Wild Pepper Capsicum annuum L. var. glabriusculum

Published December 30, 2015

REVIEW & INTERPRETATION

Wild Pepper Capsicum annuum L. var. glabriusculum: Taxonomy, Plant Morphology, Distribution, Genetic Diversity, Genome Sequencing, and Phytochemical Compounds Corina Hayano-Kanashiro, Nohemí Gámez-Meza, and Luis Ángel Medina-Juárez* ABSTRACT The fruit of the chiltepin [Capsicum annuum L. var. glabriusculum (Dunal) Heiser and Pickersgill] is considered an important genetic resource for pepper crop improvement. The chiltepin is distributed from Colombia, Central America, and Mexico to the southwestern United States. The present review provides a synopsis of the taxonomic classification of the genus Capsicum and the chiltepin and studies were conducted to explore the current genetic diversity of this resource and the urgent necessity for conservation programs. Additionally, brief information regarding the recent sequencing of the chiltepin genome is provided, which contributes important data that could assist in its genetic improvement and conservation. Furthermore, this review discusses the phytochemical compounds of the chiltepin. The revised information shows that the chiltepin is an important genetic resource for pepper crop improvement and a primary source of phytochemical compounds.

Departamento de Investigaciones Científicas y Tecnológicas de la Universidad de Sonora, Blvd. Luis Encinas y Rosales s/n. Colonia Centro, C.P. 83000, Hermosillo, Sonora, México. Mexican Research Council Consejo Nacional de Ciencia y Tecnología (CONACYT) fellowship No. 191479. Received 20 Nov. 2014. Accepted 9 Sept. 2015. *Corresponding author ([email protected]). Abbreviations: ABTS, 2,2¢-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); CATIE, Centro Agronómico Tropical de Investigación y Enseñanza; DPPH, 2,2-diphenyl-1-picrylhydrazyl; GRIN, Germplasm Resources Information Network; Hs, expected heterozygosity; LTR, long tandem repeat; PCR, polymerase chain reaction; SHU, Scoville heat units; TE, transposable elements.

C

hiltepin is a semidomesticated or wild species distributed in a range from Colombia, Central America, and Mexico to the southwestern United States (González-Jara et al., 2011). In Mexico, it is distributed along the Pacific coast from Sonora to Chiapas (Nabhan et al., 1990; Miranda-Zarazúa et al., 2010; González-Jara et al., 2011). This plant is a highly branched perennial shrub with thin stems (Fig. 1A) that often climb up other shrubs and can reach heights of up to two meters (Nabhan, 1985). Chiltepin peppers are mainly used in cooked foods (Forero et al., 2009). They produce and accumulate compounds known as capsaicinoids (Rochín-Wong et al., 2013), which are responsible for the characteristic pungent taste of these fruits (Tewksbury et al., 2008). Capsaicinoids are a group of acid amides derived from vanillylamide. This capacity to accumulate capsaicinoids determines the degree of pungency associated with particular Capsicum spp. (Aza-González et al., 2011). In 2007, ‘Bhut Jolokia’ from the Assam region of northeastern India was recognized by Guinness World Records as the world’s spiciest chili pepper (Bosland et al., 2012) with 1,001,304 Scoville heat units (SHU); in contrast, the Orange Habanero had a value of 357,729 SHU (Bosland and Published in Crop Sci. 56:1–11 (2016). doi: 10.2135/cropsci2014.11.0789 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved.

crop science, vol. 56, january– february 2016

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Taxonomical Aspects of Chiltepin

Figure 1. Chiltepin Capsicum annuum L. var. glabriusculum. (A) Fruits of chiltepin under wild conditions. (B) Chiltepin flower. (C) Left: Red stage of chiltepin fruits (mature); Right: Green stage (immature).

Baral, 2007). Another study reported that two individual plants of the ‘Trinidad Moruga Escorpion’ variety (Capsicum chinense) from Trinidad and Tobago had 2,009,231 and 2,006,598 SHU (Bosland et al., 2012). In Mexico, chiltepin is considered the second spiciest chili pepper (100,000 to 200,000 SHU) after the habanero chili pepper (C. chinense; 100,000 to 445,000 SHU; Servicio de Información Agroalimentaria Pesquera, 2010), but some varieties of C. annuum can also achieve similar levels of pungency, depending on the culture conditions (Cázares-Sánchez et al., 2005). Capsaicinoids possess antioxidant properties (Perucka and Materska, 2001). The chiltepin pepper is also a source of other antioxidant phytochemicals, such as phenolic compounds, carotenoids, tocopherols, tocotrienols, and vitamin C (Howard et al., 2000; Materska and Perucka, 2005). The chiltepin is considered to be the wild progenitor of the cultivated C. annuum var. annuum (Aguilar-Meléndez et al., 2009; González-Jara et al., 2011) and an important genetic resource for pepper crop improvement due to the discovery of wild populations of Capsicum that did not develop disease symptoms or showed light symptoms, suggesting its resistance to pathogens such as the Pepper huasteco yellow vein virus (Hernández-Verdugo et al., 2001a; González-Jara et al., 2011). This review aims to summarize the taxonomic classification, plant morphology, distribution, genetic diversity, genome sequencing, and phytochemical characteristics of the chiltepin. 2

