Carotenoid and total vitamin C content of peppers from selected Brazilian cultivars

Accepted Manuscript Title: Carotenoid and total vitamin C content of peppers from selected Brazilian cultivars Author: Tˆania da Silveira Agostini-Costa Ismael da Silva Gomes Luis Alberto Martins Palhares de Melo Francisco Jose Becker Reifschneider Cl´audia Silva da Costa Ribeiro PII: DOI: Reference:

S0889-1575(16)30237-X http://dx.doi.org/doi:10.1016/j.jfca.2016.12.020 YJFCA 2808

To appear in: Received date: Revised date: Accepted date:

17-5-2016 1-12-2016 20-12-2016

Please cite this article as: da Silveira Agostini-Costa, Tˆania., da Silva Gomes, Ismael., de Melo, Luis Alberto Martins Palhares., Reifschneider, Francisco Jose Becker., & da Costa Ribeiro, Cl´audia Silva., Carotenoid and total vitamin C content of peppers from selected Brazilian cultivars.Journal of Food Composition and Analysis http://dx.doi.org/10.1016/j.jfca.2016.12.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Original Research Article

Carotenoid and total vitamin C content of peppers from selected Brazilian cultivars

Tânia da Silveira Agostini-Costaa, Ismael da Silva Gomesa, Luis Alberto Martins Palhares de Meloa, Francisco Jose Becker Reifschneiderb, Cláudia Silva da Costa Ribeirob a

Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, PqEB, W5

norte, 70770-917, Brasília, DF, Brazil.

b

Embrapa Hortaliças, Rodovia Brasília/Anápolis BR 060 km 09, 70359-970, Gama, DF,

Brazil.

Corresponding author: Tânia da Silveira Agostini Costa Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, PqEB, W5 norte, 70770-917, Brasília, DF, Brazil E-mail: [email protected] Phone: +55 61 3448 4916 Fax: +55 61 3340-3624

Highlights   

Two pepper cvs were notable for their zeaxanthin, provitamin A and vitamin C levels; Red peppers from field had higher vitamins and zeaxanthin than those from greenhouse; Total carotenoids were identical in one yellow and one red jalapeño.

Abstract. Carotenoid and ascorbic acid content was determined in peppers of two red cultivars and three yellow lineages of “Jalapeño” (Capsicum annuum L.) plus a “sweet red biquinho” and a “red bode” cultivar (C. chinense Jacquin) from the Brazilian Breeding Program. Capsanthin (68±3 to 177±2 µg/g) was found to be the main carotenoid in the red peppers, and violaxanthin (34±3 to 100±17 µg/g) was the main carotenoid in the yellow jalapeño peppers. Red jalapeño peppers grown in the field (in summer) showed higher zeaxanthin (29±0 and 31±2 µg/g), β-cryptoxanthin (11±1 and 7±0 µg/g), provitamin A (161±2 and 81±2 µg/g) and ascorbic acid (132±2 and 129±2 µg/g) than peppers grown in the greenhouse. Peppers of one yellow jalapeño lineage (C. annuum) and of the red “bode” cultivar (C. chinense) also stood out for their very high levels of zeaxanthin (36±6 and 53±7µg/g), besides nutritional compounds, provitamin A (222±17 and 299±32 retinol activity equivalents/100g) and vitamin C (152±5 and 123±1 mg/100g). These results indicate that the selected peppers presented compounds that are beneficial for human health and that they could be used in the Brazilian pepper breeding programs to develop new commercial hybrids.

Keywords: Capsicum annuum, C. chinense, Solanaceae, capsanthin, zeaxanthin, provitamin A, ascorbic acid, food analysis, food composition, HPLC.

Chemical compounds studied in this article Capsanthin (PubChem CID: 5281228); capsorubin (PubChem CID: 5281229); violaxanthin (PubChem CID: 449438); antheraxanthin (PubChem CID: 5281223);

zeaxanthin (PubChem CID: 5280889); lutein (PubChem CID: 5281243); βcryptoxanthin (PubChem CID: 5281235); α-carotene (PubChem CID: 4369188); βcarotene (PubChem CID: 5280489); ascorbic acid (PubChem CID: 54670067).

