compounds.
Compound Compound-specific MS/MS parametersa
Retention time (min) Fragmentor voltage (V) Precursor ion (m/z) Product ion (m/z) Collision energy (V)
Quinic acid 0.52 150 191 85 20Gallic acid 0.58 90 169 125 10Catechin 0.74 150 289 245 10Protocatechuic acid 0.79 105 153 109 9Chlorogenic acid 0.80 100 353 191 10Epigallocatechin gallate 0.81 165 457 169 16Epicatechin 0.95 150 289 245 102,5-dihydroxybenzoic acid 1.03 100 153 109 9p-hydroxybenzoic acid 1.08 80 137 93 10Aesculetin 1.13 105 177 133 15Caffeic acid 1.18 100 179 135 10Vanillic acid 1.24 100 167 108 15Syringic acid 1.31 90 197 182 7p-coumaric acid 1.69 90 163 119 9Umbelliferone 1.73 120 161 133 19Scopoletin 1.77 80 191 176 8Ferulic acid 1.90 90 193 134 11Vitexin 1.90 200 431 311 22Sinapic acid 1.92 100 223 193 17Luteolin-7-O-glucoside 2.13 230 447 285 30Hyperoside 2.16 200 463 300 30Quercetin-3-O-glucoside 2.25 210 463 300 30Rutin 2.33 135 609 300 42Apiin 2.60 250 563 269 36o-coumaric acid 2.62 100 163 119 5Myricetin 2.67 150 317 179 20Quercitrin 2.75 190 447 300 27Kaempferol-3-O-glucoside 2.80 190 447 284 30Apigenin-7-O-glucoside 2.81 135 431 268 41Secoisolariciresinol 2.90 130 361 165 263,4-dimethoxycinnamic acid 2.99 110 207 103 7Baicalin 3.40 140 445 269 22Daidzein 3.43 145 253 208 31Matairesinol 3.66 130 357 122 24Quercetin 3.74 130 301 151 15Naringenin 3.87 130 271 151 16Cinnamic acid 3.91 100 147 103 5Luteolin 4.03 135 285 133 25Genistein 4.12 145 269 133 32Kaempferol 4.55 130 285 285 0Apigenin 4.71 130 269 117 25Isorhamnetin 4.79 160 315 300 21Chrysoeriol 4.82 125 299 284 20Baicalein 5.15 165 269 269 0Amentoflavone 5.78 220 537 375 35
a Common parameters for all analytes: Injection volume e 5 mL; mobile phase: A e 0.05% aqueous formic acid, B emethanol; flow rate 1 mL/min; solvent gradient: starting30% B, reaching 70% B in 6.00 min, 100% B at 9.00 min, holding until 12.00 min, post-time 3min. Column temperature 45 �C. Ion source parameters: drying gas (N2) 350 �C, 9 L/min; nebulizer gas pressure 40 psi; capillary voltage 4 kV, negative polarity.
