Free Amino Acid and Phenolic Contents and ... - ACS Publications

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Free Amino Acid and Phenolic Contents and Antioxidative and Cancer Cell-Inhibiting Activities of Extracts of 11 Greenhouse-Grown Tomato Varieties and 13 Tomato-Based Foods Suk-Hyun Choi,† Hyen-Ryung Kim,‡ Hyun-Jeong Kim,§ In-Seon Lee,§ Nobuyuki Kozukue,# Carol E. Levin,^ and Mendel Friedman*,^ †

Department of Food Service Industry, Seowon University, Cheongju-city 361-742, Republic of Korea Department of Foodservice Management and Cuisine, Gyeongju University, Gyeongju 780-712, Republic of Korea § Center for Traditional Microorganism Resources, Keimyung University, Daegu 704-701, Republic of Korea # Bio Organic Material and Food Center, Seowon University, Cheongju-city 361-742, Republic of Korea ^ Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710, United States ‡

ABSTRACT: Tomato (Solanum lycopersicum) plants synthesize nutrients, pigments, and bioactive compounds that benefit nutrition and human health. The nature and concentrations of these compounds are strongly influenced by varietal factors such as size and color as well as by processing. To better understand how these factors affect the concentration of nutrients and bioactive compounds, we analyzed 11 Korean tomato varieties grown under the same greenhouse conditions and 13 processed commercial tomato products for free amino acids and amino acid metabolites by HPLC, for individual phenolics by HPLC-MS, for total phenolics by the FolinCiocalteu method, for antioxidative activity by the FRAP and DPPH methods, and for cancer cell-inhibiting effects by the MTT assay. We also determined the protein content of the tomatoes by an automated Kjeldahl method. The results show that there is a broad range of bioactive compounds across tomato varieties and products. Small tomatoes had higher contents of bioactive compounds than the large ones. The content of phenolic compounds of processed products was lower than that of fresh tomatoes. Tomato extracts promoted growth in normal liver (Chang) cells, had little effect in normal lung (Hel299) cells, mildly inhibited growth of lung cancer (A549) cells, and first promoted and then, at higher concentrations, inhibited growth in lymphoma (U937) cells. The relationship of cell growth to measured constituents was not apparent. Dietary and health aspects of the results are discussed. KEYWORDS: fresh tomatoes, tomato-based food, amino acids, proteins, phenolic compounds, antioxidative effects, anticarcinogenic, effects, dietary significance

’ INTRODUCTION Widely cultivated and consumed tomatoes are fruits of the species Solanum lycopersicum. Tomatoes are a good source of nutrients and bioactive phenolic compounds.14 The nature and amounts of biosynthesized bioactive compounds are influenced by agricultural practices (irrigation and soil amendment), preand postharvest environmental factors (soil fertility, climate, and storage conditions), variety, and ripeness. The present study aims to better understand the contributions of variety and processing conditions on the contents of protein, free amino acids, and phenolics and on bioactivity. Because individual phenolics may exhibit different health-promoting effects, we measured both the composition of individual phenolics by HPLC and the total phenolic content by colorimetry. To determine bioactivity, we measured oxidation potentials by FRAP and DPPH assays and effects on growth of normal and cancer cells by the MTT assay. We evaluated extracts from 11 varieties of fresh tomatoes and from 13 commercially processed tomato products. To minimize the effects of environment, agricultural practice, and ripening, the Korean tomatoes were grown and harvested under identical conditions in a greenhouse. The store-bought processed samples r 2011 American Chemical Society

consisted of five whole, cut, and peeled tomatoes, one paste, one sauce, three ketchups, and three juices. Knowledge of both the composition and concentrations of bioactive compounds of tomatoes can benefit consumers. The main objective of this study was to contribute to our knowledge about the distribution and variability of tomato ingredients in widely consumed tomato varieties grown under defined conditions and in processed tomato products. The results demonstrate striking differences in the distribution of free amino acids, proteins, and phenolic compounds as well as in antioxidative and human cancer cell growth-inhibiting effects of tomatoes and tomato products.

’ MATERIALS AND METHODS Materials. Thirteen commercial tomato products comprising canned whole tomatoes, whole (cut) tomatoes, sauce, paste, juice, and bottled Received: July 12, 2011 Revised: November 8, 2011 Accepted: November 9, 2011 Published: November 09, 2011 12801

dx.doi.org/10.1021/jf202791j | J. Agric. Food Chem. 2011, 59, 12801–12814

Journal of Agricultural and Food Chemistry

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through a 0.45 μm membrane filter (Millipore, Bedford, MA) and degassed in an ultrasonic bath before use.

ketchup were purchased from a local supermarket (Cheongju, Korea). Quercetin-3-rutinoside (Q3R, lot BCBB6172, 95.3%), chlorogenic acid (5-caffeoylquinic acid, 5-CQA, lot 27H1006, 95%), 2,2-diphenyl-1picrylhydrazyl (DPPH, catalog no. D9132, lot 12K1944, g90%), and 2(3)-tert-butyl-4-hydroxyanisole (BHA, catalog no. B1253, lot 098K0242, g95%) were purchased from Sigma (St. Louis, MO) and gallic acid (lot AGM01, 98%) and tripyridyltriazine (TPTZ, lot FHL01, g98%) from Tokyo Chemical Industry (Tokyo, Japan). Folin Ciocalteu phenol reagent (lot OF1181) was from Junsei Chemical Co., Ltd. (Tokyo, Japan) and naringenin (NG, lot BCBC7827 V, 95%) from Aldrich (Milwaukee, WI). Normal liver (Chang) and lung cell lines (Hel299), lung cancer (A549), and histiocytic lymphoma (U937) cells were from the American Type Culture Collection (ATCC, Rockville, MD) and the Korean Cell Line Bank (KCLB, Seoul, Korea). The cells were maintained in minimum essential medium (MEM) supplemented with 10% of fetal bovine serum, 50 units/mL penicillin, and 50 mg/mL streptomycin at 37 C in a 5% CO2 incubator. Cell culture reagents were obtained from Gibco BRL (Life Technologies, CergyPontoise, France). Each sample was dissolved in dimethyl sulfoxide (DMSO, 2 mg/200 μL) and stored at 4 C. HPLC grade acetonitrile and formic acid were purchased from J. T. Baker (Phillipsburg, NJ) and Aldrich (Milwaukee, WI), respectively. The solvents were filtered

Sampling of Tomato Fruits and Tomato-Based Foods. Eleven varieties of Korean tomato fruits were used in this experiment. Tomato seeds were planted on October 1, 2009, in a greenhouse of Buyeo Tomato Experiment Station, Chung-Nam, Korea, and were harvested on May 2931, 2010, at their optimum maturity stage for market (Figure 1). In the greenhouse, temperatures were set in the range from 13 to 25 C during the day and from 10 to 13 C during the night. Fruits were collected, weighed, and measured for size as shown in Table 1. Each tomato fruit sample consisted of three (large size) to eight (small size) uniform-size fresh fruits. After removal of the calyx, the remainder of the fruits including flesh, seeds, and gelatinous fluid were cut into slices 45 mm thick and immediately immersed in liquid nitrogen, and then freeze-dried with an Ilsinbiobase Freeze-Dryer (model PVTFD 10R, Ilsinbiobase Co., Ltd. Korea). Water content was determined by weighing the sample before and after freeze-drying. The dried samples were ground briefly in a mortar and then in a Wiley mill to pass a 20-mesh screen. To avoid the possibility that moisture, light, temperature, and oxygen could affect freeze-dried tomato samples, we hermetically sealed the samples in a desiccator containing P2O5, which was stored in a dark freezer at 25 C. The desiccator was brought to room temperature before the samples were removed for use in the described experiments. The tomato sources and their products are listed in Table 2. Canned whole and cut tomatoes were prepared by draining the excess liquid, weighing, and slitting the tomato to allow the liquid in the cavity to drain, followed by rinsing with distilled water. Each sample was macerated in a glass mortar. Tomato ketchup, sauce, and juice were directly stirred with a glass stick. Each aliquot was used for two determinations of amino acids and three determinations for phenolic compounds. Crude Protein Content of Tomatoes. The total N content of each freeze-dried tomato powder (0.41931.801 g) was determined in duplicate by an automated Buchi Auto Kjeldahl Unit K-370 coupled with a Buchi Kjeldahl Sampler K-371 (Switzerland) according to the manufacturer’s instructions. Protein content per 100 g of dry weight was calculated by multiplying the experimental N content by 6.25. We did not determine the protein content of the processed tomato products.

Extraction of Amino Acids and Phenolic Compounds from Tomatoes and Processed Tomato Products. Each freeze-dried tomato powder (75.4144.9 mg, depending on availability) was placed into a 25 mL volumetric flask to which was added 80% methanol in water (25 mL). The flask was then placed into an ultrasonic bath for 60 min at 30 C. The filtrate was centrifuged at 18000g for 10 min at 1 C. The extracts were then passed through a 0.45 μm Millipore nylon filter

Figure 1. Photographs of 11 varieties of Korean tomato fruits used in the present study.

