Czech J. Food Sci., 35, 2017 (6): 469–475 Food Analysis, Food Quality and Nutrition doi: 10.17221/458/2016-CJFS Antioxidant Activity and Phenolic Acid Content of Selected Vegetable Broths Bilge ERTEKIN FILIZ*, Nazli KORKMAZ, Nilgun H. BUDAK, Atif C. SEYDIM, and Zeynep B. GUZEL SEYDIM Department of Food Engineering, Faculty of Engineering, Süleyman Demirel University, Isparta, Turkey *Corresponding author: [email protected] Abstract Ertekin Filiz B., Korkmaz N., Budak N.H., Seydim A.C., Guzel Seydim Z.B. (2017): Antioxidant activity and phenolic acid content of selected vegetable broths. Czech J. Food Sci., 35: 469–475. The antioxidant activity and content of phenolic substances in vegetable broths were determined. Green beans, beetroots, courgettes, onions, parsley, carrots, cabbages, celery, broccoli, spinach, cauliflowers, and tomatoes were subjected to boiling. Fresh vegetables and vegetable broths were analysed for ascorbic acid content, total phenolic content, ORAC and TEAC values. Phenolic acids were quantified using HPLC. The ascorbic acid content of vegetables ranged from 5–109 mg/100 ml, while no ascorbic acids could be detected in vegetable broths. Total phenolic content was between 17–1729 mg GAE/l for all samples. ORAC and TEAC values of vegetable broths were between 0–3 µmol TE/ml and 0–2 µmol TE/ml, respectively. Gallic, chlorogenic, caffeic, p-coumaric, and ferulic acid were detected in both fresh vegetables and vegetable broths. The highest phenolic acid content was observed in water in which beetroots were boiled. It was found that the vegetable broths of beetroots, celery stalks, cabbages, parsley and broccoli harboured remarkable antioxidant activity. Keywords: boiling; ORAC; phenolic acids; TEAC; antioxidant capacity; vegetable; cooking Vegetables are an important part of a healthy diet, vary depending on the bioactive structures of the and the principal source of natural antioxidants such vegetables, the cooking method and length, the bio- as vitamin C, α-tocopherol, and phenolic compounds availability of phenolics, temperature and resistance of (Ames et al. 1993). These antioxidants have been the structure to heat (Jimenez-Monreal et al. 2009). linked to the protection against diseases provided Although the levels of many food antioxidants are by fruits and vegetables (Wen et al. 2010; Harasym significantly diminished by the thermal process, the & Oledzki 2014). overall antioxidant properties of foods can be main- Boiling is one of the most common methods used tained or improved by various applications (Nicoli to prepare vegetables before consumption. The boil- et al. 1997). In fruits and vegetables, phytochemicals ing process can lead to changes in the physical and with antioxidant properties are bound to the plant cell chemical properties of vegetables; for example, several membranes or exist as free compounds. Antioxidants studies have shown that boiling is able to improve the can be liberated due to the thermal destruction of palatability and bioavailability of naturally occurring cell walls and subcellular compartments leading to nutrients in vegetables (Turkmen et al. 2005; Wen et stronger radical-scavenging activity, which implies al. 2010). When vegetables are subjected to boiling, higher bio-accessibility. Boiling leads to the lixivi- their phenolic composition and antioxidant activities ation phenomenon in which nutrients leach out of Supported by Scientific and Technological Research Council of Turkey (TUBİTAK), Project No. 2209. 469 Food Analysis, Food Quality and Nutrition Czech J. Food Sci. Czech J. Food Sci., 35, 2017 (6): 469–475 doi: 10.17221/458/2016-CJFS the vegetables into the cooking water (Amin et al. dophenol dye until the appearance and persistence 2004; Jimenez-Monreal et al. 2009). of pink colour. Even though a number of studies have been pub- Total phenolic content. Total phenolic content lished regarding the effects of cooking on antioxidant (TPC) of samples was determined according to the and nutritional properties of vegetables (Amin et Folin-Ciocalteu method (Singleton & Rossi 1965; al. 2004; Amin & Lee 2005; Turkmen et al. 2005; Singleton et al. 1999). Absorbance was measured Miglio et al. 2008; Xu & Chang 2008; Xu & Chang at 760 nm. The concentration of total phenolic com- 2009; Jimenez-Monreal et al. 2009; Wen et al. pounds in samples was determined as gallic acid 2010; Patras et al. 2011; Leong & Oey 2012), there equivalents (GAE) using an equation obtained from a is very little known about the retention of nutrients gallic acid (Acros, USA) standard curve. The results in water after the boiling of vegetables. The purpose are expressed as milligrams of gallic acid equivalents of this study, therefore, was to determine the total (GAE) per litre. antioxidant activity and phenolic acid content of Oxygen radical absorbance capacity (ORAC) selected vegetable broths. assay. Oxygen radical absorbance capacity (ORAC) was kinetically measured using a Biotek SynergyTM HT Multi-Detection Microplate Reader (Winooski, MATERIAL AND METHODS USA). Aliquot (25 µl) of the diluted samples and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman- Sample preparation and experimental design. 