N. Simin et al. / LWT - Food Science and Technology 54 (2013) 139e146 141
extracts contain only a very small amount of phenolics (mainly caf-feic acid, 68.81e403.5 mg g�1 of dw). Since the previously reportedpapers, concerning the flavonoid profile, pointed out that edibleonions mainly contain flavonols (Park & Lee; 1996; Price & Rhodes,1997), our research was focused on this class of the flavonoid com-pounds. Consequently, it was found that by far the most abundantflavonoid in the aerial parts extract is rutin (quercetin-3-O-rutino-side) (23.16e201.86 mg g�1 of dw), followed by isoquercitrin(quercetin-3-O-glucoside) (10.23e57.21 mg g�1 of dw), iso-rhamnetin (30-O-methylquercetin) (0.23e2.47 mg g�1 of dw) andkaempferol-3-O-glucoside (0.63e1.98 mg g�1 of dw). Flavonoidaglycones e quercetin, kaempferol and chrysoeriol e were alsopresent, but in lower amounts. Based on these results, A. flavumsubsp. flavum could be classified into flavonol (flavonol glycosides)chemotype. Furthermore, there are reports suggesting that onioncontains high amounts of quercetin glycosides (quercetin-3,40-O-diglucoside and quercetin-40-O-glucoside) (Lombard, Geoffriau, &Peffley, 2002), quercetin aglycone and isorhamnetin-40-O-gluco-side, as well as small amounts of rutin, quercetin-3-O-glucoside and
isorhamnetin (a methyl ether of quercetin) (Park & Lee, 1996; Price &Rhodes, 1997). Accordingly, the main difference between thephenolic profiles of A. cepa and A. flavum is in content of rutin, whichis the major compound in A. flavum but only a minor component ofA. cepa extract. Besides, quercetin aglycone is more abundant inonion than in A. flavum, while kaempferol-3-O-glucoside is presentonly in A. flavum. Among benzoic acid derivatives, in the aerial partsextracts of A. flavum, p-hydroxybenzoic, protocatechuic, vanillic andsyringic acids were found in considerable amounts (in the range of100e1100 mg g�1 of dw). The dominant hydroxycinnamic acid wasferulic acid (593e1377 mg g�1 of dw), followed by p-coumaric andcaffeic acids. The same phenolic acids were also found in A. cepaextract (Gorinstein et al., 2008). Quinic acid, an intermediate in plantphenolics biosynthesis, was present in large amounts in bothbulb and aerial parts extracts of A. flavum (551.1e1132 mg g�1 of dw),while coumarin aesculetin and lignan secoisolariciresinolwere also present, but in small quantities. On the other hand,the following compounds were not detected in any examinedextracts: naringenin, catechin, epicatechin, epigallocatechin gallate,
Fig. 1. Representative LC-MS/MS chromatograms of A. flavum (location Babusnica)aerial parts (a) and bulb (b) extracts, 1: quinic acid, 2: 5-O-caffeoylquinic acid, 3: p-hydroxybenzoic acid, 4: aesculetin, 5: caffeic acid, 6: p-coumaric acid, 7: ferulic acid, 8:luteolin-7-O-glucoside, 9: rutin, 10: quercetin-3-O-glucoside, 11: kaempferol-3-O-glucoside, 12: secoisolariciresinol, 13: luteolin, 14: chrysoeriol, 15: protocatechuic acid,16: quercetin, 17: kaempferol, 18: isorhamnetin.
N. Simin et al. / LWT - Food Science and Technology 54 (2013) 139e146142
o-coumaric acid, 3,4-dimethoxycinnamic acid, amentoflavone, apiin,baicalein, baicalin, daidzein, apigenin-7-O-glucoside, quercitrin,hyperoside, myricetin, genistein, umbelliferone, scopoletin andmatairesinol.
Table 2Determined concentrations of selected phenolics in the A. flavum subsp. flavum methano
Location Content of selected phenolics in the extracts (mg g
Dimitrovgrad Bab
Plant part Aerial parts Bulb Aer
Phenolic acidsp-hydroxybenzoic acid 354.2 � 11.76 46.49 � 1.54 1142,5-dihydroxybenzoic acid 33.81 � 1.45 4.24 � 0.18 NdProtocatechuic acid 402.0 � 18.61 5.38 � 0.25 446Vanillic acid 465.5 � 17.18 64.97 � 2.40 426Gallic acid 4.35 � 0.09 Nd 7.9Syringic acid 164.5 � 7.95 41.08 � 1.98 173Cinnamic acid 22.84 � 1.20 Nd Ndp-coumaric acid 584.9 � 19.24 7.43 � 0.24 133Caffeic acid 376.3 � 11.22 150.0 � 4.47 220Ferulic acid 913.7 � 38.65 29.97 � 1.27 768Sinapic acid 68.50 � 3.67 Nd 30.5-O-caffeoylquinic acid 6.99 � 0.28 1.35 � 0.06 1.2FlavonoidsApigenin 25.00 � 0.60 129.0 � 3.10 NdVitexin 3.27 � 0.09 Nd 2.4Isorhamnetin 2468 � 59.97 Nd 661Kaempferol 279.9 � 5.96 Nd 48.Kaempferol-3-O-glucoside 1651 � 28.57 0.77 � 0.01 141Chrysoeriol 14.34 � 0.33 Nd 25.Luteolin 3.75 � 0.15 Nd 1.9Luteolin-7-O-glucoside 1.65 � 0.03 Nd 1.2Quercetin 787.7 � 28.44 Nd 190Quercetin-3-O-glucoside 57,215 � 1985 Nd 32,Rutin 201,859 � 4562 Nd 80,Other phenolicsAesculetin 313.7 � 6.31 59.40 � 1.19 36.Secoisolariciresinol 46.13 � 2.09 35.48 � 1.60 16.Quinic acid 708.1 � 25.70 1132 � 41.12 551Total phenolics (mg/g)c 271.33 0.578 118
a Results are given as the concentration (mg g‒1 of extract dry weight) � standard errob Nd e not detected.c Sum of the contents of all detected phenolic compounds.
