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Effects of microwave heating on the antioxidant activities of tomato (Solanum lycopersicum)
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Effects of microwave heating on the antioxidant activities of tomato (Solanum lycopersicum) Boumendjel Mahieddinea, , Benabdallah Aminab, Samar Mohamed Faouzic, Bouras Sanac, Dalli Widedc ⁎
a
Laboratory on Biochemistry and Environmental Toxicology, Badji Mokhtar Annaba University, Algeria Department of Agronomy, SAPVESA Laboratory, University of El-Tarf, Algeria c Laboratory on Biodiversity and Ecosystem Pollution, University of El-Tarf, Algeria b
ARTICLE INFO
ABSTRACT
Keywords: Tomato Microwaves Polyphenols Lycopene Ion chelating DPPH radical scavenging
The aim of this study is to estimate phytochemical constituents and antioxidant activities of tomato slices treated by microwave heating. Two treatments time were applied, 30 and 300 s. Quantitative analyses of phenols, flavonoids, lycopene, followed by the evaluation of the antioxidant activity by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ion chelating methods, were conducted on ethanolic extracts. Results showed the presence of these bioactive phytonutrients in fresh tomato and that their biodisponibility increases with the duration of the selected heat treatment. The phytochemical content showed important levels of polyphenols (1.92, 1.96, 6.95 mg gallic acid equivalent/g fresh matter) and flavonoids (2.33, 2.57, 10.68 mg RE/g fresh matter), in addition to lycopene (5.772, 31.8654, 40.71 mg/kg fresh matter) of non-treated, treated tomatoes at 30 s and treated tomatoes at 300 s, respectively. Results also revealed a high level of antioxidant activity by both methods with IC50 of 1.69, 1.50, 1.33 µg/ml, for DPPH method and IC50 of 401, 360.5, 299.91 µg/ml, for ion chelating ability. Thus, in our study, we demonstrated that microwave treatments enhance the nutritional quality of tomato slices by enhancing the biodisponibility of some of its content. Higher treatment time gives better results.
1. Introduction Recent studies have shown that balanced diet, including daily consumption of fruits and vegetables, can repel different forms of cancer by providing many bioactive molecules such as: polyphenols, flavonoids, terpenes, tannins and various antioxidant enzymes. Tomato (Solanum lycopersicum) is one of the most frequently cultivated vegetable in the world, standing second only to potatoes in production (Tuyen et al., 2016; Umar and Abdulkadir, 2016). It is consumed fresh and processed, for its nutritional and bioactive antioxidants such as phenolics, carotenoids and vitamins C and E (Pernice et al., 2010). Phenolic compounds 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 and Savage, 2006). These constituents, especially flavonoids in tomatoes, were able to withstand industrial processing methods, being detected in a variety of tomato-based products (Stewart et al., 2000). Industrial processing including, heat treatments, induces variation in technological and biochemical quality of tomato pastes (Boumendjel
and Boutebba, 2003; Boumendjel et al., 2012). Many heating systems are also implicated in the loss of a part of tomato’s antioxidant properties (Kerkhofs et al., 2005; Chen et al., 2006; Perez-Jimenez et al., 2008; Pérez-Conesa et al., 2009). Nutritional antioxidants operate by controlling free radical rank with a related decline in the symptoms of oxidative stress (Mercedes and Navarro, 2016). This concept has led to an increased demand for substitution of synthetic additives by food industry with phyto-antioxidants for safety and various therapeutic uses (Shukla et al., 2016). Lycopene, the red pigment of tomato, phenolics and flavonoids had great interest during the last few years and have been described to provide great antioxidant and free radical scavengers able to protect the body against oxidative stress-induced cellular damage (Riahi and Hdider, 2013). The hot air drying is the main frequently used method, presenting however, some inconveniences such as the loss of time and energy (Bondaruk et al., 2007), the loss of flavor, color and nutrient values (Maskan, 2000). Due to its domestic wide use, microwave oven is now the most used method of cooking and heating in the kitchen. Therefore, we must take into consideration the effect of this method on fruit and
Peer review under responsibility of Faculty of Agriculture, Ain-Shams University. ⁎ Corresponding author. E-mail address: [email protected] (B. Mahieddine). https://doi.org/10.1016/j.aoas.2018.09.001 Received 30 May 2017; Received in revised form 18 May 2018; Accepted 10 September 2018 0570-1783/ 2018Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Mahieddine, B., Annals of Agricultural Sciences, https://doi.org/10.1016/j.aoas.2018.09.001
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vegetables by estimating their nutrients values. Based on these considerations, our purpose is to evaluate the concentration of the main tomatoes phytochemicals (polyphenols, flavonoids and lycopene) and their persistence after a heat treatment by microwaves, then to assess antioxidant abilities by DPPH and ion chelating methods.
homogenized with 4 ml of the solvent mixture of hexane/ethanol/ acetone (2/1/1, v/v), placed on the rotary mixer for 30 min, then 10 ml of distilled water was added. The absorbance was measured at 503 nm after 5 min, the lycopene concentration was calculated using a molar extinction coefficient of 31.2, and results were expressed in mg/kg of fresh matter (FM).
2. Materials and methods
2.6. In vitro antioxidant activity
2.1. Microwave treatments
The in vitro antioxidant activity of ethanolic extracts was assessed with two methods: the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and the ferrous ion chelating power.