Table 1. Dimensions, Weights, and Water and Protein (N 6.25) Contents of 11 Varieties of Tomato Fruits Grown under the Same Conditionsa tomato variety

a

color

length (cm)

width (cm)

weight (g/fruit)

H2O (%)

protein (g/100 g dry wt)

protein (g/100 g fresh wt)

Starbuck

red

7.9 ( 0.2

5.9 ( 0.3

233.9 ( 5.9

94.7

11.2

0.59

Rapide

red

7.4 ( 0.3

5.7 ( 0.1

210.4 ( 5.4

94.1

10.8

0.64

Dotaerang Gold

red

6.2 ( 0.2

5.1 ( 0.4

120.7 ( 6.4

92.6

8.2

0.61

Ho-yong

red

5.8 ( 0.0

4.9 ( 0.2

96.5 ( 2.6

90.4

9.3

0.89

Amoroso

red

4.0 ( 0.1

3.7 ( 0.0

34.9 ( 0.6

91.1

11.5

1.02

Tiger

black

3.4 ( 0.1

3.2 ( 0.0

20.6 ( 0.4

89.4

15.4

1.63

Seng Green Chorok

green

3.3 ( 0.1

3.1 ( 0.1

20.7 ( 1.0

91.5

15.3

1.30

Yo-yo Koko

red red

3.2 ( 0.0 3.1 ( 0.1

3.0 ( 0.1 3.1 ( 0.7

18.0 ( 0.3 17.1 ( 0.2

92.0 91.7

11.6 12.2

0.93 1.01

Dotori

red

2.8 ( 0.1

3.4 ( 0.1

15.2 ( 1.0

91.2

11.5

1.01

Mini Chal

red

2.5 ( 0.0

3.3 ( 0.0

12.1 ( 0.3

91.1

11.8

1.05

Length, width, and weight are average ( SD (n = 5); protein value is average of duplicate determinations. 12802

dx.doi.org/10.1021/jf202791j |J. Agric. Food Chem. 2011, 59, 12801–12814


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Table 2. Commercial Processed Tomato Products and Their Sources tomato product no.

a

tomato product name

company name, country

H2Oa (%)

TP-1

S&W whole peeled tomatoes

S&W Fine Foods Inc., USA

91.8

TP-2

Hunt’s whole tomato

Conagra Food Inc., USA

89.4

TP-3

S&W premium crushed Italian recipe tomatoes


90.0

TP-4

Tesco Italian cut tomatoes

Tesco Ltd., UK

92.1

TP-5

S&W premium ready-cut tomatoes


93.4

TP-6

Prego chunky garden tomato, onion, and garlic sauce

Campbell Soup Co., USA

86.1

TP-7

Hunt’s tomato paste

Conagra Food Inc., USA

72.3

TP-8 TP-9

Ottogi tomato ketchup Chungjungwon Jinhan ketchup

Ottogi Ltd., Korea Chungjungwon Ltd., Korea

62.9 63.6

TP-10

Heinz tomato ketchup

Heinz Inc., USA

65.4

TP-11

Yooginong tomato juice

Pureplus Ltd., Korea

88.4

TP-12

Jayeoneun tomato juice

Woongjin Ltd., Korea

88.3

TP-13

Nongjang tomato juice

Gaya Nongjang Ltd., Korea

88.5

Water content is average (%) of duplicate determinations.

(Bedford, MA) before HPLC analysis. The filtrate was used for analysis of amino acids, phenolic compounds, and antioxidant and cellular activities. Each aliquot (0.711.652 g) from 13 commercial products was placed into a 25 mL volumetric flask. The flask was brought up to volume with 80% methanol in water, placed into an ultrasonic bath for 60 min, and centrifuged at 18000g for 10 min at 1 C. The extract was then passed through a 0.45 μm Millipore nylon filter. The filtrate was used for analysis of amino acids, phenolic compound, and antioxidant and cellular activities. Analysis of Amino Acids. The analysis was carried out by an ionexchange chromatography method used previously.3 Briefly, an aliquot (10 μL) of the filtrate obtained from the above extraction was used to determine free amino acids. A Hitachi model L-8800 amino acid analyzer (Hitachi Co. Ltd., Tokyo, Japan) and a column packed with Hitachi custom ion-exchange resin 2622 (4.6 i.d. 60 mm, particle size = 5 μm) was used for the amino acids. Lithium citrate buffer and ninhydrin flow rates were 0.35 and 0.30 mL/min, respectively. The column temperature was 3070 C and the reaction coil temperature, 135 C. High-Performance Liquid Chromatography (HPLC). HPLC was carried out on a Shimadzu Prominance LC-20A (Shimadzu, Kyoto, Japan), which consists of a CMA-20A controller, a DGU-20A3 degasser, two LC-20AD solvent delivery modules, an SIL-20AC autosampler, a CTO-20A column oven, and an SPD-M20A photodiode array (PDA) detector. The flow rate was 0.8 mL/min at 30 C. UV detection was set at 340 nm. An aliquot (20 μL) was injected directly into an Inertsil ODS3 V (5 μm, 4.6 250 mm) HPLC column (GL Sciences Inc., Tokyo, Japan). The mobile phase consisted of the following gradient modes: acetonitrile (A) and 0.5% formic acid (B), A = 5% (05 min), 18% (5.130 min), 70% (30.190 min), 90% (90.1100 min), and 5% (100.1120 min). Each sample was extracted three times, and each of the three extracts was analyzed by HPLC in triplicate. Liquid ChromatographyMass Spectrometry (LC-MS). LC was carried out on an Agilent Technologies (Santa Clara, CA) 1200 series binary LC system with a photodiode array detector monitored at 340 nm. The system was coupled with a 3200 Q Trap LC-MS/MS system (Applied Biosystems Inc., Foster City, CA). An aliquot (20 μL) of the extract obtained above was directly injected into an Inertsil ODS-3 V (5 μm, 4.6 250 mm) HPLC column (GL Sciences Inc.). The mobile phase, the column temperature, and the flow rate were the same as those of the above-described HPLC system. The LC eluate was introduced into the mass spectrometer from 5 to 40 min. Mass (MS) and tandem mass spectrometry (MS/MS) were operated in the negative ion mode in the mass range of m/z 1601200. Helium was used as the collision gas for the MS/MS spectrometric procedures, followed by the isolation of

ions over a selected mass window of 2 Da. MS/MS represents multiple stages of precursor ion m/z selection followed by product ion detection for successive progeny ions. Mass selection of the analyte by m/z was followed by fragmentation and analysis of the fragments. For quantification, integrated chromatographic peak areas from the test samples were compared to peak areas of known amounts of standard phenolic compounds 5-CQA, Q3R, and NG. Total Phenolics and Antioxidant Activities. The supernatant (500 μL) obtained from the above extraction was placed in a 10 mL vial and then dried completely at 30 C under reduced pressure. Each residue was weighed and then dissolved in 10% DMSO in water (10 mL). This solution was used for the determination of total phenolic content and antioxidant activities. Total phenolic content was measured using a modified colorimetric FolinCiocalteu method.5 The extract solution (1.0 mL, 0.51.0 mg/mL) from samples of tomatoes was mixed with 10% Na2CO3 solutions (1.5 mL) and incubated at room temperature for 2 min. After the addition of 50% FolinCiocalteu phenol reagent (500 μL) and water (7 mL), the reaction tube was further incubated for 1 h at room temperature, followed by centrifugation at 5000g for 10 min and reading of the absorbance at 700 nm. Measurements were carried out in triplicate. Concentrations of total phenolic compounds expressed as gallic acid equivalents (g/100 g dry wt) are based on a standard calibration curve obtained with gallic acid. Antioxidant Power Assays by Two Methods. The supernatant (500 μL) obtained from the above extraction was placed in a 10 mL vial and dried at 30 C under reduced pressure. Each residue was weighed and then dissolved in 10% DMSO in water (10 mL). This solution was used for the determination of antioxidant activities by the two assays as described below. The FRAP assay measures the ability of antioxidants contained in the samples to reduce ferric tripyridyltriazine (Fe3+ TPTZ) to the ferrous form (Fe2+). The ferrous and ferric ion complex with TPTZ reagent is the main product of this reaction. The antioxidant capacity of the samples (10 μL) was measured at 593 nm.6 The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging effects of the samples were measured according to the method of BrandWilliams et al.7 with some modifications. Dilutions (0.8 mL, 1001000 μg/mL) of each sample were added to 0.15 mM DPPH (0.2 mL). The commercial antioxidant BHA was used as a positive reference. After 30 min of incubation at room temperature, the absorbance was read at 517 nm against a blank. The inhibition of the DPPH free radical was calculated using the following equation: DPPH scavenging effect (%) = (1 Asample/Ablank) 100, where Ablank is the absorbance of the control 12803


Journal of Agricultural and Food Chemistry reaction (containing all reagents except the test sample) and Asample is the absorbance of the test sample. The antioxidant activity of sample was expressed as the IC50, defined as the concentration of sample that inhibited formation of DPPH radicals by 50%. MTT Assay for Growth Inhibition of Cells. The MTT assay by which the yellow tetrazole 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is reduced to purple formazan in living cells was adapted from the literature.3,8 The following reagents and instruments were used: MTT reagent, 5 mg/mL in phosphate-buffered saline (PBS), protected from light, and stored at 20 C; MEM cell medium (containing 10% fetal bovine serum, 1% penicillin/streptomycin); microplate reader (Bio-Rad Co., Hercules, CA). Cell lines were seeded into a 96-well microplate (1104 cells/well) and incubated for 24 h. Next, cells were treated with four concentrations (1, 10, 50, and 100 μg/mL extract) for 48 h. The MTT solution (0.1 mg/mL) was then added to each well. After 4 h of incubation at 37 C, DMSO (200 μL) was added to each well. The absorbance (A) was then read at a wavelength of 540 nm. The decrease in absorbance in the assay measures the extent of decrease in the number of viable cells following exposure to the test substances calculated by using the following formula: % inhibition of cells = (Acontrol Atest substance)/ Acontrol 100. Three separate analyses were carried out with each extract. A blank experiment with DMSO plus cells but without the tomato product was done in each case. The method used avoids any possibility of a DMSO effect on the results. Statistical Analysis. Inhibitory concentrations at 50% values (IC50) were calculated by constructing a four-parameter logistic curve, using the values of percent inhibition, with the aid of SigmaPlot 11 (Systat Software, Inc., San Jose, CA). Pearson correlations between measured parameters were determined using Sigma Plot 11.

