Influence of Storage Conditions on Flavonoids Content and Antioxidant Activity of Selected Shallot (Allium Cepa Var

2012 vol. 77, 101-111 DOI: 10.2478/v10032-012-0019-9 ______

INFLUENCE OF STORAGE CONDITIONS ON FLAVONOIDS CONTENT AND ANTIOXIDANT ACTIVITY OF SELECTED SHALLOT (ALLIUM CEPA VAR. ASCALONICUM BACKER) HYBRID CULTIVARS

Marta PUDZIANOWSKA, Marek GAJEWSKI, Jarosław L. PRZYBYŁ, Agnieszka BURACZYŃSKA, Olga GACZKOWSKA, Marta MATUSZCZAK, Marta DZIECHCIARSKA Warsaw University of Life Sciences – SGGW Nowoursynowska 166, 02-787 Warszawa, Poland Received: December 4, 2012; Accepted: March 3, 2013

Summary The aim of this study was to determine changes in the content of flavonoids in fleshy scales of shallot bulbs in relation to different compositions of atmosphere during storage. Also antioxidant activity of the bulbs was determined. Bulbs of shallot cvs. Bonilla F1, Conservor F1 and Matador F1 were stored in two storage seasons at the normal atmosphere (NA) and controlled atmosphere (CA) of the 4 compositions: 5% CO2 + 5% O2, 5% CO2 + 2% O2, 2% o CO2 + 5% O2, 2% CO2 + 2% O2. The temperature of storage was 0-1 C and RH 65%. Before and after 7-month storage the bulbs were examined in respect of flavonoids content and antioxidant activity (AA). Flavonoids were identified in methanolic extracts with HPLC, AA was determined with FRAP and DPPH assays. Three flavonoid compounds were identified in the plants extracts. Quercetin 3,4’ di-O-glucoside and quercetin 4’-glucoside (spiraeoside) were present in the highest amounts. CA conditions influenced content of flavonoids in the bulbs. The highest content after storage was observed at gas composition of 5% CO2 + 5% O2. Two major flavonoid compounds, quercetin 3,4’-di-O-glucoside and quercetin 4’-O-glucoside (spiraeoside) showed the increasing tendency, however not all changes were significant. No significant differences were observed between cultivars, but ‘Bonilla’ F1 showed slightly higher flavonoids content than the other ones. Investigated cultivars did not differ in respect of AA, measured both with DPPH and FRAP assay. There was no significant influence of storage conditions on DPPH level, while FRAP level was influenced significantly. Bulbs from 5% CO2 + 2% O2 composition were characterized by the highest FRAP values and the bulbs stored at normal atmosphere – by the lowest. key words: shallot, controlled atmosphere storage, quercetin glucosides, antioxidant activity, FRAP, DPPH

