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Food Chemistry 130 (2012) 928–936

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Food Chemistry
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Microwave-assisted extraction of phenolics with maximal antioxidant activities in tomatoes
Hongyan Li a,b, Zeyuan Deng a,⇑, Tao Wu c, Ronghua Liu b, Steven Loewen d, Rong Tsao b,⇑
a

State Key Lab of Food Science and Technology, Institute for Advanced Study, Nanchang University, Nanchang 330047, Jiangxi, China Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9 c Xinjiang Key Laboratory of Plant Resources and Natural Products Chemistry, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China d University of Guelph Ridgetown Campus, 120 Main Street East, Ridgetown, Ontario, Canada N0P 2C0
b

a r t i c l e

i n f o

a b s t r a c t
Microwave-assisted extraction (MAE) was investigated for extraction of phenolic compounds from tomato with maximised antioxidant activities using response surface methodology (RSM) coupled with a central composite design, and in vitro antioxidant assays (FRAP and ORAC). MAE was more efficient for greater antioxidant activities and higher total phenolic contents than solvent extraction. The optimal MAE processing parameters were 96.5 °C, 2.06 min, 66.2% ethanol for FRAP, and 96.5 °C, 1.66 min, 61.1% ethanol for ORAC. The models were successfully applied to 20 tomato cultivars, whose total phenolic contents (TPC) and indexes (TPI) were 489.30–997.45 mg gallic acid equivalent/100 g dry weight (DW) and 281.34–468.52 mg/100 g DW, respectively. Eight phenolic compounds were identified. Individual phenolics were 6.10–42.73 mg/100 g DW. The FRAP, but not the ORAC value showed good correlation with the TPC or TPI. The methodologies developed and the knowledge acquired in this study will provide useful information to tomato breeders and food processors. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

Article history: Received 20 January 2011 Received in revised form 15 May 2011 Accepted 5 August 2011 Available online 10 August 2011 Keywords: Tomato Cultivar Phenolic compounds Microwave-assisted extraction Response surface methodology Antioxidant activities

1. Introduction Phenolic compounds are one of the main groups of dietary phytochemicals found in fruits, vegetables and grains. They are found in plant tissues, and frequently serve as pigments in plants to attract pollinators, or as plants’ chemical defence mechanism against infections caused by microorganisms and injuries by insects (Ballard, Mallikarjunan, Zhou, & O’Keefe, 2010; Tsao & McCallum, 2009). A significant role of phenolics that has been under active research in recent years is their possible beneficial health effects for humans. Phenolic compounds have been recognised for their antioxidant activity which has been linked to slowing down the ageing process and lowered risks of many prevalent chronic diseases such as cancer and coronary heart disease. Most of these problems are considered to be caused by an imbalance between the oxidative stress and antioxidants in the body (Karacabey & Mazza, 2010). Tomato (Solanum lycopersicum) is one of the most widely consumed fresh and processed vegetables in the world for its nutritional and bioactive antioxidants such as phenolics, carotenoids and vitamins C and E (Pernice et al., 2010). Phenolic compounds

⇑ Corresponding authors. Tel./fax: +86 791 8304402 (Z. Deng), tel.: +1 519 780 8062; fax: +1 519 829 2600 (R. Tsao). E-mail addresses: zeyuandeng@hotmail.com (Z. Deng), Rong.Cao@agr.gc.ca (R. Tsao).

in tomato have been reported to be linked to reduced risk of prostate and various other forms of cancer, as well as heart diseases (Toor, Savage, & Heeb, 2006). Phenolic compounds, especially flavonoids in tomatoes, were able to withstand industrial processing methods, being detected in a variety of tomato-based products (Stewart et al., 2000). Conventionally, phenolic compounds in tomatoes are extracted overnight under stirring or rotation (Benakmoum, Abbeddou, Ammouche, Kefalas, & Gerasopoulos, 2008; Toor, Savage, & Lister, 2006), which is a time-consuming process. Recently, interest in microwave-assisted extraction (MAE) has increased significantly as a result of its inherent advantages (reduction in extraction time, solvent volume, energy and better extraction efficiency) over conventional extraction techniques such as solid–liquid solvent extraction (Ballard et al., 2010). MAE is a process of using microwave energy to effectively heat solvents so that the analytes can be readily partitioned from the sample matrix into the solvent. MAE can significantly reduce both extraction time and solvent consumption (Pérez-Serradilla & Luque de Castro, 2011). MAE has been considered as a potential alternative to conventional solid–liquid extraction of phytochemicals including phenolic compounds from plants (Llompart et al., 1997), however, the effects on the composition and quantity of the phytochemicals of interest depend on many factors involved in MAE. Optimisation of these factors, such as microwave temperature, extraction time and solvent