’ RESULTS AND DISCUSSION Dimensions and Water and Protein Contents. Figure 1 and Table 1 show that although all of the tomatoes were ripe, the color of two varieties departed from the traditional red color for ripened tomatoes: Tiger tomato was black, and Seng Green Chorok tomato was green. The variation in weights was 19.3-fold from lowest to highest. Moisture content varied from 89.4% (Tiger) to 94.7% (Starbuck). Table 1 also shows that the protein content of the 11 tomato varieties on a dry basis varied by a factor of 1.88 and on a fresh basis by a factor of 2.76. The black-colored Tiger variety and the green-colored Seng Green Chorok variety have the highest protein contents. Table 2 shows that the moisture content of five whole and cut processed tomatoes was similar to that of the fresh tomatoes mentioned above. Tomato paste had higher solids content (72.3% water) than tomato sauce (86.1% water). Ketchups had the highest solids levels, presumably because of added sugars and salt. The values of the three juices approach those of fresh or processed tomatoes. Free Amino Acid Content of Fresh and Processed Tomatoes. Free amino acids in plant foods represent a source of nitrogen and of nutritionally essential amino acids. They may react with other food components or be substrates for microbes to form new compounds. Examples include reaction with sugars to form browning compounds, including potentially toxic acrylamide formed from Asn,9 and formation of physiologically active biogenic amines from microbial-induced decarboxylation of amino acids such as Tyr, Trp, or His to form tyramine, tryptamine, and histamine, respectively.10 Free amino acids can also affect the flavor of tomatoes. Elevated levels of free glutamine have been shown to improve tomato taste.11

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Environmental factors can affect free amino acid content. Zushi and Matsui11 found that the content of free amino acids in tomatoes increased under salinity stress, whereas under water stress, proline and γ-aminobutyric acid increased and asparagine, glutamine, and arginine decreased. In the fresh tomato samples, free amino acids contributed a large fraction, an average of 38 wt %, to the total available pool consisting of free and protein-bound amino acids. For tomato varieties, the contributions ranged from 24.4% in Yoyo to 60.2% in Mini Chal. In a related study, Sorrequieta et al.12 found up to 28% of the total protein pool in tomatoes in the form of free amino acids. Previously, we determined that the levels of free amino acids started to increase at the maturation stage at which the fruit reaches its final size, peaked when the fruit turned half red, and then decreased during the final stages of ripening.3 This is relevant because processed tomatoes may be harvested at ripeness stages different from those for tomatoes destined for fresh consumption. Tables 3 and 4 show the distribution of free amino acids in tomatoes and processed tomato products in the order of elution position (Figure 2). For correlations using statistical analysis, samples were grouped into fresh tomatoes, canned tomato products (TP-17), ketchups (TP-810), and tomato juices (TP-1113). ANOVA tests (p < 0.05) were performed both on total amino acids and on individual free amino acids as percent of total amino acids. Total free amino acid contents were not significantly different between the fresh and canned tomatoes. The ketchup and juice samples had lower levels. In ketchup, this may be due to their dilution among the other ingredients. In tomato juice, it is possible that some components may be filtered and/or precipitated from solution during production. Total essential free amino acids, the sum of His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val, measured as percent of total free amino acids, were significantly higher in the processed tomatoes. There was no significant difference in total nonessential free amino acids between groups. The most abundant amino acid, Glu, was modestly but significantly higher in fresh than in canned tomatoes. Its amide form, Gln, was present only in fresh tomatoes. The application of heat during processing and the acid content of tomatoes likely hydrolyze Gln to Glu. γ-Aminobutyric acid (4Abu or GABA), a metabolite of Glu, was the third most abundant free amino acid and, like Glu, was present in similar amounts in all of the samples. The relatively abundant Asp and Ala were all significantly higher by a factor of 23 in processed compared to fresh tomatoes. Asn, the amide of Asp, was also significantly higher in processed tomatoes by a factor of ∼2. These amino acids may have been liberated from proteins undergoing mild hydrolysis. The Asn content deserves special mention. The content of fresh tomatoes ranged from 0.30% (Tiger) to 0.62% (Mini Chal) and that in processed tomatoes from 0.67% (TP-4) to 1.1% (TP-10). The relatively low levels of Asn may due to the fact that during the maturation of tomatoes from green to red, free Asn decreases more rapidly than the other amino acids.3 By contrast, we also recently reported much higher levels of Asn in jujube fruit, but not in seeds.13 Processed tomatoes had significantly higher levels of Asn than fresh. It is possible that unripe tomatoes, which may contain more Asn, were used in the preparation of the processed products. The low content of Asn, especially in fresh ripe tomatoes, suggests that tomatoes may not contribute significantly to the formation of acrylamide. Other N-Containing Compounds. In addition to the genetically coded amino acids mentioned above, the extracts also 12804


