Determination of Major and Minor Capsaicinoids in Different Varieties of the Capsicum Fruits Using GC-MS and Their Inhibition Ef

International Food Research Journal 21(6): 2237-2243 (2014) Journal homepage: http://www.ifrj.upm.edu.my Determination of major and minor capsaicinoids in different varieties of the Capsicum fruits using GC-MS and their inhibition effect of the chilli extract on α-amylase activity 1Maokam, C., 2Techawongstien, S. and 1*Chanthai, S. 1Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand 2Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand Article history Abstract Received: 27 November 2013 The major and minor components of capsaicinoids were determined in seven main varieties of Received in revised form: the Capsicum fruits grown in Northeast of Thailand. The methanol extract of capsaicinoids was 7 April 2014 cleaned up by C18 cartridge prior to qualitative and quantitative analysis by GC-MS. The chilli Accepted: 9 April 2014 samples qualitatively comprised of nine identified compounds including nornordihydrocapsaicin, Keywords nordihydrocapsaicin, N-vanillyl-nonanamide, capsaicin, dihydrocapsaicin, N-vanillyl- decanamide, homocapsaicin, homodihydrocapsaicin isomer I and isomer II. Using full scan MS, Capsaicinoids the analytical figures of merit of the method had been validated and, based on the calibration Chilli curve of dihydrocapsaicin, both groups of the capsaicinoids could be quantitatively determined. α-amylase activity It was found that the two major capsaicinoids consisting of 88.3 – 96.6% (capsaicin, 45.2 Dinitrosalicylic acid GC-MS – 60.5% and dihydrocapsaicin, 32.2 – 44.0%) were found in higher contents in all studied samples, while some of the minor ones were still undetectable. The total contents of these capsaicinoids were ranged of 3,718.2 – 7,671.8 µg g-1 DW. Furthermore, an inhibition of α-amylase activity of the chilli extract was estimated by dinitrosalicylic acid method, resulted as a potent inhibitor (16 – 33% inhibition) against the amylase activity, potentially regarding to physiological response for a risk reduction of diabetes. © All Rights Reserved Introduction Chilli or chilli peppers (Capsicum spp.) are popular spices in many parts of the world. Their properties of color, aroma, flavor and pungency account for their extensive usage. The pungency is the most outstanding property of the so-called “hot” chilli pepper, resulting from the accumulation of capsaicin and other related compounds commonly Figure 1. Chemical structure of (a) capsaicin and (b) known as capsaicinoids. They are organic compounds dihydrocapsaicin which are only found in the Capsicum genus and have also been identified but are present at very low bioactive molecules currently relevant in medical and levels and are not thought to make any contribution food science (Caterina et al., 2000; Chu et al., 2003). greatly to the overall pungency of the capsicum fruits Its chemical structure is composed of a vanillylamide (Lopez-Hernandez et al., 1996). The capsaicinoids moiety and an acyl chain of 8-13 carbon atoms have broad bands of both properties and their (Schweiggert et al., 2006). Two major capsaicinoids corresponding applications. In particular, they are present in most varieties of hot chilli are capsaicin antimutagenic and antitumoral compounds (Surh and (trans-8-methyl-N-vanillyl-6-nonenamide) and Lee, 1996), antioxidants (Barbero et al., 2006), and dihydrocapsaicin (8-methyl-N-vanillylnona-namide) are commonly used as topical analgesics for many as shown Figure 1. In addition to the two major painful clinical conditions (Kaale et al., 2002). components, other minor capsaicinoids are also The quantitative analysis of capsaicinoids in existed in chilli, including nordihydrocapsaicin, chilli pepper, topical cream (Kaale et al., 2002), self- norcapsaicin, homocapsaicin, homodihydrocapsaicin, defense weapons (Reilly et al., 2001), aerosol defense nornorcapsaicin, nornornorcapsaicin, and nonivamide sprays (Spicer et al., 2005) and pepper sauces (Pena- (Barbero et al., 2008). Some minor capsaicinoids Alvarez, 2009) has been interest for many reasons. *Corresponding author. Email: [email protected] 2238 Maokam et al./IFRJ 21(6): 2237-2243 Extraction techniques of capsaicinoids from hot mass spectrometer (USA). An analytical column, chilli pepper have numerously been developed such ZB-5 capillary column coated with 5% diphenyl and as maceration (Titze et al., 2002), Soxhlet (Korel 95% dimethylpolysiloxane (30 m × 0.25 mm, 0.25 µm et al., 2002) and ultrasound-assisted extraction film thickness), Phenomenex (USA) was employed. (Karnka et al., 2002). The capsaicinoids extract were The inhibition of α-amylase was obtained by UV- subjected to analyze by various techniques including Visible spectrophotometer (Spectronic 15, Thermo spectrophotometry (Perucka and Oleszek, 2000) and Scientific, USA) equipped with a 1-cm quartz cell. thin layer chromatography (Materska and Perucka, A low pressure rotary evaporator, model R-200, 2005). In recent years, the popular techniques for the Buchi (Switzerland) was used to reduce the solvent analysis of capsaicinoids are high-performance liquid giving the crude residue. Hot plate stirrer, model MR chromatography (HPLC) and gas chromatography- 3001, Heidolph (Germany) and centrifuge, model mass spectrometry (GC-MS). EBA 20, Hettich Zentrifugen (Germany) were used Recently, the capsaicinoids have attracted the for sample extraction. Vortex mixer, model G-560E interest of many researchers because they show (Scientific Industries Inc., (USA) and water bath, promise of being powerful antioxidants, protecting model Isotemp 228, Fisher Scientific (UK) were used the human body from free radicals. In addition, one to investigate the α-amylase inhibitory activity. The benefit of hot chilli is that they contain bioactive C18 cartridges (500 mg in 4 cm3) were obtained from components such as polyphenol that can reduce Alltech (USA). A Pipetman P20-1000 (Pipetman, oxidative stress and modulate harmful biological France) micropipette and membrane filter, 0.45 µm pathways, thereby reducing these chronic diseases. (Whatman International, UK) were used. Various polyphenols have been reported to show radical scavenging activity (Sawa et al., 1999) as Plant samples well as an inhibiting α-amylase and α-glucosidase Hot chilli samples were obtained from Department activities (Kim et al., 2000). of Plant Science and Agricultural Resources, Faculty This study was aimed to investigate both of Agriculture, Khon Kaen University, Khon Kaen, qualification and quantification of major and minor Thailand. Seven varieties of the chilli samples with capsaicinoids in some varieties of hot chilli grown their local breeding varieties belong to the same in Northeast of Thailand using a full scan mode of species, Capsicum annuum. L., include “Jindanil 80” GC-MS. Additionally, α-amylase inhibitory activity (S1), “Numkaewtong 80” (S2), “Num Mordindang” of the chilli extracts was measured by reducing sugar (S3), “Superhot” (S4), “Yodson Khem 80” (S5), method. “Yodson Korat” (S6) and “Yodson Mordindang” (S7). The chilli fruit was dried in an oven at 60oC Methods and Materials for 48 h and ground by a kitchen grinder (Moulinex Optiblend 2000, France) to pass a 30-mesh sieve. Chemicals The ground chilli samples in sealed plastic bag were All chemicals used were of analytical grade stored in a desiccator before use (Contreras-Padilla including α-amylase from Bacillus subtilis (51.5 and Yahai, 1998). U mg-1) (Fluka, Switzerland), capsaicin and dihydrocapsaicin (Sigma, USA), 3,5-dinitrosalicylic Sample preparation acid (Fluka, China), D(+)-maltose monohydrate Extraction of capsaicinoids from hot chilli (Acros Organics, USA), potassium sodium tartrate samples was carried out. This procedure consisted of (Carlo Erba, Italy), dimethylsulfoxide (Lab Scan, two main steps: magnetic stirring extraction (MSE) Thailand), methanol both analytical and HPLC and followed by solid-phase extraction (SPE). Two grade (Lab Scan, Thailand) and sodium chloride grams of the chilli sample were macerated using (Analar, England). Tapioca power was of commercial magnetic stirring with 20 mL of methanol at 60oC for grade, purchased from local market in Khon Kaen, 2 h. The extract solution was centrifuged at 5,000 rpm Thailand. for 5 min to remove the residue of the plant sample and then filtered through a Whatman No. 42 filter Instruments paper. The solvent was evaporated to dryness using a Determination of major and minor capsaicinoids rotary evaporator, and a crude extract was obtained. in hot chilli samples was performed by GC-MS. Its The chilli extract was re-dissolved with methanol to system was controlled by Xcalibur software version make a final volume of 5 mL and then passed through 1.3, which compose of Trace GC chromatograph a C18 solid phase cartridge. Model K073(0)910 (Italy) and Finnigan Polaris Q The solid-phase cartridge was conditioned with 2 Maokam et al./IFRJ 21(6): 2237-2243 2239 mL of methanol and followed by 2 mL of deionized Determination of α-amylase inhibition activity water. 400 µL of the extract solution was diluted There are several methods for an investigation of with 600 µL deionized water and injected into the α-amylase activity (Hagberg, 1951; Park and Wang, conditioned C18 cartridge. The cartridge was then 1991, Tundis et al., 2007; Menichini et al., 2009). In rinsed with 2 mL of deionized water. Finally, the this work, the α-amylase activity was determined by capsaicinoids

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