The remarkable characteristic of the genus Capsicum is its great diversity in fruit type, color, shape, taste, size, and phytochemical content (Zhigila et al., 2014). Emphasizing taxonomy in the genus Capsicum is necessary to differentiate among species and to provide information about the potential of genetic resources to improve the quality or production of the varieties and the phytochemical compounds present in the fruits (Ibiza et al., 2012). The taxonomy and the number of species of the genus Capsicum have been the subject of debate for many years (Eshbaugh, 1975; Bosland and Votava, 2012). During the 19th century, attempts were made to clarify the taxonomy of the genus (Bosland and Votava, 2012). In 1852, the French botanist M. F. Dunal described 50 Capsicum spp., 11 of which were described for the first time (Dunal, 1852; Bosland and Votava, 2012). Currently, the number of species reported for the genus Capsicum varies depending on the authors; reports have mentioned 25 (Aguilar-Meléndez et al., 2009), 31 (Kole, 2011), 27 (Ibiza et al., 2012), and 36 species (Russo, 2012). C. annuum and C. frutescens were initially proposed by Linnaeus in 1753, confirmed by Irish in 1898 (Irish, 1898), and recognized until 1953, when Heiser and Smith categorized the genus into four species: C. annuum, C. frutescens, C. baccatum, and C. pubescens (Heiser and Smith, 1953; Bosland and Votava, 2012). However, in the 20th century, the confusing and long list of names was reduced to five domesticated species: C. annuum, C. frutescens, C. baccatum, C. pubescens, and C. chinense (Russo, 2012; Zhigila et al., 2014). An important feature observed in the Capsicum genus is the presence of capsaicin (the compound that provides the pungency) (Andrews, 1995; Russo, 2012). However, some cultivars of C. annuum var. annuum lack pungency due to human selection (Moscone et al., 2007). At present, there are particular concerns among taxonomists about the classification of chiltepin. Different names have been used for this variety: C. annuum L. var. minus (Figherhuth), C. annuum L. var. baccatum (Terpó), C. annuum L. var. minimum (Heiser and Pickersgill), C. annuum var. aviculare (D’Arcy and Eshbaugh) (Long-Solís, 1998); and C. annuum L. var. glabriusculum (Dunal) Heiser and Pickersgill (Wiersema and León, 1999). The scientific name used for chiltepin is C. annuum L. var. glabriusculum (Dunal) Heiser and Pickersgill (Heiser and Pickersgill, 1975; Wiersema and León, 1999). A similar situation exists for its common name of chiltepin. It is also named chile piquín, chiltepec, chiltepillo, chilpaya, chile de monte, and chile parado (among others) in different regions of Mexico (Araiza-Lizarde et al., 2011). In other countries, this species is known as ají (Colombia), chile pequin, chilipiquin, piquin, turkey pepper, bird pepper, American bird pepper, chiltepin pepper (Sweden; http://www.ars-grin.gov/cgibin/npgs/html/taxon.pl?102342, verified 6 Oct. 2015), and chiltepe (Guatemala) (Guzmán et al., 2005).

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Plant Morphology of Chiltepin The chiltepin is characterized by a high phenotypic plasticity due to the variation of traits such as leaf morphology and the pattern of seed germination (González-Jara et al., 2011). Furthermore, this species is considered to be the pepper with the greatest variation in the size, form, and color of its fruits (Hernández-Verdugo et al., 2001b). Due to the traits mentioned above and the broad distribution range of the chiltepin, it is important to provide a brief description of its morphology to obtain an accurate and correct authentication of this species and to enable a discussion of the genetic differences among populations. The leaves of the chiltepin have thin petioles that range from 1.0 to 2.5 cm in length; the closely winged oval leaf blade is 1 to 4 cm wide and 2 to 6 cm long (Fig. 1A). The flowers are solitary, with a 1.5- to 2-mm-long goblet and a white corolla that is 6 to 9 mm in diameter (Fig. 1B; Nabhan, 1985). The mature fruits are small, red, round berries held erect on long pedicels that are 6 to 8 mm in diameter (Votava et al., 2002). The seeds are white to yellowish and 2.5 to 3 mm long (CONAFOR, 2009). In northern Mexico, chiltepin plants reach their reproductive maturity when they are six to 10 mo old. The flowering period in northern Mexico begins in July and ends in September, and the fruiting period occurs between June and October, although these periods could change depending on the region. The fruit is dark green in its immature state and turns red on maturation (CONAFOR, 2009; Montoya-Ballesteros et al., 2010), as shown in Fig. 1C. Frugivorous birds consume chiltepin seeds; they pass unharmed through their digestive tracts and are dispersed to new microhabitats (Votava et al., 2002).

