Journal of Applied Botany and Food Quality 90, 232 - 237 (2017), DOI:10.5073/JABFQ.2017.090.029 1University of Craiova, Faculty of Horticulture, Department of Horticulture and Food Science, Romania 2University of Craiova, Faculty of Sciences, Department of Chemistry, Romania Bioactive compounds and antioxidant activity of hot pepper fruits at different stages of growth and ripening Mira Elena Ionică1, Violeta Nour1*, Ion Trandafir2 (Received January 17, 2017; Accepted May 1, 2017) * Corresponding author Summary The evolution of some bioactive compounds and antioxidant activity has been investigated during fruit growth and ripening of five pep- per cultivars: ‘Dracula’, ’Pintea’, ‘Pepperone’, ‘Bulgarian carrot’ (C. annuum) and ‘Christmas bell’ (C. baccatum var. pendulum). High- performance liquid chromatography was used to quantify the content of capsaicin in the fruit in order to determine the pungency level of analyzed peppers. Pepper fruits were collected at five stages of growth and ripening. Dry matter, soluble solids, ascorbic acid, to- tal phenolics, including total flavonoids, capsaicin content and anti- oxidant activity were determined at each stage. There were major differences among the cultivars in the accumulation of the bioactive compounds in the fruit during their growth and ripening, although the quantitative accumulation pathway of various components had a similar trend during phenophases. Antioxidant activity and ascorbic acid content increased during growth and ripening of hot peppers, the highest levels being found in the last stage of ripening. The pattern of variation of total flavonoid content was cultivar dependent. In most cultivars, an important increase of the total phenolic and total flavo- noid content was observed in the last stage of ripening. Capsaicin content recorded a maximum level in F3 or F4 depending on cultivar, and decreased afterwards until the complete ripening of the pepper fruits. ‘Dracula’ cultivar was classified as “non-pungent” (fruits are not spicy) while ‘Pintea’ was classified as “highly pungent”, the other analyzed cultivars having an average level of pungency. Keywords: hot pepper, antioxidant activity, phenolics, flavonoids, capsaicin Introduction Hot pepper was part of ancient human diet in the American continent since 7500 years BC. Its South American origin is supported for all species of Capsicum genus, which probably occurred in the region between southern Brazil and Bolivia. Hot pepper has a sweet-pungent flavor, with pungency ranging by cultivar. Peppers are good sources of phytochemicals such as poly- phenols, among them flavonoids, and carotenoids (AlvArez-PArril- lA et al., 2010) that are known to present high antioxidant activity (MAterskA and PeruckA, 2005). Phenolic compounds from pepper contribute to nutritional quality and fruit taste (OrnelAs-PAz et al., 2010). The substance that gives peppers pungency is capsaicin that is found in very small quantities in sweet pepper and in quantities of tens and hundreds of times higher in hot peppers. As the content of capsaicin in peppers increases, so does its pungent taste, which triggers an increase in their antioxidant level (usMAn et al., 2014). Therewith capsaicin controls the appetite and raises the body tem- perature (ludy and MAttes, 2011). Pugency is measured with the dilution test and expressed as Scoville Organoleptic Scale designed by Wilbur scOville in 1912. Recognized as the most pungent pepper (World-record for hottest chili of 2012) is Trinidad Moruga Scorpion, 2.000.000 Scoville Units higher than any other species. Recognized as the less spicy peppers are Pimento, Peperoncini and Banana pepper cultivars, with only 100-500 Scoville Units. In ad- dition to capsaicin, chili peppers’ taste is given by their content of essential oils. Capsaicin and dihydrocapsaicin represent about 75- 90% of capsaicinoids in peppers, compounds that also have other properties and biological effects, such as chemopreventive (chAndA et al., 2004) or antioxidant properties (zhuAng et al., 2012). The epidermal cell of the placenta seems to be the accumulation site of capsaicinoids in peppers (bArberO et al., 2014). Capsaicinoids are found only in the Capsicum genus and their amounts varies in diffe- rent pepper varieties depending on cultural practices, environmental conditions, storage conditions, etc. (usMAn