0308-8146/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.08.019

and then held at the desired temperature for a certain time period according to the experimental design. O258AAAA has a trait that intensifies the fruit pigment. Matthews. The mixture was stirred with a magnetic stirrer bar for homogeneous heating. Materials and methods 2. All other chemical reagents used were of analytical grade. The vessels were placed at the centre of the microwave apparatus and heated to the desired temperature in 5 min. in single rows. All procedures were performed in dim lighting. 2005). Kansas City. Canada). / Food Chemistry 130 (2012) 928–936 929 concentration in the MAE of phenolic compounds from tomato has not been reported. USA). such an approach has not been reported elsewhere. In brief. response surface methodology (RSM) has been used to evaluate the effects of several process variables and their interactions (Liyana-Pathirana & Shahidi. USA) in order to obtain a homogeneous puree. LA3006 is a line of high lycopene. The FRAP assay is based on the reducing power of antioxidants. These materials were stored in polyethylene tubes at À80 °C prior to analysis. Four sub-samples of at least 10 ripe fruits were collected randomly from each plot in September. To the best of our knowledge. CEM Corporation Inc.5 g) was accurately weighed and transferred into a 50 ml tube containing 25 ml of 60% ethanol.. USA). caffeic acid. 08f8056 produces purpling on the immature fruit. formic acid and ethanol were purchased from Caledon Laboratories (Georgetown. 2005). 2001. & Larondelle. The extraction was carried out on a rotary shaker (Scientific Industries Inc. a breeding line with the old gold crimson allele (ogc).20 -azobis-(2-methylpropionamidine) dihydrochloride (AAPH) were purchased from Sigma (St. & Heeb. Transplants were set in the field in May 2008. Xie. Stamford. L-ascorbic acid. MO. Tsao & Deng. 2006). & Rogez. 2009. The ferric reducing antioxidant power (FRAP) and the oxygen radical absorption capacity (ORAC) assays are the two most frequently used in vitro models for assessing the total antioxidant activities of phenolic compounds (Magalhães. The peroxyl radical is considered biologically relevant as it is involved in lipid peroxidation and other biologically significant pathways (Ou.1.. TD00-0046-1AAAAA produces pink–orange coloured fruit. 2004). 08f8005 and 08f8006 are sister lines of 08f8002. Hampsch-Woodill.2. Yang. Canada. The temperature in the vessel was directly measured using a thermocouple type thermometer. In the present study. ferulic acid.. & Prior. ON. Sodium acetate. NC. Alternatively. chlorogenic acid. and rows spaced 150 cm apart. & Khanizadeh. 15 h) at room temperature (Toor. Folin–Ciocalteu’s phenol reagent. glacial acetic acid. Extraction of phenolic compounds 2. The mixture was worked up the same way as described above. improving. 0. Twenty tomato varieties and breeding lines known for different phytochemical compositions were used in this study. Microwave-assisted extraction Phenolic compounds were extracted from tomato. Bohemia. the mixture in the extraction vessel was allowed to cool down to room temperature (in ca. Labconco. These accessions were a collection of one of the authors (SL). Q105AAAA. Westbury. NC.2. habrochaites in the pedigree. Brinkman Instruments Inc. 08f8058 is a sister line to 08f8056. naringenin. Aqueous ethanol. 2007). Trolox and 2. 08f8002 has an unknown trait that affects the ripening process.4) because the pressure differences caused by differences in solvent volume may influence the extraction efficiency (Ballard et al. O393ABAAAA consistently rates as having good flavour. Samples were extracted in triplicate. H9478 is a commercial F1hybrid cultivar developed by HeinzSeed. Reis. 2. 2. The plots were maintained according to a standard processing tomato production management system with the exception that fungicides were not applied. as well as in modelling and optimising the extraction of phenolic compounds from plants (Pompeu. A brief description for all the 20 tested tomatoes is as follows: OH7983 is a commercial openpollinated processing tomato cultivar developed by Ohio State University. Louis. The correlation between the antioxidant activities and their phenolic contents was also studied.H. Chemicals and standards Protocatechuic acid. 2010). NY. After microwave heating.3. TI01-033AAAAA has an atypical ripe fruit colour and an unusual flavour.1. A known amount of about 30 g of this puree was freeze-dried (Bulk tray dryer. ON.3.. Matthews. Li et al. Canada) in preparation for spectrophotometric and HPLC analyses. The whole tomatoes were washed with tap water. was used. sodium phosphate dibasic and HPLC-grade solvents. Q219AAAA has both Solanum pennellii. and LA1016 has the diospyros gene that originates from Solanum cheesmaniae. Tsao. USA).2-lm PTFE membrane filter (VWR International. a safe and efficient solvent for the extraction of phenolic compounds.3. fluorescein. 10 min) before centrifugation (Eppendorf centrifuge 5810R. the applicability of MAE to the extraction of phenolic compounds in tomato and optimised conditions for antioxidant activities as measured by the FRAP and ORAC assays were investigated using the central composite rotatable design (CCD) combined with the RSM. The supernatants were combined and filtered through a 0. Rogez. Silva. 2010). including methanol. A constant solvent volume of 25 ml with varying sample mass was chosen for the microwave extraction in the preliminary study (see Section 2. Sockovie. and Solanum habrochaites wild tomato species in the pedigree and has ripe fruits that appear normal red on the outside but are distinctly orange when cut.. 08f8055 is purported to have high polyphenols. using MAE system (Model MES-1000. gallic acid. an atypical pink ripe fruit colour possibly from S. cut into pieces and ground with a commercial blender (7011. Both TI01-0044BAAAA and TI01-0044BABAA have . and analysed for the phenolic composition and antioxidant activities. Plant materials The 20 tomato cultivars and breeding lines used for this study were grown in University of Guelph processing tomato breeding plots. Ontario. Rotary solid–liquid solvent extraction Dry tomato powder (0. The residue was re-extracted in the same solvent and under the same conditions. WaringÒ Laboratory Science. 2008. gentistic acid. 2. 45 cm plant spacing within the row.3. All tomatoes were harvested at commercial maturity. USA) (at 400 rpm) overnight (ca. Q49AAAA is without the crimson gene. on a Brookston silt loam soil near Ridgetown. CT.20 -azobis-(2-methylpropionamidine) dihydrochloride). NY) at 4000 rpm for 5 min.4. whereas the ORAC assay has been used to evaluate the antioxidant activity of water-soluble phytochemicals against the peroxyl radical-induced oxidation initiated by thermal decomposition of AAPH (2. Their effects on the antioxidant activity of the phenolic extracts are also not known. 2. LA1593 is a wild species (Solanum pimpinellifolium) reported to be high in phenolics.5-tri(2-pyridyl)2.6-triazine (TPTZ). MO. Savage. and optimising food processes (Karacabey & Mazza. sodium phosphate monobasic. RSM is a collection of statistical and mathematical techniques that has been successfully used for developing. USA) and ground into fine powder. 1. Silva. p-coumaric acid. Segundo. & Lima.5 g freeze-dried tomato powder was suspended in 25 ml of ethanol in a 100 ml Teflon PFA (perfluoroalkoxy)-lined extraction vessel (CEM Corporation Inc. 2008. ferric chloride hexahydrate. rutin.