12805

L-Glu

L-Gln

34.4 ( 0.4

3.3 ( 0.2

14.5 ( 0.3 36.0 ( 1.1

15.5 ( 0.9

21.1 ( 0.5

19.3 ( 0.5

13.0 ( 1.2

78.8 ( 0.9

58.9 ( 4.6

7.7 ( 1.8

22.2 ( 0.5 nd

20.5 ( 1.0

34.2 ( 0.2

42.5 ( 0.1

29.7 ( 0.4

130.1 ( 0.8

L-Ala

L-Cit

L-Cys

L-Met

L-Ile

L-Leu

L-Tyr

L-Phe

433.8

4708.6

869.8

42.0

sum essential AAb

sum all AAb

sum metabolitesb

% free AA

35.6 ( 1.6

33.3

609.3

3598.8

262.8

19.8 ( 0.6

nd

34.3 ( 0

49.2 ( 0.9

nd

30.7 ( 0.1

10.4 ( 0.4

L-Arg

L-Car

L-His

Me-His

9.1 ( 0.2

Hyl

55.8 ( 0.1

4.2 ( 0

21.7 ( 0.3

4.3 ( 0

23.3 ( 0.4

EtNH2

L-Lys

8.3 ( 0.8

575.8 ( 3.1 3.7 ( 0

12.0 ( 0

737.5 ( 3.4 7.6 ( 0.1

β-Ala

4Abu Trp

L-Val

11.1 ( 0.1

15.7 ( 0.6

L-Gly

L-Pro

38.9

700.6

3193.7

161.9

13.3 ( 0.1

nd

23.1 ( 0.7

5.2 ( 0.2

19.8 ( 0

25.6 ( 0.3

3.6 ( 0.1

627.3 ( 0.5 nd

5.5 ( 0.2

40.4 ( 0.1

11.5 ( 0.6

15.8 ( 0.4

13.9 ( 0.1

13.8 ( 0.5

11.1 ( 0.1 nd

nd

30.8 ( 0.2

36.3

817.2

3371.6

216.3

17.0 ( 1.6

nd

22.7 ( 1.9

11.2 ( 0.1

24.1 ( 0.3

34.8 ( 0.2

nd

725.0 ( 2.7 nd

8.2 ( 1.0

57.6 ( 1.0

15.2 ( 0.3

20.0 ( 0.2

21.1 ( 0.4

17.0 ( 0.2

14.4 ( 0.1 nd

nd

41.9 ( 0.4

10.5 ( 0

52.6 ( 1.6

15.8 ( 0.8 8.1 ( 0

373.2 ( 3.8

1436 ( 2

117.8 ( 0.7 15.0 ( 0.1

39.4 ( 0.3

258.7 ( 0.5

nd

38.0 ( 0.4

Ho-yong

277.2 ( 1.9

1609 ( 2

66.2 ( 0.1 11.6 ( 0.1

24.0 ( 0

288.0 ( 1.0

nd

33.4 ( 0

Dotaerang Gold

43.7

684.5

5020.5

418.5

28.9 ( 1.8

nd

43.1 ( 0.5

5.9 ( 0.4

51.0 ( 1.2

45.7 ( 1.2

1.6 ( 0.3

577.1 ( 0.6 6.5 ( 0.2

11.8 ( 0.3

137.6 ( 2.0

27.7 ( 0.9

47.4 ( 0.5

37.6 ( 0.2

26.5 ( 0.1

20.4 ( 0.2 68.2 ( 1.2

nd

106.3 ( 1.7

13.9 ( 0.1

74.1 ( 7.6

357.0 ( 0.3

2631 ( 18

84.6 ( 1.7 19.6 ( 1.0

48.4 ( 1.6

506.4 ( 0.5

nd

42.4 ( 0.4

Amoroso

387.1

46.4

1453.4

7142.3

46.2

1423.3

7062.1

517.8

65.7 ( 0.3

66.0 ( 2.0 16.6 ( 1.5

60.7 ( 0.6 64.0 ( 5.7

51.2 ( 0.8

62.8 ( 0.3

21.7 ( 0.1

4.9 ( 0

1214 ( 11 8.3 ( 0

15.4 ( 0.2

164.8 ( 1.5

29.5 ( 0.6

57.6 ( 0.9

39.1 ( 0.9

17.1 ( 1.5

18.6 ( 0.7 nd

15.8 ( 2.1

47.8 ( 2.0

13.6 ( 0.5

37.9 ( 3.1

1019 ( 12

3183 ( 25

111.4 ( 2.0 39.8 ( 0.5

88.8 ( 1.6

573.1 ( 0.8

nd

34.5 ( 5.1

Seng Green Chorok

50.5 ( 0.7

8.9 ( 0.5

36.1 ( 2.6

28.1 ( 0.8

6.6 ( 0.0

1199.0 ( 23.3 7.8 ( 0.2

24.5 ( 0.5

104.9 ( 1.9

19.2 ( 0

46.7 ( 1.0

36.0 ( 0.8

20.4 ( 0.5

30.7 ( 0.6 nd

4.8 ( 0.2

165.7 ( 3.1

14.5 ( 0.6

321.2 ( 7.9

434.2 ( 10.2

3347 ( 1

100.3 ( 3.1 21.4 ( 0.6

54.0 ( 1.4

861.6 ( 0.1

77.1 ( 1.3

40.4 ( 1.0

Tiger

24.4

381.6

2828.4

212.9

21.7 ( 0.6

nd

23.6 ( 0.9

9.9 ( 0.4

27.0 ( 0.6

28.9 ( 2.6

5.2 ( 0.1

282.9 ( 0.8 3.7 ( 0.8

6.0 ( 0.2

71.9 ( 0.4

19.7 ( 0.2

22.4 ( 0.2

17.8 ( 0.2

16.4 ( 4.2

8.5 ( 0.1 nd

7.2 ( 0.6

58.9 ( 3.4

5.3 ( 0.2

nd

207.9 ( 0.7

1591 ( 4

30.4 ( 0.3 10.8 ( 0.1

21.6 ( 0.4

288.6 ( 3.1

nd

41.5 ( 2.1

Yo-yo

25.1

661.1

3063.0

277.7

27.2 ( 1.3

nd

25.6 ( 0.3

17.2 ( 0.4

33.4 ( 1.2

40.7 ( 0.3

3.6 ( 0.2

534.7 ( 6.3 nd

10.1 ( 0.3

83.8 ( 0.8

25.1 ( 0.9

30.1 ( 0.2

26.0 ( 0.1

21.1 ( 0

16.2 ( 0.4 72.5 ( 0.9

5.8 ( 0.4

37.4 ( 0.6

8.8 ( 0

24.9 ( 0.3

403.9 ( 1.3

1235.1 ( 7.1

63.5 ( 0.7 17.1 ( 0.1

41.5 ( 0.4

208.7 ( 0.3

nd

49.0 ( 2.9

Koko

25.0

601.3

2874.0

191.5

24.7 ( 1.1

nd

21.4 ( 2.1

nd

30.2 ( 0

41.4 ( 0.4

5.0 ( 0.1

485.2 ( 8.4 nd

9.1 ( 0.9

42.8 ( 0.2

22.5 ( 2.9

28.7 ( 2.0

20.1 ( 1.3

24.1 ( 1.5

nd nd

14.3 ( 0.5

70.9 ( 1.6

9.6 ( 0.3

22.8 ( 6.8

221.1 ( 3.1

1471 ( 26

32.2 ( 0.6 11.7 ( 0.1

24.2 ( 0.5

194.8 ( 2.2

nd

46.3 ( 3.5

Dotori

60.2

615.8

7102.6

340.3

77.3 ( 0.6

nd

nd

60.6 ( 0.7

33.0 ( 0.3

29.7 ( 1.1

6.3 ( 0.2

406.0 ( 1.9 7.2 ( 0

6.9 ( 0.2

126.1 ( 0.8

19.3 ( 0.1

40.4 ( 0.4

31.1 ( 0.5

25.2 ( 0.6

14.6 ( 0.3 nd

12.8 ( 0.2

131.4 ( 0.9

10.3 ( 0

51.7 ( 1.5

1129 ( 4

3871 ( 15

62.8 ( 0.1 44.0 ( 0.1

62.7 ( 0.2

749.8 ( 2.7

57.9 ( 0

35.6 ( 0.3

Mini Chal

Amino acid abbreviation follow IUPAC standard; values are average (mg/100 g dry wt) ( SD (n = 2); nd, not detected. b Sum essential (indispensable) AA = sum of His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val; sum all AA = sum 20 amino acids; sum metabolites = sum of p-Ser, o-Pea, L-Cit, β-Ala, 4Abu, EtNH2, Hyl, Me-His, and L-Car.

a

1811 ( 26

322.5 ( 2.5

2245 ( 10

473.9 ( 1.2

L-Asn

nd

102.4 ( 1.8 13.4 ( 0.6

80.3 ( 0.2 17.6 ( 0.1

nd

44.9 ( 0.6

71.7 ( 0.4

L-Thr

L-Ser

282.2 ( 3.1

448.6 ( 4.7

L-Asp

38.6 ( 1.4

28.0 ( 1.9

39.4 ( 0.2

36.5 ( 0.2

p-Ser

Rapide

Starbuck

o-Pea

AA

Table 3. Concentrations of 20 Free Amino Acids (AA) and 8 AA-Derived Metabolites (in Bold Font) in 11 Tomato Fruits (Table 1)a