From the results obtained, it can also be concluded that thecontent of investigated phenolics was highly dependent on thehabitat conditions, while qualitative composition was stable.Hence, for further investigation of biological activity, we chose theextract of A. flavum from Babusnica location, because it had anaverage content of phenolic compounds.
The data presented in this study could be a base for furtherresearch of this Allium species and its potential application. Forexample, the high content of rutin can indicate the potential use ofA. flavum as a medicinal plant due to the valuable properties ofrutin, including its remarkable antioxidant (Rice-Evans, Miller, &Paganga, 1996), hepatoprotective (Janbaz, Saeed, & Gilani, 2002)and anti-inflammatory activity (Selloum, Bouriche, Tigrine, &Boudoukha, 2003). On the other hand, the bioavailability studieshave shown that quercetin and quercetin-3-O-b-glucoside areabsorbed rapidly in small intestine and have a long eliminationhalf-time, while bioavailability of rutin is only 20% of that of theglucoside (Hollman & Arts, 2000). That implicates that even thoughquercetin and isoquercitrin are present in extracts in loweramounts than rutin, their contribution to the overall health-bene-ficial effect of A. flavum could be much higher than that of rutin.
3.2. Antioxidant activity
Antioxidants exhibit their activity through different mecha-nisms of action, such as inhibition of oxidising enzymes, chelationof transition metals, transfer of hydrogen or single electron toradicals, singlet oxygen deactivation, or enzymatic detoxification ofreactive oxygen species (Prior, Wu, & Schaich, 2005). Since there isno single method which enables testing of all the mechanisms
l extracts from three locations in Serbia.
‒1 of dw)a
usnica Vrsacki breg
ial parts Bulb Aerial parts Bulb
.9 � 3.82 13.06 � 0.43 287.2 � 9.53 31.35 � 1.04b 2.27 � 0.10 10.75 � 0.46 2.69 � 0.12.6 � 20.68 4.48 � 0.21 403.8 � 18.69 11.32 � 0.52.1 � 15.72 20.07 � 0.74 1116 � 41.21 52.22 � 1.938 � 0.17 Nd 9.41 � 0.20 Nd.6 � 8.38 38.12 � 1.84 462.7 � 22.35 58.01 � 2.80
Nd Nd Nd.8 � 4.40 5.35 � 0.18 260.2 � 8.56 18.89 � 0.62.2 � 6.56 68.81 � 2.05 236.1 � 7.03 403.5 � 12.02.9 � 32.52 30.55 � 1.29 1377 � 58.25 57.55 � 2.4335 � 1.63 3.31 � 0.18 48.79 � 2,62 5.32 � 0.295 � 0.05 4.84 � 0.20 13.63 � 0.55 0.99 � 0.04
Nd 52.72 � 1.27 Nd5 � 0.07 Nd Nd 8.17 � 0.23.5 � 16.08 Nd 762.1 � 18.52 5.15 � 0.1380 � 1.04 Nd 160.2 � 3.41 1.47 � 0.039 � 24.56 0.67 � 0.01 1978 � 34.23 12.80 � 0.2242 � 0.58 1.34 � 0.03 17.47 � 0.40 Nd0 � 0.08 4.98 � 0.20 5.25 � 0.21 Nd7 � 0.02 14.67 � 0.23 0.91 � 0.01 Nd.9 � 6.89 Nd 457.7 � 16.52 6.80 � 0.25352 � 1122 3.14 � 0.11 17,379 � 603 279.02 � 9.68958 � 1829 4.50 � 0.10 23,159 � 523 526.42 � 11.90
47 � 0.73 20.79 � 0.42 124.3 � 2.50 59.80 � 1.2080 � 0.76 27.26 � 1.23 197.8 � 8.94 344.6 � 15.58.1 � 20.00 1050 � 38.12 641.9 � 23.30 999.2 � 36.27.93 0.270 49.82 1.89
r of repeatability (as determined by method validation).