bii is the quadratic coefficient. Randomized.5. All compounds were tested and expressed as lmol ascorbic acid equivalent (AAE) per gram dry weight tomato (lmol AAE/g DW). The antioxidant activities were expressed as lmol ascorbic acid equivalent (AAE) per gram dry weight tomato (lmol AAE/ g DW).12 ± 20. thus greater antioxidant activity.50 ± 22.50 ± 20.64 (1.930 H.03 26.39 270. 200.20 ± 20.63 22.11 26. To establish an MAE protocol for maximised extraction of antioxidative phenolic compounds in tomato.61 ± 23. VT.30 ± 17. 2008).74 ± 19.78 ± 1.89 21. USA).04 28. A calibration curve was constructed daily by plotting the calculated differences of area under the fluorescein decay curve between the blank and the sample for a series of standards of Trolox solutions (6.19 265.51 20. Antioxidant assays 2. 2.52 ± 15.78 ± 1. Trolox.57 ± 21.77 Not randomized. 2. / Food Chemistry 130 (2012) 928–936 2. 2..90 26. each included five levels (Table 1). 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3Á6H2O in the ratio of 10:1:1 (v/v/v)) and added to the wells.0868 nM) solution and incubated for 30 min at 37 °C before injection of 25 ll AAPH (153 mM) in a microplate well.09 ± 17.74 200. The regression analysis of experimental data was performed to establish the empirical second order polynomial models.2.40 ± 1. a water-soluble azo compound. The FRAP assay measures the ability of the antioxidants in the tomato phenolic extracts to reduce ferric–tripyridyl-triazine (Fe3+–TPTZ) complex to the blue coloured ferrous form (Fe2+) which absorbs light at 593 nm. a water-soluble tocopherol analogue.85 ± 22.20 28. Stronger absorption therefore indicates higher reducing power of a phytochemical. 100. 25. Shown in Eq.70 ± 0. Winooski.65 ± 1.82 ± 2.61 23.75 280.20 ± 2. The plate was incubated at 37 °C for the duration of the reaction.59 28. Oxygen radical absorption capacity (ORAC) assay The assay for the oxygen radical scavenging capacity was conducted according to Ou et al. and Xi and Xj are independent variables.22 ± 19. Inc. 12.84 254. standard or sample extract (10 ll) were mixed with Table 1 Three-factor. 50 and 100 lmol/l). The response surface methodology (RSM) with a CCD was used to identify the relationship between the response function and the process variables as well as to determine the optimal conditions for extraction (Karacabey & Mazza.51 239. Ferric reducing antioxidant power (FRAP) assay The FRAP assay was determined according to the method of Benzie and Strain (1996) which was modified for the 96-well microplate reader (Khanizadeh et al. (2001).73 237. 300 ll of ferric–TPTZ reagent (prepared by mixing 300 mM acetate buffer.43 ± 9.14 285.80 24.55 ± 20. 2009).1.. Y1 and Y2.68) 60 (0) 60 (0) 60 (0) 60 (0) Response-1 FRAPc 23. All compounds were tested and expressed as lmol Trolox equivalent (TE) per gram dry weight tomato (lmol TE/g DW). Six concentrations of 25.32 (À1.77 ± 1.84 ± 1.61 ± 22.51 266.18 (À1. five-level central composite design used for RSM and experimental data of the investigated responses of tomato phenolic extracts. A CCD was applied to determine the best combination of extraction variables for the antioxidant activities.58 ± 1..17 25.89 Response-2 ORACd 222. was used as standard and fluorescein as fluorescent probe.11 ± 2. 400 and 800 lmol/l were used to prepare the standard curve of L-ascorbic acid.00 ± 1. A breeding line O393ABAAAA was used to optimise the conditions of extraction. 2010).24 ± 0.06 27.29 251. The fluorescence was measured every minute for about 120 min until it reached zero (excitation wavelength 485 nm.37 ± 1.68) 96.75 25. The absorbance readings were taken at 593 nm at 30 min using a UV–vis microplate kinetic reader (EL 340.29 ± 2. Winooski.07 ± 1.2 mol/l Folin–Ciocalteu reagent in 96-well microplates and allowed to react for 10 min at room temperature.25.10 ± 1. A 125 ll saturated sodium carbonate (Na2CO3) solution was then added and allowed to stand for 30 min at room temper- Y ¼ b0 þ 3 X i¼ 1 bi X i þ 3 X i¼ 1 bii X 2 i þ 2 3 X X i¼1 j¼iþ1 bij X i X j ð 1Þ where Y is the measured response variable (FRAP or ORAC value). AAPH.94 ± 1. Experimental design Many factors including microwave power.68) 3. respectively. Determination of total phenolic content Briefly. pH 3. these factors were evaluated in a preliminary study to determine the most influential factors for in-depth investigations.10 251. v/v) 40 (À1) 40 (À1) 40 (À1) 40 (À1) 80 (1) 80 (1) 80 (1) 80 (1) 60 (0) 60 (0) 60 (0) 60 (0) 26. Li et al. Inc.5.68) 93. Briefly. 25 ll of blank.95 259.68) 80 (0) 80 (0) 80 (0) 80 (0) 80 (0) 80 (0) 80 (0) 80 (0) Factor 2 (X2) microwave extraction time (min) 1 (À1) 1 (À1) 3 (1) 3 (1) 1 (À1) 1 (À1) 3 (1) 3 (1) 2 (0) 2 (0) 0. extraction time.76 30.6.84 262. Bio-Tek Instruments. (1). Trolox standard or tomato extracts (in triplicate) were mixed with 200 ll fluorescein (0. can affect the extraction efficiency in MAE (Liyana-Pathirana & Shahidi. 25 ll gallic acid standard or tomato extracts (in triplicate) was mixed with 125 ll 0.36 (À1. b0 is a constant.6.4.. Standard ordera 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a b c d Run orderb 6 15 7 8 18 3 16 4 11 12 17 13 10 1 9 5 14 2 Factor 1 (X1) microwave irradiation temperature (T) 70 (À1) 90 (1) 70 (À1) 90 (1) 70 (À1) 90 (1) 70 (À1) 90 (1) 63.40 ± 10.68 (1. 2005). bij is the two factors interaction coefficient.62 ± 6. The results were expressed as lmol Trolox equivalent (TE) per gram dry weight tomato (lmol TE/g DW) (André et al.68) 2 (0) 2 (0) 2 (0) 2 (0) 2 (0) 2 (0) Factor 3 (X3) ethanol concentration (%.39 263. and the three independent variables were microwave temperature (X1). 50.34 272. composition of solvent. was used as a peroxyl radical generator. bi is the linear coefficient (main effect). microwave temperature. USA).5.29 ± 27. Briefly. . emission wavelength 528 nm) in a Bio-Tek Fluorescence Spectrophotometer equipped with an automatic thermostatic holder (PLX 800.93 228. Bio-Tek Instruments.65 30.15 31. solvent to solid ratio and extraction cycles. extraction time (X2) and ethanol concentration (X3) which were selected based on the preliminary results. VT.82 (1.5. FRAP and ORAC values were responses.82 263.

Inc. 80 and 100 lg/ml for generating the standard curves. 5. One-way analysis of variance (ANOVA) was used to compare the means. ANOVA analysis also showed that the interactive effect between ethanol concentration and microwave temperature on the FRAP value was insignificant (p > 0. .3. which displayed a similar curve effect and linear effect on the antioxidant activities by the TEAC and ORAC methods.. This suggests an enhanced degree of breakage of cell membranes in the raw material by the increasing ethanol concentrations in the beginning. 10. the polarity of the solvent changes. When the ethanol concentrations were at 40–80% and the microwave temperature at 70–80 °C. A non-significant lack of fit (p > 0. 2007).. Milford. caffeic acid. Minneapolis. As evident in Fig. MA.8. v/v/ v). 1b).1. Xie.9196.. MAE extraction Aqueous methanol or ethanol is a preferred extraction solvent system for the phenolics (Karacabey & Mazza. resulting in varied antioxidant activity of the extracts (Karacabey & Mazza. 2010). chlorogenic acid. 3.001). which confirms that the model can adequately represent the true relationship between the parameters chosen. / Food Chemistry 130 (2012) 928–936 931 ature before the absorbance of the reaction mixture was read at 765 nm using a visible–UV microplate kinetic reader (EL 340.0 (Analytical Software. but was not significantly changed by the ethanol concentration (Fig. Tallahassee. USA) (Zhang et al. Stat-Ease. and serially diluted to 0.2. Results and discussion 3.5 °C. gentistic acid. 320 and 360 nm). USA) was equipped with a diode array detector and an Ezchrom workstation for data processing. HPLC methods of phenolic compounds The Accela system (Thermo Scientific. 1. 40. 5% A to 50% in 17 min. Separation of phenolic compounds was achieved on a Waters AtlantisÒ C18 column (5 lm. Cary.05). 2010). The significant contribution of ethanol concentration and temperature to the total antioxidant activities of grape cane extracts has been reported by Karacabey and Mazza (2010). NC. 4. 95:2:3. however. The standard solutions were prepared separately by dissolving 10 mg of each compound in 5 ml DMSO and then topped up to 100 ml in a volumetric flask with methanol (final concentration 100 lg/ml). the FRAP value only increased as the temperature went higher. Using this model (Eq.The empirical relationship between the FRAP value and the extraction parameters were generated as follows (Eq. Eight phenolic standards commonly found in tomato. (2)): Y ¼ 8:754428 À 0:14629X 1 þ 0:859841X 2 þ 0:335686X 3 À 0:01215X 1 X 2 þ 0:000238X 1 X 3 þ 0:021826X 2 X 3 2 2 þ 0:002701X 2 1 À 0:24029X 2 À 0:00279X 3 ð2Þ An analysis of variance (ANOVA) was performed to determine the significance. This is favourable as the extraction time is extended at a higher microwave temperature may lead to thermal degradation of the phenolics (Chen.7. p-coumaric acid.01). Waltham. At higher temperatures. (SAS Ins. therefore they were set at 100 w.1. 1c). Statistical analyses were performed with Statistix for Windows version 9. 2010). Differences in phenolic compounds were considered significant at p < 0.. It was reported that the different solvent compositions and extraction temperature can lead to differences in the profile of phenolic compounds. The correlation coefficient (R2) of the model was 0. & Gong.1.05). (2)). respectively.2%. Solvent composition and extraction temperature have been found by others to affect the phenolic contents of grape cane extracts. protocatechuic acid. The total phenolic content (TPC) was expressed as mg gallic acid equivalent (GAE) per gram dry weight tomato (mg GAE/g DW) based on the calibration curve. & Gao. The solvent gradient in volumetric ratios was as follows: 1% A to 5% in 3 min. Statistical analysis Results were expressed as mean ± SD of three independent extractions. 95:2:3. whereas the effect of extraction time was not significant (p > 0. USA). Winooski. extraction time 2. ferulic acid. USA). The UV–vis absorbance of the peaks were collected between 200 and 620 nm using a diode array detector (DAD) and monitored at three wavelengths (280. Calibration was achieved with an aqueous gallic acid solution (50–500 lg/ml).9. Bio-Tek Instruments. RSM was performed using the Design Expert software (Version 7. the FRAP value increased initially but decreased as the ethanol concentration increased. Fig. The effects of the independent variables and their mutual interactions on the FRAP value of phenolic extracts can be seen on the three dimensional response surface curves and contour plots shown in Fig. 20. solid to solvent ratio and extraction cycles had little effect on the antioxidant activities. resulting in varied antioxidant activities (Karacabey & Mazza. the optimal extraction conditions for the FRAP value are as follows: microwave temperature 96.6 Â 250 mm) (Waters Corp.2. MA. Increased diffusion resistance due to the coagulation of proteins may also result in low FRAP value of phenolic compounds (Yang. There was a 4-min post-run which brings back to the starting conditions for reconditioning. which leads to increased impurities being extracted. At lower temperatures. 1a–c. v/v/v) and water–methanol–formic acid (B. at 25 min the gradient was increased to 100% A and held at 100% A for an additional 2 min. 2009). VT. FL. however. dissolution of the phenolic compounds can reach the equilibrium in a shorter time thus are not readily affected by changes in the extraction time. The column was thermostatically controlled at 30 °C and the flow rate was set to 1 ml/min. 2007). 60. Optimum extraction conditions based on the FRAP activity The surface response analysis for the antioxidant activities as measured by the FRAP and ORAC assays. This phenomenon is considered to be caused by the low rate of mass transfer at low temperatures. Ethanol was chosen for the simple reason that this is used in the food industry. The mobile phase consisted of two solvents: methanol–water–formic acid (A. increasing extraction temperatures led to a gradual increase in the FRAP value over time. This suggests that a higher microwave temperature and a short extraction time are more effective in extracting antioxidative phenolic compounds from tomato using MAE.H. Inc. which would require more time for the phenolic compounds to dissolve from the raw materials into the solution (Fig. 1a. 1:50 (w/v) and two cycles. Li et al. MN) and SAS V. Inc. The preliminary studies indicated that variables including microwave power.05. 3.. as the ethanol concentration continues to increase. The extraction temperature had a significant effect on the FRAP value of phenolic compounds (p < 0. rutin and naringenin were analysed using kmax at 320 nm except for protocatechuic acid (280 nm). 1c shows the interactive effects of the ethanol concentration and microwave temperature. USA).06 min and ethanol concentration 66. 2.05) showed that the quadratic model is valid to the spatial influence of variables on the response. indicated that the effects of the extraction temperature and ethanol concentration on the response were significant with a positive linear relationship (p < 0. 2. Liu.