Geographical Distribution and Ethnobotanical Aspects of Chiltepin The geographical distribution of the chiltepin provides the basis for germplasm evaluations and correlations of the genetic characterization with morphological aspects. The chiltepin is distributed from northern Peru (Russo, 2012), Colombia, Central America, and Mexico to the southwestern United States, including southern Arizona (Votava et al., 2002), New Mexico, and Texas (Nabhan et al., 1990). In Mexico, it is found along the Pacific coast from Sonora to Chiapas and around the Yucatan peninsula and the Gulf of Mexico (Bañuelos et al., 2008; Miranda-Zarazúa et al., 2010; González-Jara et al., 2011). In the north of Mexico, it is found in the states of Coahuila, Nuevo León, Sinaloa, Baja California, and Sonora (Miranda-Zarazúa et al., 2010). In the desert region, it is most common in northwest Mexico and the southwestern United States. The Papago and Pima Indians have traditionally made annual pilgrimages to harvest chiltepin (Nabhan et al., 1990). It is used not only as a condiment but also as a traditional medicine to treat sore crop science, vol. 56, january– february 2016

throats and acid indigestion by some local inhabitants of Sonora State (Nabhan et al., 1990) and can also be used to treat diseases such as earaches, rheumatism, colds, cough, gastritis, and ulcers (Bañuelos et al., 2008).

Cultivation Areas and Economic Aspects of Chiltepin The chiltepin grows in a variety of habitats with deep soils and evergreen vegetation, in xeric regions in the Sonoran desert, or associated with nurse trees, such as in the Central Mexican Plateau (González-Jara et al., 2011). The temperatures for most of the Mexican locations range between 20°C to 26°C (Kraft et al., 2014). The pepper also grows near ditches and roadsides throughout much of Latin American territory with certain weedy characteristics (Nabhan et al., 1990). In desert regions, the chiltepin grows near watercourses and in canyons in the mountainous areas; these characteristics provide higher humidity than the desert (Nabhan et al., 1990). In this context, it has been observed that the chiltepin needs the protection of a nurse tree. These trees provide the chiltepin with shade, humidity, and increased soil fertility and protect it from grazing and trampling. The nurse tree could also serve as a shelterbelt, an erosion barrier, or a hedge (Nabhan et al., 1990). In some areas (i.e., the Sonoran desert), overexploitation may have occurred for several decades and has been identified, together with habitat loss, as a cause for the decline and even extinction of local populations of chiltepin (González-Jara et al., 2011). Human exploitation of chiltepin involves the use of living fences and stands of chiltepin plants in pasture lands, along with cultivation in home gardens. During the 1980s, harvesting chiltepin became an important economic activity, mainly for rural populations (Bañuelos et al., 2008) in central and northern Mexico (González-Jara et al., 2011). The chiltepin fruit is harvested from both natural and cultivated populations as a spice and is also used as a traditional medicine (Pagán et al., 2010). The harvesting is a seasonal activity for women, men, young people, children, and older people (Bañuelos et al., 2008) and can increase their family incomes (Perramond, 2005) by as much as 45% (Montes, 2010). The green fruits of the chiltepin in northern Mexico cost up to $5 per kg, while the red fruits can reach $45 per kg (Montes, 2010). The total chiltepin harvest has been estimated to be approximately 50 metric tons per year (Votava et al., 2002; González-Jara et al., 2011). Exports to California and Arizona reach up to 6 t per year of red dry fruits (Montes, 2010). In recent years, its cultivation has increased to monocultures in small traditional fields, possibly as a result of the growing demand and a reduction in the wild populations (González-Jara et al., 2011).

