Phenolic profiles, antioxidant and antimutagenic activities of Solanum lycopersicum var. cerasiforme accessions from Mexico

ABSTRACT The fruit of 18 Solanum lycopersicum var. cerasiforme accessions from Mexico were evaluated for total phenolics (TP) by the Folin-Ciocalteau assay, phenolic profiles by high performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS), antioxidant activity (AoxA) by 2,2´-azino-bis(3-ethylbenzothiazolin)-6-sulfonic acid (ABTS), 2,2-diphenil-1-pycrilhydrazyl (DPPH), and oxygen radical absorbance capacity (ORAC), and antimutagenic activity (AmuA) by the Ames assay. TP was measured as Gallic Acid Equivalents (GAE) and the AoxA as Trolox Equivalents (TE). TP varied from 37 to 86 mg GAE 100 g−1 fresh weight (fw). The AoxA by ABTS (568-1187 µmol TE 100 g−1 fw) and DPPH (157-350 µmol TE 100 g−1 fw) correlated with TP and the levels of caffeoylquinic acids and rutin. The AmuA did not correlate with the levels of phenolics. Some accessions had higher AoxA and AmuA than those reported for commercial cultivars and also showed high levels of caffeoylquinic acids and rutin; thus, their consumption could have good health promoting effects.


Introduction
Tomato is one of the most widely consumed vegetables and an important source of bioactive compounds (e.g. vitamins A and C, carotenoids, and phenolics) that exhibit a broad range of health benefits. However, the development of the commercial tomato cultivars has reduced the levels of bioactive compounds compared to those found in traditional varieties and wild relatives (Adalid, Rosello, & Nuez, 2010;Boches, Peterschmidt, & Myers, 2011;Hanson et al., 2004). Genetic engineering has been successfully used to improve nutraceutical characteristics of tomato cultivars (Li et al., 2015;Luo et al., 2008), but the use of this technology is limited by the safety concerns of consumers about genetically modified organisms. Therefore, wild tomato relatives represent an important alternative source of compounds with nutritional and biological activities.
Mexico is a source of wild tomatoes, including the cherry tomato Solanum lycopersicum (S.l.) var. cerasiforme, which is derived from Solanum pimpinellifolium (Blanca et al., 2015) and adapts to a wide range of climatic and geographic conditions. However, few studies have documented the chemical composition and antioxidant potential of S.l. var. cerasiforme tomato from Mexico, with sampling limited to a small number of populations or accessions available in collections of international institutions (Adalid et al., 2010;Boches et al., 2011;Hanson et al., 2004). Specifically, there is no information about some nutraceutical properties such as antimutagenic activity of S.l. var. cerasiforme fruit. Wild and semidomesticated populations of S.l. var. cerasiforme collected from the states of Oaxaca, Guerrero, and Puebla showed significant variations in fruit quality traits such as lycopene and vitamin C content, and color parameters Crisanto-Juárez, Vera-Guzmán, Chávez-Servia, & Carrillo-Rodríguez, 2010;Juárez-López et al., 2009). Thus, further knowledge about the chemical composition and biological activities of S.l.
var. cerasiforme is required to promote its cultivation and consumption as well as its use for breeding functional tomatoes. The aim of the present study was to determine the levels of phenolic compounds and their possible relationship with the antioxidant and antimutagenic activities of S.l. var. cerasiforme accessions from Mexico.

Plant material
Eighteen accessions of Solanum lycopersicum (S.l.) var. cerasiforme from eight states of Mexico were provided by the germplasm banks of University of Guadalajara and Autonomous University of Sinaloa (Table 1). Seeds were germinated and the seedlings were grown in vertisol soil (pelluster) under irrigation conditions at the Faculty of Agronomy Field Station of the Autonomous University of Sinaloa, Culiacan Sinaloa, Mexico, during the winter-spring season of 2010-2011. Seedlings were planted in the field with a spacing of 1 m within a row and 1.6 m between rows. Water was applied every 7-14 days as needed from transplant until harvest. The total amounts of N, P, K, Ca, and Mg supplied were 129.9, 42.8, 132.3, 56.1, and 42.1 kg ha −1 , respectively. The insecticide abamectin (AGRIMEC ® 1.8% CE, Syngenta) was applied twice at 0.5 L ha −1 for whitefly control. The minimum and maximum temperatures averaged 12.5 ± 3.1 and 32.5 ± 2.6°C, respectively. A completely randomized design with three replications of five plants per accession was used. A total of 10 fruits per plant were harvested at the red-ripe stage (~9 days after breaker) as defined by Yamaguchi (1983). Yellow-fruited accessions were harvested based on fruit size and maximum color intensity. After washing, the whole fruits from each replicate were homogenized with a T18 basic ULTRA-TURRAX ® (IKA, Staufen, Germany), lyophilized and stored at −70°C until use.