Table 3Total antioxidant status (TAS), DPPH�, hydroxyl radical and nitric oxide scavenging activities and lipid peroxidation (LP) inhibition ability of the A. flavum subsp. flavum and A.cepa methanol extracts and BHA.
TASa, mmol Trolox eq. g�1 of dw IC50 (DPPH�)a, mg mL�1 IC50 (OH�)b, mg mL�1 IC50 (NO�)a, mg mL�1 IC50 (LP)a, mg mL�1
ExtractA. flavum, whole plant 0.356 � 0.006 117.38 � 6.52 0.72 � 0.029 5.369 � 0.787 0.778 � 0.029A. flavum, arial parts 1.487 � 0.256 42.12 � 0.53 n. e.c 1.463 � 0.124 0.253 � 0.068A. cepa, whole plant 1.368 � 0.187 31.22 � 1.33 0.28 � 0.011 4.690 � 0.468 0.123 � 0.019StandardBHA 5.433 � 0.356 11.08 � 0.09 1.51 � 0.06d n. a.e 0.024 � 0.002
a Results are given as mean � SD of three measurements.b Results are given as IC50 values � standard error of repeatability (as determined by method validation).c n. e. e not evaluated.d Reported by Babovic et al. (2010).e n. a. e 50% of inhibition is not achieved even at a concentration of 10 mg mL�1.
N. Simin et al. / LWT - Food Science and Technology 54 (2013) 139e146 143
mentioned, the evaluation of antioxidant activity of extracts oftencombines several different methods. Therefore, the extract ofA. flavum was examined with regard to scavenging capacitytowards DPPH�, ABTS�þ, hydroxyl radical and nitric oxide and LPinhibition ability. Synthetic antioxidant BHA and methanol extractof the official drug A. cepa (WHO, 1999) were also tested forcomparative purposes. Concerning that antioxidant activity ofexamined extract in all applied tests was concentration-dependent,the results were expressed as IC50 values, except for the TAS assay,were the results are given in mmol of Trolox eq. per gram of dryweight (Table 3). According to the results of all the assays applied, itcan be concluded that the aerial parts extract of A. flavum possesseshigh antioxidant capacity, higher than the whole plant extract andcomparable to that of the A. cepa extract. Considering that pheno-lics are mostly located in the aerial parts of A. flavum, it can beassumed that at least partially these compounds are responsible forfree radical scavenging activity.
However, the antioxidant potential of examined extracts wasstill lower than the activity of synthetic antioxidant BHA. Only intwo assays (NO and OH radical scavenging ability tests) BHAexpressed low antioxidant activity, which can be attributed to thelow solubility of BHA in aqueous buffers.
Overall, our results demonstrate that A. flavum is a moderateantioxidant agent in comparison to BHA. However, considering thatsynthetic antioxidants often have adverse effects (Park, Chang, &Cha, 1990), the usage of natural instead of synthetic antioxidantsis of particular importance. Thus, the estimated antioxidant activityof A. flavum, especially towards OH radicals which are extremelytoxic in biological systems, can contribute to the benefits of thisspecies as a food or food supplement.