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Genetic Characterization of the Chiltepin The combined molecular, phenotypical, and geographical data for chiltepin provide reliable information for the assessment of the amount of the species’ genetic diversity (De Vicente et al., 2005). The levels and patterns of genetic diversity can facilitate the reliable classification of accessions for crop improvement programs (Mohammadi and Prasanna, 2003). Although studies of genetic variability are important, limited research has been performed on chiltepin populations (González-Jara et al., 2011). The genus Capsicum is considered for conservation with high global priority. Thus, IPGRI (International of Plant Genetic Resources Institution) and the Centro Agronómico Tropical de Investigación y Enseñanza (CATIE) in Costa Rica formed a global plan for Capsicum in 1979 and determined three priority areas for exploration and collection (Kole, 2011). The first was in Latin America (including Mexico and the Central and South American countries); the second was in Central and South Asia and the Mediterranean; and the third was Southeast Asia, China, and Africa (IBPGR, 1983). There are three networks for Capsicum germplasm collection that provide accessible computerized databases for passport data, characterization, evaluation, and data distribution: Germplasm Resources Information Network (GRIN), The World Vegetable Center, and CATIE (Kole, 2011). For example, GRIN, operated by the USDA, has 3437 accessions of Capsicum annuum L. as of June 2015, of which 66 accessions are C. annuum L. var. glabriusculum (http://www.ars-grin.gov/ cgi-bin/npgs/html/taxon.pl?8904, verified 6 Oct. 2015). Genetic characterization studies have been performed for C. annuum L. var. glabriusculum to explore the genetic diversity and relationships among accessions from different regions around the world using enzyme systems and polymerase chain reaction (PCR)-based markers. One study examined evolutionary relationships using nine enzyme systems that represented 20 loci from 192 accessions belonging to Capsicum (domesticated, semidomesticated, and wild; Loaiza-Figueroa et al., 1989) and 12 accessions from outside of Mexico (Costa Rica, Peru, and New Mexico). There were 69 wild accessions used in this study; these accessions came mostly from the northern states of Mexico (Loaiza-Figueroa et al., 1989). This analysis identified a low level of expected heterozygosity (Hs) in the wild populations (Hs = 0.025), followed by the domesticated (Hs = 0.012) and semicultivated (Hs = 0.007) populations. This finding was in contrast to the study by Hernández-Verdugo et al. (2001b), in which the Hs was larger for the wild (Hs = 0.474) and the cultivated species (Hs = 0.434). Loaiza-Figueroa et al. (1989) demonstrated that genetic differentiation was correlated with geographical isolation, as observed in C. annuum L. var. glabriusculum, where populations from the northeastern and northwestern areas are separated by an arid region 4

known as the Central Mexican Plateau. This result was consistent with a more recent study by Aguilar-Meléndez et al. (2009), which also reported geographic structuring among the semicultivated and cultivated Capsicum species. The genetic diversity of two collections of chiltepin has been examined: in situ populations from Arizona and ex situ germplasms obtained from Guatemala and the north of Mexico (Votava et al., 2002). No differences were detected among chiltepin seeds from Arizona based on random amplification of polymorphic DNA (RAPD) analysis. The accessions from Sonora and Chihuahua were the most genetically diverse of the North American accessions. The greatest genetic diversity out of all of the accessions was seen with the accessions from Guatemala (Votava et al., 2002). Another study of PCR-based marker amplified fragment length polymorphisms (AFLP) used 74 different accessions of Capsicum spp. from different states in Guatemala (Guzmán et al., 2005). The accessions belonged to C. annuum L. (such as C. annuum L. var. annuum and C. annuum var. glabriusculum), C. pubescens, C. chinense, and C. frutescens. Interestingly, 18 of these accessions belonged to C. annuum L. var. glabriusculum, out of which 82% were grouped in a cluster. Moreover, the results suggest the existence of gene flow between Capsicum spp. such as C. annuum, C. frutescens, C. annuum L. var. annuum, and C. annuum L. var. glabriusculum (Guzmán et al., 2005). A similar study of genetic diversity performed using distinct accessions of semiwild (C. annuum L. var. glabriusculum) and cultivated Capsicum spp. (C. annuum var. annuum) from central and southeastern Mexico was conducted by Aguilar-Meléndez et al. (2009). The authors analyzed nucleotide sequence diversity at three nuclear loci (Dhn, G3pdh, and Waxy) and found that the domesticated peppers exhibited a loss of 10 to 17% of genetic diversity compared with the semiwild populations. They also detected a geographic structure associated with genetic differentiation in the analyzed populations of Capsicum spp. LoaizaFigueroa et al. (1989) reported a similar result in a study investigating an isoenzyme polymorphism. Interestingly, the authors identified the greatest nucleotide diversity in both Dhn and G3pdh in the Yucatan peninsula region, suggesting that this region was an important center for pepper domestication and diversity. González-Jara et al. (2011) analyzed the effect of human management on the genetic variation of the chiltepin in wild, managed wild, and cultivated populations, along with those available from local markets in Mexico. The authors used nine microsatellite markers (simple sequence repeats [SSRs]) to determine the changes in genetic diversity in chiltepin populations. They found that the highest genetic variation was found within the Yucatan populations and the lowest within the Sonora populations (northwest of Mexico), again suggesting that the Yucatan State was a center of diversity for these peppers. The study

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also suggested that cultivated populations exhibited a significant decrease in genetic variation compared with the wild-type populations, although there were no phenotypic differences among them. An important observation was that the genetic variation was reduced up to 50% in the chiltepin populations (González-Jara et al., 2011). Another study conducted in landraces (Cascabel, Cola de rata, and Tequila types), hybrids (Anaheim, Guajillo, Serrano, and Poblano), and wild pepper C. annuum var. glabriusculum populations from northwestern Mexico identified an excess of homozygous individuals based on SSR analysis. Seven microsatellite primer pairs were used to analyze the variability and genetic structure of the wild and landrace populations to understand the domestication process (Pacheco-Olvera et al., 2012). The authors identified a higher level of genetic differentiation among domesticated populations compared with wild populations. Additionally, they found greater genetic variation compared with a previous study by Loaiza-Figueroa et al. (1989) characterizing Capsicum populations from Mexico using isozyme markers. An interesting result reported by these authors was the identification of barriers that separated most of the wild populations through their distribution (Pacheco-Olvera et al., 2012).

