Characterization of Capsaicinoids and Antioxidants in ...

Plant Foods Hum Nutr DOI 10.1007/s11130-013-0386-5

ORIGINAL PAPER

Characterization of Capsaicinoids and Antioxidants in Hot Peppers as Influenced by Hybrid and Harvesting Stage Qumer Iqbal & Muhammad Amjad & Muhammad Rafique Asi & Agustin Ariño

# Springer Science+Business Media New York 2013

Abstract Studies were conducted to investigate the accumulation pattern of capsaicinoids and antioxidants such as carotenoids, ascorbic acid and phenolic compounds in three hot pepper hybrids at five different harvesting stages: immature green, mature green, color break, red ripe and dried fruit. Capsaicin and dihydrocapsaicin contents were maximum at mature green stage in both Sky Red (61.30 and 43.76 mg/100 g) and Wonder King (43.93 and 26.16 mg/100 g) hybrids, whereas Maha hybrid reached top values at color break stage (39.13 and 24.20 mg/100 g). The accumulation of total carotenoids showed an 8-fold increase from red ripe (12 mg/100 g) to dried fruit stage (96 mg/100 g), while a noticeable decline by 76 % was observed for ascorbic acid at same harvesting stages (150 vs. 36 mg/100 g, respectively). The three hot pepper hybrids showed great variations in the evolution of total phenolic contents during harvesting stages. Overall, the mature green stage was ideal to acquire maximum pungency due to capsaicinoids, while peppers at red ripe stage were best sources of ascorbic acid and dried fruits contained higher levels of total carotenoids. Electronic supplementary material The online version of this article (doi:10.1007/s11130-013-0386-5) contains supplementary material, which is available to authorized users. Q. Iqbal (*) : M. Amjad Institute of Horticultural Sciences, University of Agriculture, Faisalabad 38040, Pakistan e-mail: [email protected] M. R. Asi Nuclear Institute for Agriculture and Biology (NIAB), Jhang Road, Faisalabad 38000, Pakistan A. Ariño Veterinary Faculty, University of Zaragoza, 50013 Zaragoza, Spain Present Address: Q. Iqbal Nuclear Institute for Agriculture and Biology (NIAB), Jhang Road, Faisalabad 38000, Pakistan

Keywords Hot peppers . Capsaicinoids . Carotenoids . Ascorbic acid . Phenolic compounds . Harvesting stage

Introduction Hot pepper is an important agricultural crop, not only because of its economic importance, but also due to the nutritional and medicinal value of its fruits and spices used as food colorings and flavorings. A wide spectrum of capsaicinoids, carotenoids, antioxidant vitamins and phenolic compounds are present in hot pepper fruits. Levels of all these antioxidants can vary with hybrid, maturity stage at harvest and agro-climatic conditions, as well as during storage and processing [1, 2]. The consumption of hot peppers is mainly due to their pungent flavor which is determined by the production of capsaicinoids such as capsaicin and dihydrocapsaicin within pod. Capsaicinoids possess physiological, pharmacological and antimicrobial activities, used in the treatment of several painful and inflammatory conditions [3, 4]. A variety of vegetables are good dietary sources of carotenoids, especially those with orange, yellow, red and dark green colors [5]. While capsicums all have carotenoids the amounts they have differ between hybrids and level of maturity [6]. Ascorbic acid is a nutritional and functional component of hot pepper fruits recognized as an antioxidant and biologically active compound [7–9]. Hot pepper has higher ascorbic acid concentration as compared to other fruits and vegetables which are commonly recognized as rich sources of this antioxidant, and great variations have been documented due to agronomic and physiological conditions [10]. In recent years, phenolic compounds have attracted the interest of researchers because of their antioxidant activity that can protect the human body from free radicals. Hot peppers were found to be a good source of phenolic compounds and are ranked fourth after broccoli, spinach and onion with respect to total phenolic

Plant Foods Hum Nutr

contents [11]. The concentration of polyphenols is generally reported to increase as the peppers reached maturity [12, 13], while other authors observed the opposite trend [14]. In summary, while capsicums all have phytochemicals and antioxidant nutrients, the types and the amounts they have differ between hybrids and harvesting stage. However, most previous research in peppers has been only done in two maturing stages, green and red fruits. The aim of the present work was to complete the knowledge on Capsicum antioxidants during all five maturing stages, namely immature green, mature green, color break, red ripe and dried fruit. An experimental study was designed to quantify the distribution pattern of capsaicinoids and antioxidants in hot pepper fruits as influenced by hybrid and maturation level, which could assist producers to decide the optimum harvest date to maintain the quality of produce and increase the health benefits of these compounds.