Journal of Agricultural and Food Chemistry ARTICLE


12806

79.1 ( 0.2

100.0 ( 0.3

122.9 ( 1.3

133.2 ( 0

L-Ser

34.2 ( 0.7

40.2 ( 0.5

42.1 ( 0.5

37.4 ( 1.0

120.2 ( 0.5 8.9 ( 1.0

561.1 ( 0.7

10.4 ( 0.2

19.5 ( 1.2

45.6 ( 2.9

57.9 ( 0.5

58.6 ( 0.1

47.5 ( 1.4

188.1 ( 1.7 15.0 ( 1.0

679.1 ( 1.7

10.9 ( 0.1

L-Cys

L-Met

L-Ile

L-Leu

L-Tyr

β-Ala

4Abu

Trp

L-Arg

701.3

5238.8

823.6

sum essential AAb

sum all AAb

sum metabolitesb

821.7

4463.9

472.8

46.2 ( 0.2

79.6 ( 1.8

L-Car

L-His

60.3 ( 0.5 181.5 ( 2.8

nd

57.9 ( 0.1

89.3 ( 0.1

nd

47.6 ( 3.7

57.1 ( 0.3

nd

88.4 ( 0.5 42.4 ( 0.6

Me-His

L-Lys

Hyl

EtNH2

L-Phe

nd

28.4 ( 0.1

39.6 ( 0.9

L-Val

L-Ala

nd

nd

23.7 ( 0.3 231.3 ( 0.1

1099

5774.4

665.7

87.1 ( 0.4

75.3 ( 0.6 214.4 ( 4.5

nd

83.0 ( 1.6

59.3 ( 1.5

nd

11.2 ( 0.1

767.2 ( 7.4

171.0 ( 2.6 17.0 ( 0.8

45.4 ( 1.3

58.5 ( 1.6

60.6 ( 1.2

44.1 ( 2.0

25.3 ( 1.0

42.9 ( 0.4

34.0 ( 0.1 397.5 ( 3.9

43.8 ( 4.2

nd

33.5 ( 0.4 354.4 ( 9.4

L-Pro

L-Gly

L-Glu

50.5 ( 0.9 2319 ( 23

35.7 ( 0

1797 ( 4

45.9 ( 0.1

2022 ( 1

L-Asn

146.74 ( 0.6

119.1 ( 1.2

903.7 ( 9.1

nd nd

41.5 ( 3.5

TP-3

1365

7850.5

728.4

83.0 ( 1.8

87.0 ( 0.5 256.3 ( 6.9

nd

71.7 ( 0.1

79.0 ( 0.3

nd

18.4 ( 0.4

954.4 ( 1.7

207.1 ( 1.8 17.6 ( 1.0

67.4 ( 1.5

55.6 ( 0.1

54.7 ( 0.5

50.3 ( 3.0

67.1 ( 2.3

38.0 ( 1.0

31.6 ( 0.4 214.3 ( 9.7

nd

3703 ( 1

52.7 ( 0.1

133.0 ( 0.7

145.6 ( 1.6

1406 ( 1

nd nd

57.7 ( 1.6

TP-4

1160

6213.4

886.9

91.0 ( 3.3

104.3 ( 0.3 125.2 ( 1.7

nd

103.0 ( 0.4

94.7 ( 0.8

nd

14.9 ( 0.4

881.5 ( 2.9

222.7 ( 0.2 24.6 ( 0.9

68.2 ( 0.5

76.6 ( 1.2

74.6 ( 0.8

61.3 ( 1.3

39.4 ( 1.7

55.9 ( 0.6

40.2 ( 0.1 457.0 ( 1.4

nd

2261 ( 4

70.2 ( 0.2

208.5 ( 0.8

173.6 ( 0.1

1388 ( 2

nd nd

34.3 ( 1.2

TP-5

1010

5130.3

615.8

251.3 ( 1.7

71.2 ( 1.2 180.2 ( 5.2

nd

94.8 ( 0.6

60.9 ( 1.2

nd

6.9 ( 0.1

647.0 ( 1.6

151.1 ( 1.0 38.8 ( 0.4

57.9 ( 1.3

62.8 ( 0.2

64.3 ( 1.1

46.2 ( 2.2

55.1 ( 0.7

48.4 ( 0.1

26.2 ( 0.1 357.4 ( 0.4

nd

1750 ( 4

54.2 ( 0.3

139.8 ( 1.9

141.3 ( 1.5

813.0 ( 3.0

nd 52.0 ( 1.7

30.6 ( 9.2

TP-6

1237

6266.2

695.1

68.8 ( 0.3

66.0 ( 0.8 77.8 ( 1.7

nd

73.5 ( 1.1

382.2

2119.9

230.5

24.2 ( 0.9

378.0

2080.2

203.0

21.5 ( 0.1

21.9 ( 0.1 20.7 ( 1.1

3.2 ( 0.3 27.1 ( 0.2 37.1 ( 1.4

20.6 ( 0.3 1.5 ( 0

15.7 ( 0.2

1.7 ( 0

nd

227.5 ( 3.5

52.8 ( 1.2 24.6 ( 0.3

16.4 ( 0.1

18.2 ( 0.3

22.1 ( 0.1

13.9 ( 0.4

8.6 ( 0.9

14.1 ( 0.1

7.8 ( 0 63.8 ( 1.5

nd

1002 ( 2

19.6 ( 0.2

42.5 ( 1.1

39.4 ( 1.3

316.8 ( 1.0

5.2 ( 0.2 57.0 ( 1.3

22.4 ( 0.8

TP-9

24.6 ( 0.2

13.9 ( 0.5

0.5 ( 0.1

5.6 ( 0.2 41.5 ( 1.5

3.4 ( 0.1

242.3 ( 0.1

61.6 ( 0.1 23.5 ( 0.3

17.9 ( 0.2

19.4 ( 0.1

24.9 ( 1.6

12.3 ( 1.5

9.1 ( 1.2

15.4 ( 0.8

10.2 ( 0.3 100.8 ( 1.9

12.4 ( 2.1

878.7 ( 10.5

20.8 ( 0.5

49.4 ( 0.4

41.8 ( 0.2

nd

825.0 ( 1.8

179.0 ( 0.2 80.1 ( 1.8

52.1 ( 1.4

68.1 ( 0.2

83.6 ( 0.6

35.7 ( 0.5

nd

58.9 ( 1.1

37.5 ( 0.8 491.6 ( 1.9

41.6 ( 1.2

2278 ( 5

56.0 ( 0.2

162.4 ( 1.1

130.3 ( 1.1

383.7 ( 1.4

3.7 ( 0.3 40.5 ( 2.4

1146 ( 4

19.2 ( 0.4

47.2 ( 0.3

TP-8

13.2 ( 0.5 146.7 ( 4.0

TP-7

343.6

1394.5

164.3

19.7 ( 1.1

nd 35.3 ( 1.0

18.9 ( 0.6

19.9 ( 0

17.9 ( 0.5

nd

nd

201.4 ( 0.8

45.7 ( 0.4 18.6 ( 0.2

13.9 ( 1.5

15.8 ( 0.5

24.0 ( 0.8

12.4 ( 2.8

nd

14.8 ( 0.4

9.5 ( 0.2 112.5 ( 1.3

nd

435.1 ( 3.9

15.3 ( 0.1

35.6 ( 0.8

31.7 ( 0.8

245.0 ( 0.5

nd 33.1 ( 2.0

18.4 ( 3.7

TP-10

362.2

2529.9

250.6

27.9 ( 1.0

25.4 ( 2.5 27.6 ( 0.8

nd

20.2 ( 0.6

34.5 ( 2.6

nd

nd

236.6 ( 0.7

60.2 ( 0.6 24.7 ( 0.8

22.1 ( 1.7

27.1 ( 0.1

37.2 ( 1.4

19.5 ( 2.5

41.1 ( 4.6

17.4 ( 0.1

9.8 ( 0.2 95.0 ( 0.2

nd

1152 ( 7

21.5 ( 0.2

50.8 ( 0.6

43.6 ( 0.1

497.0 ( 3.6

nd nd

38.8 ( 2.0

TP-11

419.1

1870.9

199.0

16.2 ( 0.2

17.9 ( 0.2 131.3 ( 3.3

nd

13.7 ( 0

54.5 ( 0.5

nd

nd

180.7 ( 1.4

47.3 ( 0.6 21.7 ( 1.1

28.7 ( 2.0

20.3 ( 1.7

34.2 ( 3.8

17.7 ( 1.7

38.5 ( 1.8

16.7 ( 0.8

8.0 ( 0 63.0 ( 1.1

nd

743.8 ( 2.4

15.8 ( 0.1

35.3 ( 0

31.2 ( 0.3

303.5 ( 0.9

nd nd

30.9 ( 0.1

TP-12

436.7

1639.9

140.9

10.4 ( 0.4

13.9 ( 2.0 177.9 ( 5.1

nd

12.3 ( 0.4

51.5 ( 2.6

nd

nd

170.1 ( 2.6

35.3 ( 0.1 15.8 ( 0.1

10.0 ( 0.8

13.1 ( 0.8

14.5 ( 1.3

12.4 ( 1.9

27.8 ( 5.4

13.4 ( 1.2

8.5 ( 0 77.5 ( 2.4

nd

599.3 ( 0.7

11.5 ( 0.1

31.1 ( 0.2

26.0 ( 0.4

286.2 ( 2.3

nd nd

21.4 ( 2.0

TP-13

TP-113 are commercial processed tomato products shown in Table 2. Values are average (mg/100 g dry weight) ( SD (n = 2); amino acid abbreviations follow IUPAC standard; nd, not detected; Gln and Cit were not present in any of the extracts. b Sum of essential (indispensable) AA = sum of His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val; sum all AA = sum of 19 amino acids; sum metabolites = sum of p-Ser, Tau, o-Pea, β-Ala, 4Abu, EtNH2, Hyl, Me-His, and L-Car.

a

853.9 ( 2.4

979.4 ( 1.0

L-Thr

22.6 ( 2.4

30.0 ( 1.6

L-Asp

Tau o-Pea

nd nd

TP-2

TP-1

nd nd

p-Ser

AA

Table 4. Concentration of 19 Free Amino Acids (AA) and 9 Metabolites (in Bold Font) in 13 Commercial Processed Tomato Products (Table 2)a




ARTICLE

Figure 2. HPLC amino acids in the extract of Rapide tomato fruit (A) and tomato ketchup, TP-8 (B).

contain the following metabolites, which have either been formed post-translationally from amino acids or independently synthesized in the plant: β-alanine (β-Ala), 4-aminobutyric acid (4Abu), carnitine (Car), citrulline (Cit), ethanolamine (EtNH2), hydroxylysine (Hyl), N-methyl-histidine (Me-His), o-phosphoethanolamine (o-Pea), phosphoserine (p-Ser), and taurine (Tau). No significant differences were found in the sum of N-containing compounds between groups. This was due in part to the large variation in the content of many of these compounds. Except for 4Abu, L-Car, o-Pea, and Hyl, none contributed >2% to the total free amino acids. 4Abu, the third most abundant free amino acid, was found consistently in all samples. There was no significant difference in the content of 4Abu, but there was more variation in the fresh than in processed samples. Levels ranged from 5.7% (Mini Chal) to 21.5% (Ho-young). L-Car, the next most abundant metabolite, was present in all processed samples, but in only two of the fresh tomatoes. Hyl was found in all of the samples. The amounts of L-Car and Hyl were significantly higher in juice than in the other samples. o-Pea was found in some samples but not others. None was found in any of the juices or canned tomatoes. The three ketchups, the sauce, and paste all contained o-Pea. p-Ser and β-Ala were present in all samples in variable amounts. Met-His was present in all of the fresh tomatoes and the ketchups, but was absent in all canned products and juices. L-Cit was present only in fresh tomatoes at a level not exceeding 0.5%. Low levels of EtNH2 were present in 10 of the 11 fresh tomatoes and in 3 processed samples: tomato paste and two ketchups. Low levels of Tau were present in the same processed samples, but was absent in the others. These patterns of the distribution of the

amino acid metabolites imply that some of the metabolites are formed and some are degraded by different processing conditions used to prepare tomato-based foods. In related studies Meher et al.14 validated a GC method for determining free and protein-bound amino acids and metabolites in red tomato fruit. These authors suggest that a large database on amino acids and metabolites for tomato genotypes might be a useful tool in quality breeding to improve resistance and quality of functional food. Identification of Phenolic Compounds in Tomatoes and Processed Tomato Products. Structural identification of individual compounds in extracts was performed by comparing the HPLC retention times and the UVvis and mass spectra data (Figures 3 and 4; Tables 5 and 6) with available standards and with studies from other investigators.3,7 The 11 fruits contained the following 11 phenolic compounds: caffeic acidhexose isomer (I) (CHI), caffeic acidhexose isomer (II) (CHII), 3-caffeoylquinic acid (3-CQA), 5-caffeoylquinic acid (5-CQA), caffeoylquinic acid isomer (CQAI), quercetin-3-apiosylrutinoside (Q3AR), quercetin-3-rutinoside (Q3R), dicaffeoylquinic acid (di-CQA), tricaffeoylquinic acid (tri-CQA), naringenin chalcone (NGC), and naringenin (NG). We did not have a standard for 3-CQA. We established that this compound exists in tomatoes (peak 3 in the HPLC chromatogram) on the basis of the HPLC, MS, and MS/MS measurements and the related observations reported by Moco et al.15 In the MS and MS/MS data, we could find no clear evidence for the presence of kaempferol, ferulic, sinapic, and pcoumaric acid derivatives in the analyzed samples as found in 12807



Figure 3. HPLC chromatograms of phenolic compounds in Dotaerang Gold (A), Yo-yo (B), and Koko (C) Korean tomato fruits. Peaks: 1, CHI; 2, CHII; 3, 3-CQA; 4, 5-CQA; 5, CQAI; 6, Q3AR; 7, Q3R; 8, diCQA; 9, tri-CQA; 10, NGC; 11, NG.

other tomato varieties by Ferreres et al.16,17 Peaks 10 and 11 showed the same MS and MS/MS ion fragments. However, the UVvis spectra and retention times were different (naringenin chalcone, RT = 62.70 min, λmax = 366 nm; naringenin, RT = 63.44 min, λmax = 288 nm). On the basis of these results and mass spectral data of standard naringenin, peak 10 was identified as naringenin chalcone and peak 11 as naringenin.15 Naringenin chalcone was not detected in any processed tomato products. Peaks 1215 were present in chromatograms of TP-6, the tomato sauce product containing onions and garlic. Peak 15 was also found in TP-13, a tomato juice. No other samples included these peaks. Peak 12 showed a [M H] ion of m/z 625.3 with retention time at 38.92 min. The fragment ion of m/z 301.1 (quercetin moiety) in MS/MS was produced by the natural loss of two glucosyl units ([M H] m/z 625.3 463.2 = 162.1; [M H] m/z 463.2 301.1 = 162.1). From these results and related data,3 peak 12 was identified as quercetin-3,40 -diglucoside. Peak 13 showed a [M H] ion of m/z 563.3 with a retention time at 47.19 min on HPLC. The fragment ion of m/z 269.4 (apigenin moiety) in MS/MS was produced by the loss of hexose (162 Da) and pentose (131.9 Da). From these results and related data,18 peak 13 was identified as apigenin-glucosidepentoside. Peak 14 showed a [M H] ion of m/z 463.3 with a retention time at 48.96 min on HPLC. The fragment ion of m/z 301.1 (quercetin moiety) in MS/MS was produced by the loss of

ARTICLE

Figure 4. HPLC chromatograms of phenolic compounds in 6 of 13 commercial tomato products. Peaks: 1, CHI; 2, CHII; 3, 3-CQA; 4, 5-CQA; 5, CQAI; 6, Q3AR; 7, Q3R; 8, di-CQA; 9, tri-CQA; 11, NG; 12, Q3,40 G; 13, AGP; 14, Q40 G; 15, RA. Peak 10, NGC, was not detected in any of the processed tomato products.