Introduction Species of Capsicum, from the Solanaceae family, are widely produced and consumed around the world (Giuffrida et al 2013, Ornelas-Paz et al 2013), and Brazil is an important center of diversity for this genus. Capsicum annuum L. is the species most cultivated in Brazil, and C. chinense Jacquin is the Brazilian species with the highest diversity in the Amazon Basin (Ribeiro and Reifschneider 2008); both species are intercrossable and can produce fertile and heterogeneous hybrids (Wahyuni et al 2011). Jalapeño is one of the most popular varieties (Alvarez-Parrila et al 2011) of C. annuum, while “Bode” and “Biquinho” are highly flavored varieties of C. chinense (Garruti et al 2013) that are much sought after by Brazilian farmers (Ribeiro and Reifschneider 2008). Peppers are a rich source of phytochemicals such as vitamins (Wahyuni et al 2011, Zhuang et al 2012), capsaicinoids (Aguiar et al 2016), carotenoids (Giuffrida et al 2013, Ha et al 2007, Rodríguez-Burruezo et al 2010, Wall et al 2001) and polyphenols (Wahyuni et al 2011, Zhuang et al 2012). Carotenoids are lipophilic C40 isoprenoids with polyene chains and different end groups (β, ε, κ) (Britton et al 1995a). The presence of numerous conjugated double bonds and cyclic end groups is crucial for their roles in light harvesting in photosynthetic organisms and (photo) protection in all living organisms (Britton 1995, Niranjana et al 2015). Carotenoids are classified into oxygenfree carotenes, such as α-carotene and β-carotene and oxygen-containing xanthophylls, such as β-cryptoxanthin, zeaxanthin, violaxanthin and capsanthin (Britton et al 1995a).

They are the main determinants of pepper color in several species of the Capsicum genus (Ha et al 2007), and the red-colored fraction contains two distinctive κxanthophylls known as capsanthin and capsorubin, which are pigments exclusive to this genus (Hornero-Méndez et al 2000). Some carotenoids show provitamin A activity (IOM 2001); some such as capsanthin, β-carotene, zeaxanthin and lutein have been confirmed as antioxidants (Müller et al 2011) with health benefits in the prevention or alleviation of various diseases (Bian et al 2012, Dhingra and Bansal 2014, Niranjana et al 2015). Ripe red peppers present a higher antioxidant capacity than green fruits (Cervantes-Paz et al 2012, Kim et al 2011). Ascorbic acid is the main vitamin C compound and is another functional and nutritional constituent of pepper fruit (Teodoro et al 2013, Topuz and Ozdemir 2007); it is well known as an antioxidant and bioactive compound, particularly in ripe peppers (Kim et al 2011, Topuz and Ozdemir 2007). Jalapeño peppers have shown antioxidant properties (Alvarez-Parrila et al 2011) and demonstrated effective action against food lipid and human LDL cholesterol oxidation (Alvarez-Parrilla et al 2012) due to several bioactive compounds. The influence of genetic differences (Wall et al 2001, Zhuang et al 2012) and ofenvironmental conditions (Ornelas-Paz et al 2013) can influence the content of secondary metabolites of peppers and may have implications for their biological effects (Alvarez-Parrilla et al 2012). The objective in this study was to determine the content of carotenoids,provitamin A and vitamin C in two red cultivars (BRS Garça and BRS Sarakura) and three yellow lineages (CNPH 25.313; 25.324 and 25.296) of Jalapeño peppers (C. annuum L.), and in two red and flavored cultivars (BRS Moema and BRS Seriema) of C. chinense Jacquin grown in Brazil’s Central region. Additionally, some differences observed due to their growing environment were also evaluated. The results