Genome Sequencing of Chiltepin In 2014, the genome sequences of C. annuum var. glabriusculum and the inbred pepper cultivar Zunla-1 were released (Qin et al., 2014). The seeds of C. annuum var. glabriusculum were collected in north-central Mexico in the state of Queretaro. The authors reported that considerable genetic differences were found between chiltepin accessions from Arizona in the U.S. and Guatemala (Qin et al., 2014). The genome size of the chiltepin was estimated to be 3.07 Gb using Illumina technology (Qin et al., 2014). More than 81% of the chiltepin genome was composed of transposable elements (TE), which is a higher percentage than that reported for potato (Solanum tuberosum L.) and tomato (Solanum lycopersicum L.). The TE in the chiltepin genome included 70.1% long tandem repeat (LTR) retrotransposons and 4.87% DNA transposons. In the pepper, the LTR retrotransposons contributed to genome expansion to a higher degree, suggesting that the large genome size of the pepper is characterized by LTR expansion (Qin et al., 2014). To facilitate the gene annotation of the chiltepin genome, Qin et al. (2014) mapped the amino acid sequences of the genes annotated in Zunla-1 (cultivar C. annuum L.), S. tuberosum, and S. lycopersicum onto the chiltepin genome using blastp. Sequences with more than one small intron, stop codons, and regions where >50% of the coding sequences were annotated as TE were removed. After filtering the sequences, 34,476 genes were identified in the chiltepin genome, and 90% of the predicted


genes were supported by RNA-seq entries, homologous proteins, and expressed sequence tags (Qin et al., 2014). To explore diversity during the transition from wild to cultivated populations with different fruit shapes, sizes, colors, and pungencies, Qin et al. (2014) selected 18 cultivated accessions of C. annuum L., two semiwild (C. chinense) and wild accessions (C. eximium), the cultivated inbred line Zunla-1, and the chiltepin (C. annuum var. glabriusculum). In this study, the wild accessions possessed higher genetic diversity than the cultivars. The authors detected an average of 9,826,526 single-nucleotide polymorphisms for all pepper accessions, and an average of 237,509 small insertions and deletions (Qin et al., 2014).

Phytochemical Studies Plants have developed various defense strategies against adverse conditions, such as biotic and abiotic stresses. They synthesize secondary metabolites to avoid being eaten by herbivores or attacked by microorganisms; additionally, these metabolites play roles in response to competition, exposure to sunlight, and lack of nutrients (SepúlvedaJiménez et al., 2003). The secondary metabolites are synthesized in small quantities. Some of these compounds are not widespread, with production often restricted to a particular plant genus, family, or even a specific species (Ávalos-García and Pérez-Urria Carril, 2009). These compounds are produced by plants as a means of communication with the environment in which they grow. They are considered to be nonessential for cells but have been observed to be involved in ecological functions (De La Cruz-Chacón et al., 2013). Capsicum fruits are a rich source of phenolic compounds, flavonoids, capsaicinoids, carotenoids, tocopherols, ascorbic acid (Wahyuni et al., 2013), and volatile compounds (Forero et al., 2009).

Phenolic Compounds A wide variety of phenolic compounds are synthesized from aromatic amino acids during secondary metabolism in plants as a defense against attack by pathogens. Additionally, they contribute to the flavor, color, and astringency of the plant (Chitindingu et al., 2007). The phenolic compounds have various biological activities, the most important being their antioxidant capacity. They are considered potent inhibitors of the oxidation of low-density lipoproteins and polyunsaturated fatty acids, and thus may reduce the risk of cardiovascular disease; they also have greater stability than ascorbic acid (Serrano et al., 2006). One study showed that chiltepin was an important source of total phenolic compounds (Rochín-Wong et al., 2013). These authors showed that chiltepin fruits collected in northwest Mexico had a content of 663.26 ± 33.91 mg of chlorogenic acid equivalents/100 g of dry sample. The phenolic compounds identified in the chiltepin were the gallic, chlorogenic, and ferulic phenolic acids. In the case

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of total flavonoids, a content of 424.58 ± 10.22 mg of catechin equivalents/100 g dry sample was reported; catechin, luteolin, and resveratrol were also identified. RodríguezMaturino et al. (2012) reported 485 mg/100 g of total phenolic compounds in the chiltepin and 592 mg/100 g in the habanero chili. These chili fruits were purchased from regional markets in Mexicali, Mexico. Howard et al. (2000) reported 431.6 mg/100 g of total phenolic compounds and 0.55 mg/100 g of total flavonoid content in the habanero chili. Medina-Juárez et al. (2012) reported the total phenolic compound amounts of five commercially important peppers (Capsicum annuum L.), including the Anaheim (97.99 mg/100 g), Caribe (154 mg/100 g), Jalapeno (59.34 mg/100 g), bell (103.26 mg/100 g), and Serrano (94.58 mg/100 g). These fruits were collected in northwest Mexico, and all showed lower values of total phenolic compounds than the compounds found in the chiltepin.