3.3. Anti-inflammatory activity
Anti-inflammatory potential of A. flavum extracts was studied bymeasuring their inhibitory effect on arachidonic acid metabolites
Fig. 2. COX and 12-LOX branch
production in COX-1 and 12-LOX pathways. Eicosanoids formed inthese pathways (Fig. 2) are significant mediators of inflammation(Smith, 1989). The products of COX-1 pathway e 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid (12-HHT), thromboxane B2(TXB2) and prostaglandin E2 (PGE2), and LOX-12 pathway prod-ucts e 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE) were quantified by a highly sensitive and specificLC-MS/MS technique.
The results obtained are shown in Table 4 and Fig. 3. The extractsof A. flavum as well as onion extract demonstrated dose-dependentinhibitory activity towards both COX-1 and 12-LOX pathway en-zymes, although it was lower than the activity of a well-knownpotent inhibitor of COX-1, aspirin (IC50 ¼ 0.005 mg/mL for COX-1metabolites) and 12-LOX, quercetin (IC50 ¼ 0.0074 mg/mL for 12-HETE). However, the inhibitory activity of aerial parts extract ofA. flavum was comparable to the previously published results forwell-known anti-inflammatory drug Plantago major L.(IC50¼ 0.65 mg/mL for 12-HHTand IC50¼ 1.73mg/mL for 12-HETE)(Beara et al., 2010). As in the antioxidant assays, the aerial partsextract of A. flavum expressed higher anti-inflammatory activitythan both A. cepa extract and A. flavum whole plant extract, espe-cially on inhibition of 12-LOX (IC50 ¼ 0.078 mg mL�1,IC50 ¼ 1.033 mgmL�1 and IC50 ¼ 2.925mgmL�1, respectively). Thatsuggests that the phenolic compounds present in aerial parts ofA. flavum, are probably responsible for the activity.
From the doseeinhibition curves (Fig. 3), it was not possible todetermine the particular enzymes of arachidonic acid metabolismwhich were inhibited by the whole plant extract of A. flavum.However, based on the good match between all of the four curves,the two ways of action can be assumed: the initiating step ofarachidonic acid metabolism e the reaction catalyzed by phospho-lipase A2 (PLA2)ewas inhibited, or several other enzymes catalyzingthe formation of the monitored metabolites (12-LOX, COX-1, orthromboxane synthase and PGE synthase) (Fig. 2) were inhibited. Incase of the aerial part extract (Fig. 3), the doseeinhibition curve of
es of eicosanoid pathway.
Table 4IC50 values for COX-1 and 12-LOX assay of A. flavum subsp. flavum extracts and standard compounds.
IC50 values (mg mL�1)a
12-HHT production TBX2 production PGE2 production 12-HETE production
ExtractA. flavum, whole plant 2.671 � 0.230 3.114 � 0.044 4.358 � 0.069 2.925 � 0.272A. flavum, aerial parts 0.785 � 0.067 0.766 � 0.011 0.660 � 0.010 0.078 � 0.007A. cepa, whole plant n. e.b 1.866 � 0.026 n. e.b 1.033 � 0.096StandardAspirin 0.005 � 0.0004 0.005 � 0.0001 0.0055 � 0.00002 n. a.c
Quercetin 0.022 � 0.002 0.054 � 0.0002 0.0127 � 0.0001 0.0074 � 0.005
a Results are given as IC50 values � standard error of repeatability (determined by method validation).b n. e. e not evaluated.c n. a. e 50% of inhibition is not achieved even at a concentration of 24 mg mL�1.
N. Simin et al. / LWT - Food Science and Technology 54 (2013) 139e146144
12-HETE production does not match the curves for COX-1 pathwaymetabolites (12-HHT, TXB2, PGE2), indicating that compounds fromthe extract are affecting both 12-LOX and some of COX-1 pathwayenzymes (COX-1, or thromboxane synthase and PGE synthase)(Fig. 2). It should also be noted that inhibitory potential of the aerialparts extract towards 12-LOX is higher than for COX-1 branch en-zymes. Consequently, the aerial parts extract of A. flavum in con-centrations up to 0.25 mg mL�1 could act as selective 12-LOXinhibitor. This result is particularly significant considering the fact
0
25
50
75
100
125
0 2 4 6 8 10 12 14
Inhi
bitio
n (%
)
Final concentration (mg/mL)
-25
0
25
50
75
100
125
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Inhi
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A
B
Fig. 3. Inhibition of production of 12-HHT (full diamonds, A), 12-HETE (full squares,-), PGE2 (full triangles, :) and TXB2 (full circles, C) by whole plant extract (A) andaerial parts extract (B) of A. flavum.