Rio grande tomato varieties were obtained from the local market of El-Tarf department (North Eastern of Algeria) and were harvested at their red-ripe stage. The experiments were carried out in a Whirlpool JC213WH microwave oven of 1000 W capacity and 30 L inner volume, used at the maximum power level. The weight of the tomato slices during the heating process was specified with a digital scale of 100 g capacity and ± 0.01 g precision. Tomatoes were initially cut into slices of approximately the same shape and weight (44 g). The first slice represents the control. The second one is treated by microwave for 30 s (which represent the minimum time for heating by microwaves) and the third one for 300 s (which is equal to the maximum time frequently used for reheating a meal). This experiment was repeated three times.
2.6.1. DPPH radical scavenging assay The DPPH radical-scavenging capacity was measured by the method reported in Bouaziz et al. (2008). In this study, 300 µl of each ethanol extract was mixed with 900 µl of 0.4 mM DPPH methanolic solution. The reaction was allowed to stand at room temperature in the dark for 30 min and the absorbance was recorded at 517 nm. The scavenging activity (SA) was estimated using the following equation: SA (%) = (Acontrol − Asample/Acontrol) × 100, where Acontrol is the absorbance of the control reaction (containing all reagents except the test sample) and Asample is the absorbance of the tested sample. The concentration of extract that could scavenge 50% of the DPPH radicals (IC50) was calculated. Trolox and BHT were used as positive references.
2.2. Ethanolic extract preparation In order to evaluate the phytochemical concentrations, each tomato samples were extracted with ethanol (70% v/v) and macerated for 24 h with agitator maintained at room temperature. The extract was filtered and stored in a brown bottle at 4 °C prior to further analysis.
2.6.2. Ferrous ion chelating activity The ferrous ion chelating activities of ethanolic extracts were measured according to a standard method (Yan et al., 2006). In this study, 300 µl of different concentrations of each extract were added to 300 µl of FeSO4 solution (0.1 mM) and left for incubation at room temperature for 10 min. Then, the reaction was initiated by adding 300 µl of ferrozine (0.25 mM). The mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance of the solution was measured at 562 nm against a methanol blank. The ability of methanolic extracts to chelate ferrous ion was calculated using the following formula: Chelating effect (%) = [(Acontrol − Asimple/ Acontrol) × 100]. Where Acontrol is the absorbance of the control sample (consisting of methanol, iron and ferrozine) and Asimple the absorbance of the tested sample. Results were expressed as IC50 (efficient concentration corresponding to 50% ferrous iron chelating). EDTA (Ethylendiamin Tetra-acetic) was used as a positive control.
2.3. Determination of total polyphenols concentration The total polyphenol concentrations were determined using the Folin–Ciocalteu method (Singleton and Rossi, 1965). 500 µl of dilute extract from each sample was mixed with 2 ml Folin–Ciocalteu reagent (diluted 10 times with distilled water). After 5 min, 2.5 ml of sodium carbonate solution (7.5%) was added and the mixture was allowed to stand for 90 min with intermittent shaking. The absorbance of the resulting solution was measured at 760 nm. The phenol concentrations were expressed in terms of milligrams of Gallic acid equivalent per gram of fresh weight (mg GAE/g fresh matter). 2.4. Determination of total flavonoids concentration
2.7. Statistical analysis
The total flavonoid concentrations were estimated according to the aluminum chloride colorimetric method (Koolen et al., 2013). Briefly, 500 µl of diluted extract was mixed with 500 µl of 2% AlCl3 ethanolic solution. After incubation at room temperature for 40 min, the absorbance was measured at 430 nm. Flavonoid concentrations were calculated from a calibration curve of rutin and expressed as milligrams of rutin equivalent per gram of fresh weight (mg RE/g FM).
The data were expressed as the mean ± standard deviation (SD) of triplicate independent experiments and analyzed using one-way analysis of variance (ANOVA) and Student’s t-test p < 0.05 was considered to be statistically significant. We performed a PCA (principal component analysis) and obtained a dendrogram of variations with R software.
2.5. Determination of lycopene concentration
3. Results and discussion
Lycopene is the main antioxidant present in tomatoes. It is therefore important to carry out its estimation before and after heat treatment in order to have an evaluation of the effect of the microwave treatment on the concentration of the fruit in carotenoid terpenes. Methods of estimating the lycopene level require a measurement of a chromatic component to the colorimeter. The determination of the level of lycopene by spectrophotometry at 503 nm, which is correlated with the lycopene level (Grolier et al., 2000), remains more reliable than the measurement of the red color by chromatometry. At this wavelength, lycopene is responsible of 90% of the absorbance, while β-carotene less than 10% (Grolier et al., 2000). Lycopene was extracted before measured (Sadler et al., 1990). Briefly, 1 g of each tomato samples was
3.1. Total phenolic, flavonoid and lycopene concentrations The results of total phenolic, flavonoid and lycopene concentrations are given in Table 1. As it can be seen, the highest amounts of total phenolic, flavonoid and lycopene concentration are observed for tomato sample T300 s (6.95 mg GAE/g FM, 10.68 mg RE/g FM), followed by tomato sample T30 s (1.96 mg GAE/g FM, 2.57 mg RE/g FM), while minimum values were observed for tomato sample control (1.92 mg GAE/g FM, 2.33 mg RE/g FM) for the total phenolic and flavonoid concentrations, respectively. For total phenols concentration, our results are higher than those obtained by other authors (George et al., 2004) on fresh tomatoes with 2
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Table 1 Effect of microwave heating on total phenolics, flavonoids and lycopene concentration of tomatoes samples.