Capsaicinoids Chili pepper fruits are unique in their natural synthesis of capsaicinoids. These compounds are responsible for the pungent sensation experienced by mammals when they bite into Capsicum fruits (Tewksbury et al., 2008). Capsaicinoids are a group of acid amides derived from vanillylamine. They are synthesized and accumulate in the epidermal cells of placental tissue, where they are secreted towards the outer cell wall and finally stored within structures named “blisters” located on the placental surface (Aza-González et al., 2011). There are more than 10 different capsaicinoids. Their chemical structure consists of a phenolic nucleus attached to a fatty acid via an amide bond. Capsaicin and dihydrocapsaicin constitute >90% of all capsaicinoids in peppers; other structures include nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin. Capsaicinoid production and relative abundance levels of capsaicinoids in various Capsicum fruits mostly depend on the chili pepper species, genotype, growth conditions (Aza-González et al., 2011; Wahyuni et al., 2013), and maturity stage. Red fruits showed higher amounts of capsaicin and dihydrocapsaicin than green fruits (Conforti et al., 2007). These increases occur concomitant with advancing growth and fruit development (Siddiqui et al., 2013). Capsaicinoids have diverse biological properties with beneficial effects to human health. They have the capacity to stimulate the cardiovascular system (Govindarajan and Sathyanarayana, 1991) and possess antiinflammatory (Anogianaki et al., 2007) and antioxidant properties (Si et al., 2012) and analgesic and antiobesity activities (Luo et al., 2011). The studies conducted by Montoya-Ballesteros et al. (2010) and Rochín-Wong et al. (2013) reported 4.2 to 8.2 mg/g and 4.17 ± 0.11 mg of capsaicinoids/g of dry fruit collected in northern Mexico, respectively. Dihydrocapsaicin was not present in sufficient amounts for detection. Other peppers grown in the central region of Mexico 6

showed higher values of capsaicinoids than those of chiltepin: 53.71 mg/g of dry piquin (Capsicum annuum var. aviculare) and 80.52 mg/g of dry chile de arbol (Capsicum annuum var. annuum; Contreras-Padilla and Yahia, 1998). Blanco-Rios (2013) reported 43.70 mg/g of capsaicinoids in Anaheim red fruits and 102.07 mg/g of capsaicinoids in Jalapeno peppers (Capsicum annuum L.). These fruits are economically important in Mexico, and all showed lower capsaicinoid contents than the chiltepin.

Capsinoids Capsinoids are similar in structure to the capsaicinoids, but possess an ester group instead of an amide moiety (Singh et al., 2009). These phytochemicals were first characterized structurally in fruits of the nonpungent pepper ‘CH-19 Sweet’, where two novel compounds were isolated: capsiate and dihydrocapsiate (Kobata et al., 1998). Capsinoids may offer similar benefits to capsaicinoids with a milder flavor. Capsinoids are associated with enhanced adrenal catecholamine secretion, promotion of energy metabolism, suppression of body fat accumulation, and antioxidant activity, indicating that they provide health benefits and are of importance for both the pharmaceutical and food industries (Singh et al., 2009). Few reports have documented the presence of capsinoids in Capsicum spp. Singh et al. (2009) quantified and identified capsinoids in different varieties of Capsicum spp. using the high-performance liquid chromatography method.

Carotenoids Carotenoids are essential constituents of the photosynthetic machinery and are involved in many aspects related to photosynthesis, such as light harvesting, photoprotection, photomorphogenesis, and nonphotochemical quenching (DellaPenna and Pogson, 2006). They are also responsible for the attractive colors of many flowers and fruits involved in attracting pollinators and seed dispersers (Hirschberg, 2001). Capsicum fruit color can be attributed to several pigments, including chlorophylls, carotenoids, and flavonoids (Lightbourn et al., 2008). Capsicum is considered one of the richest sources of carotenoids among the vegetable crops (Wahyuni et al., 2011). From a nutritional point of view, the interest in these tetraterpenes is due to their provitamin A activities (b-carotene, a-carotene and b-cryptoxanthin; Abushita et al., 2000). Carotenoids with provitamin A activities have been proposed to be bioactive compounds, along with other carotenoids such as lycopene, lutein, and zeaxanthin that possess antioxidant activity amongst other biological activities (Rao and Rao, 2007). The conjugated double bonds within their structures act as stabilizers of free radicals and other reactive oxygen species (Abushita et al., 2000). Compounds such as b-carotene, b-cryptoxanthin, and zeaxanthin are transformed into antheraxanthin and violaxanthin,