that 12-HETE, a product of 12-LOX, is involved in the progression ofvarious human diseases like cancers (Nie & Honn, 2002), psoriasis(Kragballe, Desjarlais, Duell, &Voorhees, 1986), atherosclerosis(Nakao, Ooyama, Chang, Murota, & Orimo, 1982) and rheumatoidarthritis (Liagre, Vergne, Rigaud, & Beneytout, 1997). Thus, theextract of the aerial parts of A. flavum could be beneficial forthe treatment of pathological conditions related to the excessive12-HETE generation.
3.4. Cytotoxic activity
The activity of A. flavum extracts on cell growth was evaluatedin vitro by SRB assay using HeLa, MCF7, HT-29 and MRC-5 cell lines.For the purpose of comparison we also investigated the activity ofA. cepa extract and the activity of a highly potent cytotoxic agentpodophyllotoxin. At the same time, we evaluated cytotoxicity offerulic and caffeic acids and flavonoids quercetin, kempferol andrutin, which are present in extracts of A. flavum and tried tocorrelate the activity of the examined extracts with their compo-sition and to identify active principles. The results obtained areshown in Table 5.
Treatment of all of the four cell lines by A. flavum extractsresulted in a considerable, dose-dependent inhibition of cellgrowth. Extracts of A. flavum exhibited the overall higher activitycompared to onion extract. A. cepa extract affected only the growthof HeLa cell line, but at a high concentration (IC50 ¼ 722 mgmL�1). Itis in accordance with the previously reported results of Votto,Domingues, and Souza (2010), where the onion extract signifi-cantly inhibited cell growth of erythroleukemic cells only in con-centrations higher than 4 mg mL�1. The strongest inhibitory effectswere obtained by the extract of aerial parts of A. flavum (FBH),reaching low IC50 values in MCF7, MRC-5 and HeLa cell lines(25.44 mg mL�1, 30.37 mg mL�1 and 36.39 mg mL�1, respectively).The A. flavum whole plant extract exhibited a somewhat loweractivity towards these cell lines indicating that the aerial partsextract of A. flavum the highest concentrations of antiproliferativecompounds are found in. However, the whole plant extract ofA. flavum showed selective action towards the cervix epithelioidcarcinoma and colon adenocarinoma cell lines. Namely, eventhough this extract affected the growth of the human fetal lung cellline (MRC-5), it reached 50% of growth inhibition at a higher con-centration (105 mg mL�1) then in the case of HeLa and HT-29 cells(71 mgmL�1 and 81 mgmL�1, respectively), suggesting its respectiveselective action towards cancer cells.
In comparison to a highly potent cytotoxic agent podophyllo-toxin, the examined extracts showedmuch lower activity. Cytotoxiceffect of kempferol, quercetin and caffeic acid was high in all of thefour cell lines (with the exception of activity of caffeic acid on HT-29cells, which was low, IC50 ¼ 274.93 mg mL�1), suggesting that thepresence of these compounds in A. flavum extract can be related to
Table 5Extracts and standards concentrations required to inhibit cell growth by 50% (IC50) after 48 h of exposure.
IC50 (mg/mL)a
MRC-5 HeLa MCF7 HT-29
ExtractA. flavum, whole plant 105.99 � 12.89 71.45 � 7.49 104.46 � 9.53 81.11 � 8.41A. flavum, aerial parts 30.37 � 3.58 36.39 � 6.38 25.44 � 4.59 134.59 � 9.36A. cepa, whole plant >1000 722.36 � 35.00 >1000 >1000StandardKaempferol 3.20 � 0.11 2.84 � 0.62 11.10 � 2.96 14.08 � 3.15Quercetin 11.74 � 2.98 12.77 � 1.23 12.22 � 0.60 38.91 � 5.89Caffeic acid 34.16 � 1.77 19.73 � 4.93 75.02 � 10.42 274.93 � 7.91Ferulic acid 202.69 � 15.43 >500 114.35 � 16.45 271.79 � 24.60Rutin >500 >500 >500 >500Podophyllotoxin 0.005 � 0.0001 0.004 � 0.0001 0.001 � 0.0001 0.003 � 0.0001
a Values are means � SD of eight measurements.