Corresponding author: e-mail: [email protected] © Copyright by InHort

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INTRODUCTION product quality, including its biological value (Bartz & Brecht 2003). The Allium vegetables are well known for objective of the study was to identify their biological activity resulting from flavonoids in bulbs of hybrid cultivars the presence of sulphur compounds in of shallot (Allium cepa var. ascalo- the bulbs, however other compounds, nicum Backer) grown from seeds, namely flavonoids and other polyphe- examine their antioxidant activity nols, have been in the area of re- (AA) and influence of storage under searchers interest lately. Flavonoids different atmosphere compositions on show wide range of biological activi- flavonoids content and AA. ty, mainly connected with beneficial effect on cardiovascular system and MATERIALS AND METHODS with their antioxidant activity (Hollman & Katan 1999). Flavonoids The experiment was carried out show several different ways of antiox- in storage seasons 2009/2010 and idant action, such as free radicals 2010/2011. For the investigation three scavenging, breaking the chain of hybrid cultivars of shallot: Conservor their formation, inhibition of enzymes F1, Bonilla F1 and Matador F1 (Bejo and bounding the metal ions catalyz- Zaden, Holland) were chosen. Bulbs ing oxidation processes and stimula- of ‘Bonilla’ F1 are of garlic-like tion of critic gene for glutathione tran- shape, have light brown and yellow scription resulting in increased con- dry and fleshy scales. ‘Conservor’ F1 centration of intracellular glutathione and ‘Matador’ F1 are characterized by (Moskaug et al. 2005). Flavonoids are red and brown tints of dry scales, of divided into several groups: flavones, different intensity, and pink tint of flavonoles, flavanoles, flavanones, inner scales. ‘Matador’ F1 bulbs are of isoflavones and anthocyanins. Allium garlic-like shape, with light red-brown vegetables are rich in flavonoles, pri- dry scales. ‘Conservor’ F1 bulbs have marily quercetin, and among the on- red-brown dry scales and elongated, ion varieties the most abundant in spindle shape. The plants were culti- quercetin are shallots and red onions vated from seedlings sowing in the (Di Carlo et al. 1999; Kyle & Duthie experimental field in Warsaw, on a 2006). Several investigations were medium-loam soil. Mature bulbs were performed to recognize the changes of harvested in the third week of August flavonoids during storage of shallot and cured in a shed, at ambient tem- bulbs (Horbowicz & Kotlińska 2001, perature, for one month. Bulbs were Horbowicz 2006, Tendaj & Mysiak stored for 7 months in CA cold stor- 2010), however the influence of con- age at the temperature between 0 and trolled atmosphere storage was not 1ºC, 65% RH. studied. CA storage is recommended Five atmosphere compositions in literature for long-term storage of were studied: (1) normal atmosphere onions and several other vegetable 0% CO2 + 21% O2, (2) 5% CO2 + crops, as a method for better keeping 5% O2, (3) 5% CO2 + 2% O2, (4) 2%

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CO2 + 5% O2, (5) 2% CO2 + 2% O2. matograph equipped with autosampler Because of lack of recommendations SIL-20, photodiode array detector for shallot, CA conditions recom- SPD-M10A VP DAD and Class VP mended for common onion were cho- 7.3 chromatography software (Shi- sen. The experiment was established in madzu, Kyoto, Japan). A modern C- three analytical replications, with thirty 18 reversed-phase column with core- bulbs in each. Each analytical replica- shell technology (Phenomenex Ki- tion was prepared by mixing bulbs netex® 2.6 μm, C18, 100A, 100×4.60 from three experimental replications. mm i.d.) was used as solid phase. Chemical analyses were performed Binary gradient of mobile phase A before and after storage period. For the (deionised water acidified with phos- analyses good quality, healthy bulbs phoric acid 0.1 %) and B (acetonitrile were chosen. For each cultivar antioxi- acidified with phosphoric acid 0.1%) dant activity (AA) was determined and was used as follows: 0.01 min, 18% identification of flavonoids by HPLC B; 1.4 min, 95% B; 1.5 min, 95% B; was performed. To estimate AA two 1.51 min, 18% B. The following con- assays were applied: FRAP and DPPH. ditions were applied: injection vol- Reagents: ume: 1 μl, flow rate 1.2 ml·min-1, Methanol, acetic acid, iron (III) chlo- oven temperature 30ºC, total time of ride hexahydrate were purchased in analysis 8 minutes, UV-spectra were Chempur; HPLC grade methanol, ace- recorded between 190 and 450 nm. tonitrile, 2,2-Diphenyl-1-picrylhydrazyl Peak identification was con- (DPPH), 6-Hydroxy-2,5,7,8- tetra- firmed by comparison of retention methylchromane-2-carboxylic acid time and spectral data with adequate (Trolox) and ferrous sulfate were ob- parameters of standards purchased tained from Sigma-Aldrich. from ChromaDex (ChromaDex, Ir- Extracts preparation vine, USA). For quantitation of inves- 5 g of plant fresh material was ex- tigated compounds the 5-point cali- tracted with 100 ml of methanol, us- bration curve method was used. ing Büchi B-811 extraction system Methanol stock standard solutions (modified Soxhlet extractor, Büchi were prepared according to the Chro- Labortechnik AG, Postfach, Switzer- maDex’s Tech Tip 0003: Reference land). After solvent evaporation, the Standard Recovery and Dilution. The residue was dissolved in 10 ml of following concentrations of standards methanol. were applied: quercetin 3,4' di-O- HPLC analysis: glucoside: 10.0, 20.0, 40.0, 100.0, Extracts prepared as it is described 200.0 µg·ml-1; quercetin 3-O- above were filtered with Supelco Iso- glucoside: 49.6, 99.2, 198.4, 496.0, Disc™ Syringe Tip Filter Unit, PTFE 992.0 µg·ml-1; quercetin 4'-glucoside: membrane, diameter 25 mm, pore size 21.4, 42.8, 85.6, 214.0, 428.0 µg·ml-1. 0.20 μm, and subjected to HPLC. The solutions were applied on a col- Identification and quantification of umn in triplicate. The peak table and flavonoids in shallot extracts were spectra library (190-450 nm) of indi- performed using a Shimadzu chro- vidual compounds were created. De-