T control T 30 s T 300 s
Total phenolics concentration (mg GAE g−1 FM)
Flavonoid concentration (mg RE g−1 FM)
Lycopene concentration (mg/ kg FM)
1.92 ± 0.098 1.965 ± 0.152 6.955 ± 0.228*
2.337 ± 0.009 2.576 ± 0.049 10.682 ± 0.098**
5.772 ± 0.725 31.865 ± 2.774* 40.715 ± 7.744**
Table 2 Effect of microwave heating on the antioxidant activities of ethanol extract of tomatoes samples.
T control T 30 s T 300 s
DPPH IC50 (mg/ml)
Chelating ion ability IC50 (µg/ml)
1.697 ± 0.003 1.501 ± 0.001 1.333 ± 0.001
401.000 ± 2.081 360.166 ± 1.166 299.916 ± 0.740
Values are given as mean ± SD (n = 3). Synthetic antioxidant: IC50 values for the Trolox: 0.115 mg/ml. IC50 values for the EDTA: 6.5 ± 0.2 μg/ml (Chelating ability).
In each column: * indicate p > 0.05 and ** indicate P > 0.01. T: control without any heat treatment; T 30 s: tomatoes treated for 30 s by microwave; T 300 s: tomatoes treated for 300 s by microwave. GAE: Gallic acid equivalent; FM: fresh matter; RE: Rutin equivalent.
3.3. DPPH radical scavenging assay The DPPH stable purple radicals react with suitable reducing agents or hydrogen atom donor (A-H), during which the electrons become paired off, yielding a stable diamagnetic molecule (yellow-colored diphenylpicrylhydrazine); the solution loses color, depending on the number of electrons taken up (Biswas et al., 2014). The DPPH antioxidant assay is based on the ability DPPH, a stable free radical, to decolorize, in the presence of antioxidants (Benabdallah et al., 2016), thus a low value corresponds to a good scavenging ability. The results were expressed as IC50 value that is the amount of antioxidant necessary to decrease by 50% the initial DPPH radical concentration. The results indicated that T300 s was the most efficient (1.333 mg/ml), followed by T30 s (1.501 mg/ml), whereas control case (1.697 mg/ml) was the least effective. However, all extracts recorded low radical scavenging abilities when compared to that reported for Trolox used as positive controls.
0.22 mg GAE/g FM. Moreover, values obtained by our experiments showed also higher values for fresh tomatoes than those reported in the literature ranged from 0.154 to 0.162 mg GAE/g FM (Riahi and Hdider, 2013), and with total phenols of 0.14 mg GAE/g FM (Brat et al., 2006), and varied from 0.6 to 0.9 mg GAE/g FM (Aldrich et al., 2010). Concerning flavonoids concentration, reported lower values ranged from 0.106 to 0.512 mg RE/g FM (Ilahy et al., 2009). Moreover, our results are higher than those ranging from 0.109 to 0.113 mg RE/g FM (Riahi and Hdider, 2013), for Tunisian samples. The data in the scientific literature are quite contradictory. Some authors have studied the compounds themselves and not their rates in heat-treated products. Some tomatoes phenolic compounds, such as hydroxycinnamic acids (acids: p-coumaric, ferulic, caffeic and chlorogenic), are not affected by heat treatments (Re et al., 2002). The levels of caffeic and chlorogenic acid increase during the heat treatments. They concluded that for polyphenols, heat treatment helped to release them from the cell matrix (Chanforan, 2010). Methods for the quantitative measurement of lycopene are difficult due to its sensitivity to light, oxygen, metals and strong acids (Goula et al., 2006). This is due to the structure of this terpene, rich in ethylenic bonds. Therefore, it is recommended to replace metallic fry by non-metallic frying and to conduct measurements in the absence of oxygen and light. The addition of antioxidants in solvents, such as ammonium acetate in the organic phase, makes a better lycopene concentration measurement and does not interfere with the assay of the molecule (Nierenberg and Nann, 1992; Nguyen and Schwartz, 1999; Nguyen and Schwartz, 1998; Rao and Agrawal, 1998). Some techniques use antioxidant molecules such as BHT in order to limit the oxidation phenomena of polyunsaturated isoprene hydrocarbons. Lycopene concentration was significantly different between the three studied samples (Table 1). The highest concentration was obtained from tomatoes samples T300 s with 40.7156 mg/kg FM, followed by T30 s with 31.8654 mg/kg FM. Untreated samples showed the lowest concentration with 5.772 mg/kg FM. Our results are in agreement to the literature (Caris-Veyrat et al., 2004), these authors worked on organic tomatoes grown in a plastic tunnel, and found lycopene concentration ranged from 36 to 42 mg/kg FM. But it is still lower than those reported concentration ranging from 44.7 to 87.6 mg/kg of FM (Hdider et al., 2007), or of 49.5 to 106.5 mg/kg FM (Perkins-Veazie, 2007), for conventional field tomatoes. Variation in lycopene concentration depends on the maturity stage (Ilahy et al., 2011), the year of production, the growing season (Garcia and Barrett., 2006), the use of fertilizers and the location (Lenucci et al., 2006).