Preparation of the methanol extracts
Methanol extracts were prepared according to Gómez-Romero, Segura-Carretero, and Fernández-Gutiérrez (2010) with some modifications. Lyophilized fruit tissue was ground to a fine powder and 1 g was mixed with 10 mL of methanol, sonicated for 15 min, centrifuged (28,620 × g/15 min/20°C) and the supernatant was recovered. The extraction procedure was repeated two times with the residue. Supernatants were mixed and 3 mL were used for analyses of phenolic compounds and antioxidant activity, the rest was concentrated under vacuum (40°C) with a rotary evaporator (BÜCHI R-124, Brinkmann Instruments, USA) and stored at −20°C until use.

Total phenolics (TP)
TP content was determined by the Folin-Ciocalteu reaction (Waterhouse, 2002). Methanol extract (20 µL) was mixed with 1.58 mL of distilled water and 0.1 mL of the Folin-Ciocalteu reagent, gently stirred for 5 min and added with 0.3 mL of a sodium carbonate saturated solution. The mixture was incubated in darkness for 30 min at 40°C and the absorbance was measured at 765 nm. A calibration curve of Gallic acid was prepared and the results were calculated as milligrams of Gallic acid equivalents (GAE) per 100 g on a fresh weight basis (mg GAE 100 g −1 fw). Phenolic profiles by high performance liquid chromatography (HPLC)-diode array detection (DAD) and mass spectrometry (MS) analysis HPLC analysis was according to Moco et al. (2006) with some modifications. The methanol extract was passed through a syringe filter (polyvinylidene fluoride membrane, 0.45 µm, HPLC certified, Thermo Scientific, Germany) and 0.005 mL were injected into a HPLC-DAD system (ACCELA, Thermo Scientific, USA). The separation was carried out in a Fortis C18 HPLC column (3 µm, 50 × 2.1 mm) (Fortis Technologies Ltd, UK) using a linear gradient of 1% (v/v) formic acid (A) and acetonitrile (B), 0.5 to 60% of B in 40 min at a flow rate of 0.2 mL min −1 . The detection was done at 280, 320, and 350 nm. Protocatechuic acid and daidzein were used as internal standards. The identification of phenolics was based on the UVspectra, MS fragmentation, and by comparisons with MS data generated with commercial standards and reported in the literature (Gómez-Romero et al., 2010;Moco et al., 2006). Calibration curves of rutin, chlorogenic, and caffeic acid (Sigma Chemical Co., St. Louis MO, USA) were used for quantification. Flavonoids were reported as Rutin Equivalents (mg RE 100 g −1 fw) and phenolics as Caffeic Acid Equivalents (mg CAE 100 g −1 fw) or Chlorogenic Acid Equivalents (mg CGAE 100 g −1 fw). The HPLC-DAD was coupled to a mass spectrometer with an electrospray ionization source (LTQ XL, Thermo Scientific, USA). The analysis was carried out in negative mode and full scan spectra were obtained in the m/z range of 50-1500. The ions used for MS n experiments were fragmented by collision induced dissociation applying 10-45 V. The parameters of the capillary tube were 35 V and 300°C. Nitrogen and helium gases were used for drying and collision, respectively. The results were analyzed with the Xcalibur 2.2 software (Thermo Scientific, USA).

Statistical analysis
Data were analyzed by one and two way analysis of variance and the means were compared by the Fisher test (α = 0.05) by using the software STATGRAPHIC plus version 5.1 (Statistical Graphics Corporation ™ , USA). Correlation analysis was performed with PASW Statistics version 18 (SSPS Inc., Chicago, IL). The results are the mean value of three replicates.