may help to know more about the nutritional and health potential of some important Brazilian pepper cultivars, associated with their growing environment. They may also help Brazilian pepper-breeding programs to develop new commercial hybrids at produce fruits enriched with health-related compounds. Material and Methods Plant materials Peppers were grown at the experimental area of Embrapa Vegetable Crops, Gama, DF, Brazil (latitude: 15.8oW; longitude: 47.9o S; altitude: 990 m) according to Ribeiro et al. (Ribeiro et al 2008) and harvested in the summer and spring of 2014 (total rainfall: 507 mm in the summer and 58 mm in the spring). Climatic conditions as described by the National Institute of Meteorology (Brasil 2014) are represented in Figure 1.Two red cultivars (BRS Sarakura and Garça) and three yellow lineages (CNPH 25.313, 25.324 and 25.296) were of Jalapeño peppers (C. annuum L.); another two red cultivars (BRS Moema -“sweet biquinho pepper” and BRS Seriema -“bode pepper”) were of C. chinense Jacquin. For red Jalapeño pepper, one sample (about 30 fruits) was taken from each cultivar grown in the field. Another sample of 30 fruits was taken from plants grown in the greenhouse. The latter regime aimed to protect the samples from high incidences of summer rain and sunlight, but the environmental conditions, such as temperature, luminosity and humidity, were not optimized. Of the greenhouse samples, five plants per cultivar were harvested in the summer (February 2014); from each fieldgrown cultivar, ten plants were harvested in the spring (October 2014), as shown in Table 1.

One sample of each lineage of yellow Jalapeño pepper was harvested from

the field in the summer (CNPH 25.313 and 25.296), and another sample was taken in the spring (CNPH 25.313 and 25.324). One sample of each cultivar, BRS Moema and BRS Seriema, of C. chinense was harvested from the field in the spring. In the

greenhouse, red peppers were all harvested at the same time but at three successive maturity stages based on their color (green, partially purple and fully red), aiming to make ascorbic acid valuations. However,for carotenoid analysis, only fully red peppers were used. Red and yellow fruits from the field were always fully ripe, but not overripe. In the Laboratory, the fruits were carefully selected to ensure that they were free of defects, totaling about 20 fruits per sample. These fruits were washed, dried with paper towels, frozen in liquid nitrogen, wrapped in plastic bags and stored at -200C until the time of extraction. Carotenoid extraction Three analytical repetitions were conducted for each sample. Procedures were carried out according to (Minguez-Mosquera and Hornero-Méndez 1993) with adaptations. Carotenoids were extracted using addition of antioxidant (BHT 0.1 %Sigma-Aldrich, Steinheim, Germany), protection from light and exclusion of oxygen at air-conditioned room temperature (21oC). Peppers were devoid of seeds and cut into small pieces. The carotenoids from 3 g of homogenized peppers were extracted in a Polytron homogenizer (Kinematica, Lucerne, Switzerland) for 2 min at 20,000 rpm, using 2 g of celite and 4050 mL of cold acetone (4 ºC),four to five times, until there was a complete lack of color in the residue. The extract of free carotenoids was vacuum filtered, and the pigments were carefully transferred from the acetone to 100mL of ethylic ether (Riedel-de Haen, Seelze, Germany). The organic phase was dried over anhydrous sodium sulfate and saponified under nitrogen atmosphere using an equal volume of 20% KOH in methanol (J. T. Baker, Phillipsburg, NJ, USA) for 1 hour at room temperature. Saponified extract was transferred to a separatory funnel and carefully washed with distilled water, until it reached a pH near to neutral, using alcoholic phenolphthalein. The extract was dried over anhydrous sodium sulfate and vacuum evaporated (33oC); the pigments were