2-carboxylic acid; Acros, USA) calibration solutions Green beans (Phaseolus vulgaris), courgettes (Cucur- were added to wells of a 96-well bottom reading bita pepo L.), parsley (Petroselinum crispum), carrots microplate (BD Falcon, USA). After the addition of (Daucus carota L.), tomatoes (Lycopersicon esculentum 150 µM fluorescein (Acros, USA) stock solution to Mill. L.), celery (Apium graveolens L), broccoli (Brassica each well the microplate was incubated at 37°C for oleracea var. italica), onions (Allium cepa L.), beetroots 30 minutes. Then, 2,2'-azobis(2-amidinopropane) (Beta vulgaris L. subsp. conditiva), spinach (Spinacia dihydrochloride (AAPH; Acros, USA) solution was oleracea), white cabbages (Brassica oleracea L. var. added to start the reaction. The microplate reader was capitata f. alba), and cauliflowers (Brassica oleracea programmed to record the fluorescence reading with L. var. botrytis) were purchased from a local fresh an excitation-emission wavelength of 485–520 nm market at their mature stage. After washing with tap using Gen 5TM software (Budak & Guzel-Seydim water, inedible parts were removed. Fresh vegetable 2010). Results were expressed as µmol TE (Trolox samples were obtained using a juicer (HR1821/10; Equivalents) per ml. Philips, The Netherlands). For vegetable broth (VB) Trolox equivalent antioxidant capacity (TEAC) extraction, beetroots, courgettes, onions, carrots, cab- assay. 2,2'-Azinobis (3-ethlybenzthiazolin-6-sulfonic bages, root celery, and tomatoes were sliced into 5-mm acid) diammonium salt (ABTS+; Acros, USA) radical thick slices. Parsley, spinach, stalk celery, cabbages, cation inhibition against Trolox was spectrophoto- broccoli, and cauliflowers were broken into pieces. metrically measured at 734 nm (Shimadzu Scientific Pre-treated vegetables were boiled in 10× volumes of Instruments Inc., Japan) (Re et al. 1999). TEAC values water (w/v) until they become soft enough to easily of samples was calculated from the Trolox standard poke with a fork. Each boiling experiment was carried curve and expressed as Trolox equivalents per mil- out in duplicate on three separate occasions. Fifty ml lilitre of sample. of fresh vegetable and VB samples were transferred HPLC determination of phenolic acids. Phe- ® to Falcon test tubes in an ice bath. Samples were nolic acids were evaluated by reversed-phase high- then stored in a deep freezer (–40°C). The data are performance liquid chromatography (RP-HPLC, expressed as mean ± standard error. The percentage Shimadzu SCL-10A; Scientific Instruments Inc., of leaching into VB was assessed in relation to fresh Japan). Detection and quantification were carried vegetables. out with a diode array detector at a wavelength of Ascorbic acid content. Ascorbic acid content was 198 nm. Separation of gallic, chlorogenic, caffeic, determined using a 2,6-dichlorophenol (DIP) titri- p-coumaric, and ferulic acid (Sigma Chemical Co., metric method (AOAC 1990). Five ml of homogenised Belgium) was conducted at 30°C on a C18 column sample was diluted with 25 ml of 6% oxalic acid (Gemini C18, 150 × 3 mm, 5 µm, 110A; Phenom- solution and titrated with the 2,6-dichlorophenolin- enex, USA). Elution was performed at a flow rate 470 Czech J. Food Sci., 35, 2017 (6): 469–475 Food Analysis, Food Quality and Nutrition doi: 10.17221/458/2016-CJFS FV VB * Figure 2. Total phenolic content (mgGAE/l) in( phenoliccontent 2.Total Figure beetroot lowest. the had Ascorbic acid was detected not while Parsley highest hadthe ascorbic acid content 1). (Figure ml mg/100 5 ± 109 ranged to 0.3 ± 5 from coefficient. correlation Pearson the calculating TPC, ORAC andTEAC results was evaluated by be statistically significant. between relationship The bles VB. and assess between differences freshvegeta results for independent The error. standard expressed data as were mean(version 16).The ± method. standard was carried external an against comparison by out times with standards. retention of Quantification tion ofphenolicacids was based onacomparison adjustedacid Identifica were 4.5with 1NNaOH. to of mobilephases 50 mM containing values pH 3min). next (for inwater acetonitrile 18% firstthe min)and 3 (for 15% acetonitrileinwater of 0.8ml/min,using as mobilephase of amixture capacity values matrix 1.Correlation ofantioxidant Table P =0.005;** (mg GAE/l) vegetable of samples ascorbic acid content The Data analysis 1 1 2 4 8 2 6 0 0 0 00 00 00 0 0 0 TEAC ORAC ORAC TPC TPC TEAC RESULTS AND DISCUSSIONRESULTS ( Green beans A ) P Courgette =0.001 P P Parsley to considered values were 0.05 < of . Results were analysed using SPSS Results. analysed were usingSPSS ORAC Carrot 0.646 0.280 0.297 0.399 1 Tomato 1 * Celery root Celery stalk 0.820 TEAC 0.646 0.565 0.280 1 Broccoli 1 t -test was used to ** Onion * * o Red beet -phosphoric 0.820 0.565 0.297 0.399 Spinach TPC A 1 1 ) fresh vegetables and( Cabbage ** * - Cauliflower - (mg/100 ml) infreshvegetables 1.Ascorbic acid content Figure derivatives ( cinnamoyl flavonols of hydroxyl isand source a coli activities.