3.4. Ferrous ion chelating activity The transition metal, iron, is capable of generating free radicals by Fenton reactions. Ferrozine can quantitatively form complexes with Fe2+. In the presence of other chelating agents, the complex formation is disrupted with the decrease of the red color as a result. Metal chelating capacity was significant since the extract reduced the concentration of the catalyzing transition metal (Ebrahimzadeh et al., 2009). The chelating activity of samples on metal ions was determined by measuring the absorption of ferrozine-Fe2+ complex at 562 nm. The results of the chelating iron indicated that tomato T300″ had the largest ability to chelate iron (299.92 µg/ml FM), followed by T30″ (360.17 µg/ml), while tomato control showed the lowest ability with 401 µg/ml (Table 2). Previous works evaluated antioxidant activities of tomatoes from different regions of the world and by different methods. Some authors assessed the antioxidant properties of tomato extracts from Spain, by the Trolox Equivalent Antioxidant Capacity (TEAC) and FRAP assays and, reported synergistic action between the lycopene and phenolic concentration of tomato (García-Alonso et al., 2015). Moreover, other references analyzed three varieties of tomato from Indonesia, using different polarity solvents (which were n-hexane, ethyl acetate and ethanol) for their total phenolic, flavonoids and carotenoid concentrations and their antioxidant abilities, reported a high correlation between TPC with IC50 of DPPH and FRAP (Ferric Reducing Antioxidant Power) capacity (Fidrianny et al., 2015). Furthermore, those measuring the effect of storage time and temperature on physicochemical properties and antioxidant activities (by DPPH assay) of different Tomato cultivars from Portugal, observed an increase in the phenolics, ascorbic acid, lycopene concentrations and antioxidant activity with temperature and storage duration (Vinha et al., 2013). In addition, studies comparing antioxidant properties of fresh, freeze-dried and hot-air-dried tomatoes from Taiwan, by three methods (DPPH, FRAP, ion chelating ability), indicated that the drying method could enhance the nutritional value of tomatoes by increasing parts of total flavonoids, phenolics and lycopene concentrations (Chang et al., 2006). These last ones are considered as antioxidant components,
3.2. Antioxidant activity The antioxidant activity of tomato extracts was evaluated by measuring, the free radical scavenging activity (DPPH) and the ferrous ion chelating power. All the extracts exhibited a noticeable effect, which varied significantly among samples (Table 2). 3
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rate of hydrophilic and lipophilic compounds released by heat treatment. For total phenolics concentration, the tomatoes slices exhibit values from 1.92 ± 0.098 for control to 1.965 ± 0.152 and 6.955 ± 0.228 (mg GAE g−1 FM) for 30 s and 300 s respectively. We also notice that both axis 1 and 2 showed a very high correlation (90% and 9%) between phenolic concentration and flavonoids. In tomato fruits, flavonoids characterize the main constituent of the total phenol concentration (Toor et al., 2005). Variations of lycopene concentration showed same scenario. The release of this carotenoid enhances with microwave treatment ranging from 5.772 ± 0.725 for control to 31.865 ± 2.774 and 40.715 ± 7.744 (mg/kg FM) for 30 s and 300 s respectively. Lycopene, the predominant carotenoid in tomatoes, exhibits the highest antioxidant activity and singlet oxygen quenching ability of all dietary carotenoids (Tuyen et al., 2016). Following dendrogram of variation of antioxidant activities and chemical components in microwave treated tomato slices (Fig. 2), which gives more information about these correlations and explains the relations between chemical compounds and the specific antioxidant activity. We noticed that variations of polyphenols and flavonoids are the most correlated to antiradical scavenging activity (DPPH), while lycopene explained a part of these correlations in addition to Ion chelating activity. Microwave treatment of T300 s gives the best results among the considered cases by releasing the maximum rates of both phenolics and carotenoids concentrations of tomatoes slices.
Fig. 1. Principle component analysis of antioxidant activities vs chemical components (phenols, flavonoids and lycopene).
4. Conclusion The aim of the present study is to assess the effect of microwave heat treatments on the chemical composition (polyphenols, flavonoids and lycopene) in relation with antioxidant activities of tomato fruits (DPPH and Ion chelating). Two treatments are applied (30 s and 300 s) and compared to control case. Results show the presence of these bioactive phytonutrients in fresh tomato and that their biodisponibility increases with the duration of the selected heat treatment. Moreover, analysis of obtained results indicates an enhancement in the ethanolic rates of polyphenols, flavonoids and specially lycopene. These variations are strongly correlated to the variation of antioxidant activities evaluated by both DPPH and Ion Chelating methods. Phenolic compound explains the major part of DPPH scavenging variations, where lycopene mainly explains the Ion chelating activity. As a result of our study, we demonstrate that microwave treatments enhance the nutritional quality of tomato slices by enhancing the biodisponibility of some of its hydrophilic and lypophilic concentrations. Microwave treatment of T300 s, in this experiment, is better than T30 s for heating tomatoes by releasing the maximum rates of both phenolics and carotenoids concentration of tomatoes slices. More time intervals and treatments levels should be evaluated to validate the most appropriate heating time in order to give more conclusions about the efficiency of microwave ovens.
Fig. 2. Dendrogram of variation of antioxidant activities and chemical components in microwave treated tomato slices.
therefore they released to the increase of antioxidant abilities. However, it is difficult to compare results of antioxidant abilities because of the variation of numerous parameters such as locality, concentration, varieties, solvents and methods used to dry or to obtain samples. In addition to methods used to assess antioxidant abilities. Microwave energy is quickly absorbed by water molecules, which, consequently, results in rapid evaporation of water and therefore higher drying rates and energy savings, for that reason microwave drying give significant energy savings, with a potential reduction in drying times in addition to the inhibition of the surface temperature of the treated material.