N. Simin et al. / LWT - Food Science and Technology 54 (2013) 139e146 145
its anti-cancer properties. Furthermore, the fact that A. flavumexhibited both the high antiproliferative activity and high 12-LOXinhibitory activity suggests that inhibition of 12-LOX, the enzymethat plays a significant role in proliferation of tumor cells (Nie &Honn, 2002), can be one of the possible mechanisms of action ofA. flavum extract cytotoxicity. The antiproliferative activity of ferulicacid was moderate towards MCF7, HT-29 and MRC-5, whilethis compound did not affect the growth of HeLa cells(IC50 > 500 mgmL�1). Moreover, rutinwas inactive against all of thetested cell lines even at 500 mg mL�1, indicating that huge amountsof rutin present in the extract of A. flavum do not contribute to itscytotoxic activity. The different activity of flavonoid aglycone e
quercetin e and quercetin glycoside e rutin e implicates that freehydroxyl group at C-3 carbon of quercetin plays an important rolein the antiproliferative activity.
The results obtained in this study are in a good correlation to thepreviously reported results concerning good inhibitory effect ofA. flavum methanolic extract on growth of HCT-116 colorectal car-cinoma cells (Curcic et al., 2012). However, we have shown for thefirst time, that active compounds from A. flavum also have thepotential to inhibit the growth of cervix epithelioid carcinoma,colon adenocarcinoma and breast adenocarinoma cells. Accord-ingly, A. flavum species could be regarded as possible anti-canceragents and a possible source of new therapeutics.
4. Conclusions
This study is a contribution to the overall knowledge onphytochemistry and bio-potential of the Allium species, most ofwhich are widely used in human diet as spices and vegetables andas medicinal plants. In this study, the phenolic profile, anti-inflammatory activity and cytotoxic activity on cervix epithelioidcarcinoma, colon adenocarcinoma and breast adenocarinoma cellsof A. flavum subsp. flavum are reported for the first time. We foundthat A. flavum is rich in phenolic compounds, especially flavonolglycosides (rutin, quercetin-3-O-glucoside and kaempferol-3-O-glucoside) and phenolic acids (ferulic acid, p-coumaric, caffeic, p-hydroxybenzoic, vanillic, protocatechuic and syringic acid), whichpredominantly are distributed in aerial parts of the plant, andwhose concentration in plant material depends on the habitatconditions. Accordingly, the methanol extract of aerial parts of theplant exhibited a considerable antioxidant activity, comparable tothe onion (A. cepa) extract, but lower than the synthetic antioxidantBHA. Also, the aerial parts extract expressed higher ability to inhibit12-lipooxygenase activity than well-known anti-inflammatorydrug Plantago major L., but still lower than quercetin. Furthermore,the extracts of A. flavum inhibited growth of four human cell lines ina dose-dependent manner, where the whole plant extract showed
selective action towards the cervix epithelioid carcinoma and thecolon adenocarcinoma cell lines. In conclusion, the overall results ofthis study indicate that A. flavum subsp. flavum has notable contentof phenolic compounds, consequently possessing high bio-potential, that strongly support traditional use of A. flavum as afood. Moreover, this study also point out that A. flavummight be aninteresting candidate for the development of newantioxidant, anti-inflammatory and cancer chemopreventive dietary supplements orphytopharmaceuticals.
Acknowledgements
We sincerely thank to the Institute for Blood Transfusion ofVojvodina, Novi Sad for providing the platelets. Wewish to thank toGordana Vlahovic, English-language editor, for editing our manu-script. The Ministry of Education and Science, Republic of Serbia(Grant No. 172058) supported this research work.
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