Materials and Methods Plant Material Commercially available hot pepper hybrids (Capsicum annuum L.) namely Sky Red, Maha and Wonder King were grown in plastic tunnels under drip irrigation system at the Institute of Horticultural Sciences, University of Agriculture (Faisalabad, Pakistan) and harvested at five different stages: immature green (15–18 days after flowering), mature green (≈ 40 days, fully expanded completely green), color break stage (≈ 48 days, fully expanded 50 % green, red), red ripe (≈ 54–65 days, fully expanded completely red) and dried fruit (≈ 90–95 days after flowering, fruits were allowed to dry on plant). After harvest, fruits of each hybrid were placed in polyethylene bags and stored immediately in a freezer at −20 °C until analysis. Each sample had three replications. Determination of Capsaicin and Dihydrocapsaicin Samples were analyzed by using the method of Collins et al. [15] with slight modifications. Hot pepper pods were ovendried at 60 °C for 2–5 days, cooled, and then ground to dried pepper powder. A mixture of sample:acetonitrile in a ratio of 1:10 was placed in 120 ml glass bottles with Teflon-lined lids, capped and placed in a water bath at 80 °C for 4 h and swirled manually every hour. The bottles were removed from water bath and cooled at room temperature. The supernatant content of samples (2–3 ml) was filtered through 0.45 μm (Millex®-HV filter) using a 5 ml disposable syringe (Millipore, Bedford, MA) into a HPLC sample vial. Standards of capsaicin and dihydrocapsaicin were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Liquid Chromatographic Analysis of Capsaicinoids An HPLC system (LC-10, Shimadzu, Japan) equipped with SPD-10A UV–vis detector (set at 280 nm wavelength), and SCL-10A system controller unit with Class LC-10 software was used. The analysis was carried out at 30 °C column temperature, and a flow rate of isocratic mobile phase (acetonitrile:water, 60:40) set at 1 ml/min. The column was Discovery C18 (250×4.6 mm, 5 μm) supplied by Supelco (Bellefonte, PA, USA). A 20 μl aliquot was injected and capsaicinoids were identified with reference to retention time of standards and quantified using linear equation. The capsaicinoid concentrations in samples were expressed as mg/100 g. Fig. 1 shows UV chromatograms of a standard solution (a) and a hot pepper sample (b). Total Carotenoids Based on AOAC official method 970.64 [16], 2 g of hot pepper sample was ground using mortar and pestle and transferred to a 100 ml flask covered with stopper. The sample was blended for 1 min with a mixture of 30 ml hexane:acetone: ethanol:toluene (10:7:6:7). For hot saponification, 2 ml of 40 % methanolic KOH were pipetted into flask, swirled for 1 min and placed in 56 °C water bath for 20 min. The sample was cooled for 1 h in the dark and then 30 ml hexane was pipetted into flask, swirled 1 min, diluted to volume with 10 % anhydrous Na2SO4 solution and shaken vigorously for 1 min. Upper phase was 50 ml. Absorbance was measured at 436 nm using IRMECO UV–vis spectrophotometer Model U2020 with β-carotene as standard. Total carotenoids were expressed as mg/100 g. Ascorbic Acid Ascorbic acid was quantitatively determined according to 2,6dichlorophenolindophenol AOAC official method 967.21 [16]. A sample of hot peppers (10 g) was blended with 2.5 ml of 20 % metaphosphoric acid and distilled water was then added up to 100 ml mark. 10 ml of the suspension was titrated with freshly prepared standard of 2,6-dichlorophenolindophenol dye until light but distinct rose pink color persisted for 15 s. Ascorbic acid concentration in each sample was then calculated as mg/100 g. Total Phenolic Contents Total phenolic contents of hot peppers were analyzed using the modified Folin-Ciocalteu reagent method [17]. About 0.5 g sample was macerated in 3 ml 80 % aqueous acetone with a mortar and pestle. The extracts were placed into tightly sealed micro-tubes and maintained in darkness at 4 °C overnight. Samples were centrifuged at 1000 rpm for 2 min. A

Plant Foods Hum Nutr Fig. 1 UV chromatograms of capsaicinoid compounds: (a) standard solution, and (b) Sky Red hot pepper sample

mixture of 135 μl H2O, 750 μl 1/10 dilution Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) and 600 μl 7.5 % (w/v) Na2CO3 was added to 50 μl of extract in 1.5 ml micro-tubes. After vortexing for 10 s, the mixture was incubated at 45 °C in a water bath for 15 min. Samples were allowed to cool at room temperature before reading the absorbance at 765 nm using IRMECO UV–vis spectrophotometer Model U2020. A blank was prepared from 50 μl 80 % aqueous acetone. Gallic acid standard curve was prepared from a freshly made 1 mg/ml gallic acid (Acros Organics, Belgium) in 80 % aqueous acetone. The total phenolic contents in samples were expressed as mg/100 g. Statistical Analysis Analysis of variance of the data from each attribute was computed using the STATISTICA Computer Program. The Least Significant Difference test at 5 % level of probability was used to check the differences among mean values.