et al., 2014). Literature mentions that dry matter and soluble solids content of pepper and different compounds in pepper, such as ascorbic acid and capsa- icinoids vary during growing and ripening of the fruit (bArberO et al., 2014; MArtinez et al., 2007). This work was conducted to investigate the evolution of some bio- active compounds and antioxidant activity of five pepper varieties grown in Dolj County, Romania during fruit growth and ripening. This work also aims to estimate the evolution and the level of capsa- icin in fruit and to determine the pungency level of analyzed peppers. Materials and methods Plant material Fruits of five pepper cultivars (Capsicum annuum and Capsicum baccatum): ‘Dracula’, ‘Pintea’, ‘Pepperone’, ‘Bulgarian carrot’ (C. annuum) and ‘Christmas bell’ (C. baccatum var. pendulum), were collected at five stages of development and ripening i.e. F1-14 days after flowering, F2-20 days after flowering, F3-30 days after flowe- ring, F4-40 days after flowering, F5-50 days after flowering (physio- logical maturity) from Dabuleni, Dolj county, Romania and analyzed in terms of chemical characteristics and antioxidant activity. The selection of the fruits was based on the number of days after flowe- ring. Dabuleni is a sandy area in the South of Romania close to the Danube River. It has a strong continental climate with mild Mediterranean in- fluence and an average temperature of 11 °C. The region gets severe drought from July to September and average rainfalls in May and June. The orchard management was consistent with cultural practice (10-12 watering sessions with 300-350 m3/ha, 100-100-50 NPK fer- tilization, breaking the tip of plant, pests and diseases control). The planting was carried out in sandy soil, with the experiment being set up as randomized block design in 3 replicates with 30 plants per cultivar. From each cultivar 10 fruits in 3 replicates were collected in order to perform chemical analysis. Evolution of bioactive compounds during growth and ripening of hot peppers 233 Analytical methods Several analyses have been performed: dry matter, soluble solids, ascorbic acid, total phenolic and total flavonoid content, capsaicin content and antioxidant activity. The dry matter content was deter- mined by removing water from the sample in an oven at 105 °C and the result was expressed in percentages. The soluble solids were measured with a digital refractometer (Hanna Instruments, Woon- socket, USA) from the juice pressed from the fruit, the results being expressed in percentages. Ascorbic acid content Ascorbic acid was extracted and analyzed by reversed phase HPLC. Fresh pepper homogenate (5 g) was mixed and diluted with 2% HCl. After 20 minutes the solution was centrifuged at 4200 rpm for 10 min. The supernatant was filtered through 0.2 mm pore size fil- ter. The ascorbic acid in the sample test solution was separated by reversed phase chromatography on a 250 mm × 4.6 mm i.d., 5 μm particle Hypersil Gold aQ Analytical Column, of which was detected by absorbance and quantified with external calibration graph. The detector was set at λ=254 nm. This setting was chosen since ascor- bic acid has its maximum optical absorbance close to 254 nm. The HPLC analysis was performed with a Surveyor Thermo Electron system (Thermo Electron Corporation, San Jose, CA, USA) com- prising a vacuum degasser, Surveyor Plus LCPMPP pump, Surveyor Plus ASP autosampler and diode array detector with 5 cm flow cell. Integration, data storage and processing were performed by Chrom Quest 4.2 software. The determinations were made in isocratic con- ditions, at 10 °C, using a mobile phase made of 50 mM phosphate so- lution filtered through a polyamide membrane (0.2 μm) and degassed in a vacuum. The flow rate of the mobile phase was 0.7 mL/min for all the chromatographic separations. The volume injected was 5 μL for either prepared sample or standard solution. The results were expressed in mg kg-1 fresh weight (fw). All reagents were acquired from Sigma-Aldrich, Germany and ultrapure water was obtained from a Milli-Q water purification system (TGI Pure Water Systems, USA). Total phenolic content Total phenolic content was assessed by using the Folin-Ciocalteu phenol reagent method (singeltOn and rOssi, 1965). Folin-Ciocal- teu reagent (2 N, Merck), gallic acid (99% purity, Sigma-Aldrich), anhydrous sodium