Antioxidant and antimutagenic activities
The AoxA of the methanol extracts showed a great variability with significant differences among the 18 accessions (Table 1). ABTS values varied from 568.4 µmol TE 100 g −1 in Placharosa (Sinaloa) to 1187.3 µmol TE 100 g −1 in Tumbisca (Michoacan). The AoxA by DPPH varied from 157.2 µmol TE 100 g −1 in F. Ruiz (Chiapas) to 349.9 µmol TE 100 g −1 in Tumbisca. The ORAC values of the accessions showed almost a fivefold variation and the highest activity was observed in Villamaza (Sinaloa). The average fruit weight of the accessions varied from 2.42 to 2.64 g (Table 1) and it was not expected to affect significantly the activities and components evaluated. The methanol extracts from the 18 accessions were neither toxic nor mutagenic on the tester strain up to 1000 µg/plate (data not shown). The AmuA of these extracts was assayed at a single concentration (100 µg/plate) (Table 1) and the values showed a three-fold variation (19.7-61.1%). Rincon (Oaxaca) showed a strong inhibition of the mutagenic activity (61.1%), whereas most of the other accessions showed positive antimutagenicity (40-60%).

Phenolics content and its correlation with the antioxidant and antimutagenic activities
Total phenolics (TP) of the methanol extracts varied from 37.1 to 85.6 mg GAE 100 g −1 (Table 1) and showed positive correlations with the AoxA determined by ABTS (r = 0.80, p < 0.001) and DPPH (r = 0.64, p < 0.01) but not with the AoxA by ORAC and the AmuA.

Antimutagenic activity: concentration-response effect
The antimutagenicity of methanol extracts from 10 selected accessions was evaluated in the range 0-1000 µg/plate ( Table 4). The AmuA increased significantly with the extract concentration and showed the highest value in the accession Llanos. The AmuA varied from positive to strong (54.6-85.8%) at 250-1000 µg/plate and it was positive for most of the accessions at 100 µg/plate, whereas at 50 µg/plate this activity was negative or weak (Table 4).

Antioxidant activity
The AoxA values of the tomato accessions (Table 1) were higher than those reported for commercial varieties. For instance, the ORAC values of the accessions were higher than those reported for some tomatoes (216-2026 µmol TE 100 g −1 fw) (Kevers et al., 2007;Ninfali, Mea, Giorgini, Rocchi, & Bacchiocca, 2005). The same was observed for the activities determined by ABTS and DPPH, where the values reported range from 274 to 562 µmol TE 100 g −1 fw and 84-180 µmol TE 100 g −1 fw, respectively (Kaur et al., 2013;Kevers et al., 2007). These results suggest that the tomato accessions analyzed in this study represent good sources of antioxidants. The present study showed positive correlation between TP and the AoxA (ABTS, DPPH) of the methanol extracts from the accessions as it has been reported previously for commercial varieties (Kevers et al., 2007;Ninfali et al., 2005). The lack of correlation between phenolics content and the AoxA by ORAC has been reported previously by other authors and has been attributed mainly to differences in the reaction mechanisms, assay conditions, and the presence of other compounds in the extract (Li et al., 2012). ORAC is a hydrogen atom transfer (HAT)-based assay, whereas ABTS and DPPH are commonly classified as mixedmode methods (i.e. Electron transfer and HAT) (Apak, Özyürek, Güçlü, & Çapanoğlu, 2016).