a glucosyl unit (162 Da). These MS/MS data confirmed the assigned structure to quercetin-40 -glucoside. Peak 15, with a retention time at 50.74 min, a [M H] ion of m/z 359.2, and the fragment ion of m/z 161.0 in MS/MS, was identified as rosmarinic acid. Because these four compounds were not found in any of the fresh tomatoes and were present in only two processed products, one of which contains onions and garlic, we suspect that they are not derived from the tomatoes. It is more likely they are contributions from the added seasonings, such as onions. Thus, these compounds were not included in the analysis of tomato phenolics described below. Phenolic Content of Fresh Tomatoes and Tomato-Based Food. Tables 7 and 8 list the individual and total contents of phenolics in extracts of the tomato samples separated by HPLC (Figures 3 and 4). Their values range widely. Total phenolics, excluding onion-like compounds, in fresh tomatoes ranged from 73.4 to 627.5 mg/100 g dry wt. The corresponding range from 22.2 to 100.5 mg/100 g dry wt in processed tomatoes was both lower and wider, indicating that processing may adversely affect phenolic levels. The broad range of phenolic content is noteworthy for NGC. NGC is present in very large amounts in some 12808



ARTICLE

Table 5. Phenolic Compounds Identified by LC-PDA, MS, and MS/MS in the Pulp Extracts from 11 Varieties of Tomato Fruits (Table 1) HPLC peak

a

retention timea (min)

UVvis (nm)

[M H] (m/z)

MS/MS fragments

identification

1

23.91 ( 0.01

292, 244

341.2

179.2, 135.0

caffeic acidhexose isomer (I) (CH I)

2 3

27.58 ( 0.01 28.80 ( 0.01

316, 248 326, 248

341.3 353.3

221.3, 179.2, 135.0 191.2

caffeic acidhexose isomer (II) (CH II) 3-caffeoylquinic acid (3-CQA)

4

30.31 ( 0.02

326, 248

353.0

191.0

5-caffeoylquinic acid (5-CQA)

5

31.28 ( 0.02

326, 248

353.2

273, 204, 191.0

caffeoylquinic acid isomer (CQAI)

6

40.62 ( 0.03

354, 254

741.1

300.2

quercetin-3-apiosylrutinoside (Q3AR)

7

43.31 ( 0.01

354, 256

609.1

300.1

quercetin-3-rutinoside (Q3R)

8

49.60 ( 0.01

328, 250

515.4

354.0, 173.2

dicaffeoylquinic acid (di-CQA)

9

57.00 ( 0.01

328, 250

677.1

353.0, 173.2

tricaffeoylquinic acid (tri-CQA)

10 11

62.70 ( 0.02 63.44 ( 0.01

366, 250 288, 251

271.1 271.1

151.1, 119.0 151.1

naringenin chalcone (NGC) naringenin (NG)

Average ( SD (n = 3).

Table 6. Phenolic Compounds Identified by LC-PDA, MS, and MS/MS in the Extracts from 13 Commercial Processed Tomato Products (Table 2) HPLC peak

UVvis (nm) (λmax)

[M H] (m/z)

MS/MS fragments

identification

1

23.95 ( 0.05

293, 243

341.2

179.1, 135.0

caffeic acidhexose isomer (I) (CHI)

2

27.17 ( 0.06

315, 284, 247

341.2

179.0, 135.1

caffeic acidhexose isomer (II) (CHII)

3

28.88 ( 0.07

318, 248

353.3

191.0


4

30.38 ( 0.06

326, 248

353.1

191.0


5 6

31.36 ( 0.05 40.69 ( 0.03

327, 249 352, 251

353.2 741.3

191.0 300.0

caffeoylquinic acid isomer (CQAI) quercetin-3-apiosylrutinoside (Q3AR) quercetin-3-rutinoside (Q3R)

7

43.35 ( 0.01

354, 254

609.3

300.1

8

49.68 ( 0.01

329, 250

515.3

353.0, 179.0, 173.2

dicaffeoylquinic acid (di-CQA)

9

57.12 ( 0.04

328, 250

677.4

353.0, 173.2

tricaffeoylquinic acid (tri-CQA) naringenin (NG)

10

a

retention timea (min)

nd

naringenin chalcone (NGC)

11

63.49 ( 0.04

288, 252

271.1

151.0

12

38.92 ( 0.03

342, 250

625.3

463.2, 301.1

quercetin-3,40 -diglucoside (Q3,40 G)

13 14

47.19 ( 0.01 48.96 ( 0.01

336, 266 361, 250

563.3 463.3,

269.4 301.2

apigenin-glucoside-pentoside (AGP) quercetin-40 -glucoside (Q40 G)

15

50.74 ( 0.21

329, 249

359.2

161.0

rosmarinic acid (RA)

Average ( SD (n = 3); nd, not detected.

fresh tomato samples but is absent in others. When present, the levels often approach the combined levels of all other phenolics. Ho-yong is an exception, as NGC is present only as a minor component. NG was present only in samples that had exceptionally high levels of NGC. The processed products had lower but still significant levels of NGC and no NG. Q3R and 5-CQA were present in all samples in significant quantity. Q3R was the predominant phenolic compound in 12 of the 13 processed products. One ketchup sample contained mostly 5-CQA. Q3R plus 5-CQA comprises about 50% of the total phenolics in processed products. In the fresh tomato samples, these two compounds were generally the most prevalent phenolics after NGC. However, content was more variable than for processed products, ranging from 16 to 67% of total amounts. In fresh tomatoes, the sum of Q3R, 5-CQA, and NGC comprised 4392% of the total. The small tomatoes are richer in phenolic content per unit weight (p < 0.05) than the large ones. Total Phenolic Content of Tomatoes and Tomato-Based Foods. The first column in both Tables 9 and 10 lists the total phenolic content in terms of gallic acid equivalents of 11 freeze-dried

tomato extracts and 13 processed tomato products, respectively, determined by the FolinCiocalteu colorimetric method. The values (in mg/100 g dry wt) in fresh tomatoes range from 320.0 (Rapide) to 663.1 (Tiger), a 2.07-fold difference from lowest to highest value. The values (in mg/100 g dry wt) of processed tomato products range from 344.4 (TP-10) to 606.8 (TP-4), a 1.76-fold difference from lowest to highest value. The total phenolic content values obtained according to the FolinCiocalteu method are much higher than those obtained by summing the individual phenolics determined by HPLC. This difference was more pronounced in the processed tomato products than in the fresh ones, for which the total polyphenol content was 822 times higher than the sum of individual phenolics. For fresh tomatoes, the range was from 1 to 8 times higher. The tomato variety Koko was the only sample in which the two values matched. The two measures of phenolics were only weakly correlated with each other, with an r value of 0.5 (p < 0.05). A possible explanation is that the reagent likely reacts with other components, both naturally present in tomatoes and formed during processing. 12809