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tection wave applied: quercetin 3,4' Vis Shimadzu 1700 PharmaSpec di-O-glucoside 254 nm, quercetin 3- spectrophotometer was measured. The O-glucoside 353 nm, quercetine 4'- total antioxidant capacity of shallot glucoside 365 nm. The content of the extracts was determined against a determined compounds was calcu- standard of known FRAP value, fer- lated in mg·100 g-1 dry matter (DW). rous sulphate (1000 μM). The method was elaborated and vali- Statistical analysis dated especially for this purpose in Numerical data from both seasons Department of Vegetable and Medici- were subjected to analysis of variance nal Plants (Warsaw University of Life with Statgraphics Plus 4.1TM software Sciences). (Statistical Graphics Corp. 1999). DPPH (2,2-diphenyl-1-picrylhydra- Analysis draw a distinction between zyl) assay combinations and therefore Tukey’s DPPH• scavenging activity of exam- HSD test was used to compare the ined extracts (prepared as it is de- means, at the level of significance scribed above) was carried out accord- P=0.05. The data presented in tables ing to Yen and Chen (1995) method. are means of both seasons of the ex- To 1 ml of examined extract and 3 ml periment. Homogenous groups were of methanol 1 ml of DPPH• methan- marked in the tables only in case of olic solution (120 mg·ml-1) was added. significant differences. Absorbance was measured on the UV- Vis Shimadzu 1700 PharmaSpec spec- RESULTS AND DISCUSSION trophotometer after 10 minutes at 517 nm. In the blind test extract was re- Three flavonoids were identified placed by 1 ml of methanol. Trolox in in examined extracts obtained from concentrations 0.3-4.9 μg·ml-1 was shallot bulbs: quercetin 3,4’-di-O- used to estimate standard curve. glucoside, quercetin 3-O-glucoside FRAP (Ferric Reducing/ Antioxi- (isoquercetin) and quercetin 4’- dant Power) assay glucoside (spiraeoside). Quercetin FRAP was examined according to 3,4’-di-O-glucoside and spiraeoside Benzie and Strain (1996, 1999). were present in the highest amounds FRAP reagent was prepared from 300 (Table 2 & 3). Bonaccorsi et al. (2008) mM acetic buffer (pH 3.6), 10 mM identified 7 flavonol glucosides in TPTZ (4,6-tripyridyl-s-triazine) and onion and shallot bulbs, the main fla- 20 mM FeCl3·6H2O solutions mixed vonoid constituents of shallots were before analysis in 10:1:1 proportion. 3 quercetin 3,4’-di-O-glucoside 4’- ml of freshly prepared FRAP reagent glucoside (spiraeoside), just as in this was warmed up to 37°C, its absorb- study. Their concentration was also ance was measured, then 100µl of similar to those identified in our study. extract (prepared as it is described Quercetin 3,4’-di-O-glucoside and above) was added and after 4 minutes spiraeoside were found also in onion of incubation at 37°C change in ab- bulbs by Caridi et al. (2007) and Ro- sorbance caused by reduction of Fe3+ drigues et al. (2011). Lachman et al. to Fe2+ in examined sample using UV- (2003) found spiraeoside, rutin and