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Table 1. Carotenoids, chlorophylls, and tocopherols contents in green and red chiltepin fruit (C. annuum L. var. glabriusculum) in dry weight.† Total carotenoids, mg/g b-carotenoid, mg/g Zeaxanthine, µg/g Lutein, µg/g Chlorophyll a, µg/g Chlorophyll b, µg/g a-Tocopherol, mg/100g g-Tocopherol, mg/100g

Green

Red

12.81 ± 0.7a 4.93 ± 0.3a nd‡ 90.03 ± 2.7 31.48 ± 1.6 40.63 ± 1.3b 20.37 ± 1.7a 7.97 ± 0.3a

33.23 ± 0.3b 7.69 ± 0.2b 3.98 ± 0.2 nd nd 12.12 ± 0.3a 30.23 ± 2.3b 9.08 ± 0.6b

The values in each row with different letters show a significant difference (p < 0.05).

a,b

†

Values are mean ± standard deviation of triplicates.

‡

nd, not detected.

which are substrates for capsanthin-capsorubin synthase. This enzyme is exclusively present in the Capsicum genus (Kevrešan et al., 2009) and is critical for the production of the major carotenoids in red-fruited peppers, such as capsanthin and capsorubin (Wahyuni et al., 2011). The main carotenoids found in red chiltepin fruits collected in northwest Mexico were b-carotene and zeaxanthine, while in green chiltepin fruits, the main carotenoids were lutein and chlorophylls a and b (Table 1; Rochín-Wong, 2012). These results are similar to those reported by Howard et al. (2000) and Wahyuni et al. (2011), who established variation in the carotenoid content as a function of maturity. Blanco-Rios (2013) reported lower values of total carotenoids in the Anaheim pepper (approximately 6 mg/g) than those reported for the red chiltepin fruits (Rochín-Wong et al., 2013).

Tocopherols Mixtures of tocopherols and tocotrienols (vitamin E) are recognized for their effective inhibition of lipid oxidation in food and biological systems. They consist of two primary structural components: a complex aromatic ring and a long side chain. The tocopherols are found in four forms: a-tocopherol, b-tocopherol, g-tocopherol, and d-tocopherol. These forms differ in the number and position of the methyl groups in the aromatic ring. a-Tocopherol accumulates in the pericarp tissue, whereas g-tocopherol is found in the seeds of pepper fruits (Wahyuni et al., 2011). Capsicum fruit, especially in the dehydrated form, is an excellent source of tocopherols (vitamin E). The chloroplast transformation to chromoplasts during fruit ripening in Capsicum annuum L. is associated with a series of events, such as the massive accumulation of carotenoids and the increased synthesis of a-tocopherol (Arango and Heise, 1998). Chiltepin is usually consumed in a dehydrated form. This process is accomplished using a traditional method of exposure to sunlight for 32 h at temperatures ranging from 34 to 40°C with 37% relative humidity. During this drying process, the total a- and g-tocopherol contents crop science, vol. 56, january– february 2016

increased from 30.23 ± 2.29 and 9.08 ± 0.6 mg/100 g dry weight of fresh chiltepin to 38.63 ± 1.05 and 10.26 ± 0.22 mg/100 g of dried chiltepin, respectively (Rochín-Wong, 2012). Additionally, the a- and g-tocopherol contents increased with maturity (from green to red; Table 1).

Antioxidant Capacity It is very important to evaluate the antioxidant capacity of phytochemical compounds in fruits and vegetables because natural antioxidants, such as phenolic compounds, capsaicinoids, carotenoids and tocopherols, are becoming increasingly necessary in our diets to maintain health and to reduce the incidence rates of cardiovascular disease and cancer (Maoka et al., 2001; Oude Griep et al., 2010; LópezAlarcón and Denicola, 2013). The interest in the pepper fruit (Capsicum annuum L.) is due to its use as coloring, flavoring, and pungent agents in sauces, processed meats, snacks, candies, and alcoholic beverages. Furthermore, the interest is also in large part due to the presence of bioactive compounds and their importance as dietary antioxidants (Materska and Perucka, 2005; Alvarez-Parrilla et al., 2011). Different chemical methods are used to evaluate the antioxidant activities of the phytochemical compounds present in fruits and vegetables (Ozgen et al., 2006). These chemical tests are widely used to determine the antioxidant potentials of vegetable products and nutraceutical preparations (Blasa et al., 2011). Trolox equivalent antioxidant capacity is based on the capacity of the antioxidants to reduce the 2,2¢-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS·+), and the 2,2-diphenyl-1-picrylhydrazyl (DPPH·) radicals (Arnao et al., 1999; Brand-Williams et al., 1995). The capacity of phenolic compounds to neutralize the radicals ABTS·+ and DPPH· has been reported by other authors (Alvarez-Parrilla et al., 2011; Kim et al., 2011). Rochín-Wong et al. (2013) reported the capacity of the phenolic compounds of chiltepin to inhibit the radicals ABTS·+ (93.36 ± 1.48 mmol Trolox equivalent/g dry weight) and DPPH· (24.04 ± 0.78 mmol Trolox equivalent/g dry weight). The correlations between the total phenol content of the chiltepin and its antioxidant capacity were significant (P < 0.05, r = 0.93 for ABTS and r = 0.98 for DPPH; Rochín-Wong et al., 2013). Furthermore, chlorogenic acid showed a significant correlation (P < 0.05) in both assays (ABTS, r = 0.91; DPPH, r = 0.9). Significant correlations between the total phenol content of chiltepin and the antioxidant activity were previously reported for the Jalapeño and Serrano peppers (Alvarez-Parrilla et al., 2011). Thus, the phenolic compounds present in the chiltepin could play important roles as antioxidants. With respect to the antioxidant capacity of chiltepin capsaicinoids, Rochín-Wong et al. (2013) reported antioxidant capacities of 12.68 ± 0.39 mmol Trolox equivalent/g dry weight by ABTS and 7.11 ± 0.19 mmol Trolox equivalent/g dry weight by DPPH. The correlations