2.6 ( 0.0

6.0 ( 0.1

Mini Chal

10.3 ( 0.1

4.4 ( 0.0

2.2 ( 0.0

5.1 ( 0.1

2.3 ( 0.0

6.3 ( 0.1 5.0 ( 0.1

2.7 ( 0.0

4.32 ( 0.1

3.7 ( 0.1

2.3 ( 0.1

3-CQA

39.7 ( 0.2

68.6 ( 0.1

80.1 ( 0.2

80.5 ( 0.2

60.0 ( 0.2

12.4 ( 0.1 45.7 ( 0.1

53.4 ( 0.1

44.0 ( 0.1

27.6 ( 0.3

8.5 ( 0.0

5-CQA

15.0 ( 0.1

16.5 ( 0.1

8.4 ( 0.0

15.7 ( 0.2

5.4 ( 0.1

7.1 ( 0.0 9.2 ( 0.1

8.2 ( 0.0

8.5 ( 0.1

7.4 ( 0.1

5.1 ( 0.1

CQAI

8.4 ( 0.0

10.3 ( 0.0

11.1 ( 0.0

18.9 ( 0.0

7.7 ( 0.0

4.8 ( 0.2 23.4 ( 0.1

12.9 ( 0.1

11.2 ( 0.0

4.7 ( 0.1

5.2 ( 0.0

Q3AR

39.3 ( 0.2

9.0 ( 0.2

9.1 ( 0.1 30.8 ( 0.1

57.9 ( 0.3

20.1 ( 0.1

4.4 ( 0.0

2.6 ( 0.0 13.1 ( 0.1

5.3 ( 0.0

3.8 ( 0.1

3.0 ( 0.0

1.7 ( 0.0

di-CQA

45.3 ( 0.0

88.8 ( 0.3

15.3 ( 0.1

18.4 ( 0.1 28.3 ( 0.1

17.3 ( 0.1

13.5 ( 0.0

3.6 ( 0.0

3.9 ( 0.1

Q3R

9.7 ( 0.1

23.1 ( 0.1

5.1 ( 0.1

15.0 ( 0.1

1.6 ( 0.0

1.8 ( 0.0 11.2 ( 0.1

2.3 ( 0.0

1.6 ( 0.0

2.0 ( 0.0

1.8 ( 0.0

tri-CQA

248.8 ( 1.1

323.6 ( 1.0

453.4 ( 0.5

227.8 ( 0.5

nd

50.0 ( 0.2 244.0 ( 1.0

0.9 ( 0.0

nd

nd

43.5 ( 0.2

NGC

3.1 ( 0.5

4.4 ( 0.0

3.8 ( 0.1

2.0 ( 0.1

nd

nd 2.9 ( 0.2

nd

nd

nd

nd

NG

391.9

552.2

627.5

484.9

111.7

114.8 397.4

114.1

101.1

73.4

79.3

sum

12810

0.3 ( 0.0

0.7 ( 0.0

0.3 ( 0.0

nd

1.7 ( 0.0

3.9 ( 0.0

1.9 ( 0.1

2.1 ( 0.0

TP-10

TP-11

TP-12

TP-13

0.5 ( 0.0

0.8 ( 0.0

2.0 ( 0.1

4.7 ( 0.0

TP-8

TP-9

0.8 ( 0.0

5.3 ( 0.1

3.5 ( 0.1

4.7 ( 0.2

0.6 ( 0.2

2.5 ( 0.0

1.0 ( 0.0

8.9 ( 0.1

4.5 ( 0.0

10.7 ( 0.1 11.7 ( 0.1

14.1 ( 0.1

1.1 ( 0.2

1.5 ( 0.0

1.6 ( 0.1

3.2 ( 0.1 5.8 ( 0.0

4.8 ( 0.1

1.2 ( 0.1

1.2 ( 0.1

1.8 ( 0.0

0.8 ( 0.0

2.5 ( 0.0

1.6 ( 0.0

3.6 ( 0.0 4.5 ( 0.1

3.5 ( 0.0

3.9 ( 0.1

5.2 ( 0.0

1.7 ( 0.1

2.7 ( 0.0

CQAI

2.7 ( 0.0

2.2 ( 0.1

2.9 ( 0.0

1.5 ( 0.0

1.4 ( 0.0

3.6 ( 0.0

6.0 ( 0.3 8.1 ( 0.2

1.4 ( 0.1

10.2 ( 0.2

4.3 ( 0.0

2.8 ( 0.0

nd

Q3AR

15.5 ( 0.1

10.7 ( 0.1

5.9 ( 0.1

11.0 ( 0.1

4.6 ( 0.1

15.2 ( 0.0

30.1 ( 0.4 40.7 ( 0.1

10.1 ( 0.0

32.3 ( 0.2

22.8 ( 0.1

16.8 ( 0.0

10.2 ( 0.0

Q3R

0.4 ( 0.0

0.5 ( 0.0

0.4 ( 0.0

0.3 ( 0.0

1.0 ( 0.0

0.6 ( 0.0

4.1 ( 0.1 1.6 ( 0.0

nd

0.7 ( 0.0

3.4 ( 0.0

nd

nd

di-CQA

0.6 ( 0.0

1.1 ( 0.0

0.8 ( 0.0

0.3 ( 0.0

0.8 ( 0.0

0.6 ( 0.0

2.4 ( 0.1

2.4 ( 0.1

nd

4.4 ( 0.1

1.1 ( 0.0

8.3 ( 0.0

3.6 ( 0.1 19.9 ( 0.2 21.0 ( 0.2

nd

5.0 ( 0.2

8.2 ( 0.4

4.3 ( 0.1

2.4 ( 0.1

NGC

1.4 ( 0.0 1.6 ( 0.0

nd

0.7 ( 0.1

nd

nd

tri-CQA

nd

nd

nd

nd

nd

nd

16.3 ( 0.0 nd

nd

nd

nd

nd

nd

Q3,40 G

nd

nd

nd

nd

nd

nd

44.3 ( 0.9 nd

nd

nd

nd

nd

nd

AGP

nd

nd

nd

nd

nd

nd

23.3 ( 0.3 nd

nd

nd

nd

nd

nd

Q40 G

2.9 ( 0.1

nd

nd

nd

nd

nd

13.1 ( 0.3 nd

nd

nd

nd

nd

nd

RA

33.9

24.4

22.2

23.8

27.3

38.5

182.0 100.5

31.4

69.5

68.7

35.3

27.0

sum

Values are average (mg/100 g of dry wt) ( SD (n = 3). nd, not detected. The values of CHI, CHII, 3-CQA, 5-CQA, CQAI, di-CQA, tri-CQA, and RA are expressed as 5-CQA. Q3,40 G, Q3AR, AGP, and Q40 G are expressed as Q3R. NGC is expressed as NG. Abbreviations: CHI, caffeic acidhexose isomer (I); CHII caffeic acidhexose (II); 3-CQA, 3-caffeoylquinic acid; 5-CQA, 5-caffeoylquinic acid; CQAI, caffeoylquinic acid isomer; Q3,40 G, quercetin-3,40 -diglucoside; Q3AR, quercetin-3-apiosylrutinoside; Q3R, quercetin-3-rutinoside; AGP, apigenin-glucoside-pentoside; Q40 G, quercetin40 -glucoside; di-CQA, dicaffeoylquinic acid; tri-CQA, tricaffeoylquinic acid; RA, rosmarinic acid; NGC, naringenin chalcone.

a

7.6 ( 0.1

3.9 ( 0.1

nd

2.2 ( 0.1 1.1 ( 0.0

1.3 ( 0.1

3.8 ( 0.1 4.4 ( 0.0

TP-5

nd

TP-6 TP-7

9.1 ( 0.1

4.9 ( 0.1

2.0 ( 0.1

3.2 ( 0.0

3.4 ( 0.1

TP-3

5.0 ( 0.0 2.9 ( 0.1

TP-4

3.4 ( 0.0 2.6 ( 0.3

nd

0.9 ( 0.0

3.3 ( 0.1

5-CQA

3.3 ( 0.1

3-CQA

TP-1

CHII

TP-2

CHI

tomato product

Table 8. Concentration of Phenolic Compounds (Table 6) in the Extracts from 13 Commercial Processed Tomato Products (Table 2)a

a

Values are average (mg/100 g of dry wt) ( SD (n = 3). nd, not detected. The values of CHI, CHII, 3-CQA, 5-CQA, CQAI, di-CQA, and tri-CQA are expressed as 5-CQA. Q3AR is expressed as Q3R. NGC is expressed as NG. Abbreviations: CHI, caffeic acidhexose isomer (I); CHII caffeic acid-hexose (II); 3-CQA, 3-caffeoylquinic acid; 5-CQA, 5-caffeoylquinic acid; CQAI, caffeoylquinic acid isomer; Q3AR, quercetin-3-apiosylrutinoside; Q3R, quercetin-3-rutinoside; di-CQA, dicaffeoylquinic acid; tri-CQA, tricaffeoylquinic acid; NGC, naringenin chalcone; NG, naringenin.

1.7 ( 0.0

8.6 ( 0.0 2.8 ( 0.0

13.1 ( 0.0

Seng Green Chorok

Yo-yo

7.3 ( 0.0

1.9 ( 0.0 2.4 ( 0.0

10.2 ( 0.0 10.4 ( 0.5

Amoroso Tiger

9.8 ( 0.1

1.2 ( 0.0 4.2 ( 0.0

9.3 ( 0.0

Koko

3.2 ( 0.1 1.8 ( 0.0

11.0 ( 0.1

Dotaerang Gold

Ho-yong

Dotori

1.7 ( 0.0 2.7 ( 0.0

5.6 ( 0.0

18.7 ( 0.2

Starbuck

CHII

CHI

Rapide

tomato variety

Table 7. Concentration of Phenolic Compounds (Table 5) in the Extracts from 11 Varieties of Tomato Fruits (Table 1)a




ARTICLE

Table 9. Total Phenolic Content (FolinCiocalteu) and Antioxidant Activity by FRAP and DPPH in Extracts of 11 Varieties of Tomato Fruitsa

Table 10. Total Phenolic Content (FolinCiocalteu) and Antioxidant Activity by FRAP and DPPH in Extracts of 13 Commercial Processed Tomato Productsa

FRAP value total polyphenolb tomato variety

(mg/100 g dry wt)

(mM Fe2+/ 100 g dry wt)

FRAP value DPPH value,

tomato b

IC50 (μg/mg)

product

total polyphenolc

(mM Fe2+/

DPPH value,

(mg/100 g dry wt)

100 g dry wt)

IC50 (μg/mg)

Starbuck

484.2 ( 35.2

1.5 ( 0.6

439.6 ( 3.9

TP-1

454.6 ( 34.0

0.2 ( 0.9

550.3 ( 4.5

Rapide

320.0 ( 11.7

1.7 ( 0.5

301.6 ( 1.2

TP-2

465.1 ( 5.2

0.1 ( 0.5

720.9 ( 5.2

Dotaerang Gold Ho-yong

430.6 ( 18.5 445.8 ( 3.5

1.6 ( 0.1 1.2 ( 0.2

419.4 ( 4.8 496.8 ( 5.7

TP-3 TP-4

555.8 ( 14.2 606.8 ( 45.4

1.7 ( 0.1 1.9 ( 0.2

371.7 ( 1.7 366.3 ( 4.9

Amoroso

489.1 ( 5.9

2.1 ( 0.4

434.3 ( 3.8

TP-5

485.5 ( 10.2

0.4 ( 0.7

585.0 ( 6.6

Tiger

663.1 ( 8.4

4.5 ( 0.7

194.2 ( 5.2

TP-6

482.6 ( 5.9

0.6 ( 0.8

432.0 ( 1.0

Seng Green Chorok

470.6 ( 40.2

1.7 ( 0.6

189.1 ( 2.2

TP-7

591.9 ( 42.4

1.4 ( 0.8

526.4 ( 5.8

Yo-yo

571.7 ( 5.2

3.2 ( 0.4

145.0 ( 4.4

TP-8

350.6 ( 3.0

1.2 ( 1.0

1552.6 ( 0.4

Koko

462.8 ( 10.6

2.0 ( 0.7

260.9 ( 1.2

TP-9

371.4 ( 7.5

0.5 ( 0.9

1167.2 ( 11.7

Dotori

560.3 ( 8.9

4.1 ( 2.3

159.2 ( 3.9

TP-10

344.4 ( 6.3

0.5 ( 1.2

2940.6 ( 21.3

Mini Chal standard BHA

538.2 ( 6.4

2.8 ( 0.4

280.1 ( 4.8 4.0 ( 0.2

TP-11 TP-12

380.8 ( 4.8 375.9 ( 9.5

0.5 ( 0.6 0.7 ( 0.4

1227.6 ( 13.9 813.1 ( 8.8

TP-13

423.1 ( 11.4

a Values are average ( SD (n = 3). b Total polyphenol content is expressed as gallic acid equivalent.

