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Capsaicinoids Content in Habanero Pepper (Capsicum Chinense Jacq.)

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Capsaicinoids Content in Habanero Pepper (Capsicum Chinense Jacq.) HORTSCIENCE 43(5):1344–1349. 2008. Materials and Methods For this study, 18 accessions of Habanero Capsaicinoids Content in Habanero pepper (Capsicum chinense) (Table 1) from a germplasm bank established in the Centro de Pepper (Capsicum chinense Jacq.): Investigacio´n Cientı´fica de Yucata´n (Scien- tific Research Center of Yucatan) were used. Hottest Known Cultivars Of these, 13 accessions were of the ‘‘orange type’’, four of the ‘‘red type’’, and one ‘‘yel- Adriana Canto-Flick, Eduardo Balam-Uc, Jerico´ Jabı`n Bello-Bello, low type’’. The plants were cultivated and Carlos Lecona-Guzma´n, Daniela Solı´s-Marroquı´n, Susana Avile´s-Vin˜as, maintained under protected conditions and ´ ´ 1 the fruit was harvested when it had ripened on Eunice Gomez-Uc, Guadalupe Lopez-Puc, and Nancy Santana-Buzzy the plant. Thirty fruits per plant were taken Unidad de Bioquı´mica y Biologia Molecular de Plantas, Centro de from 10 plants of each accession. Investigacio´n Cientı´fica de Yucata´n, Calle 43 # 130, Chuburna´ de Hidalgo, This work was carried out in the munic- Me´rida, Yucata´n, CP. 97200 Me´xico ipality of Ixil, Yucatan, located between the parallels 21°09# and 21°19# lat. north and the Lourdes Georgina Iglesias-Andreu meridians 89°25# and 89°34# long. west at an Lab. Biotecnologı´a y Ecologı´a Aplicada (LABIOTECA), Universidad average height of 9 m above sea level. The Veracruzana. Campus para la Cultura, las Artes y el Deporte, Xalapa, climate of the region is hot, subhumid with rain in the summer and a dry season from Veracruz, C.P. 91001, Me´xico January to May. The average annual temper- Additional index words. pungency, capsaicinoids, HPLC, Scoville Units, germplasm, ature fluctuates between 24.6 and 27.7 °C and Habanero pepper, Capsicum chinense, Jacq. annual rainfall between 600 and 900 mm. The predominant soil type in this region is Abstract. The aim of this study was to determine the pungency level of different accessions litosols or superficial stony soil in accordance of Habanero peppers. The high-performance liquid chromatography (HPLC) technique with the classification by the Food and Agri- was used to evaluate the content of total capsaicinoids in the whole fruit, placenta, and culture Organization of the United Nations. pericarp of 18 accessions of Habanero pepper from the germplasm bank of the Capsicum Determination of capsaicinoid content. chinense species maintained in the Scientific Research Center of Yucatan [Centro de The capsaicinoid content in placenta, peri- Investigacio´n Cientı´fica de Yucata´n (CICY)]. Thirteen of these accessions belonged to the carp, and whole fruit of each selected acces- ‘‘orange type’’, four to the ‘‘red type’’, and one to the ‘‘yellow type’’. During the study, the sion was evaluated. They were frozen plants were cultivated and maintained under greenhouse conditions and the fruit was immediately in liquid N2 and lyophilized to harvested only when it was completely ripe on the plant. The results show considerable carry out the capsaicinoid analysis. Capsai- intraspecific diversity for this characteristic as well as the existence of cultivars of this cinoids were quantified with HPLC accord- species that surpass the levels of pungency reported for Habanero peppers under the ing to the method reported by Collins et al. conditions evaluated. (1995) but slightly modified. Three repeti- tions of 1 g (fresh weight) lyophilized sam- ples of whole fruit, placenta, and pericarp were evaluated. These samples were mixed Peppers (Capsicum spp.) are well known 90% of the pungency (Govidajaran, 1986; with acetonitrile (1 : 40) and maintained at for their ability to cause an intense organo- Iwai et al., 1979; Kawada et al., 1985; 80 °C for 4 h with constant shaking before leptic sensation of heat when consumed. Pep- Kosuge and Furata, 1970). Capsaicinoids are they were allowed to cool to room tempera- pers probably originated in Bolivia, as this unique to the genus Capsicum (Govidarajan ture. The mixtures were then filtered through area contains many of the 20 to 27 recognized et al., 1987 ; Govindarajan and Sathyanar- a Wathman filter (Wathman Intl LTD., Maid- species of Capsicum (Andrews, 1999; ayana, 1991). The level of capsaicinoids can stone, England) and subsequently centrifu- Hunziker, 2001;Walsh and Hoot, 2001). In be determined using chemical, instrumental, ged for 10 min at 10,000 g. Supernatant was 2006, C. chinense cv. Red Savina was recog- or sensorial methods. The Scoville organic extracted and filtered through a nylon mem- nized as the hottest chili pepper on record with test, invented by Scoville in 1912, is a sub- brane (0.45 mm) into a 5-mL glass sample a heat level of 577,000 Scoville Units (SHU) jective measure of chili pungency. Several vial covered from the light and stored at 5 °C (Guinness Book of World Records, 2006). different methods have been used for the until analysis. A 