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Effects of microwave heating on the antioxidant activities of tomato (Solanum lycopersicum) Boumendjel Mahieddinea, , Benabdallah Aminab, Samar Mohamed Faouzic, Bouras Sanac, Dalli Widedc ⁎
a
Laboratory on Biochemistry and Environmental Toxicology, Badji Mokhtar Annaba University, Algeria Department of Agronomy, SAPVESA Laboratory, University of El-Tarf, Algeria c Laboratory on Biodiversity and Ecosystem Pollution, University of El-Tarf, Algeria b
ARTICLE INFO
ABSTRACT
Keywords: Tomato Microwaves Polyphenols Lycopene Ion chelating DPPH radical scavenging
The aim of this study is to estimate phytochemical constituents and antioxidant activities of tomato slices treated by microwave heating. Two treatments time were applied, 30 and 300 s. Quantitative analyses of phenols, flavonoids, lycopene, followed by the evaluation of the antioxidant activity by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ion chelating methods, were conducted on ethanolic extracts. Results showed the presence of these bioactive phytonutrients in fresh tomato and that their biodisponibility increases with the duration of the selected heat treatment. The phytochemical content showed important levels of polyphenols (1.92, 1.96, 6.95 mg gallic acid equivalent/g fresh matter) and flavonoids (2.33, 2.57, 10.68 mg RE/g fresh matter), in addition to lycopene (5.772, 31.8654, 40.71 mg/kg fresh matter) of non-treated, treated tomatoes at 30 s and treated tomatoes at 300 s, respectively. Results also revealed a high level of antioxidant activity by both methods with IC50 of 1.69, 1.50, 1.33 µg/ml, for DPPH method and IC50 of 401, 360.5, 299.91 µg/ml, for ion chelating ability. Thus, in our study, we demonstrated that microwave treatments enhance the nutritional quality of tomato slices by enhancing the biodisponibility of some of its content. Higher treatment time gives better results.
1. Introduction Recent studies have shown that balanced diet, including daily consumption of fruits and vegetables, can repel different forms of cancer by providing many bioactive molecules such as: polyphenols, flavonoids, terpenes, tannins and various antioxidant enzymes. Tomato (Solanum lycopersicum) is one of the most frequently cultivated vegetable in the world, standing second only to potatoes in production (Tuyen et al., 2016; Umar and Abdulkadir, 2016). It is consumed fresh and processed, for its nutritional and bioactive antioxidants such as phenolics, carotenoids and vitamins C and E (Pernice et al., 2010). Phenolic compounds 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 and Savage, 2006). These constituents, especially flavonoids in tomatoes, were able to withstand industrial processing methods, being detected in a variety of tomato-based products (Stewart et al., 2000). Industrial processing including, heat treatments, induces variation in technological and biochemical quality of tomato pastes (Boumendjel
and Boutebba, 2003; Boumendjel et al., 2012). Many heating systems are also implicated in the loss of a part of tomato’s antioxidant properties (Kerkhofs et al., 2005; Chen et al., 2006; Perez-Jimenez et al., 2008; Pérez-Conesa et al., 2009). Nutritional antioxidants operate by controlling free radical rank with a related decline in the symptoms of oxidative stress (Mercedes and Navarro, 2016). This concept has led to an increased demand for substitution of synthetic additives by food industry with phyto-antioxidants for safety and various therapeutic uses (Shukla et al., 2016). Lycopene, the red pigment of tomato, phenolics and flavonoids had great interest during the last few years and have been described to provide great antioxidant and free radical scavengers able to protect the body against oxidative stress-induced cellular damage (Riahi and Hdider, 2013). The hot air drying is the main frequently used method, presenting however, some inconveniences such as the loss of time and energy (Bondaruk et al., 2007), the loss of flavor, color and nutrient values (Maskan, 2000). Due to its domestic wide use, microwave oven is now the most used method of cooking and heating in the kitchen. Therefore, we must take into consideration the effect of this method on fruit and
Peer review under responsibility of Faculty of Agriculture, Ain-Shams University. ⁎ Corresponding author. E-mail address: [email protected] (B. Mahieddine). https://doi.org/10.1016/j.aoas.2018.09.001 Received 30 May 2017; Received in revised form 18 May 2018; Accepted 10 September 2018 0570-1783/ 2018Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Mahieddine, B., Annals of Agricultural Sciences, https://doi.org/10.1016/j.aoas.2018.09.001
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vegetables by estimating their nutrients values. Based on these considerations, our purpose is to evaluate the concentration of the main tomatoes phytochemicals (polyphenols, flavonoids and lycopene) and their persistence after a heat treatment by microwaves, then to assess antioxidant abilities by DPPH and ion chelating methods.
homogenized with 4 ml of the solvent mixture of hexane/ethanol/ acetone (2/1/1, v/v), placed on the rotary mixer for 30 min, then 10 ml of distilled water was added. The absorbance was measured at 503 nm after 5 min, the lycopene concentration was calculated using a molar extinction coefficient of 31.2, and results were expressed in mg/kg of fresh matter (FM).
2. Materials and methods
2.6. In vitro antioxidant activity
2.1. Microwave treatments
The in vitro antioxidant activity of ethanolic extracts was assessed with two methods: the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and the ferrous ion chelating power.