5 2.5 ve e xtra c 1.0 25. A mean value of 32.5 °C. Li et al.0 28. Verification of predicted extraction parameters To validate the predictability of the established models. The good correlation between these results confirmed that the response models were adequate in predicting the optimised conditions.5 22. Optimum extraction conditions based on the ORAC activity Similarly.0 24.0 2.5 2.0 2.5 30.05). which were in close agreement with the predicated values (32. 1f indicates that the optimum ethanol concentration can be different depending on the temperature of the 3. the ORAC value increased as the ethanol concentration became higher.0 micr o wa 1. / Food Chemistry 130 (2012) 928–936 28. . indicating that the model can adequately represent the real relationship between the parameters chosen.0 27. 1e).0 ntra e 40 v tio n o wa micr ORAC 250 240 230 80 3.66 lmol TE/g DW (n = 3) were found in the actual FRAP and ORAC experiments. an increase in the ethanol concentration from 40% to 80% caused a 10.5 3.66 min and ethanol concentration. but the interactive effect of temperature and ethanol concentration was not significant (p > 0.0 ratio n i me io n t tract x e ve o wa micr 1. 3.0 tio n tim e 31.5 24.64 lmol AAE/g DW and 290. The coefficient of determination (R ) was 0.0 time no l c tio n 1.0 tio n tim e 80 85 ORAC 260 250 240 90 85 80 e r u ra t 75 mp e ve te 70 o wa r ic m 90 (b) 70 75 o wa ic m r re rat u mpe ve te (e) 3. extraction time. In general.0 2.05) further confirmed the validity of the model. 96. 1. but at higher temperatures.5 28. Response surface plots showing the effects of variables on FRAP assay (a–c) and ORAC assay (d–f) of phenolic compounds.1%.0 80 3. the relationship of ORAC was similar to what was observed in the FRAP assay.5 21. the optimised parameters were tested in an experiment using the breeding line O393ABAAAA.0 2.0 70 Et ha 60 no l c o 50 (a) (d) 280 270 nc e n t 40 1.5 70 Et ha 60 2. (3)).0 80 70 Et ha no l 60 co n 50 ce n tr atio 40 n ORAC 265 245 225 205 (c) 90 85 t ure 80 a r e 75 mp e te 70 wav o r mic 80 70 Et ha 60 no l c 50 o nce ntra 40 tio n (f) 90 85 80 erat ure p 75 t em 70 ro wav e mic Fig.83% increase in the ORAC value (Fig.3.5 FRAP 26.932 H.4.8727 for this equation.5 o nce 50 xt rac e 1. At a fixed extraction time of 2 min.55 lmol AAE/ g DW and 290. the final predictive equation for the relationship between the ORAC value and the extraction parameters is obtained (Eq. the ORAC value decreased as the ethanol concentration rose when the concentration was over 60%.5 2.8% increase in the ORAC value (Fig. 1d).5 ve e xt rac 1. The optimal ORAC value was found to be at the following extraction conditions: microwave temperature. At lower temperatures.0 270 260 FRAP 26.0 micr o wa 1.62 lmol TE/ g DW) using the respective model Eqs. (2) and (3). Fig. and the increase in microwave temperature from 70 to 90 °C led to a 9. 61.5 285 FRAP 26. Y ¼ 7:721339 À 1:34226X 1 þ 21:58616X 2 þ 7:256877X 3 À 0:16775X 1 X 2 À 0:0362X 1 X 3 À 0:17736X 2 X 3 2 2 þ 0:031769X 2 1 þ 0:21467X 2 À 0:02809X 3 2 ð 3Þ extraction process.0 23. 1. A non-significant lack of fit (p > 0.