20-mL aliquot was used for However, Bosland and Baral (2007) recently quantification of capsaicinoids from peppers each HPLC injection. An HPLC instrument, reported that ‘‘Bhut Jolokia’’, a natural inter- and oleoresins, including organoleptic Agilent 1100 (Agilent Technologies, Ger- specific hybrid between Capsicum chinense methods (Govindarajan et al., 1977; Scoville, many) series, equipped with a Zorbax and Capsicum frutescens, is in fact the world’s 1912), spectrophotometry (Anan et al., 1996; (ODS)-C18 reversed-phase column of 4.6 hottest known chili pepper with a heat level of Awasthi and Singh, 1973; Bajaj, 1980; Bajaj mm i.d. · 250 mm, with a detector set at 879,953 to 1,001,304 SHU. and Kaur, 1979; Mori, 1976), thin-layer 280 nm was used. The mobile phase was The heat sensation is incited by a group of chromatography (Sankarikutty et al., 1978), capsaicinoids, alkaloids found only in chili gas–liquid chromatography (Todd et al., Table 1. Accessions of Habanero pepper used for pepper (Zewdie and Bosland, 2000). Capsa- 1977), and high-performance liquid chroma- the analysis of pungency by high-performance icin and dihydrocapsaicin are responsible for tography (Weaver and Awde, 1986). Of liquid chromatography. these, high-pressure liquid chromatography Accession Color Accession Color (HPLC) is considered the most reliable and RUX Red NP1EG Yellow rapid method (Yao et al., 1994) available for Received for publication 13 Nov. 2007. Accepted Xaman Orange AL Orange the identification and quantification of cap- Cuza Orange AJ Orange for publication 5 Mar. 2008. saicinoids. The aim of this study was to We thank the Yucata´n-Produce Foundation and Campn Orange AI Orange SINAREFI for funding our research, M.C. Rosa determine the content of total capsaicinoids REX Red AG Orange Marı´a Galaz Avalos for technical assistance, and in the whole fruit, placenta, and pericarp of Btz Red AF Red Ing. Eleazar Xool for the use of his greenhouse. 18 accessions of Habanero pepper collected SiQR Orange AC Orange 1To whom reprint requests should be addressed; in the Peninsula of Yucatan using the HPLC NP4EC Orange Csc Orange e-mail [email protected] technique. NP3EC Orange AK Orange 1344 HORTSCIENCE VOL. 43(5) AUGUST 2008 BREEDING,CULTIVARS,ROOTSTOCKS, AND GERMPLASM RESOURCES isocratic with 70% solvent B (100% metha- Results study also highlighted the considerable dif- nol) and 30% solvent A (10% methanol ference in capsaicinoid content among the solution v/v). HPLC operating conditions to The determination of capsaicinoid con- different parts of the fruit. The placenta pro- determine total capsaicinoids included: tent in the fruit of peppers, using HPLC, is duced the greatest number of peaks cor- ambient temperature, flow rate of 1 mLÁmin, based only on the period of retention and on responding to capsaicinoids, whereas the and run duration of 10 min. All solvents were the size of the peak of each capsaicinoid fewest were detected in the pericarp. How- filtered and degassed using a 47-mm, all glass present, which was identified by comparison ever, the pericarp apparently registered the filter holder. Capsaicin and dihydrocapsaicin with the retention periods of the commercial highest number of peaks corresponding to were identified and quantified using stand- standards for each composite. The chromato- pigments. These results corroborate those ards of both compounds (Fluka, purity was grams obtained for whole fruit, placenta, and presented by Iwai et al. (1979), Susuki and 98% for capsaicin and 90% for dihydrocap- pericarp of Habanero pepper showed two Iwai (1984), and Bagdathoglu (2002), who saicin). Standard curves were prepared in major peaks, identified as capsaicin (Cap) reported that capsaicinoids synthesize and 100% methanol using serial dilution of and dihydrocapsaicin (Dhc), which regis- accumulate in the vesicles of placenta tissue 10, 25, 50, 100, 500, and 1000 ppm by a tered a difference of 2.03 min between the in the fruit. dilution of a 2000 pm stock solution.(r2 = retention periods of the capsaicin (6.83 min) Pungency levels obtained for whole fruit 0.996). Quantification of unknown capsaici- and the dihydrocapsaicin (8.86 min). With generally varied ranging from 405,228 to noids was registered by the external standard these results, we can assume that the separa- 892,719 SHU (Table 1). In this study, besides method. tion of the major capsaicinoids was efficient. detecting a considerable variation in pun- The content of capasaicinoids was con- Figure 1 shows the chromatograms of the gency levels for the cultivars included in verted from parts per million (ppm) to SHU RUX accession, red-type Habanero. Simi- the collection evaluated, it was also possible by multiplying the parts per million by 16 larly, it was possible to detect minor capsai- to observe that these levels were superior to (Helrich, 1998). The collected data were cinoids such as norhydrocapsaicin (Ndc) and those already reported for Habanero pepper processed with the program STATISTICA homodihydrocapsaicin (Hc) as well as other (Bosland and Baral, 2007).
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