diluted in 25 mLof petroleum ether 40-60 oC (Riedel-de Haen, Seelze, Germany) for total carotenoid evaluation. About 2-4 mL of extract was evaporated under nitrogen, dissolved in 2-3mL of acetone and filtered in a 0.45 μm PTFE syringe filter (Millipore) before HPLC analysis. HPLC analysis The analysis was done according to (Azevedo-Meleiro and Rodriguez-Amaya 2009). HPLC analysis was carried out in a ProStar Varian system (Mulgrave, VIC, Australia) equipped with ternary pump, autosampler and photodiode array detector (PDA PS240/PS-410 / Galaxie PS-335/Software 1.9). The column used was a monomeric C18 Spherisorb Waters (ODS-2, 3 µm, 150 x 4.6 mm Waters, Ireland). Briefly, 10 μL of the extract was automatically injected into the HPLC system. The mobile phase consisted of HPLC grade acetonitrile (J. T. Baker Phillipsburg, NJ, USA), containing 0.05 % of triethylamine purum (Riedel-de Haen, Seelze, Germany); HPLC grade methanol (J. T. Baker, Phillipsburg, NJ, USA) and ethyl acetate LC-MS chromasolv (Riedel-de Haen, Seelze, Germany) from 95:5:0 to 60:20:20 in 20 min, maintaining the latter proportion until the end of the run. Flow rate was 0.5 mL.min-1. Detection was at wavelengths of maximum absorption of the carotenoids in the mobile phase. The carotenoids were identified according to: a) their retention time, using carotenoid standards analyzed in the same chromatographic conditions; b) their maximum absorbed wavelength (λmax) on diode array detector; and c) their fine structure, which is% III/II expressed by the ratio between the peak height of the longestwavelength absorption band (III) and the middle absorption peak, generally λmax (II), making the minimum between the two peaks the baseline, multiplied by 100. Standards of β-carotene (97 % by HPLC), lutein (95%) and zeaxanthin (mix of isomers 75% trans-zeaxanthin and 25% cis-zeaxanthin) were obtained from DSM

product (France).Standards of α-carotene (96 % by HPLC), capsanthin (97 %), capsorubin (98 %) and neoxanthin (84 %) were obtained from Carotenature (Ostermundigen, Switzerland).Violaxanthin (> 97 %), lutein and zeaxanthin were repurified according to (Rodriguez-Amaya 1999) on calcium hydroxide and magnesium oxide - hyflosupercel (1:2) open-column, using an acetone gradient in petroleum ether as mobile phase. Only the main portion of the band was collected. The concentrations were determined spectrophotometrically, using tabled A1%1cm (Britton et al 1995b), and corrected according to purities obtained. The ultraviolet visible spectrophotometer was a Lambda 25 Perkin Elmer. The quantification was carried out using an external calibration curve, which was linear using six concentrations of lutein (r = 0.9998), αcarotene (r = 0.9999), β-carotene (r = 0.9999), zeaxanthin (r = 0.9999), capsorubin (0.9999),capsanthin (0.9997) andviolaxanthin (0.9992). Theprovitamin A was calculated according to the conversion factor (IOM 2001), in which 12 μg of β-carotene and 24 μg of α-carotene correspond to 1 RAE (retinol activity equivalents).

Total vitamin C The total vitamin C was determined after dehydroascorbic acid reduction by TCEP-HCI (tris 2-carboxyethyl-phosphine hydrochloride), and the ascorbic acid was determined by HPLC as described by (Fontanaz et al 2006) with adaptations. The extraction was done using 10 g of pepper and 40 mL of reducing agent TCEP-HCI (>98 Fluka Biochemika Sigma Aldrich St Louis, USA 250 μg/mL); the volume was completed up to 100 mL with 1% trichloroacetic acid (TCA >99 Vetec Quimica Fina Sigma-Aldrich, Duque de Caxias, RJ, Brazil). The extract was analyzed by HPLC Varian equipped with a quaternary pump, automatic injector and photodiode array detector controlled by Galaxie software, using LiChospher column RP-18, 5 μm, 4.6x250 mm; isocratic

elution 1 mL/min; detection at 265 nm; injection volume of 20 μL. Mobile phase: 1.6 g of decylamine (pairing agent >95% Sigma-Aldrich, Steinheim, Germany): 80 mL of HPLC grade acetonitrile (J. T. BakerPhillipsburg, NJ, USA): 100 mL of 0.25 M sodium acetate pH 5.4: water up to 1000 mL, adding 50 mg of TCEP-HCl in the final solution. The ascorbic acid identification was done by co-chromatography with standard, and also through comparison of the spectral profile provided by a diode array detector (between 230 and 330nm). The quantification was carried out using an external calibration curve, which was linear using six concentrations of ascorbic acid (Sigma>99%) ranging from 0.3 to 1.5 mg mL-1 (r= 0.9999); (CV < 2 %); recovery 101.7%.