carbonate (99% Sigma-Aldrich) were used. One g dried pepper homogenate was extracted with 15 mL ethanol in an ultrasonic bath for 60 min at ambient temperature. After extraction, the samples were centrifuged for 5 min at 4200 rpm and supernatants were filtered through polyamide membranes with pore diameter of 0.45 μm and stored at a temperature of -20 °C. Hundred μL of each pepper ethanolic extract were mixed with 5 mL of distilled water and 500 μL of Folin-Ciocalteu reagent. After 30 sec to 8 min, 1.5 mL of sodium carbonate (20% v/v) was added. The reaction mixture was diluted with distilled water to a final volume of 10 mL. The prepara- tion of the standard solution of gallic acid followed the same proce- dure. The absorbance at 765 nm of each mixture was measured on a Varian Cary 50 UV spectrophotometer (Varian Co., USA) after incubation for 30 min at 40 °C and results were expressed in mg gal- lic acid (GAE) kg-1 dry weight (dw). Antioxidant activity The antioxidant activity (AOX) was measured in the ethanolic ex- tract using the DPPH (2,2-diphenyl-1-picrylhydrazil) assay. Ethanol (Merck, Germany), DPPH (2,2-diphenyl-1-picrylhydrazil) (Sigma- Aldrich, Germany) and Trolox (Merck, Germany) were employed. The extraction of samples was made according to the same proto- col described for total phenolic content. The free radical scavenging ability of the extracts against DPPH free radical was evaluated as described by OliveirA et al. (2008), with some modifications. Each ethanol pepper extract (50 μL) was mixed with 3 mL of a 0.004% (v/v) DPPH methanolic solution. The mixture was incubated for 30 min at room temperature in the dark and the absorbance was measured at 517 nm on Varian Cary 50 UV-VIS spectrophotometer. The DPPH free radical scavenging ability was calculated in refe- rence to Trolox (6-hydroxy-2,3,7,8-tetramethylchroman-2-carboxylic acid), which was used as standard reference to convert the inhibition capability of each extract solution to the mmol Trolox equivalent an- tioxidant activity/L. The radical was freshly prepared and protected from light. A blank control of methanol/water was used in each assay. All assays were conducted in triplicate and results were expressed in mmol Trolox kg-1 dw. Total flavonoid content Total flavonoids content was assessed by the spectrophotometric method described by MOhAMMAdzAdeh et al. (2007) based on the color reaction of this class of compounds with the ions of Al (III). Pepper homogenate (1 g dry matter) was extracted with 15 mL etha- nol in an ultrasonic bath for 60 min at ambient temperature. After extraction, the samples were centrifuged for 5 min at 4200 rpm and supernatants were filtered through polyamide membranes with a pore diameter of 0.45 μm and stored at -20 °C. 0.5 mL filtrate was placed in a polyethylene test tube, together with a 0.1 mL 10% aque- ous solution of aluminum nitrate, 0.1 mL aqueous 1 M sodium ace- tate, 4.3 mL methanol, mixed well and left to react for 40 min at room temperature. After completion of the reaction, the absorbance of the mixture was measured at 415 nm on a Varian Cary 50 UV-VIS spectrophotometer (Varian Co., USA). The quantification of flavo- noids was carried out on the basis of a calibration curve using quer- cetin as standard reference in the range of 0-100 mg/L. The results were expressed as mg of quercetin equivalents kg-1 dw. Capsaicin content Identification and quantification of capsaicin were carried out by HPLC following the method described by zhuAng et al. (2012), with some modifications. For sample preparation, fresh matter (pulp transformed into a paste by a disintegrator) was dried in an oven at 55 °C to a constant weight and the dried matter was ground finely in a blender (Polytron). An amount of 1 g of the dried matter was extracted with 20 mL of etha- nol 95%. After 30 min, the extract was kept in an ultrasonic bath for 60 min at 60 °C and filtered through a 0.2 mm pore size filter. HPLC-DAD analysis was performed on a Finnigan Surveyor Plus system (Thermo Electron Corporation, San Jose, CA, USA) coup- led with diode array detector (PDA5P with cell flow in 5 cm). The separation was performed using a Hypersil Gold aQ column (250 × 4.6 mm) with a particle size of 5 μm. Capsaicin was detected at 278 nm and quantified (mg kg-1 dw) using standard compound. The determinations were made in isocratic conditions at 25 °C, using a mobile phase made of 50 mM acetonitrile solution (water:acetonitrile 57:43) filtered through a polyamide membrane (0.2 μm) and degassed in a vacuum. The flow rate of the mobile phase was 1 mL min-1 for all the chromatographic separations. The volume injected was 5 μL for either prepared sample or standard solution. For device control, data acquisition and processing, Chrom Quest 4.2 software was used. Statistical analysis Significance of differences between cultivars was determined by per- forming a one-way ANOVA test using Statgraphics Centurion XVI Software (StatPoint Technologies, Warenton, VA, USA). 