Carotenoid and Polyphenol Content of Different Tomato Cultivars and

Chapter 29

Downloaded by MONASH UNIV on February 26, 2016 | http://pubs.acs.org Publication Date: June 19, 2003 | doi: 10.1021/bk-2003-0851.ch029

Carotenoid and Polyphenol Content of Different Tomato Cultivars and Related Antioxidant Activity 1

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P. G . Pietta , P. I. Mauri1, M. Minoggio , C . Gardana , I. Iemoli , and P. Simonetti 2

1ITB-CNR, V.le F.Ili Cervi, 93 20090 Segrate (MI), Italy 2Department of Food Science and Microbiology, Via Celoria, 2-20133 Milan, Italy

Different tomato phenotypes were analysed for the content of total polyphenols, selected flavonoids, including flavonols (rutin and a rutin-derivative), flavanones (naringenin and the related chalcone) and carotenoids (lycopene and β-carotene). The identity of these components was based on their chromatographic, ultraviolet and mass spectrometric behavior. All phenotypes were surveyed for their total antioxidant activity (TAA), and correlations betweens T A A values and the content of each group of constituents.

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© 2003 American Chemical Society In Food Factors in Health Promotion and Disease Prevention; Shahidi, Fereidoon, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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Biological activities of flavonoids and related cinnamic acid derivatives have become well known in recent years. Many studies suggest that these polyphenols have beneficial effects on human health, due to their antioxidant capacity (7) and their ability to modulate the activity of different enzymes (2-4), interact with specific receptors (5), exert vasodilatory effects (6) and chelate metal ions such as those of Cu and Fe (7). Based on their daily intake [about l g (8, 9)], which exceeds largely that of other antioxidants (vitamin C, 70-100 mg/day; vitamin E , 7-10 mg/day; βcarotene, 2-3 mg/day), dietary polyphenols may represent an important exogenous defence against the imbalance between prooxidants and antioxidants, that is the oxidative stress. Indeed, polyphenols play an active role in diminished formation of reactive oxygen species (ROS), since they affect enzymes that catalyse redox reactions, including mitochondrial succinoxidase, NADH-oxidase and enzymes involved in arachidonic acid metabolism (10). In addition, the highly oxidizing reactive oxygen species (ROS) are reduced by dietary polyphenols, which in turn are transformed in less aggressive aroxyl radicals (77). Unfortunately, most positive evidence of these activities has been reached using isolated compounds for in vitro studies. Thus, it is difficult to extend this evidence to the complex matrix of foods and beverages and in vivo. The occurrence of dietary polyphenols in largely consumed vegetables and their bioavailability need to be known for a proper evaluation of their potential health benefits. In the past years many studies on the uptake and the metabolism of dietary polyphenols have been published, and presently a better information on the absorption and fate of polyphenols from regular food, beverages and supplements is available. B y contrast, the polyphenol content of different vegetables consumed in large amounts and with beneficial properties is still poorly defined. This is the case of tomatoes, which represent an important part of the mediterranean diet and are thought to diminish the risk of certain chronic diseases (72, 73). Different factors may influence the polyphenol content of tomatoes, the major being the cultivar followed by climatic conditions and maturity degree. Therefore, it was of interest to evaluate the polyphenol pattern of tomato cultivars selected among those mostly preferred by consumers or processed by food industry. To this purpose, thirty-one different tomato cultivars were analyzed by spectrophotometric, chromatographic and mass spectrometric methods. Firstly, the content of flavonols (rutin and a rutin-pentoside), flavanones (naringenin and its chalcone), cinnamic acid derivatives (chlorogenic acid, caffeic acid, ferulic acid and a chlorogenic acid analogue), carotenoids (lycopene, β-carotene and lutein) and total polyphenols were determined. Furthermore, the total antioxidant activity (TAA) of the selected cultivars was assayed, and the obtained T A A values were correlated with the content of tomato components.

In Food Factors in Health Promotion and Disease Prevention; Shahidi, Fereidoon, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Materials and methods


Chemicals Rutin, naringenin were purchased from Extrasynthese (Genay, France). Ferulic acid, caffeic acid, chlorogenic acid, lycopene, β-Carotene and lutein were piurchased from Sigma-Aldrich (Steinheim, Germany). Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid, Aldrich Chemical Co., Gillingham, U K ) was used as the antioxidant standard. A B T S [2,2'-azinobis-(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt] was obtained from Sigma-Aldrich (Dorset-UK). Methanol, acetonitrile and water were H P L C grade from Merck (Darmstadt, Germany).