Data analysis The levels of bioactive compounds among different accessions and cultivars of peppers, environment (field and greenhouse) and season (summer and spring) were compared using descriptive statistical analysis, followed by ANOVA and/or test t (p value <0.01). The free-access R program was used for all statistical analyses.

Results and discussion Red jalapeño peppers Red jalapeño peppers accumulated β-β-carotene, β-β-xanthophylls and derivatives of two distinctive κ-xanthophylls, capsanthin and capsorubin. However, no β-ε-carotene or β-ε-xanthophylls were found in red jalapeños. Indeed, ten main carotenoids (Figure 2) were identified, highlighting a single β-β-carotene and nine β-β-xantophylls: two hydroxy-β-β-carotenes (β-cryptoxanthin and zeaxanthin); two epoxy-hydroxy-β-βcarotenes, mutatoxanthin and antheraxanthin; and five keto-κ-carotenoids, capsanthin,

cis-capsanthin, capsanthin-5,6-epoxide, capsanthone and capsorubin. Furthermore, traces of violaxanthin and small amounts of the tentatively identified capsanthin 3,6epoxide were detected, mainly in red peppers harvested in the summer. According to the t-test, red cv Sarakura and Garça did not differ significantly with respect to the mean values of ascorbic acid (Figure 3a). However, cv Sarakura presented higher total carotenoids (304±92 μg/g), capsanthin (146±35 μg/g), β-carotene (9±3 μg/g), provitamin A (103±44 μg RAE/100g), antheraxanthin and mutatoxanthin than cv Garça. Capsanthin tends to be found at the polar surface of plasma lipoprotein (consisting of phospholipids and apoprotein), and increased intake of capsanthin-rich foods could be beneficial for health (Kim et al 2009). Among red jalapeño cultivars harvested only in the summer, peppers grown in the field showed significantly higher zeaxanthin (30±1 μg/g); β-cryptoxanthin (9±2 μg/g), provitamin A (122±43 μg RAE/100g) and ascorbic acid (131±6 mg/100g) than peppers grown in the greenhouse (Figure 3a). Ascorbic acid from other Jalapeño peppers grown in a greenhouse in Texas,USA, ranged between 69 and 119 mg/100g (Bae et al 2014). Fruits of two American red bell pepper cultivars (Russo and Howard 2002) produced zeaxanthin only if grown in the field (2-6 μg/g), and peppers from a field-grown Cayenne group also had higher levels of zeaxanthin (5 μg/g) and βcryptoxanthin (6 μg/g) than peppers grown in an optimized glasshouse environment. Carotenoid levels tend to increase in plants in response to stress, especially due to drought, less-than-optimum temperature and exposure to long periods of illumination; however, this effect differs among cultivars (Russo et al.2002). One bell pepper and two non-bell pepper cvs had the highest levels of zeaxanthin (30-33 μg/g), β-cryptoxanthin (11-42 μg/g) and provitamin A (38-339 RAE) when cultivated in the glasshouse, with optimized temperature and ambient light, under 12 h of illumination (Russo et al.2002).