234 M.E. Ionică, V. Nour, I. Trandafir Results and discussion The data obtained concerning the dry matter, soluble solids and ascorbic acid content of the hot peppers are presented in Tab. 1. The dry matter content (%) in hot peppers followed an upward trend during growth and ripening except the F2 (20 days after flowering) where a small absolute decrease was noticed. However, the dry mat- ter content varied little in immature stages but showed high increase in ripening stages with a maximum in the physiological maturity, corresponding to the ripening process of fruits, which was mani- fested through the synthesis of metabolites with complex molecular structure. The same changes were reported by niklis et al. (2002). There were also differences among cultivars, the highest content of dry matter being found in the ‘Pepperone’ cultivar while the ‘Pintea’ cultivar registered the lowest content. The differences among culti- vars were maintained during all the stages of growth and ripening. Regarding the soluble solids content (%), it was found that it had the same upward trend during growth and ripening except the ‘Pintea’ cultivar which registered a small decrease in F2 and ‘Bulgarian car- rot’ cultivar which registered the same decrease in F3. The highest rate of accumulation of soluble substances was found in the last sta- ges with similar values to those found by dOrji et al. (2005). dOrji et al. also found that the highest changes in soluble solids happened in the last stages of ripening and in the firm red stage, respectively. There were differences among cultivars: the highest content of solu- ble solids being found in the ‘Bulgarian carrot’ (9.82%) followed by ‘Pepperone’ (9.65%), ‘Dracula’ (8.4%), ‘Pintea’ (8.1%) and ‘Christ- mas bell‘ (7.40%). In all cultivars the ascorbic acid content was positively correlated with the dry matter and the soluble solids content, which is in accor- dance with the data presented by niklis et al. (2002). Considering that the glucose is the precursor in the ascorbic acid syn- thesis (dAvey et al., 2000; niklis et al., 2002) it is understandable why there is a positive correlation between dry matter, soluble solids and ascorbic acid content. The highest accumulation of ascorbic acid was recorded between the first phenophases (F1-F2). In this period in all cultivars high plant growth rates were observed. The data obtained are comparable to data presented in the literature, the upward trend being also described by kuMAr and subbA tAtA (2009) and MArtinez et al. (2005). There were significant differen- ces among cultivars, the highest content of ascorbic acid being found in the ‘Bulgarian carrot’ cultivar (1606.47 mg kg-1 fw) and the lowest content in the ‘Pintea’ (1161.35 mg kg-1 fw) cultivar. According to the classification of hot pepper cultivars depending on the content of ascorbic acid described by khAdi et al. (1987) and siMOnne et al. (1997), the analyzed cultivars fall into the category of an average content of ascorbic acid (1010-2000 mg kg-1 fw) even if analyzing in terms of species, chilli is considered a species with high ascorbic acid content. The results obtained on the content of phenolics, total flavonoids, and antioxidant activity (AOX) in hot peppers are shown in Tab. 2. Antioxidant activity is an important parameter to establish the health functionality of a food product and there are many methods used for its measurement (kAur and kAPOOr, 2001). AOX increased during growth and ripening of the hot peppers, the highest levels being found in the last stage (F5). There were differences among cultivars, the highest AOX level being found in the ‘Pintea’ cultivar (20.04 mmol Trolox kg-1 dw) and the lowest in the ‘Dracula’ cultivar. The