were completely separated in 30 min in this method except for peaks 2 and 3 (chlorogenic acid and ferulic acid. Li et al. values are expressed as lmol ascorbic acid equivalent (AAE) per gram dry weight tomato (lmol AAE/g DW). the overall intraand inter-day variations (RSD) of the eight standards were less than 1.20 and 100 lg/ml.85% and 2. n = 3. Hernández-Jover. values are means ± SD. The LOD and LOQ The total phenolic index (TPI).6. Peaks: 1 = Protocatechuic acid. (B) ORAC assay. separation of the two was actually better at 360 nm (see insert in Fig. 5 = pCoumaric acid. respectively). Soliva-Fortuny. respectively. 6 = Ferulic acid.H. regression and linear ranges of eight standards were performed using HPLC. 2 = Chlorogenic acid. Correlation between the concentration and the peak area of these phenolic compounds was highly linear (R2 > 0. 2. which is the sum of concentrations of all phenolics detected (Tsao & Yang. 3 and Table 2. & Fernández-Gutiérrez. Their concentrations were expressed in naringenin equivalents (Raffo. 1 10 mAU 140 120 100 80 60 (a) 40 20 0 0 (b) 5 10 20 200 100 2 0 0 16. Right: UV spectra of uk1 (1) and uk2 (2). FRAP and ORAC values of phenolic compounds in 20 tomato cultivars and breeding lines. and environmental conditions (Luthria. in addition to other factors such as storage conditions.93%. 3 = Gentistic acid. Malfa. The recoveries were between 94% and 105%.99) between 0.05). respectively. 8 = Naringenin. values are expressed as lmol Trolox equivalent (TE) per gram dry weight tomato (lmol TE/g DW). 4 = Caffeic acid. 2). Segura-Carretero. 3. There were two significant but unknown peaks with UV spectral features similar to typical phenolic compounds in all cultivars studied (Fig. All eight phenolic compounds found in tomato (Gómez-Romero. Odriozola-Serrano. & Martín-Belloso.25 and 0. values followed by the same letter in the same assay are not significantly different (p < 0. enlarged peaks of chlorogenic acid and gentistic acid at 360 nm. 2009). The FRAP and ORAC values.1 17. Fogliano. 2010.7 17. Mixed standards (a) and a typical HPLC chromatogram of phenolic compounds extracted from tomato (O393ABAAAA) (b) at 320 nm. 2b).5 3. Although quantification of these two compounds was based on signals at 320 nm. 2006). The two peaks in actual samples are in fact separated completely (Fig. (A) Q105AAAA O258AAAA H9478 OH7983 LA1593 LA1016 Q49AAAA (B) a b b c c c d d d d e ef ef g fg g g g g gh h samples LA3006 08f 8006 T101-0044BAAAA 08f 8055 O258AAAA 08f 8058 TD00-0046-1AAAA O393ABAAAA 08f 8056 T101-0044BABAA T101-033AAAAA LA1593 LA1016 Q105AAAA OH7983 Q49AAAA H9478 08f 8005 Q219AAAA 08f 8002 a ab bcd bc cde def ef ef g ef gh f ghi f ghi j ghi j k hi j kl i j kl m i j kl m kl m kl m lm m j kl m 0 100 200 300 400 TD00-0046-1AAAA T101-0044BABAA samples 08f 8005 Q219AAAA LA3006 O393ABAAAA 08f 8055 T101-0044BAAAA 08f 8058 08f 8002 08f 8006 T101-033AAAAA 08f8056 0 15 30 45 60 75 FRAP value (µmol AAE /g DW) ORAC value ( µmol TE/ g DW) Fig. was analysed using the above HPLC method for all samples. The method had good accuracy and repeatability. 7 = Rutin. Inserts: left. 2). . The discrepancy is not surprising because genotype can play an important role in the phenolic profiles of tomato. The phenolic content and antioxidant activities of 20 tomato cultivars and breeding lines 300 nm 350 400 200 250 1 uk1 56 3 4 7 2 uk2 7 4 5 6 8 8 2. & Quaglia.3 1 15 20 25 30 Fig.5. Validation of HPLC method The linearity. / Food Chemistry 130 (2012) 928–936 60 933 50 40 30 300 were less than 0. All samples were extracted separately using the two different extraction conditions optimised for the FRAP and ORAC antioxidant activities. 3. Maiani. (A) FRAP assay. Both TPCs and TPIs were significantly different among the 20 tomato cultivars. extraction procedure.75 lg/ml. 2003). TPC and TPI of the 20 tomato cultivars and breeding lines are shown in Fig.