Rio grande tomato varieties were obtained from the local market of El-Tarf department (North Eastern of Algeria) and were harvested at their red-ripe stage. The experiments were carried out in a Whirlpool JC213WH microwave oven of 1000 W capacity and 30 L inner volume, used at the maximum power level. The weight of the tomato slices during the heating process was specified with a digital scale of 100 g capacity and ± 0.01 g precision. Tomatoes were initially cut into slices of approximately the same shape and weight (44 g). The first slice represents the control. The second one is treated by microwave for 30 s (which represent the minimum time for heating by microwaves) and the third one for 300 s (which is equal to the maximum time frequently used for reheating a meal). This experiment was repeated three times.
2.6.1. DPPH radical scavenging assay The DPPH radical-scavenging capacity was measured by the method reported in Bouaziz et al. (2008). In this study, 300 µl of each ethanol extract was mixed with 900 µl of 0.4 mM DPPH methanolic solution. The reaction was allowed to stand at room temperature in the dark for 30 min and the absorbance was recorded at 517 nm. The scavenging activity (SA) was estimated using the following equation: SA (%) = (Acontrol − Asample/Acontrol) × 100, where Acontrol is the absorbance of the control reaction (containing all reagents except the test sample) and Asample is the absorbance of the tested sample. The concentration of extract that could scavenge 50% of the DPPH radicals (IC50) was calculated. Trolox and BHT were used as positive references.
2.2. Ethanolic extract preparation In order to evaluate the phytochemical concentrations, each tomato samples were extracted with ethanol (70% v/v) and macerated for 24 h with agitator maintained at room temperature. The extract was filtered and stored in a brown bottle at 4 °C prior to further analysis.
2.6.2. Ferrous ion chelating activity The ferrous ion chelating activities of ethanolic extracts were measured according to a standard method (Yan et al., 2006). In this study, 300 µl of different concentrations of each extract were added to 300 µl of FeSO4 solution (0.1 mM) and left for incubation at room temperature for 10 min. Then, the reaction was initiated by adding 300 µl of ferrozine (0.25 mM). The mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance of the solution was measured at 562 nm against a methanol blank. The ability of methanolic extracts to chelate ferrous ion was calculated using the following formula: Chelating effect (%) = [(Acontrol − Asimple/ Acontrol) × 100]. Where Acontrol is the absorbance of the control sample (consisting of methanol, iron and ferrozine) and Asimple the absorbance of the tested sample. Results were expressed as IC50 (efficient concentration corresponding to 50% ferrous iron chelating). EDTA (Ethylendiamin Tetra-acetic) was used as a positive control.
2.3. Determination of total polyphenols concentration The total polyphenol concentrations were determined using the Folin–Ciocalteu method (Singleton and Rossi, 1965). 500 µl of dilute extract from each sample was mixed with 2 ml Folin–Ciocalteu reagent (diluted 10 times with distilled water). After 5 min, 2.5 ml of sodium carbonate solution (7.5%) was added and the mixture was allowed to stand for 90 min with intermittent shaking. The absorbance of the resulting solution was measured at 760 nm. The phenol concentrations were expressed in terms of milligrams of Gallic acid equivalent per gram of fresh weight (mg GAE/g fresh matter). 2.4. Determination of total flavonoids concentration
2.7. Statistical analysis
The total flavonoid concentrations were estimated according to the aluminum chloride colorimetric method (Koolen et al., 2013). Briefly, 500 µl of diluted extract was mixed with 500 µl of 2% AlCl3 ethanolic solution. After incubation at room temperature for 40 min, the absorbance was measured at 430 nm. Flavonoid concentrations were calculated from a calibration curve of rutin and expressed as milligrams of rutin equivalent per gram of fresh weight (mg RE/g FM).
The data were expressed as the mean ± standard deviation (SD) of triplicate independent experiments and analyzed using one-way analysis of variance (ANOVA) and Student’s t-test p < 0.05 was considered to be statistically significant. We performed a PCA (principal component analysis) and obtained a dendrogram of variations with R software.
2.5. Determination of lycopene concentration
3. Results and discussion
Lycopene is the main antioxidant present in tomatoes. It is therefore important to carry out its estimation before and after heat treatment in order to have an evaluation of the effect of the microwave treatment on the concentration of the fruit in carotenoid terpenes. Methods of estimating the lycopene level require a measurement of a chromatic component to the colorimeter. The determination of the level of lycopene by spectrophotometry at 503 nm, which is correlated with the lycopene level (Grolier et al., 2000), remains more reliable than the measurement of the red color by chromatometry. At this wavelength, lycopene is responsible of 90% of the absorbance, while β-carotene less than 10% (Grolier et al., 2000). Lycopene was extracted before measured (Sadler et al., 1990). Briefly, 1 g of each tomato samples was
3.1. Total phenolic, flavonoid and lycopene concentrations The results of total phenolic, flavonoid and lycopene concentrations are given in Table 1. As it can be seen, the highest amounts of total phenolic, flavonoid and lycopene concentration are observed for tomato sample T300 s (6.95 mg GAE/g FM, 10.68 mg RE/g FM), followed by tomato sample T30 s (1.96 mg GAE/g FM, 2.57 mg RE/g FM), while minimum values were observed for tomato sample control (1.92 mg GAE/g FM, 2.33 mg RE/g FM) for the total phenolic and flavonoid concentrations, respectively. For total phenols concentration, our results are higher than those obtained by other authors (George et al., 2004) on fresh tomatoes with 2
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Table 1 Effect of microwave heating on total phenolics, flavonoids and lycopene concentration of tomatoes samples.