diffe- rences between cultivars were also reported by zhuAng et al. (2012), who mentioned some pepper extracts as effective electron donors. Hot peppers contain several flavonoids in different forms including glycosides (bAe et al., 2012). The data presented in Tab. 2 show im- portant variations in the content of phenolics and total flavonoids in hot peppers. The total phenolic content increases during growth and ripening with the highest level in the last stage of ripening, the results being in accordance with the data presented by zhuAng et al. (2012) and deePA et al. (2007). righettO et al. (2005) mentioned that the content of phenolics is Tab. 1: Dry matter, soluble solids and ascorbic acid content in hot peppers during growth and ripening. Different letters within the same row indicated significant differences (P < 0.05) among cultivars Dracula Pintea Pepperone Bulgarian carrot Christmas bell Dry matter (%) F1 10.11±1.41c 11.38±1.09b 11.80±1.41ab 12.39±1.36a 12.53±1.62a F2 9.99±1.19c 10.65±1.17bc 10.50±1.29b 10.12±1.11bc 12.13±1.49a F3 10.86±1.75c 11.47±1.37b 13.31±1.73a 10.57±1.26c 12.73±1.52ab F4 15.96±2.01b 14.17±1.73bc 15.42±2.15b 18.00±2.52a 13.66±1.77c F5 17.43±2.26b 16.16±2.02c 18.71±2.6a 18.09±2.48a 17.53±2.38ab Soluble solids (%) F1 4.45±0.53b 5.45±0.65a 4.70±0.56ab 3.72±0.40c 3.86±0.41c F2 4.67±0.61ab 4.37±0.48c 4.54±0.49b 5.10±0.58a 4.66±0.51ab F3 5.64±0.69bc 5.82±0.62b 7.10±0.91a 4.57±0.54c 6.00±0.72ab F4 7.77±0.93ab 4.80±0.52d 7.60±0.98b 8.32±1.05a 6.66±0.86c F5 8.40±1.09b 8.10±1.03b 9.65±1.32ab 9.82±1.27a 7.40±0.96c Ascorbic acid (mg kg-1 fw) F1 170.32±15.40a 152.80±12.80b 169.85±14.20ab 173.62±13.60a 105.43±90.80c F2 1186.54±108.70a 1102.38±97.30d 1188.21±99.10a 1178.89±86.70b 1158.40±109.20c F3 1104.62±100.40c 1123.19±104.10bc 1191.05±110.50b 1289.21±116.90a 1259.47±114.90a F4 1211.20±109.60c 1149.52±98.30de 1176.23±105.80d 1297.58±113.70b 1393.62±121.40a F5 1383.09±118.70c 1161.35±106.10d 1387.11±120.80bc 1603.57±147.50a 1446.47±137.30b Evolution of bioactive compounds during growth and ripening of hot peppers 235 affected by the type and cultivar of pepper, maturity and cultural conditions which are in accordance with the data presented in Tab. 2, the highest content in phenolics (F5) being found in the ‘Pintea‘ cul- tivar (8331.27 mg GAE kg-1 dw) and the lowest in the ‘Bulgarian carrot’ cultivar (2964.25 mg GAE kg-1 dw). The total flavonoids con- tent presented different variation patterns from cultivar to cultivar but 30 days after flowering (F3) a decrease is recorded as compared with F1 in all cultivars. Except for ‘Bulgarian carrot’ cultivar, to- tal flavonoids content grew strongly in the last stage of ripening. hOWArd et al. (2000) and MedinA-juArez et al. (2012) found the same variation associated to pepper maturity, cultivar and growing conditions. In the last stage of ripening (F5) the total flavonoids content was sig- nificantly higher in the ‘Pepperone’ cultivar (7666.095 mg quercetin kg-1 dw) followed by ‘Pintea’ and ‘Dracula’. The data regarding the capsaicin content of the hot pepper are pre- sented in Tab. 2. In the early days after flowering (14 days) the cap- saicin was found in peppers in very small amounts (undetectable in the ‘Bulgarian carrot’ and ‘Christmas bell’ cultivars). During growth and ripening, the fruit content of capsaicin increased to a maximum level that was recorded depending on the cultivar precocity in F3 (30 days after flowering) in: ‘Dracula’, ‘Pintea’ and ‘Pepperone’ (early cultivars) either F4 (40 days after flowering) in ‘Bulgarian carrot’ and ‘Christmas bell’. After reaching this peak, the capsaicin content decreased until the full ripening of the pepper fruits. bArberO et al. (2014) mentioned that the maximum relative content of capsaicin is reached on day 20 of fruit maturation, earlier than other capsaicinoids, and between day 40 and 50 of maturation the relative content of capsaicin represented only 52% of the total capsaicinoids. The same trend of capsaicin accumulation in fruits was reported by estrAdA et al. (2000) and kirschbAuM-titze et al. (2002). There are major differences between cultivars regarding the content of capsaicin, the highest level being found in the ‘Pintea’ cultivar (2229.81 mg kg-1 dw) with an equivalent pungency of 35676.96 SHU followed by the ‘Bulgarian carrot’ and ‘Pepperone’. The lowest level of capsaicin