47 ± 11.64 ± 0.49 hijk 63.40 def 8.30 ± 14.18 k 8.74 ghij 9.58 ± 1.31 d 395.50 ± 7.97 ± 2.97 b 21.98 ± 1.16 k 16.33 c 08f8055 7.10 ± 0.59 ± 3.09 a 31.39 fg 16.74 a 15.15 cd 9.36 ± 0.35 d 358.10 ef 9.44 ± 21.56 hi 25.35 ± 8. d TPI: total phenolic index (sum of individual phenolic concentrations).95 ± 1.63 ± 11.66 min and 61.11 a 7.75 a 6.03 ± 0.39 bc 8.33 ± 9.09 ± 0.89 de 12.45 ± 12.89 jk 218.46 ± 2.69 a 419.31 ef 57.31 e 503.26 f 19.73 ± 12.28 d 16.13 ± 2.83 ab 8.00 ± 0.65 ± 11.23 ± 2.72 ± 0.73 ± 1.11 ± 1.08 ± 0.95 e 6.58 ± 0.78 a 42.28 ± 16.11 ± 0.12 ghi 7.54 ± 10.66 ± 0.20 ± 3.65 ± 1.96 a 42.10 ab 7.10 ghi 8.45 ± 0.69 ± 0.35 h 332.99 hi 18.89 a 8.56 ± 2.76 ± 9.18 efgh 22.85 a 9.93 ± 0.08 a 30.81 ± 0. c Concentrations of unknown compounds are express as mg naringenin equivalent/100 g dry weight.52 ± 0.39 ± 0.61 ± 0.72 def 14.85 ± 0.18 hij 7.07 ± 0.24 ± 0.08 g 17.36 gh 36.59 ± 0.10 d O258AAAA 7.12 i 7.69 ± 0.78 bc 15.01 a 7.07 ± 9.12 a 30.75 ± 0.26 J 19.47 ± 12.27 bc 374.70 hijk 216.65 a 15.64 ± 18.25 a 6.27 ± 0.17 c 12.75 efgh 21.66 ± 2.09 ± 0.57 ± 1.10 b 29.63 ± 0.29 ± 2.18 hij 69.06 ± 0.63 ± 10.38 ± 0.78 a 180.21 ± 2.11 a Q49AAAA 7.10 ± 1.25 e 18.19 ± 11.72 b 88.02 ± 8.51 ± 1.79 b 223.06 b 468.1% ethanol).28 h 19.84 ± 0.09 ± 1.05 ± 0.08 ± 1.33 a 413.54 ± 0.70 b 732.21 bc 67.90 a 8.27 cd 199.16 l 323.46 ± 0. / Food Chemistry 130 (2012) 928–936 TD00-0046-1AAAA 7.51 ± 24.91 ± 1.83 ± 4.40 e 18.07 ab 30.13 e O393ABAAAA 7.28 b 11.79 ab 9.08 ± 0.13 b 27.33bc 17.84 ± 11.42 ± 18.88 cdef 36.51 ef 382.04 ± 0.46 cd 58.44 h 119.11 ± 0.22 ± 0.65 ± 0.46 a 39.58 ± 0.07 ± 8.08 ± 2.06 fgh T101-033AAAAA 7.85 ± 0.26 c 7.57 ± 0.37 fg 413.92 a 8.09 a 31.16 def 16.08 efg 8.11 ab 8.97 g 7.66 ± 17.51 ± 0.09 ± 16.94 ± 0.72 cde 59.57 ± 0.20 bc 38.83 ± 1.94 ± 0.54 efg 150.68 ± 1.44 fg 565.24 ± 21.11 b 31.17 ± 0.09 bcd 63.25 f 366.69 c 11.76 a 10.79 ± 1.91 ± 5.34 ± 7.23 a 10.69 ± 1.25 h 575.60 de TD00-0046-1AAAA 7.52 ± 1.01 ± 6.46 cdef 463. e TPC: total phenolic content (mg GAE/100 g dry weight).16 ± 9.36 bc 61.94 hi 302.88 ± 0.67 ± 2.53 fghi 9.09 ghi 7.40 ± 1.51 ± 26.88 ± 0.53 ± 0.03 def 26.66 ± 1.12 i 15.17 a 10.12 b 29.88 ghi 270.23 ± 0.73 ± 1.18 a 8.67 ij 197.40 ± 1.05).05 ± 0. Li et al.55 c 10.24 b 86.43 ± 0.12 a 29.00 ± 0.31 f 672.66 ± 13.51 ± 0.25 g 18.75 ± 15.84 de 8.97 ± 0.18 J 9.13 ef 8.29 ± 0.5 J 9.44 cd 9.33 hij 14.46 ± 0.85 ± 0.08 ± 0.17 cdef 355.31 b 54.57 ± 9.05 cdef 58.97 ± 1.04 ± 1.23 ± 0.79 ± 15.57 ± 0.74 fg 24.10 a 30.09 c 15.18 g 18.13 b 257.18 c 7.44 h 6.33 ± 0.53 i 17.87 ± 12.80 gh 18.44 ± 0.86 h 444.15 b 21.06 ± 0.42 ± 1.06 k 15.64 gh 23.79 ± 0.72 b 330.06 ± 12.13 jk T101-0044BAAAA 7.74 ± 0.96 fg 14.91 ± 5.31 gh 7.54 ± 0.19 f 692.13 a 30.49 b 10.03 fgh 262.92 a 7.10 def 7.71 k 117.81 ± 6.75 gh 13.68 d 726.87 ± 0.03 ± 19.07 a 6.77 ± 9.84 ± 1.48 ± 2.94 a 7.37 ± 0.12 k 16.51 ab 8.39 ± 1.24 ± 0.64 ± 0.08 b 29.77 c 11.08 b 30. .47 a 257.10 a 7.88 fg 8.42 jk LA1016 7.42 ± 15.72 g 21.46 def Q219AAAA 7.75 d 15.45 f 17.37 ± 3.97 ± 18.86 de 28.11 g 8.72 c 954.61 ghi 08f8006 7.67 ab 7.39 efg 9.63 ± 0.01bcdef 10.24 b 414.77 ± 0.33 fgh H9478 7.21 ef 9.01 cdef 25.14 ± 0.48 fg 66.62 fgh 10.13 a 29.92 a Q49AAAA 7.19 ab 7.07 ± 0.93 ± 0.54 ab 6.54 ± 1.02 bcd 29.37 ± 0.96 ± 0.66 ± 11.05 ± 0.69 def 54.86 ± 16.10 ± 1.61 ± 0.96 ab 6.80 e 333.70 ± 12.50 J 109.75 ghi 14.22 ± 0.13 hi 7.28 b 8.09 a 31.84 c 14.06 ± 2.12 ± 0.38 J 602.24 def 7.10 b 32.19 ± 0.73 ± 0.25 k 638.76 ± 1.86 ± 1.16 ab 30.84 b 30.26 fgh 7.03 ghij 10.57 ± 0.93 ± 1.21 ± 0.14 b 30.38 d 513.35 a 8.10 d 08f8002 7.06 ± 2.95 cd 125.42 fgh 15.84 e 694.35 ± 0.22 de 7.86 ± 0. Caffeic acid p-Coumaric acid Ferulic acid Rutin Naringenin Unknown 1c Unknown 2 TPId TPCe Protocatechuic acid Chlorogenic acid Gentistic acid Cultivar OH7983 7.20 ef 381.55 ± 1.23 ± 2.24 ± 0.26 ± 2.61 ± 0.83 ef 13.58 fg 239.95 a 7.94 ± 0.14 a 32.12 ± 0.02 fgh 26.47 ± 0.09 ± 0.04 ± 2.28 ef 177.95 i 203.23 ± 1.53 ± 2.64 ± 0.34 a 44.66 ± 0.42 e 681.05 ± 0.77 de 16.47 ± 0.39 ± 12.12 b 30.18 ± 1.10 ± 0.59 jk 9.42 ± 1.96 ± 9.07 ± 14.16 k 489.84 ± 0.28 fg 688.91 ± 0.63 bc 34.30 ef 7.12 h 15.17 ± 0.30 bcd 52.19 fg 10.08 ghi 8.24 hij O393ABAAAA 7.81 ± 16. b Phenolic composition of extracts obtained using optimised MAE extraction conditions for FRAP (96.47 jk 53.13 a 30.09 ± 0.95 b LA1593 7.63 fg 10.89 fg 16.23 ± 0.41 ± 0.65 ± 22.20 f 08f8058 7.18 gh 281.87 ± 0.07 ± 26.45 fg Q105AAAA 7.87 ± 1.28 i 323.53 fg 11.12 h 21.30 ± 0.07 ± 0.26 f 8.88 ± 2.90 hijk 191.73 ± 8.20 c LA3006 7.02 ± 0.60 ± 0.11 ef 08f8058 