T control T 30 s T 300 s
Total phenolics concentration (mg GAE g−1 FM)
Flavonoid concentration (mg RE g−1 FM)
Lycopene concentration (mg/ kg FM)
1.92 ± 0.098 1.965 ± 0.152 6.955 ± 0.228*
2.337 ± 0.009 2.576 ± 0.049 10.682 ± 0.098**
5.772 ± 0.725 31.865 ± 2.774* 40.715 ± 7.744**
Table 2 Effect of microwave heating on the antioxidant activities of ethanol extract of tomatoes samples.
T control T 30 s T 300 s
DPPH IC50 (mg/ml)
Chelating ion ability IC50 (µg/ml)
1.697 ± 0.003 1.501 ± 0.001 1.333 ± 0.001
401.000 ± 2.081 360.166 ± 1.166 299.916 ± 0.740
Values are given as mean ± SD (n = 3). Synthetic antioxidant: IC50 values for the Trolox: 0.115 mg/ml. IC50 values for the EDTA: 6.5 ± 0.2 μg/ml (Chelating ability).
In each column: * indicate p > 0.05 and ** indicate P > 0.01. T: control without any heat treatment; T 30 s: tomatoes treated for 30 s by microwave; T 300 s: tomatoes treated for 300 s by microwave. GAE: Gallic acid equivalent; FM: fresh matter; RE: Rutin equivalent.
3.3. DPPH radical scavenging assay The DPPH stable purple radicals react with suitable reducing agents or hydrogen atom donor (A-H), during which the electrons become paired off, yielding a stable diamagnetic molecule (yellow-colored diphenylpicrylhydrazine); the solution loses color, depending on the number of electrons taken up (Biswas et al., 2014). The DPPH antioxidant assay is based on the ability DPPH, a stable free radical, to decolorize, in the presence of antioxidants (Benabdallah et al., 2016), thus a low value corresponds to a good scavenging ability. The results were expressed as IC50 value that is the amount of antioxidant necessary to decrease by 50% the initial DPPH radical concentration. The results indicated that T300 s was the most efficient (1.333 mg/ml), followed by T30 s (1.501 mg/ml), whereas control case (1.697 mg/ml) was the least effective. However, all extracts recorded low radical scavenging abilities when compared to that reported for Trolox used as positive controls.
0.22 mg GAE/g FM. Moreover, values obtained by our experiments showed also higher values for fresh tomatoes than those reported in the literature ranged from 0.154 to 0.162 mg GAE/g FM (Riahi and Hdider, 2013), and with total phenols of 0.14 mg GAE/g FM (Brat et al., 2006), and varied from 0.6 to 0.9 mg GAE/g FM (Aldrich et al., 2010). Concerning flavonoids concentration, reported lower values ranged from 0.106 to 0.512 mg RE/g FM (Ilahy et al., 2009). Moreover, our results are higher than those ranging from 0.109 to 0.113 mg RE/g FM (Riahi and Hdider, 2013), for Tunisian samples. The data in the scientific literature are quite contradictory. Some authors have studied the compounds themselves and not their rates in heat-treated products. Some tomatoes phenolic compounds, such as hydroxycinnamic acids (acids: p-coumaric, ferulic, caffeic and chlorogenic), are not affected by heat treatments (Re et al., 2002). The levels of caffeic and chlorogenic acid increase during the heat treatments. They concluded that for polyphenols, heat treatment helped to release them from the cell matrix (Chanforan, 2010). Methods for the quantitative measurement of lycopene are difficult due to its sensitivity to light, oxygen, metals and strong acids (Goula et al., 2006). This is due to the structure of this terpene, rich in ethylenic bonds. Therefore, it is recommended to replace metallic fry by non-metallic frying and to conduct measurements in the absence of oxygen and light. The addition of antioxidants in solvents, such as ammonium acetate in the organic phase, makes a better lycopene concentration measurement and does not interfere with the assay of the molecule (Nierenberg and Nann, 1992; Nguyen and Schwartz, 1999; Nguyen and Schwartz, 1998; Rao and Agrawal, 1998). Some techniques use antioxidant molecules such as BHT in order to limit the oxidation phenomena of polyunsaturated isoprene hydrocarbons. Lycopene concentration was significantly different between the three studied samples (Table 1). The highest concentration was obtained from tomatoes samples T300 s with 40.7156 mg/kg FM, followed by T30 s with 31.8654 mg/kg FM. Untreated samples showed the lowest concentration with 5.772 mg/kg FM. Our results are in agreement to the literature (Caris-Veyrat et al., 2004), these authors worked on organic tomatoes grown in a plastic tunnel, and found lycopene concentration ranged from 36 to 42 mg/kg FM. But it is still lower than those reported concentration ranging from 44.7 to 87.6 mg/kg of FM (Hdider et al., 2007), or of 49.5 to 106.5 mg/kg FM (Perkins-Veazie, 2007), for conventional field tomatoes. Variation in lycopene concentration depends on the maturity stage (Ilahy et al., 2011), the year of production, the growing season (Garcia and Barrett., 2006), the use of fertilizers and the location (Lenucci et al., 2006).