Extraction of polyphenols fraction from tomatoes Twenty grams of minced tomatoes were extracted for 3h at 65°C with 200 ml of acetone:water (4:1, v/v). The mixture was filtered and the residue was extracted twice again using the same amount of acetone:water (4:1, v/v). The combined filtrates were evaporated to dryness under vacuum and the residue was dissolved in 10 ml of methanol. The resulting solution was filtered through a 0.22 pm filter and stored at -20°C.

Total polyphenols The content of total polyphenols was detennined by colorimetric assay with Folin-Ciocalteu's phenol reagent, according to Singleton and Rossi (14).

Flavonols, flavanones and phenolic acids The H P L C separation was performed using a Waters 625 L C System (Milford, M A ) connected to a Waters model 996 photodiode array detector, equipped with a Rheodyne injector (loop 50 μΐ) and a Waters Millenium workstation. The column was a 5 pm X-Terra C (150x2.1 mm, i.d.) from Waters. The eluents were: (A) 0.1% acetic acid and (B) acetonitrile. Linear gradient was 5% to 35% Β in 30 min. Flow-rate was 0.4 ml/min. Acquisition was set at 320 and 350 nm (Spectral acquisition in the range 200-400 nm). Flavonols, flavanones and phenolic acid standards were dissolved in methanol (1 mg/ml) and stored at î 8


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0°C. Aliquots of standard solutions in the range of 5-200 pg/ml were injected in H P L C apparatus.

Lycopene and β-carotene Sample preparation and determination of lycopene and β-carotene was performed according Riso and Porrini (75).


Mass spectrometry Samples were analysed by LCQ ca ion trap niass spectrometer (Termofinnigan, Milan, Italy) equipped with an electrospray interface (ESI-MS). ESI-MS conditions were optimized by flow injection of rutin standard solution. Analyses were carried out in positive scan mode from mlζ 150 to 1000. For M S / M S experiments the collision energy was 30%. De

Total antioxidant activity The T A A of the polyphenol extracts was measured by the A B T S radical cation decolorisation assay, according to Miller and Rice-Evans (16).

Results and discussion Figure 1 shows a typical H P L C cromatogram of a tomato extract. Chlorogenic acid, caffeic acid, ferulic acid, rutin, a chlorogenic acid analogue, naringenin and its chalcone were identified on the basis of their chromatographic and U V characteristics. Concerning peak X , the U V spectrum suggested a rutinlike structure, which was confirmed by comparing its mass spectrum with that of rutin. A s indicated by the fragmentation pattern shown in Figure 2, peak X differs from rutin for the presence of a pentose moiety. The nature and the binding site of this pentose is still under investigation, and presently peak X can be assigned as a rutin-pentoside. The content of rutin, its analogue, naringenin and its chalcone in the examined cultivars is shown in Table I. The levels ranged from 0.07 to 2.35 mg/100g for rutin, from 0.03 to 1.38 mg/100g for rutinpentoside and from 0.04 to 4.90 mg/100g for naringenin and its chalcone. A similar variability was found for the phenolic acids, which were mainly represented by chlorogenic acid ranging from 0.03 to 0.58 mg/100g followed by caffeic acid with a content of up to 0.1 mg/100g (Table II). Total polyphenols of the examined cultivars were present at higher concentrations (13.15 ± 1.15 mg/100 g, range 4.43 - 25.84 mg/100g), and this may be due to the lack of specificity of the Folin assay.


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Figure 5. Flavonols and flavanones in tomato cultivars with low and high lycopene content


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However, in the case of a cultivar producing almost exclusively high levels of lycopene, the trend observed for most cultivars is reversed, in the sense that also rutin/rutin-pentoside and naringenin/naringenin chalcone levels are high. B y contrast, another cultivar synthetising mainly β-carotene was poor in both mtin/rutin-pentoside and naringerjin/naringenin chalcone.


Conclusions Based on the results, it may be concluded that total antioxidant activity (TAA) of the examined tomato cultivars is mainly correlated to the content of total polyphenols, and this reinforces the need for further studies on the chemical identity of all phenolics present in tomatoes (as well as in other common vegetables).

Acknowledgements The authors acknowledge the contribution of Enrico Rosti to this work. C O P O M is acknowledged for its support.

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Carotenoid and Polyphenol Content of Different Tomato Cultivars and

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