Comparing only peppers grown in the field, the compounds mentioned above, besides antheraxanthin, were also significantly higher in the peppers harvested in the summer than in the peppers harvested in the spring. Both cultivars of red peppers harvested from the field only in the summer provided very high zeaxanthin levels (> 20 μg carotenoid / g, according to Britton and Khachik 2009). Zeaxanthin is an important bioactive compound, especially for eye health, associated with a decreased risk of agerelated macular degeneration (Bian et al 2012). This sharp increase in zeaxanthin levels (about three fold) in peppers from the field could be tentatively explained because zeaxanthin is an important carotenoid for photoprotection through quenching excess excitation energy in plants (Qin et al 2015). In contrast, capsanthin did not change significantly, and capsorubin was higher in peppers from the greenhouse than in peppers from the field; these two xanthophylls are biosynthesized from zeaxanthin. The capsanthin/capsorubin ratio ranged from 14 to 16 (field) and from 6 to 8 (greenhouse) in the summer, and from 8 to 11 (field) in the spring, showing a tendency for capsorubin biosynthesis to increase in the greenhouse (probably due to lower stress levels). R/Y ratios (R, total red κ-xanthophylls, and Y, total yellow isochromic fractions) were about 1.5 for red jalapeño peppers from the field in the summer and about 3.0 for peppers from the greenhouse in the summer and from the field in the spring. The capsanthin/zeaxanthin ratios were 3.2 and 5.8, respectively, for cv Garça and Sarakura from the field in the summer, increasing to about 9 for both cultivars in the spring and to about 15 for both cultivars from the greenhouse. These results indicate higher carotenoid levels from the yellow fractions, especially zeaxanthin, for peppers from the field in the summer, probably due to higher environmental (biotic and abiotic) stress. Therefore, red jalapeño grown under stressful conditions could produce peppers with a higher content of nutritive and health compounds.

Yellow jalapeño peppers In contrast to the red jalapeño peppers, one β-ε-carotene (α-carotene) and two β-εxanthophylls (α-cryptoxanthin and lutein) were found in fully ripe yellow Jalapeño peppers. Indeed, yellow peppers presented ten main carotenoids (Figure 2), highlighting two carotenes (α-and β-carotene) and eight other xanthophylls: four hydroxy-carotenes (α–cryptoxanthin and β-cryptoxanthin, zeaxanthin and lutein) and four epoxy-hydroxycarotenes (anteraxanthin, violaxanthin, c-violaxantin and luteoxanthin). Furthermore, traces ofcapsorubin were detected, mainly in the lineage CNPH 25.296. While capsanthin was found as the main carotenoid in the red peppers, violaxanthin was the main carotenoid in the yellow jalapeño peppers. Capsanthin and capsorubin are biosynthesized from antheraxanthin and violaxanthin respectively (Ha et al 2007). As far as we know, no previous data have been reported regarding the profile of carotenoids in yellow Jalapeño peppers. However, some yellow cultivars of C.chinense (Bian et al 2012, Ha et al 2007) presented lutein, β-carotene, α-carotene, zeaxanthin and β-cryptoxanthin, while a yellow bell pepper from Brazil (Azevedo-Meleiro and Rodriguez-Amaya 2009) also provided violaxanthin as the main carotenoid. Among the three yellow jalapeños evaluated (Figure 3b), the lineage CNPH 25.313 presented the highest levels of some carotenoids, such as β-cryptoxanthin, βcarotene and α-carotene. Its provitamin A levels (219±38 μg RAE/100g in the summer and 222±17 μg RAE/100g in the spring) were higher than values found in the other yellow jalapeño accessions of this study (149 and 85 μg RAE/100g) and higher than the levels shown in the literature (Rodríguez-Burruezo et al 2010) for yellow accessions of C. baccatum, C. pubescens and C. annuum from Bolivia (20 to 102 μg RAE/100g). This yellow lineage also stood out due to its very high zeaxanthin levels (28±3 μg/g in the spring and 36±6 μg/g in the summer). Zeaxanthin has been found only as a minor

carotenoid in yellow bell peppers cultivated in Brazil (Azevedo-Meleiro and RodriguezAmaya 2009) and Spain (Rodríguez-Burruezo et al 2010). However, unlike what was observed for red peppers, the seasons, summer and spring, did not significantly affect the carotenoids or the provitamin A values in the yellow CNPH 25.313 jalapeño peppers. These results and the literature data (Russo and Howard 2002) indicate that the effect of biotic and abiotic stress on the carotenoid levels is also related to the pepper cultivars, which shows the importance of analyzing carotenoids in different plant materials and environmental conditions. However, the extent to which genetic or environmental factors alone or in combination contribute to this biosynthetic pathway is not clear and needs additional studies.