7.27 ab 7.49 cd 8.74 bc 10.41 ± 11.53 ± 1.34 bcd 16.13 ± 3.09 ab 30.43 ijk T101-0044BABAA 7.15 gh 8.10 ± 0.66 ± 1.08 e 08f8006 7.06 min and 66.37 i 646.5 °C.69 ± 1.61 ± 0.05 efgh 25.03 ± 0.46 ± 2.29 fgh 69.32 gh LA1016 7.46 ± 27.68 ijk 46.89 ± 1.32 b 12.92 ghi 347.88 ± 1.30 def 9.11 ± 0.91 i 60.67 de 10.21 ± 0.92 gh 10.09 e T101-033AAAAA 7.06 b 30.70 ± 2.42 ± 26.20 ± 0.15 de 107.69 ± 12.17 ± 0.11 ± 0.88 e 687.94 f 696.99 efgh 23.74 efgh 24.22 e 192.19 ± 1.78 i 36.27 e b 7.22 ± 0.28 d 360.85 ± 20.26 ± 0.49 a 8.12 a 29.28 ± 14.23 ± 1.57 i 16.33 ± 2.07 ± 0.26 ± 0.75 ± 0.25 ± 0.15 ± 0.38 a 9.11 g 15.13 ± 0.04 ± 1.50 i 617.53 i 21.32 a 12.08 i 08f8056 7.26 ± 21.63 def 24.06 ± 0.07 e 16.39 b 68.62 ± 0.07 a 10.19 i a Values are mean ± SD.44 c 464.17 i 612.28 ± 1.58 ± 21.77 ± 13.55 ± 0.04 ± 0.72 ± 0.55 ± 2.10 b LA1593 7.94 ± 0.22 ± 2.51 ± 11.06 gh 17.24 de 233.24 i 128. f Phenolic composition of extracts obtained using optimised MAE extraction conditions for ORAC (96.00 ab 7.32 h T101-0044BABAA 7.48 ± 0.94 ± 0.47 ± 0.09 ± 0.82 c 17.55 m 550.53 ± 1.09 fg 9.01 ± 2.14 ± 0.48 a 32.79 fg 16.98 ± 0.52 J 357.15 ± 8.00 ± 5.72 fg 10.51 ± 23.80 fg 371.18 ± 0.22 lm 353.16 ab 7. 1.68 ± 0.95 ± 18.62 def 29.24 l Cultivarf OH7983 7.48 de 48.90 ij 19.11 ± 1.19 d 424.58 ± 1.93 defg 9.15 ± 0.07 a 30.02 ± 2.78 ± 1.37 ± 1.17 a 30.63 ef 7.12 ± 0.24 f 62.84 ± 0.99bcdef 33.12 ± 0.51 ef 5.93 ± 0.64 ± 0.86 ± 2.57 m 368.09 ab 30.73 ± 9.89 h 8.34 ijk 08f8005 7.51 ± 2.58 ± 0.81 ± 0.46 ± 0.03 ± 4.13 ± 2.73 h 536.61 ± 5.06 ± 1.22 de 9.42 ± 2.24 hi 16.00 c 175.47 ± 2.04 ± 0.37 i 42.89 cde 57. n = 3.88 J 168.08 ab 30.15 ± 17.89 ± 0.85 gh 183.38 ef 44.67 e 883.39 l 186.48 def 284.61 ± 11.46 b 11.54 de 11.92 ij 11.47 ± 2.16 b 6.13 d 808.43 ± 0.36 ± 9.94 ± 9.34 ± 0.97 ab 9.90 fg 9.23 ± 0.42 ± 0.97 ± 2.43 ± 0.72 ± 1.86 d 11.61 ± 1.82 ± 1.41 ± 0.32 ± 0.71 ab 7.95 gh 76.68 h 350.44 ± 19.42 ± 0.90 ± 1.05 ghij 211.35 ± 1.29 ± 23.27 b 219.51 ± 0.10 i 17.49 ef 68.36 ± 12.95 ef 40.65 ± 1.22 b 17.41 c 08f8055 7.43 d 664.08 ± 5.91 ± 0.13 ± 2.32 ± 0.88 ± 1.09 ij 7.16 ghi 7.55 ± 0.47 d 10.30 ± 1.03 ± 0.34 ± 0.16 ef 9.33 ± 0.16 e Q219AAAA 7.17 cd 74.934 Table 2 Contents (mg/100 g DW) of phenolic compounds in different cultivars of tomatoes for the optimisation extraction conditions for FRAC and ORAC assays (n = 3)a.41 ± 0.43 ± 2.47 ± 0.81 ± 13.09 a 31.23 J 7.05 ± 0.50 i 312.41 ± 0.17 ± 8.09 ± 1.99 ± 2.30 ± 0.46 ± 0.25 J 8.07 ± 22.04 ± 1.31 l 490.42 ± 1.98 e 424.79 ef 30.08 a 31.64 ± 9.5 °C.59 ef 20.95 bc 47.89 ± 0.32 ± 0.98 bc 17.16 a 409.32 fg 10.09 ± 8.75 ± 0.12 ± 0.17 ghij 43.14 fgh 12.62 fg 127.72 ± 16.11 b 6.71 ghij 137.11 ± 0.12 fg 16.39 ± 3.84 ± 0.21 hi 7.13 ± 0.04 ± 0.31 ± 0.13 ± 0.85 gh 14.25 c 10.00def 24.09 ± 0.02 efgh 26.12 ± 0.03 i 10.39 ijk 185.72 b 22.10 ab 31.15 ± 17.11 ± 0.06 ± 0.10 ± 0.55 ± 0.78 ijk 10.93 ± 0.95 bcde 26.04 ± 1.27 a 32.41 ± 0.25 ± 13.10 ± 1.14 ± 0.95 ± 8.26 ± 0.34 gh 9.28 ± 0.58 l 08f8056 7.19 ± 1.14 a 30.56 ± 1.35 fg 08f8002 7.53 b 12.27 ± 1.25 i 582.40 ± 0.99 ± 19.06 ± 12.50 g 604.17 a 11.09 ± 16.53 gh 10.31 ± 16.31 n 308.18 ± 11.97 ab 8.89 ± 0.14 ± 1.93 g 323.36 ± 2.46 ± 0.07 ab 30.14 ± 17.58 ± 1.22 ± 0.90 ± 2.83 ijk 259.08 h T101-0044BAAAA 7.64 ± 1.23 ± 2.25 c 493.76 cde 30.28 k 44.04 ab 8.04 cd 24.37 ± 6.44 ± 0.78 ± 1.61 jk 67.31 ± 25.18 a 49.08 ± 1.14 ± 3.10 ± 0.13 hij 8.72 f 694.42 ± 2.11 ± 2.28 ± 0.95 b 6.28 ± 12.31 ± 0.18 ± 0.78 ± 6.72 ± 21.25 ef 63. Values followed by the same letter in the same row are not significantly different (p < 0.36 ± 3.06 ± 0.07 kl H9478 7.80 k 412.07 ± 3.07 g 20.96 ± 0.08 e 08f8005 7.68 l 217.64 ± 2.30 g 18.97 ± 1.90 ef 198.14 ± 0.87 ± 0.74 c 199.39 c H.63 ± 0.82 c 997.45 ± 20.60 ± 0.13 ± 11.32 ± 1.47 ± 22.46 a 667.14 ab 8.19 f 526.64 d LA3006 7.15 a 649.46 de 137.83 ± 1.77 fg 9.61 ± 0.79 ± 16.26 ± 0.27 ± 9.37 a 62.81 ± 0.86 ± 9.06 ± 5.69 d 346.66 ijk Q105AAAA 7.15 ± 2.76 ± 0.44 ± 0.08 fg O258AAAA 7.98 i 8.51 a 8.10 ab 7.43 h 15.84 ± 0.98 ab 9.01 ± 0.73 ± 1.82 d 746.30 ± 0.32fghi 8.11 b 30.58 ± 0.16 bcd 33.50 g 610.38 ± 0.08 a 32.61 ± 17.80 gh 8.14 a 31.72 k 532.12 a 8.12 ± 0.76 def 122.98 i 27.20 ± 1.49 ± 0.94 ± 1.31 ± 0.91 ± 2.25 b 722.81 ± 8.42 g 295.02 ± 6.17 c 183.15 ± 11.89 defg 23.25 J 608.55 c 337.41 ± 0.02 ± 0.47 ± 6.68 ± 23.09 ± 1.34 ± 2.28 h 654.59 ± 2.07 ± 0.08 b 30.09 b 29.95 f 16.25 d 18.52 ± 10.06 a 30.03 ± 0.59 ± 7.64 ± 12.51 ± 2.2% ethanol).08 f 18.48 a 11.36 cde 59.72 c 7.19 a 213.79 ± 27.00 ± 1.61 ± 0.95 ± 0.73 ± 9.27 ± 0.00 bced 35.30 ab 12.60 ± 8. 2.42 f 17.