Phenolic profiles
The main phenolics in the accessions were chlorogenic acid and its derivatives di-and tri-caffeoylquinic acids as well as the flavonoids rutin and rutin-pentoside ( Figure 1; Table 2). These results agree with those obtained for the methanol extracts of three varieties of tomato (Gómez-Romero et al., 2010). Chlorogenic acid and rutin were the most abundant compounds in the methanol extracts (Table 3) and their contents in some of the accessions were similar to those reported by Boches et al. (2011) in five S.l. var. cerasiforme accessions selected from a core collection from Cornell University. As previously observed by these authors, high levels of di-and tri-caffeoylquinic acids (1-8 mg 100 g −1 fw) were also found in the present study, being higher than those reported for the cherry tomato cultivars Micro-Tom (0.19-0.28 mg g −1 dw) (Luo et al., 2008) and CLS (0.018-0.024 mg g −1 dw) (Li et al., 2015), considering that cherry cultivars contain 90-92% water. The association between the phenolics content and the antioxidant activity has been reinforced with data generated with transgenic tomato fruit; Micro-Tom fruit expressing the AtMYB12 transcription factor increased up to 35-fold the levels of di-and tri-caffeoylquinic acids (4.2-6.6 mg g −1 dw) (Luo et al., 2008), whereas the expression of AtMYB11 in the peel of the CLS cultivar increased up to 100-fold the levels of these compounds (0.25-2.39 mg g −1 dw) (Li et al., 2015). The higher levels of the phenolic compounds in both transgenic tomatoes were associated with three to five-fold increases in hydrophilic antioxidant activity (ABTS). The levels of di-and tri-caffeoylquinic acids in some of the accessions used in the  Moco et al. (2006), Ribas-Agustí et al. (2012) present study (Table 3) were close to those of the transgenic Micro-Tom fruit and similar to those of the transgenic CLS peels, suggesting these materials represent a nontransgenic alternative to improve this trait in tomato. Based on the positive correlation between ABTS values and phenolics content, chlorogenic acid, rutin, and their derivatives dicaffeoylquinic acid and rutin-pentoside could be the main compounds responsible for the antioxidant activity of the methanol extracts. In the case of flavonoids, their antioxidant activity has been associated with some structural characteristics, such as the ortho-dihydroxy (cathecol) in the B ring, 2,3 double bond in conjugation with a functional 4-oxo in the C ring, as well as hydroxyl groups at positions 3 and 5 that allow the formation of hydrogen bonds with the 4-oxo group. Based on these characteristics, the flavonoids with the best antioxidant activity should be quercetin and myricetin (Prochazkova, Bousova, & Wilhelmova, 2011). Rutin, the quercetin conjugate found at high concentrations in methanol extracts of the tomato accessions showed a good correlation with the antioxidant capacity by ABTS and DPPH.

Antimutagenic activity
To our knowledge, this is the first report showing the antimutagenic potential of S.l. var. cerasiforme. The methanol extracts (100 µg/plate) of some accessions showed higher inhibition values (Table 1) (Table 4) were higher than that obtained in a methanol extract from common beans (Phaseolus vulgaris) evaluated at 500 µg/plate (35%) using  Tabla 3. Perfiles de fenólicos por HPLC de extractos metanólicos del fruto de accesiones de Solanum lycopersicum var. cerasiforme a .
the same assay (González De Mejía, Castaño-Tostado, & Loarca-Piña, 1999). The AmuA of tomato fruit has been attributed mainly to the presence of carotenoids (Rauscher, Edenharder, & Platt, 1998). However, the AmuA of pure lycopene was lower than that of a tomato extract (Polívková, Šmerák, Demová, & Houška, 2010), suggesting that other metabolites were contributing to this activity. In addition, the methanol extracts from accessions with yellow fruit exhibited high antimutagenic values (Tables 1 and 4) and they have very low levels of lycopene. Moreover, tomato fruit is also an important source of phenolic compounds that have AmuA (Agarwal & Rao, 2000;Middleton, Kandaswami, & Theoharides, 2000). Yamada and Tomita (1996) evaluated the AmuA of caffeic acid and chlorogenic acid at 1000 µg/plate on the mutagens Trp-P-1, Glu-P-2, 4-NQO, and AF-2 using the strain TA98 of S. enterica serovar Typhimurium. The inhibition values of chlorogenic acid were 25%, 64%, 3%, and 15%, respectively; whereas those of caffeic acid were 33%, 73%, 5%, and 20%, respectively. As indicated before, the main phenolics found in the methanol extracts of the accessions studied herein were chlorogenic acid and derivatives, as well as rutin and its derivative rutin-pentoside. However, we only observed a moderate but non-significant correlation (r = 0.37, p = 0.1) between the levels of rutin and dicaffeoylquinic acid and the AmuA of the extracts evaluated at 100 µg/plate. The concentrationresponse effect for the antimutagenicity of methanol extracts showed the presence of active compounds; the lack of correlation between the antimutagenic activity and the content of specific phenolic compounds suggest that the activity was due either to nonidentified pure compounds or to the combined effect of different compounds.

Conclusions
Some of the Solanum lycopersicum var. cerasiforme accessions showed high content of phenolics, as well as high antioxidant and antimutagenic activities. The antioxidant activity of the accessions appears to be associated mainly with the levels of caffeoylquinic acids and rutin. Accessions with high levels of these compounds could be used as sources of antioxidants in the diet or for breeding functional tomatoes. Further studies are required to identify compounds associated with the strong antimutagenicity of some tomato accessions.

Disclosure statement
No potential conflict of interest was reported by the authors.