Results and Discussion Capsaicin and Dihydrocapsaicin Concentration Capsaicinoids are alkaloid compounds that produce the hot flavor or pungency associated with eating chillies [15]. Capsaicin and dihydrocapsaicin accounts for more than 90 % of the capsaicinoids in hot peppers and contribute most to pungency [18]. Analysis of variance revealed significant differences (P ≤ 0.01) for capsaicinoids concentration in all hot pepper hybrids, harvesting stages and their interactions. The pattern of capsaicin and dihydrocapsaicin concentrations in hot pepper hybrids at different harvesting stages is presented in Fig. 2 and 3 (online resource ESM_1), respectively. Results revealed that capsaicin and dihydrocapsaicin contents were maximum at mature green stage in both Sky Red (61.30 and 43.76 mg/100 g) and Wonder King (43.93 and 26.16 mg/100 g), whereas Maha reached top values at color break stage (39.13 and 24.20 mg/100 g). Overall, concentrations of both capsaicinoids evolved in a similar fashion as they were lower in immature green stage

and highest at mature green stage, and then decreased progressively to sun dried fruit stage in all the hybrids. The reduction percentage in capsaicin and dihydrocapsaicin levels from mature green to dried fruit stage was highest in Sky Red (46 and 56 %), followed by Maha (44 and 45 %) and Wonder King (38 and 43 %), respectively. The average ratio capsaicin:dihydrocapsaicin was 1:6, similar to other values reported in Capsicum species [19]. Changes during harvesting stages indicate that differences in biochemical factors within hybrids can influence the biosynthesis and stability of capsaicinoids with the advancement of maturity and drying. The decrease in capsaicinoids concentrations during maturation and senescence of hot peppers has been related to the activity of peroxidase [20], in which hot peppers hybrids differ significantly from each other [6]. The degree of pungency in Capsicum species is highly variable, and strongly depends on the hybrids [19]; capsaicinoids concentrations can be also affected by the developmental stage of the fruit and the environmental conditions during cultivation [21]. Our results are in line with other authors in that the accumulation of capsaicinoids starts at an early stage of fruit development, reaches maximum values at fruit maturity, and then decreases up to 60 % during drying [22]. Materska and Perucka [13] observed non-significant changes in capsaicinoids content during fruit maturation in one hot pepper cultivar while higher amounts in red fruits than in green fruits in other cultivar under similar growing conditions. Total Carotenoids The carotenoids abound in many yellow to red vegetables and they have been related with numerous health benefits such as the prevention of vitamin A deficiency, anti-ulcer properties, anti-aging effects and general antioxidant activity [23, 24]. The intense red color of the ripe hot peppers and their processed products are due to the presence of carotenoid pigments. Analysis of variance revealed significant differences (P ≤0.01) for total carotenoids concentration among hot pepper hybrids, harvesting stages and their interactions. The variations of total carotenoid concentrations at different harvesting stages in all three hybrids are given in Fig. 4 (online


resource ESM_1). Like other fruit, capsicums change color as they ripen and hence the carotenoids content evolves during the ripening process. All hot pepper hybrids were statistically at par for total carotenoid concentration at immature green, mature green and color break stages but carotenoid levels increased significantly at red ripe and dried fruit stage so that the concentration of total carotenoids was higher at dried fruit stage in all hybrids. The total carotenoid concentration tended to increase mildly during pepper fruit ripening from immature green to red ripe stage. However, carotenoid concentrations increased by 8-fold at dried fruit stage in all three hybrids, reaching 112.39 mg/100 g in Wonder King, 91.08 mg/100 g in Sky Red and 83.59 mg/100 g in Maha, respectively. The concentration of carotenoids in capsicum fruit depends on hybrid type, growing conditions and maturity at harvest, the latter being the most important factor. Capsicum is a carotenogenic fruit and during ripening the transformation of the chloroplast into chromoplast occurs, chlorophylls disappear and more carotenoids are formed [18]. This explains the highly significant differences observed in our study during harvesting stages. Similar results were observed by Kandlakunta et al. [23], and Zhuang et al. [24] pointing that total carotenoids concentration was significantly higher in red chillies than in green chillies. Minguez-Mosquera and Hornero-Mendez [25] also reported an increase in the carotenoids content in the Spanish Capsicum Bola variety occurring during the drying step. From these results, it can be concluded that red chillies contain 4-fold more total carotenoids than green chillies, so as far as carotenoid levels are concerned, the potential contribution to overall human health is better from red fruit. Additionally, either raw or cooked, capsicums deliver some potentially great health benefits, but cooking will even increase the bioavailability of carotenoids.