& Krizek. Tsao & Yang.88%.. lower correlation coefficients between the TPC and ORAC may be related to the kinetic action of antioxidants in the ORAC assay. Xia. Comparison between MAE and the conventional solvent extraction The average FRAP. 263. 1848–1864.05). CA. 2010). and the models generated were successfully applied to the 20 tomato cultivars and breeding lines. F. LA1016 and LA1593 was provided by The Tomato Genetics Resource Center. & O’Keefe. M. S.. The optimised MAE parameters generated by Eq. 66. Analytical Biochemistry. 2003). The FRAP values varied from 26. 2009). 70(1). Li et al. A. 2010).52 mg/100 g DW.-Y.. . ferulic acid and unknown 1. J.52 mg/100 g DW. & Gerasopoulos. 3). 0. L. & Fernández-Gutiérrez. no correlation was found between the ORAC value and the TPC or TPI (p > 0. Benzie. ORAC and TPC values of the 20 cultivars and breeding lines were 38. 0.56). Pavasant.. while the ORAC values ranged from 235. 239(1). Mallikarjunan. respectively)) than the ORAC value which was only related to gentistic acid (r = 0.75. which have shown that applying MAE can increase the contents of antioxidants and reduce extraction time significantly as compared to conventional extraction methods (Ballard et al. (2009). and the Sustainable Productions Program of the University of Guelph/Ontario Ministry of Agriculture. H.-F.30– 997. TPI in the ORAC model. Xie. Zhou. Medina-Remón. The strong positive correlation between the TPC and FRAP.34–468. Hayat et al. 2003).81 lmol TE/g DW and 602. which promotes the destruction of sample surface and in turn the exudation of the chemical substances within the cells into the surrounding solvents takes place (Hayat et al. U. 61. overnight in the solid–liquid extraction). This increase can be simply due to the higher extraction efficiency of the MAE. (1996). This explanation is also supported by data from the 20 tomato varieties in the present study.69–468.84 lmol AAE/g DW with the highest value being twofolds of the lowest. Food Chemistry. (2007)...H.30 to 997. TPIs of the 20 tomato varieties were between 281.42 to 351. 1.61. 6. The greater TPC values as compared with the TPI values can be caused by the incomplete quantification of all peaks in the HPLC method. Acknowledgements This project was funded by the A-Base research (Project 205) of Agriculture & Agri-Food Canada. In conclusion. 517–524.1% ethanol for ORAC) were obtained.. F. which occurs due to the dipole rotation of the solvent in the microwave field. & Gong. University of California.7. MAE can enhance the product recovery because of its heating effect. The FRAP antioxidant activity showed good correlation with the TPC and TPI.19 lmol TE/g DW. However.05). Szydłowska-Czerniak. & Lamuela-Raventós. whereas the irradiation time did not significantly affect the activities (p > 0. Karlovits. which were higher than the TPIs (Table 2). & Shotipruk. 280. Microwave-assisted extraction of phenolic antioxidant compounds from peanut skins. Microwave-assisted extraction used for the isolation of total triterpenoid saponins from Ganoderma atrum. One of the obvious advantages of the MAE is the significantly reduced extraction time (2 min in the MAE vs. MAE was proven a more effective technique with reduced extraction time.. microwave temperature and ethanol concentration. and 96.. although their absolute identities need to be confirmed by other techniques such as mass spectrometry and nuclear magnetic resonance. Regression analysis using the Pearson’s correlation coefficients (r) indicated that the antioxidant activity as measured by the FRAP assay positively correlated with the TPC and TPI (p < 0.86 to 57. Two independent variables. M. (2010).. B. This was explained by the fact that phenolics are not the only compounds with antioxidant potential in tomato. Y. The increases were 8. whereas no clear correlation was found between the ORAC value and TPC or TPI. Separation and Purification Technology. 81(1).71. The solubility of the phenolic compounds increases with rapid rise of the solvent temperature... S. (2009). Davis. Hoffmann. using the response surface methodology with a central composite design. 2010.. D. TPC in the FRAP model. Valorisation of low quality edible oil with tomato peel waste. Moreover. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of ‘‘Antioxidant Power’’: The FRAP assay. The FRAP value was found to correlate with more numbers of the individual phenolics (gentistic acid. Gómez-Romero.93 and 0.). Trokowski. as compared to 35. higher total phenolic contents and greater antioxidant activities compared to conventional solvent extraction. which might explain the discrepancy between the results obtained with the ORAC assay and those obtained with other assays (Szydłowska-Czerniak et al. J. This finding is consistent with those reported in the literature. 110(3). K.16% and 7. 2009).2% ethanol) were very similar to those by Eq. TPC in the ORAC model.18 mg/100 g DW. 2010. (2010). S.10 to 42. Oufir. 2..01) on both the FRAP and ORAC values of the phenolic extracts of tomato. A. M. the TPC and TPI obtained using the two sets of parameters were also highly similar and showed no significant difference (TPI in the FRAP model. / Food Chemistry 130 (2012) 928–936 935 Mukhopadhyay. T.05). Also. Benakmoum. (2008).. J.. The TPCs as measured by the Folin–Ciocalteu method were from 489.5 °C. The two unknown compounds might be glycosidic esters of some phenolic acids which have been reported for tomatoes (Gómez-Romero et al. 2010. 284.45 mg GAE/100 g DW.. The antioxidant activities also differed significantly (p < 0. & Strain. solvent heating by microwave occurs when molecules of the polar solvent could not align themselves quickly enough to the high frequency electric field of microwave. Ammouche. Journal of Food Composition and Analysis. 490. K. Larondelle. Ding. had significant effects (p < 0. Hayat..06 min. (3) for ORAC (96..66 min.2% ethanol for FRAP.91 mg/100 g. Segura-Carretero. 1185–1192.95 mg/100 g DW. 162–170.07 lmol TE/g DW and 646. This causes the solvent molecules to dissipate the absorbed energy in the form of heat (Hemwimon.79–954.. (2) for FRAP (96. 2006).. 63–70. Hausman. respectively. Vallverdú-Queralt. P. Food and Rural Affairs (OMAFRA).-F. Microwave irradiation also accelerates cell rupture by sudden temperature rise and internal pressure increase inside the cells of plant sample.. I. 22(6). 66. 61.5 °C. 120(4).. P. A. Abbas. Abbeddou.40 mg GAE/100 g.21%.. ORAC and TPC values. Original seed of LA3006.05) among the 20 tested tomato cultivars in both the FRAP and ORAC assays (Fig..39 lmol AAE/g DW. 2. respectively. et al. et al. References André. Optimized microwave-assisted extraction of phenolic acids from citrus mandarin peels and evaluation of antioxidant activity in vitro.5 °C. 70–76.5 °C. for the MAE study. respectively.34–464.06 min. The antioxidant activities and the total phenolic contents were in general higher in extracts by MAE than that by the conventional solvent extraction. Hussain. Ballard. The methodologies developed and the knowledge acquired in this study will provide useful information to tomato breeders and food processors. respectively. Farooq. C. X.. (r = 0. Y.26 lmol AAE/g DW.05). Chen.. 684–690. with correlation coefficients (r) at 0. Food Chemistry. 71(16).57. 1. 281. and the potential interferences by other components in the TPC method (Tsao & Yang.45 mg/100 g DW) (p > 0. 2010.73 mg/100 g DW (Table 2). Journal of Food Engineering. S. A. AndrésLacueva. for the average FRAP. Influence of environment and genotype on polyphenol compounds and in vitro antioxidant capacity of native Andean potatoes (Solanum tuberosum L.66 min.. Rogez. Kefalas. and the low or lack of correlation between the TPC and ORAC have been observed by others (Samec et al.. Jáuregui.1% ethanol). S. 2007). for the conventional solvent extraction method. M. Metabolite profiling and quantification of phenolic compounds in methanol extracts of tomato fruit. & Szłyk. 3. Phytochemistry. with individual phenolic compounds ranging from 6. 489. S.. optimal MAE parameters (96.

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