3.4. Ferrous ion chelating activity The transition metal, iron, is capable of generating free radicals by Fenton reactions. Ferrozine can quantitatively form complexes with Fe2+. In the presence of other chelating agents, the complex formation is disrupted with the decrease of the red color as a result. Metal chelating capacity was significant since the extract reduced the concentration of the catalyzing transition metal (Ebrahimzadeh et al., 2009). The chelating activity of samples on metal ions was determined by measuring the absorption of ferrozine-Fe2+ complex at 562 nm. The results of the chelating iron indicated that tomato T300″ had the largest ability to chelate iron (299.92 µg/ml FM), followed by T30″ (360.17 µg/ml), while tomato control showed the lowest ability with 401 µg/ml (Table 2). Previous works evaluated antioxidant activities of tomatoes from different regions of the world and by different methods. Some authors assessed the antioxidant properties of tomato extracts from Spain, by the Trolox Equivalent Antioxidant Capacity (TEAC) and FRAP assays and, reported synergistic action between the lycopene and phenolic concentration of tomato (García-Alonso et al., 2015). Moreover, other references analyzed three varieties of tomato from Indonesia, using different polarity solvents (which were n-hexane, ethyl acetate and ethanol) for their total phenolic, flavonoids and carotenoid concentrations and their antioxidant abilities, reported a high correlation between TPC with IC50 of DPPH and FRAP (Ferric Reducing Antioxidant Power) capacity (Fidrianny et al., 2015). Furthermore, those measuring the effect of storage time and temperature on physicochemical properties and antioxidant activities (by DPPH assay) of different Tomato cultivars from Portugal, observed an increase in the phenolics, ascorbic acid, lycopene concentrations and antioxidant activity with temperature and storage duration (Vinha et al., 2013). In addition, studies comparing antioxidant properties of fresh, freeze-dried and hot-air-dried tomatoes from Taiwan, by three methods (DPPH, FRAP, ion chelating ability), indicated that the drying method could enhance the nutritional value of tomatoes by increasing parts of total flavonoids, phenolics and lycopene concentrations (Chang et al., 2006). These last ones are considered as antioxidant components,
3.2. Antioxidant activity The antioxidant activity of tomato extracts was evaluated by measuring, the free radical scavenging activity (DPPH) and the ferrous ion chelating power. All the extracts exhibited a noticeable effect, which varied significantly among samples (Table 2). 3
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rate of hydrophilic and lipophilic compounds released by heat treatment. For total phenolics concentration, the tomatoes slices exhibit values from 1.92 ± 0.098 for control to 1.965 ± 0.152 and 6.955 ± 0.228 (mg GAE g−1 FM) for 30 s and 300 s respectively. We also notice that both axis 1 and 2 showed a very high correlation (90% and 9%) between phenolic concentration and flavonoids. In tomato fruits, flavonoids characterize the main constituent of the total phenol concentration (Toor et al., 2005). Variations of lycopene concentration showed same scenario. The release of this carotenoid enhances with microwave treatment ranging from 5.772 ± 0.725 for control to 31.865 ± 2.774 and 40.715 ± 7.744 (mg/kg FM) for 30 s and 300 s respectively. Lycopene, the predominant carotenoid in tomatoes, exhibits the highest antioxidant activity and singlet oxygen quenching ability of all dietary carotenoids (Tuyen et al., 2016). Following dendrogram of variation of antioxidant activities and chemical components in microwave treated tomato slices (Fig. 2), which gives more information about these correlations and explains the relations between chemical compounds and the specific antioxidant activity. We noticed that variations of polyphenols and flavonoids are the most correlated to antiradical scavenging activity (DPPH), while lycopene explained a part of these correlations in addition to Ion chelating activity. Microwave treatment of T300 s gives the best results among the considered cases by releasing the maximum rates of both phenolics and carotenoids concentrations of tomatoes slices.
Fig. 1. Principle component analysis of antioxidant activities vs chemical components (phenols, flavonoids and lycopene).
4. Conclusion The aim of the present study is to assess the effect of microwave heat treatments on the chemical composition (polyphenols, flavonoids and lycopene) in relation with antioxidant activities of tomato fruits (DPPH and Ion chelating). Two treatments are applied (30 s and 300 s) and compared to control case. Results show the presence of these bioactive phytonutrients in fresh tomato and that their biodisponibility increases with the duration of the selected heat treatment. Moreover, analysis of obtained results indicates an enhancement in the ethanolic rates of polyphenols, flavonoids and specially lycopene. These variations are strongly correlated to the variation of antioxidant activities evaluated by both DPPH and Ion Chelating methods. Phenolic compound explains the major part of DPPH scavenging variations, where lycopene mainly explains the Ion chelating activity. As a result of our study, we demonstrate that microwave treatments enhance the nutritional quality of tomato slices by enhancing the biodisponibility of some of its hydrophilic and lypophilic concentrations. Microwave treatment of T300 s, in this experiment, is better than T30 s for heating tomatoes by releasing the maximum rates of both phenolics and carotenoids concentration of tomatoes slices. More time intervals and treatments levels should be evaluated to validate the most appropriate heating time in order to give more conclusions about the efficiency of microwave ovens.
Fig. 2. Dendrogram of variation of antioxidant activities and chemical components in microwave treated tomato slices.
therefore they released to the increase of antioxidant abilities. However, it is difficult to compare results of antioxidant abilities because of the variation of numerous parameters such as locality, concentration, varieties, solvents and methods used to dry or to obtain samples. In addition to methods used to assess antioxidant abilities. Microwave energy is quickly absorbed by water molecules, which, consequently, results in rapid evaporation of water and therefore higher drying rates and energy savings, for that reason microwave drying give significant energy savings, with a potential reduction in drying times in addition to the inhibition of the surface temperature of the treated material.
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