standard BHA

0.2 ( 0.4

568.7 ( 0.3 4.0 ( 0.2

Values are average ( SD (n = 3). b TP-113 are tomato products shown in Table 2. c Total polyphenol content is expressed as gallic acid equivalent. a

Antioxidative Activity. Reactive oxygen species (ROS) have been postulated to contribute to the causes of chronic diseases, including cancer and arteriosclerosis, through oxidative damage of enzymes and tissues. Phenolic compounds can reduce ROS levels by trapping reactive free electrons (free radicals).19 Antioxidative activity is generally measured by two distinct methods: direct determination of oxidative damage and indirect determination of levels of known reactive species.19 We determined the antioxidative effects of the 11 tomato fruit by one direct (FRAP) and one indirect (DPPH) method. FRAP monitors the reduction of a ferric ion complex to the ferrous form.6 Addition of antioxidants reduces the production of the oxidation products and changes the color of the solution. The FRAP values for tomatoes (Table 9) range (in mM Fe2+/100 g dry wt) from 1.2 (lowest effect) for Ho-yong fruit to 4.5 (highest effect) for Tiger, a 3.75-fold variation from highest to lowest activity. The corresponding values for processed tomato products (Table 10) range from 1.2 (TP-8) to 1.9 (TP-4). The table also shows that values for four foods were negative, suggesting pro-oxidant effects. The DPPH method is a convenient and rapid indirect assay for screening plant samples for radical scavenging activity known to be involved with oxidation.20 As antioxidants react with the stable, highly colored free radical DPPH, the absorbance of the solution decreases. Table 9 lists the IC50 values for the antiradical activities as well as values for the reference antioxidant BHA. The calculated IC50 values (in μg/mg) range from 145.0 (highest activity) for Yo-yo fruit to 496.8 for Ho-yong fruit (lowest activity), about a 3.43-fold variation from the lowest to highest values. The data also show that the IC50 value for BHA of 4.0 is much lower (the BHA shows higher activity) than the corresponding values for the 11 tomato fruits. These results indicate that the antioxidative effects of different tomato varieties determined by two independent methods vary widely, that the negative values obtained by FRAP imply prooxidant effects, and that the standard antioxidant BHA is about 50100 times more active than the tomato extracts. We have no obvious explanation for the apparent discrepancy of the results obtained by the two methods.

In related studies, (a) Martínez-Valverde et al.21 found that the antioxidant potential of tomatoes varied with variety and the assay method; (b) García-Alonse et al.22 found that, except for vitamin C, storage and packaging did not affect antioxidant compounds of tomato juices; (c) Durazzo et al.23 reported that there were no differences in biological effects in cell assays between organic and conventional tomatoes and that bioactive compounds could have a pro-oxidant effect at concentrations >17 μM; and (d) Vallverdu-Queralt et al.24 found that processing of tomatoes results in decreases in both antioxidant and phenolic profiles. Cancer Prevention. In previous studies we reported that (a) the tomato glycoalkaloid α-tomatine and the potato glycoalkaloids α-chaconine and α-solanine were strong inhibitors of human cancer cells;25,26 (b) the inhibition of cancer cells by high α-tomatine green tomato extracts was much higher than that by red tomato extracts lacking α-tomatine;3,27 and (c) long-term feeding of low amounts of α-tomatine protected fish (trout) against dibenzo[a,l]pyrene-induced colon cancer.28 To test the anticarcinogenic potential of red tomatoes lacking tomatine, in the present study we examined the inhibition of lung (A549) and lymphoma (U937) cancer cells at three concentrations and compared them to normal cell lines of liver (Chang) and lung (Hel299). The inhibitions were much lower than the values for tomatine-rich green tomatoes.3 The tomatoes and tomato products induced growth in the Chang normal cell line at all concentrations (Figure 5A,B). The normal Hel299 cell line was not significantly affected, and the lung cancer cell line, A549, was mildly inhibited by the tomato extracts. The results of the lymphoma cell line, U947, varied by product. Some of the extracts caused an initial growth enhancement followed by inhibition at higher concentrations, especially for the fresh tomatoes. Overall, the processed tomato products were more inhibitory than the fresh, but tomato paste (TP-7) was growth stimulating at all three concentrations. Stimulation of cell growth 12811



ARTICLE

Figure 5. Cell inhibition by three concentrations of (A) fresh tomato extracts and (B) processed tomato products. Chang and Hel299 are normal cell lines. A549 and U947 are lung cancer and lymphoma cell lines, respectively.

may be due to nutrients in the extracts in combination with low levels of, or low susceptibility to, active compounds. The growthsimulating extracts appear to be more dose-dependent, indicating that those samples contain rate-limiting amounts of the active component(s). Some of the extracts with the highest response, such as the three ketchups and the paste, were not dose-dependent, indicating that the active component(s) reached a maximum effective concentration in the 10 μg/mL sample. We found poor, or no, correlations between inhibitory activity and measured components. The results of our previous and present studies suggest that consumption of both green and red tomatoes may have an additive anticarcinogenic effect compared to that of green or red tomatoes alone. Because green tomatoes turn red rapidly after harvest, to our knowledge high-tomatine green tomatoes are available only as pickled green tomatoes.3 Our findings are mostly consistent with several studies that indicate that tomato ingredients and tomato-based foods may reduce the risks of some cancers. These include the following selected recent observations: (a) naringenin induced apoptosis in

human lung cancer A549 cells;29 (b) naringenin chalcone suppressed inflammation and tumorigenesis in several cancers;30 (c) a dietary tomato supplement prevented prostate cancer in mice;31 and (d) tomato-based food products may help prevent prostate cancer in humans.32 Differences in the composition of bioactive compounds could explain the observations in the present and related studies33 that different tomato varieties do not exhibit the same anticarcinogenic effects in cell assays and that, unlike soy-based diets, tomato-rich diets had a minor effect on cell-signaling biomarkers associated with breast cancer risk in postmenopausal women.34 The reason results of reported studies have not always been consistent may be due to differences in the content of bioactive compounds in the tomatoes used in cell, animal, and human studies. Additional Statistical Correlations. In addition to the abovedescribed statistical treatment of the data, statistical treatment was also carried out with a combined tomatoes and tomato product data set (n = 24). To determine if the measured contents of the tomatoes and tomato products are related to observed 12812


Journal of Agricultural and Food Chemistry antioxidative or cell-inhibiting effects, we calculated the Pearson product moment correlation coefficients (r) and p < 0.05 regarded as significant. The analysis shows that antioxidative data determined by FRAP correlated with phenolic content measured by both HPLC (r = 0.744) and the FolinCiocalteu method (r = 0.750). DPPH data had a weaker inverse correlation with these measures, r = 0.447 for HPLC and r = 0.590 for FolinCiocalteu. FRAP and DPPH were inversely correlated with each other (r = 0.681). We previously reported similar results by FRAP and DPPH for jujube phenolics.13 The cells growth inhibition by tomato fruit and tomato products was weakly correlated (r) with several parameters: Chang cell inhibition was inversely correlated with CHII (0.428), CQAI (0.471), di-CQA (0.474), tri-CQA (0.504), NGC (0.533), total HPLC phenolics (0.513), and FRAP (0.446) and positively correlated with essential amino acids (0.615); Hel299 cell inhibition was correlated with 5-CQA (0.532), CQAI (0.436), Q3AR (0.418), NGC (0.408), and FRAP (0.433); A549 cell inhibition was correlated with essential amino acids (0.407); and U937 cell inhibition was inversely correlated with 3-CQA (0.543), free amino acids (0.596), FolinCiocalteu phenolics (0.438), and nitrogen-containing metabolites (0.494). It is difficult to conclude whether any of these measured parameters have an effect on inhibition of cell growth. Conclusions and Research Needs. Tomatoes and tomatobased foods possess nutritional and potential medicinal properties. The major objective of the present study was to relate the composition of the 11 tomato varieties grown and harvested under the same conditions and 13 commercial tomato-based foods to potential beneficial effects in the human diet. Free amino acid content in plant foods has an important role in nutrition because free amino acids contribute to protein nutritional quality. It is also an important factor in food safety because asparagine is the major precursor of toxic acrylamide formed during food processing and cysteine reduces its toxicological potency.35,36 Our data show that tomatoes contain variable low levels of both asparagine and cysteine. There was nearly a 3-fold difference in crude protein content among the 11 tomato varieties. This aspect assumes importance in view of the recent report that the combination of sweet corn and tomatoes produces a balanced amino acid profile found in the highest quality proteins for optimum human nutrition.37 Tomatoes with a high protein content merit inclusion in this and other food combinations. For example, the black Tiger and Seng Green Chorok tomato varieties have the highest protein contents, and the red Starbuck tomato has the highest essential free amino acid content. The evaluated tomatoes and tomato-based foods contained a large number of antioxidative phenolic compounds that together with lycopene and β-carotene, which were not determined in this study, may contribute to health benefits. We found that on a dry weight basis, the phenolic content in the processed tomato products was lower than in the fresh tomatoes. The distribution of individual phenolics also varied among tomato varieties and less so in the foods. Our findings emphasize the substantial variation in free amino acid, protein, and individual and total phenolic contents found among the test substances. The data suggest that consumers have a choice in selecting varieties and foods with a high content of phenolic compounds and high antioxidative and anticarcinogenic effects. The observed wide variation in the content of bioactive compounds in the different tomato varieties and foods implies

ARTICLE

that clinical and epidemiological studies should include the content of nutritional and bioactive ingredients.

’ AUTHOR INFORMATION Corresponding Author

*Phone: (510) 559-5615. Fax: (510) 559-6162. E-mail: mendel. [email protected].

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’ NOTE ADDED AFTER ASAP PUBLICATION This paper published November 17, 2011 with an incorrect version of Figure 2. The correct version published November 22, 2011.

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Free Amino Acid and Phenolic Contents and ... - ACS Publications

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