content was found in the ‘Dracula’ cultivar (24.85 mg kg-1 dw) with an equivalent pungency of 397.68 SHU. Similar dif- ferences between cultivars were reported by usMAn et al. (2014), verA-guzMán et al. (2011) and gnAyfeed et al. (2001). To express the pungency level of the studied cultivars, capsaicin con- tent (grams of capsaicin per grams pepper dry weight) was converted to Scoville Heat Units by multiplying it by the coefficient of the heat value (1.6 × 107) (tilAhun et al., 2013). The data obtained are shown in Tab. 3. Scoville score analysis showed that the ‘Dracula’ cultivar is classi- fied as ‘non-pungent’ (fruits are not spicy) while ‘Pintea’ is classified as ‘highly pungent’, the other analyzed cultivars being classified as ‘moderately pungent’. Although the ‘Pintea’ cultivar registered the highest capsaicin con- tent and the highest level of pungency, it is very far from the first places on the Scoville scale (Scoville units between 200,000 and 300,000 − Capsicum chinense, while very spicy peppers from Thai- land only reach 100,000). Tab. 2: Phenolics, total flavonoids, capsaicin content and antioxidant activity in hot peppers during growth and ripening. Different letters within the same row indicated significant differences (P < 0.05) among cultivars Dracula Pintea Pepperone Bulgarian carrot Christmas bell Antioxidant activity (mmol Trolox kg-1 dw) F1 5.26±0.49c 6.94±0.58a 5.95±0.53bc 7.08±0.68a 6.24±0.60b F2 7.47±0.61b 8.64±0.611a 7.36±0.71b 8.89±0.86a 7.14±0.68c F3 9.01±0.88c 18.03±1.74a 11.58±1.08b 11.85±1.94b 6.91±0.67d F4 9.49±0.90c 20.04±1.99a 12.55±1.92bc 13.87±1.26b 8.44±0.82d F5 10.51±0.97cd 19.56±1.73a 17.50±1.67b 17.23±1.66b 11.55±1.09c Total phenolics (mg GAE kg-1 dw) F1 3074.32±284.12b 3210.27±301.23a 3087.00±287.65ab 3299.95±301.11a 2768.23±265.34c F2 3358.95±297.25b 3253.23±298.73bc 3462.78±300.87b 3779.28±369.30a 2878.89±279.61c F3 3450.00±300.13cd 4965.69±405.87a 3525.13±312.65c 3758.11±358.49b 3267.67±311.42d F4 3645.58±315.09d 7026.63±691.45a 4435.05±403.12c 6476.10±609.28b 3248.35±311.73d F5 5146.45±463.67c 8331.27±786.70a 6433.51±503.84b 2964.25±275.33c 3744.84±350.22d Total flavonoids (mg quercetin equivalents kg-1 dw) F1 1896.90±176.55c 1853.85±171.33c 2218.33±199.76bc 4532.89±432.05a 2415.15±230.17b F2 1422.61±139.68d 1996.33±183.74c 2286.09±202.11b 4569.75±408.17a 2001.43±198.67b F3 1868.20±183.15b 1819.65±167.95bc 1849.44±169.81b 3397.92±311.26a 1774.00±163.50c F4 1632.97±156.70d 2726.10±222.56a 2340.39±201.54b 2382.73±202.88b 1844.23±187.34c F5 5461.93±497.78b 5247.27±497.88bc 7666.09±700.80a 1435.57±122.86d 2848.81±256.40c Capsaicin (mg kg-1 dw) F1 4.98±0.45c 37.57±3.28b 45.16±5.03a 0 0 F2 5.19±0.49c 705.90±56.91a 429.55±40.89b 11.10±0.98d 38.07±3.77e F3 24.85±2.89e 2229.81±220.37a 777.49±75.69b 438.46±39.69c 126.78±11.59d F4 16.89±1.56e 1814.86±179.65a 405.90±38.45c 990.37±93.15b 187.90±17.73d F5 18.64±1.72e 1797.52±17.05a 501.07±48.71c 656.13±62.99b 30.46±2.86d 236 M.E. Ionică, V. Nour, I. Trandafir However we can say that the fruits of ‘Pintea’ cultivar are swifter than the Mexican Jalapeno or the Italian Peperoncino cultivars that barely reach a score of 500-5,000 Scoville Units (MAthur et al., 2000). bAe, h., jAyAPrAkAshA, g.k., jifOn, j., PAtil, b.s., 2012: Variation of anti- oxidant activity and the levels of bioactive compounds in lipophilic and hydrophilic extracts from hot pepper (Capsicum spp.) cultivars. Food Chem. 134(4), 1912-1918. DOI: 10.1016/j.foodchem.2012.03.108 bArberO, g.f., ruiz, A.g., liAzid, A., PAlMA, M., verA, j.c., bArrOsO, c.g., 2014: Evolution of total and individual capsaicinoids in peppers during ripening of the Cayenne pepper plant (Capsicum annuum L.). Food Chem. 153, 200-206. DOI: 10.1016/j.foodchem.2013.12.068 chAndA, s., erexsOn, g., riAch, c., innes, d., stevensOn, f., Murli, h., bley, k., 2004: Genotoxicity studies with pure trans-capsaicin. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 557(1), 85-97. DOI: 10.1016/j.mrgentox.2003.10.001 dAvey, M.W., MOntAgu, M.v., inzé, d., sAnMArtin, M., kAnellis, A., sMirnOff, n., fletcher, j., 2000: Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J. Sci. Food Agr. 80(7), 825-860. DOI: 10.1002/(SICI)1097-0010(20000515)80:7<825::AID-JSFA598>3.0. CO;2-6 de jesús OrnelAs-PAz, j., MArtínez-burrOlA, j.M., ruiz-cruz, s., sAntAnA-rOdríguez, v., ibArrA-junquerA, v., OlivAs, g.i., Pérez- MArtínez, j.d., 2010: Effect of cooking on the capsaicinoids and pheno- lics contents of Mexican peppers. Food Chem. 119(4), 1619-1625. DOI: 10.1016/j.foodchem.2009.09.054 deePA, n., kAur, c., geOrge, b., singh, b., kAPOOr, h.c., 2007: Anti- oxidant constituents in some sweet pepper (Capsicum annuum L.) geno- types during maturity. LWT-Food Science and Technology 40(1), 121- 129. DOI: 10.1016/j.lwt.2005.09.016 dOrji, k., behbOudiAn, M.h., zegbe-dOMinguez, j.A., 2005: Water rela- tions, growth, yield, and fruit quality of hot pepper under deficit irri- gation and partial rootzone drying. Sci. Hort. 104(2), 137-149. DOI: 10.1016/j.scienta.2004.08.015 estrAdA, b., bernAl, M.A., díAz, j., POMAr, f., MerinO, f., 2000: Fruit development in Capsicum annuum: Changes in capsaicin, lignin, free phenolics, and peroxidase patterns. J. Agric. Food Chem. 48(12), 6234- 6239. DOI: 10.1021/jf000190x gnAyfeed, M.h., dAOOd, h.g., biAcs, P.A., AlcArAz, c.f., 2001: Content of bioactive compounds in pungent spice red pepper (paprika) as affec- ted by ripening and genotype. J. Sci. Food Agr. 81(15), 1580-1585. DOI: 10.1002/jsfa.982 hOWArd, l.r., tAlcOtt, s.t., brenes, c.h., villAlOn, b., 2000: Chan- ges in phytochemical and antioxidant activity of selected pepper cul- tivars (Capsicum species) as influenced by maturity. J. Agric. Food Chem. 48(5), 1713-1720. DOI: 10.1021/jf990916t juárez, l.á.M., quijAdA, d.M.M., sánchez c.l.d.t., águilAr, g.A.g., MezA, n.g., 2012: Antioxidant activity of peppers (Capsicum annuum L.) extracts and characterization of their phenolic constituents. Inter- ciencia: Revista de ciencia y tecnología de América 37(8), 588-593. kAur, c., kAPOOr, h.c., 2001: Antioxidants in fruits and vegetables – the millennium’s health. Int. J. Food Sci. Tech. 36(7), 703-725. DOI: 10.1111/j.1365-2621.2001.00513.x khAdi, b.M., gOud, j.v., PAtil, v.b., 1987: Variation in ascorbic acid and mineral content in fruits of some varieties of chilli (Capsicum annuum L.). Plant Foods Hum. Nutr. 37(1), 9-15. DOI: 10.1007/BF01092295 kirschbAuM-titze, P., hiePler, c., Mueller-seitz, e., Petz, M., 2002: Pungency in paprika (Capsicum annuum). 1. Decrease of capsaicinoid content following cellular disruption. J. Agric. Food Chem. 50(5), 1260- 1263. DOI: 10.1021/jf010527a kuMAr, O.A., tAtA, s.s., 2009: Ascorbic acid contents in chili peppers (Capsicum L.). Not. Sci. Biol. 1(1), 50. DOI: 10.15835/nsb.1.1.3445 ludy, M.j., MAttes, r.d., 2011: The effects of hedonically acceptable red pepper doses on thermogenesis and appetite. Physiol. Behav. 102(3), 251-258. DOI: 10.1016/j.physbeh.2010.11.018 MArtínez, s., currOs, A., berMúdez, j., cArbAllO, j., frAncO, i., 2007: The composition of Arnoia peppers (Capsicum annuum L.) at different Tab. 3: The pungency of the hot peppers Cultivar Maximum content Scoville Units Pungency of capsaicin SHU mg kg-1 dw Dracula 24.85±2.89 397.68 Non-pungent (0-700 SHU) Pintea 2229.81±220.37 35676.96 Highly pungent (25000-70000 SHU) Pepperone 777.49±75.69 12439.92 Moderately pungent (3000-25000 SHU) Bulgarian carrot 990.37±93.15 15846 Moderately pungent (3000-25000 SHU) Christmas bell 187.90±17.73 3006.48 Moderately pungent (3000-25000 SHU) Conclusions There are major differences among cultivars in the accumulation of the bioactive compounds in the fruit during their growth and ripe- ning, although the quantitative accumulation pathway of various components had a similar trend during phenophases. The dry matter content varied little in immature stages but a large increase was observed at ripening stages with a maximum at the physiological maturity. The soluble solids content had the same upward trend during growth and ripening and the ascorbic acid content was positively correlated with the dry matter and the soluble solids content. Antioxidant activity increased during growth and ripening of hot peppers, the highest levels being found in the last stage of ripening. Although the pattern of variation of total flavonoid content was affected by the cultivar, a lower value was recorded 30 days after flowering (F3) as compared with F1 in all cultivars. Also, in most cultivars, an important increase of the total phenolic and total flavo- noid content was observed in the last stage of ripening. Cultivar’s greatest influence on the accumulation of bioactive com- pounds was observed regarding the content of capsaicin