Comparing with red cultivars of jalapeño peppers, total carotenoids produced by the yellow lineage CNPH 25.313 (246±44 μg/g in the summer and 181±24 μg/g in the spring) did not differ from the values produced by red cvs Sarakura and Garça. These comparable values between yellow and red cultivars are uncommon in the literature, which reports that for non-red peppers of another Capsicum, total carotenoids do not increase and remain low during ripening (Ha et al 2007), and that total carotenoids are usually higher in red than in yellow peppers (Ornelas-Paz et al 2013). The high total carotenoid value found in CNPH 25.313 is mainly due to its levels of xanthophylls, such as violaxanthin and antheraxanthin, which were not always found in the yellow peppers of other reports. With respect toprovitamin A and ascorbic acid (Vitamin C), CNPH 25.313 showed similar (219±38 μg RAE/100g and 136±6 mg/100g in the summer) or higher (222±17 μg RAE/100g and 152±5 mg/100g in the spring) values than both red cultivars. These results suggest that the yellow lineage CNPH 25.313 of

jalapeño peppers could provide health and nutritional compounds such as the antioxidant zeaxanthin, provitamin A and vitamin C. Red peppers C. annuum cv Jalapeño and C. chinense cv Seriema and Moema The two cultivars (BRS Seriema and Moema) of C. chinense harvested from the field in the spring presented a similar carotenoid profile (Figure 4) to that of red jalapeño peppers (C. annuum). However, capsanthin/capsorubin ratios were higher for C. chinense peppers (> 20) than for C. annuum peppers (about 10). Although the peppers of BRS Moema (C. chinense) presented lower vitamin A, mainly due to the low levels of the yellow carotenoids, these flavored peppers are sweet and could provide very good levels of capsanthin (127±8μg/g) and vitamin C (107±3 mg/100g). According to the t-test, capsanthin levels did not differ significantly between the two cultivars (BRS Garça and Sarakura) of C. annuum species and cv BRS Seriema (C. chinense). However, in contrast to cv Moema, peppers of cv Seriema stood out for their higher levels of some yellow carotenoids, mainly zeaxanthin (53±6 μg/g) and βcryptoxanthin (25±2 μg/g), plus their higher provitamin A (299±32 μg RAE/100g) and vitamin C (123±1 mg/100g) content. Seeing that the BRS Seriema provides small, appealing and aromatic “bode” peppers (Aguiar et al 2016, Garruti et al 2013), generally consumed as pickles, these results reinforce the health potential of this cultivar to provide very high levels of antioxidant carotenoids, such as capsanthin, β-carotene and zeaxanthin, besides very good levels of vitamin C and provitamin A.

Conclusions These data confirm the influence of genotype and environment on the bioactive compounds of the Capsicum peppers evaluated. For the red jalapeño (C. annuum), the peppers of BRS Sarakura presented higher total carotenoids and provitamin A, while the

peppers grown in the field showed higher zeaxanthin, provitamin A and vitamin C than the peppers from the greenhouse; and the peppers harvested in the summer showed higher zeaxanthin and vitamin C than in the spring. The peppers of only one yellow jalapeño showed similar (in the summer) or higher (in the spring) values of provitamin A and vitamin C. For C. chinense, the peppers of BRS Seriema were notable for their higher levels of zeaxanthin, provitamin A and very good levels of vitamin C. These results indicate that peppers of Brazilian cultivars, mainly those grown in the field, could contribute to improving human health with beneficial compounds, especially zeaxanthin, capsanthin, provitamin A and vitamin C.

Acknowledgments The authors acknowledge the Empresa Brasileira de Pesquisa Agropecuária (Embrapa) for supporting this study (Project Macroprograma 2 No 02.12.02.007.00.00).