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quercetin as the major flavonoid con- compositions (Table 2). Quercetin 3- stituents of onions. Investigated culti- O-glucoside (isoquercetin) and vars didn’t differ significantly in con- quercetin 4’-O-glucoside (spiraeoside) tent of identified flavonoids, however contents were significantly higher ‘Bonilla’ F1 showed a slightly higher only in the case of 5% CO2 + 5% O2 mean content of these compounds. (2) variant (Table 3 & 4). Horbowicz This cultivar was also characterized and Kotlińska (2001) observed de- by the highest dry matter content (Ta- crease of flavonols in shallot bulbs ble 1). Bulbs of investigated cultivars after 8 months of storage in a cold examined before storage showed big- store. However, in the case of shorter ger differences in flavonoids content, period of curing, increase of flavonols compared to their mean values. Lack during the first two months of storage, of significant differences in average followed by further decrease and re- amount of quercetin glycosides be- peated increase at the end of storage tween cultivars is a result of their period, was observed. Increase of diverse performance in various at- quercetin content in onions at the be- mosphere compositions. According to ginning of storage period under normal Horbowicz and Kotlińska (2001), and atmosphere was reported by Patil et al. Bonaccorsi et al. (2008) vegetatively (1995), especially for higher tempera- propagated populations of shallot ture, but onions stored in CA (99% N2 examined after harvest shows differ- + 1% O2) showed no significant ences in flavonoids content. Also changes during storage. Increase of many studies performed on common spiraeoside and rutin was also reported onion reveal differences between cul- by Lachman et al. (2003) during 36 tivars in respect to flavonoids content weeks of storage at 4°C and 22°C. (Patil et al. 1995, Prakash et al. 2007, Content of quercetin in bulbs of two Bonaccorsi et al. 2008, Beesk et al. Polish onion cultivars, Błońska and 2010, Rodrigues et al. 2011). There is Sochaczewska, was higher after five a lack of information about influence months of storage in a cold store than of CA conditions on flavonoids after curing, however during the stor- changes in shallot bulbs during storage period some drops were visible, age. In our study increase of quercetin depending on cultivar and month of 3,4’-di-O-glucoside was observed storage (Horbowicz 2001). during the storage in all atmosphere

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Table 1. Dry matter content in shallot bulbs in relation to storage conditions and the cultivar, means of the two seasons of the experiment (%)

Storage (A) Cultivar (B) Before Storage conditions Means storage 1 2 3 4 5 Conservor 14.0cde 8.86a 12.9c 14.1cde 13.0c 13.3cd 12.7A Matador 13.4cd 9.93ab 13.3cd 13.4cd 13.4cd 13.1cd 12.7A Bonilla 16.4e 15.3cde 15.2cde 16.9e 15.9de 12.6bc 15.4B Means 14.6C 11.4A 13.8BC 14.8C 14.1BC 13.0B LSD0.05 AxB 2.33

1 - normal atmosphere (control); 2 - 5% CO2 + 5% O2; 3 - 5% CO2 + 2% O2; 4 - 2% CO2 + 5% O2; 5 - 2% CO2 + 2% O2 Means which differ significantly according to HSD Tukey's test at P < 0.05 are marked with different letters (The main effects are marked with capital letters, interactions with small letters).

Table 2. Quercetin 3,4'-di-O-glucoside content in shallot bulbs in relation to storage conditions and the cultivar, means of the two seasons of the experiment (mg·100 g-1 DW)

Storage (A) Cultivar (B) Before Storage conditions Means storage 1 2 3 4 5 Conservor 251 853 842 687 512 353 583 Matador 329 586 866 752 476 364 562 Bonilla 335 515 766 770 593 531 585 Means 305B 651AC 825A 736A 527ABC 416BC LSD0.05 AxB n. s.