Ascorbic Acid Hot peppers are a richer source of ascorbic acid than other vegetables and fruits commonly recognized as best suppliers for this substance. Significant differences were observed for ascorbic acid concentration in all hot pepper hybrids, harvesting stages (P ≤0.01) and their interactions (P ≤0.05). Distribution of ascorbic acid concentration in three hybrids at different harvesting stages is presented in Fig. 5 (online resource ESM_1). Harvesting stage is considered as one of the major factor that determines the compositional quality of fruits and vegetables. There was a progressive increase in ascorbic acid concentration with the advancement of fruit maturation and ripening but it decreased quickly as fruit dries. Considerable variations existed within the hybrids examined and the level of ascorbic acid varied from immature fruit (between 65.56 and 92.86 mg/100 g) to fully expanded red fruits (from 131.63 to 163.46 mg/100 g), respectively.

Both Sky Red and Wonder King hybrids behaved statistically alike at all harvesting stages except red ripe stage, whereas hybrid Maha differed significantly from the other two hybrids at all maturity stages showing lower concentrations. Similarly, ascorbic acid concentrations were 50 % higher in red ripe pepper fruits as compared to green peppers [6, 26] and Marin et al. [27] also indicated that as the maturing advanced until red ripe fruits, ascorbic acid concentration increased in hot peppers. It is reported that red peppers contain highest ascorbic acid concentration among other important plant food materials including spinach and broccoli [28]. The results of the present study revealed that as the ripening advanced in hot peppers, the ascorbic acid concentration reached its maximum towards the red ripe stage and then declined by an average of 76 %. The longer fruits are maintained on the plant, the more physiological changes and loss of cellular integrity occur causing a switch toward senescence and altered nutritive components [20]. The observed loss of ascorbic acid with the advent of dried fruits might be due to biochemical changes during final harvesting stage. The above results are quite interesting in view of the dietary importance of ascorbic acid content in hot peppers, being that red ripe fruits are more beneficial to meet the recommended daily allowance of ascorbic acid.

Total Phenolic Contents In recent years, phenolic compounds have attracted the interest of researchers because of their powerful antioxidant properties that can protect the human body from free radicals [29]. Hot peppers are a good source of phenolic compounds and are ranked fourth after broccoli, spinach and onion with respect to total phenolic contents [11]. Analysis of variance revealed significant differences (P ≤0.01) for total phenolic contents among hot pepper hybrids, harvesting stages and their interactions. The stage of fruit harvest is one of the major factors that determine the total phenolic contents in fruits and vegetables [12]. The distribution of total phenolic contents in all hot pepper hybrids at different harvesting stages is shown in Fig. 6 (online resource ESM_1). All hot pepper hybrids showed great variations in the accumulation of total phenolic contents. Total phenolic contents in Sky Red tended to decrease gradually from immature green stage (114.10 mg/100 g) to dried fruit stage (59.23 mg/100 g), whereas an increasing trend was observed in Maha from immature green (58.30 mg/100 g) to red ripe stage (101 mg/100 g) and then a sharp decline in dried fruits (70 mg/100 g). Similarly in Wonder King maximum concentration of total phenolics was observed at mature green stage (88.06 mg/100 g) which was statistically at par with color break stage. Overall, maximum phenolic contents were observed at color break (90.92 mg/100 g) and mature green


stage (88.25 mg/100 g) but both harvesting stages did not differ statistically. Generally, concentration of total soluble phenolics increases as peppers reached maturity regardless of analytical method employed [12]. Materska & Perucka [13] recorded higher concentrations of phenolic compounds in green fruits than red fruits. Conforti et al., [14] and Ghasemnezhad et al., [30] also reported that concentration of total phenolic compounds in peppers varies depending on maturity level. They also observed a decreasing trend of total phenolics in peppers with maturity while Deepa et al., [31] showed increase in total phenolic content from green to red stage. It can be concluded from present results that level of total phenolic contents in hot peppers depend on individual hybrid and stage of harvest, probably related to the physiological changes that take place during maturity.

Conclusions On the basis of the data in this study, significant differences were found in capsaicinoids, total carotenoids, ascorbic acid and total phenolic contents among hot pepper hybrids and harvesting stages. Results revealed that mature green stage was ideal to acquire maximum pungency, while peppers at red ripe stage were best sources of ascorbic acid. Dried fruits contained higher levels of total carotenoids in all hybrids tested. At the same time, consumers should be educated about the benefits of including fresh and dried hot pepper fruit in their daily diet for the potential health advantages. Acknowledgements The authors are grateful for being provided laboratory facilities at the Nuclear Institute for Agriculture and Biology, Pakistan Atomic Energy Commission, and financial support by the Higher Education Commission, Islamabad, Pakistan, to complete the research work. The corresponding author is also highly grateful for the support of the University of Agriculture, Faisalabad, Pakistan. Conflict of Interest The authors declare that they have no conflict of interest.

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