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250 200 Harvesting month

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Figure 1. Climatic conditions (Temp: temperature; Insol: insolation and R. Humidity: relative humidity) in summer 2014 (Sum) and spring (Spr) in Brasília, DF (Brasil 2014).

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c) Figure 2. HPLC chromatograms and spectral profiles of main carotenoids from a) red jalapeño peppers cv BRS Sarakura harvested from field in the summer; b) red “bode” peppers cv BRS Seriema harvested from field in the spring; c) yellow jalapeño peppers lineage CNPH 25.313 harvested from field in the summer. 1: capsorubin; 2: capsanthin-5,6-epoxide; capsanthin-3,6-epoxide; 4: capsanthin; 5: capsanthone; 6:cis-capsanthin; 7: antheraxanthin; 8: mutatoxanthin; 9: zeaxanthin; 10: β-cryptoxanthin; 11: β-carotene; 12: cis-violaxanthin; 13: violaxanthin; 14: luteoxanthin; 15: lutein; 16: α-cryptoxanthin; 17: α-carotene.

Concentration of bioactive compound

300

Red jalapeño peppers (C. annuum)

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50 0 Sk Ga Sk Ga Sk Ga Sk Ga Sk Ga Sk Ga Sk Ga Sk Ga Sk Ga Sk Ga Sk Ga CSO

a)

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b) Figure 3. Carotenoids (μg/g – fr wt), provitamin A (RAE/100g – fr wt) and ascorbic acid (mg/100g – fr wt) in a) red cultivars (BRS Sarakura: Sk; BRS Garça: Ga) and b) yellow lineages (CNPH 25.296: J296; 25.313: J313; 25.324: J324) of jalapeño peppers grown in the field (red and yellow peppers) or in the greenhouse (GH; only for red peppers) in the summer (Sum) and in the spring (Spr). Each bar is a result of three analytical replicates. CSO: capsorubin; CSA: capsanthin; V: violaxanthin; c-V: cis-violaxanthin; AX: antheraxanthin; M: mutatoxanthin; Z: zeaxanthin; β-cry: β-cryptoxanthin; β-C: β-carotene; L: lutein; αCRY: α-cryptoxanthin; α-C: α-carotene; R: total red κ-xanthophylls; Y: total yellow isochromic fractions; Vit A: provitamin A; AA: ascorbic acid (vitamin C).

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Red peppers (C. annuum and C. chinense)

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Figure 4. Carotenoids (μg/g fr wt), provitamin A (RAE/100g fr wt) and ascorbic acid (mg/100g fr wt) in red peppers of species C. annuum (cultivars BRS Sarakura: Sk; BRS Garça: Ga) and C. chinense (cultivars BRS Seriema: Se and BRS Moema: Mo) grown in the field and harvested in the spring. Each bar is a result of three analytical replicates. CSO: capsorubin; CSA-e: capsanthin 5,6-epoxyde; CSA: capsanthin; c-CSA: cis-capsanthin; AX: antheraxanthin; M: mutatoxanthin; Z: zeaxanthin; β-cry: β-cryptoxanthin; β-C: β-carotene; R: total red κ-xanthophylls; Y: total yellow isochromic fractions; TC: total carotenoids; Vit A: provitamin A; AA: ascorbic acid (vitamin C).

Table 1. Selected peppers fromBrazilian pepper-breeding programs from Embrapa Vegetable Crops, Gama, DF, Brazil Species

C. anuumm C. anuumm C. anuumm C. anuumm C. anuumm C. chinense C. chinense

Pepper

Jalapeño Jalapeño Jalapeño Jalapeño Jalapeño Biquinho Bode

Color / Cultivar (or Lineage)

Red BRS Sarakura Red BRS Garça Yellow CNPH 25.313 Yellow CNPH 25.296 Yellow CNPH 25.324 Red BRS Moema Red BRS Seriema

Initials

Sk Ga J313 J296 J324 Mo Se

2014 crop Field summer

Field Spring

   

     

Greenhouse summer  