that was found in peppers in very small amounts (undetectable in the ‘Bulga- rian carrot’ and ‘Christmas bell’ cultivars) in F1. During growth and ripening, fruit content of capsaicin increased to a maximum level (F3 or F4), then declined until the full ripening of the pepper fruits. Scoville score analysis showed that the ‘Dracula’ cultivar is classified as “non-pungent” (fruits are not spicy) while ‘Pintea’ is classified as “highly pungent”, the other analyzed cultivars having an average level of pungency. Acknowledgement This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS/CCCDI – UEFISCDI, project number PN-III-P2-2.1-BG-2016-0019, within PNCDI III. References AlvArez-PArrillA, e., de lA rOsA, l.A., AMArOWicz, r., shAhidi, f., 2010: Antioxidant activity of fresh and processed Jalapeno and Serrano peppers. J. Agric. Food Chem. 59(1), 163-173. DOI: 10.1021/jf103434u Evolution of bioactive compounds during growth and ripening of hot peppers 237 stages of maturity. Int. J. Food Sci. Nutr. 58(2), 150-161. DOI: 10.1080/09637480601154095 MArtínez, s., lóPez, M., gOnzález-rAurich, M., bernArdO AlvArez, A., 2005: The effects of ripening stage and processing systems on ascor- bic acid content in sweet peppers (Capsicum annuum L.). Int. J. Food Sci. Nutr. 56(1), 45-51. DOI: 10.1080/09637480500081936 MAterskA, M., PeruckA, i., 2005: Antioxidant activity of the main phenolic compounds isolated from hot pepper fruit (Capsicum annuum L.). J. Ag- ric. Food Chem. 53(5), 1750-1756. DOI: 10.1021/jf035331k MAthur, r., dAngi, r.s., dAss, s.c., MAlhOtrA, r.c., 2000: The hottest chilli variety in India. Curr. Sci. 79(3), 287-288. MOhAMMAdzAdeh, s., shArriAtPAnAhi, M., hAMedi, M., AMAnzAdeh, y., ebrAhiMi, s.e.s., OstAd, s.n., 2007: Antioxidant power of Iranian propolis extract. Food Chem. 103(3), 729-733. DOI: 10.1016/j.foodchem.2006.09.014 niklis, n.d., siOMOs, A.s., sfAkiOtAkis, e.M., 2002: Ascorbic acid, soluble solids and dry matter content in sweet pepper fruit: change during ripe- ning. J. Vegetable Crop Prod. 8(1), 41-51. DOI: 10.1300/J068v08n01_06 OliveirA, i., sOusA, A., ferreirA, i.c., bentO, A., estevinhO, l., PereirA, j.A., 2008: Total phenols, antioxidant potential and antimicrobial activ- ity of walnut (Juglans regia L.) green husks. Food Chem. Toxicol. 46(7), 2326-2331. DOI: 10.1016/j.fct.2008.03.017 righettO, A.M., nettO, f.M., cArrArO, f., 2005: Chemical composition and antioxidant activity of juices from mature and immature acerola (Malpighia emarginata DC.). Food Sci. Technol. Int. 11(4), 315-321. DOI: 10.1177/1082013205056785 scOville, W.l., 1912: Note on capsicums. J. Am. Pharm. Assoc. 1(5), 453- 454. DOI: 10.1002/jps.3080010520 siMOnne, A.h., siMOnne, e.h., eitenMiller, r.r., Mills, h.A., green, n.r., 1997: Ascorbic acid and provitamin A contents in unusually co- lored bell peppers (Capsicum annuum L.). J. Food Comp. Anal. 10(4), 299-311. DOI: 10.1006/jfca.1997.0544 singletOn, v.l., rOssi, j.A., 1965: Colorimetry of total phenolics with phos- phomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16(3), 144-158. tilAhun, s., PArAMAguru, P., rAjAMAni, k., 2013: Capsaicin and ascor- bic acid variability in Chilli and Paprika cultivars as revealed by HPLC analysis. J. Plant Breed. Genet. 1(2), 85-89. usMAn, M.g., rAfii, M.y., isMAil, M.r., MAlek, M.A., lAtif, M.A., 2014: Capsaicin and dihydrocapsaicin determination in chili pepper genotypes using ultra-fast liquid chromatography. Molecules 19(5), 6474-6488. DOI: 10.3390/molecules19056474 verA-guzMán, A.M., chávez-serviA, j.l., cArrillO-rOdríguez, j.c., lóPez, M.g., 2011: Phytochemical evaluation of wild and cultivated pepper (Capsicum annuum L. and C. pubescens Ruiz & Pav.) from Oa- xaca, Mexico. Chil. J. Agr. Res. 71(4), 578. DOI: 10.4067/S0718-58392011000400013 zhuAng, y., chen, l., sun, l., cAO, j., 2012: Bioactive characteristics and antioxidant activities of nine peppers. J. Funct. Foods 4(1), 331-338. DOI: 10.1016/j.jff.2012.01.001 Address of the authors: Mira Elena Ionica, Violeta Nour: University of Craiova, Faculty of Horti- culture, Department of Horticulture and Food Science, 13 A.I.Cuza Street, 200585, Romania E-mail: vionor@yahoo.com Ion Trandafir: University of Craiova, Faculty of Sciences, Department of Chemistry, 107 Calea Bucuresti Street, 200529, Romania © The Author(s) 2017. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/).