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between the concentration of capsaicinoids and the antioxidant activity were high for both tests (r = 0.9 and r = 1.0, respectively). This result indicates that capsaicinoids could contribute to the antioxidant activity of this pepper.

Volatile Compounds The volatile composition in Capsicum fruits is an important characteristic for the differentiation of types of peppers to guarantee their origin and to improve their quality. Therefore, the food industry is interested in obtaining peppers with concentrated aromas for flavored products (Pino et al., 2011). Nearly 125 volatile compounds in fresh and processed Capsicum fruits have been identified, but the flavor significance of these compounds is not clearly understood (Pino et al., 2007). In Capsicum annuum var. glabriusculum, 140 volatile compounds have been found (Forero et al., 2009). Aliphatic esters were found in both the green and red fruits, with notable abundance during the green stage. The highest levels of hexyl isopentanoate (29.2 mg/kg), hexyl 2-methylbutanoate (8.3 mg/kg), g-himachalene (6.6 mg/kg), hexyl isohexanoate (6.2 mg/kg), (E)-2-hexenal (5.9 mg/kg), hexyl isobutanoate (4.3 mg/kg), and (Z)-3-hexenyl isopentanoate (4.0 mg/kg) were found in chiltepin during the green stage, chemicals that have powerful and fruity odor notes (Forero et al., 2009). In contrast, the major chemical classes such as alcohols (in green 3.0 mg/kg, in red 4.0 mg/kg), carbonyls (in green 15.0 mg/kg, in red 18.0 mg/ kg), and terpenes (in green 23.0 mg/kg, in red 25.0 mg/ kg) showed a tendency to increase with maturation. The higher amount of volatiles found during the green stage of the pepper compared with the mature stage suggests that the green stage is better for flavor (Forero et al., 2009). In contrast, the habanero pepper (Capsicum chinense Jacq.) is considered a highly aromatic pepper (Pino et al., 2007). Fifty-three volatile compounds were identified in green and orange habanero peppers. The main volatile compounds were: hexyl isopentanoate (14.8% in green; 33.1% in orange), 3,3-dimethyl cyclohexanol (16.7% in green; 21.7% in orange), hexyl pentanoate (20.3% in green; 17.7% in orange), and (Z)-3-hexenyl isopentanoate (14.8% in green; 0.7% in orange; Cuevas-Glory et al., 2015). Similar results were found in 10 cultivars of habanero peppers at the mature stage. Out of the 65 volatile compounds identified in this pepper, the most abundant volatile compounds were hexyl isopentanoate (1.924 mg/ kg dry fruit in average) and 3,3-dimethyl cyclohexanol (1.298 mg/kg dry fruit in average; Pino et al., 2007).

CONCLUSIONS AND FUTURE PROSPECTS The chiltepin is considered an important genetic resource for pepper crop improvement due to the virus resistance found in some varieties. The taxonomic classification for 8

this species needs to be clarified, and a thorough analysis of chiltepin populations from different geographical regions is urgently required. Moreover, the chiltepin is an important source of phytochemicals, such as phenolic compounds, capsaicinoids, carotenoids, tocopherols, and volatile compounds. Therefore, more detailed analyses pertaining to the antioxidant capacities of these secondary metabolites are required. Finally, with the availability of whole-genome sequences of the chiltepin, future studies should focus on the molecular mechanisms that regulate the biochemical pathways involved in the production of secondary metabolites in the chiltepin. Acknowledgments The authors thank M.Sc. Carmen Sarai Rochin Wong and C. B. Claudia Celeste Molina Dominguez for their contribution to this work. C. H.-K. thanks the Mexican Research Council Consejo Nacional de Ciencia y Tecnología (CONACYT) for fellowship No. 191479.

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