Evaluation of Phenolic Compounds and Pigments Content in Yellow Bell Pepper Wastes

Bell peppers are one of the most important species consumed and cultivated in Spain. Peppers are a source of carotenoids and phenolic compounds widely associated with biological activities such as antimicrobial, antiseptic, anticancer, counterirritant, cardioprotective, appetite stimulator, antioxidant, and immunomodulator. However, undersized and damaged fruits are usually wasted. Thus, in order to evaluate the phenolic content, a Box–Behnken design has been carried out to optimize the extraction from Capsicum annuum yellow pepper by ultrasound-assisted extraction (UAE). The independent factors were time (min), ethanol/water (% v/v) and solvent/sample ratio (v/w). The model was validated by ANOVA and confirmed. Furthermore, the whole pepper and the pepper without peduncles and seeds were extracted using optimal conditions and characterized by HPLC-ESI-TOF-MS. Moreover, their antioxidant activities, measured by three different methods (DPPH, ABTS, and FRAP), carotenoid composition, assessed by HPLC-MS, and chlorophyll content, assessed by a spectrophotometric method, were compared. A total of 38 polar compounds were found of which seven have been identified in pepper fruit extracts for the first time. According to the results, whole pepper (WP) samples presented higher content in phenolic acids; meanwhile, the edible portion (EP) was higher in flavonoids. No differences were found in the antioxidant activity except for the FRAP assay where the WP sample showed higher radical scavenging activity. EP samples showed the highest content of carotenoids and WP ones in chlorophylls.


Introduction
Capsicum peppers from the Solanaceae family are a diverse genus including more than 31 species from which five are domesticated: Capsicum baccatum, Capsicum annuum, Capsicum pubescens, Capsicum frutescens, and Capsicum chinense. Between them, C. annuum bell peppers are the most important species consumed and cultivated in Spain for their pungency and unique flavor. Further, from 2008 to 2019, the bell pepper consumption ranged from over 210 million kilograms to around 240 million kilograms in Spain. Furthermore, the community of Andalusia reached the highest amount of produced fresh peppers in Spain in 2019 with a total volume of more than 979.8 thousand metric tons of fresh peppers produced (Statista). Furthermore, it is a vegetable of great importance in nutrition because it is a source of pigments, vitamins, and phenolic compounds widely associated with biological activities such as antimicrobial, antiseptic, anticancer, counterirritant, cardioprotective, appetite stimulator, antioxidant, and immunomodulatory [1,2]. The extraction step is of significant importance for the isolation and separation of those phenolic compounds and depends on factors such as extraction time, temperature, and solvent. In Capsicum fruits, different traditional methods and solvents have been evaluated previously as maceration and Soxhlet methods; however, low recoveries were obtained [3]. Furthermore, recently, there has been an increment in the demand for extracting techniques that reduce extraction times and consumption of organic solvents. Therefore, among other extraction methods, ultrasound-assisted extraction is considered an excellent green alternative that has been widely used for the extraction of phenolic compounds in other matrices [4,5]. Some authors have reported using ultrasound technology in pepper matrices but with different approaches such as for extracting hydroxy-sanshool compounds [6], capsaicinoids [7][8][9][10][11][12], capsinoids [13], β-cyclodextrin [14], minerals (Ca, Fe, K, Mg, Mn, P, and Zn) [15], oils [16], oleoresin [17], or alkenylbenzenes [18], or with other uses such as for removing the residual solvent from fragrant oils [19] or for improving drying kinetics [20][21][22].
During the phase of selection of bell peppers, some of them are discharged because they are damaged or undersized. However, they contain bioactive compounds that could be used for animal feed or as nutraceutical, cosmeceutical, and functional food ingredients. Thus, the present study focused on developing and optimizing the ultrasound-assisted extraction of total phenolic compounds from bell pepper wastes and to compare the phenolic, antioxidant, carotenoid, and chlorophyll content in whole pepper (including peduncles and seeds) and in the edible portion.

Sample Preparation
Samples were obtained from two companies located in Almería (Spain) that are clients of Vellsam. There were two samples of 3 kg of Capsicum annuum var. Kioto yellow pepper ( Figure 1) which were coded as 1 and 2 corresponding to different companies. The samples were lyophilized entire (WP) (whole pepper including edible portion, peduncles, and seeds) and only the edible portion (EP) After the lyophilization, the samples were milled and sieved with an average particle size of 0.2 mm. They have been named as given in Table 1.

Experimental Design
A Box-Behnken design was established to optimize the extraction of phenolic compounds with high antioxidant activity from pepper edible parts. It is composed of 15 experiments structured in three blocks with three levels (−1, 0, 1). Each experiment was carried out in duplicate. The independent variables were time (5,45, and 85 min), ratio ethanol/water (0, 50, and 100% v/v), and ratio solvent/sample (10, 105, and 200 v/w) and the dependent variable considered the total phenolic content measured by the Folin-Ciocalteu method. It was adjusted to a second-order polynomial model equation and ANOVA was performed to evaluate the adjustment of the model. Response surface methodology (RSM) was used to select the optimal conditions. All data were processed using STATISTICA 7.0 (2002, StatSoft, Tulsa, OK, USA).

Ultrasound-Assisted Extraction
The extractions were performed by using an ultrasonic bath (Bandelin, Sonorex, RK52, Berlin, Germany) operating at a frequency of 35 kHz. Briefly, each extraction was carried out by adding ethanol/water solution (10 mL) with a determined amount of pepper and sonicated for a fixed time according to the established by the model. After the extraction, the samples were centrifuged at 3500 rpm for 15 min and the supernatant was stored at −18 • C until the analysis.

Determination of Total Phenolic Content (TPC)
TPC of pepper extracts was determined by the Folin-Ciocalteu spectrophotometric method [23]. Thus, 100 µL of the extract was added to 500 µL of the Folin-Ciocalteu reagents and 6 mL of bi-distilled water. The flask was agitated for a minute. After that, 2 mL of 15% (w/v) Na 2 CO 3 was added and made up to 10 mL with bi-distilled water. The flasks were kept in darkness. After 2 h, measures at 750 nm and 25 • C were carried out with a UV-visible spectrophotometer (Spectrophotometer 300 Array, UV-Vis, single beam, Shimadzu, Duisburg, Germany). Gallic acid was used for the calibration curve from 1 to 500 ppm. Results are expressed as mg gallic acid equivalents (GAE)/g dry weight (d.w.).

Antioxidant Activity: DPPH, ABTS and FRAP
Three different assays were used to determine the antioxidant capacity of the pepper extracts obtained with the optimal conditions. In all assays, Trolox was used as the standard for the calibration curves and the results were expressed in mg of Trolox equivalents (TE)/g of dry weight (d.w.). For the DPPH assay, 0.1 mL of the samples was added to 2.9 mL of 100 µM DPPH methanol solution and measured after 30 min at 517 nm at 25 • C [24,25]. For the ABTS as described previously by Pellegrini et al. [26], the assay consisted of adding 10 µL of extracts to 1 mL of ethanol diluted ABTS reagent and measuring the decrease in absorbance after 30 min at 734 nm at 30 • C. Finally, the FRAP assay was performed according to a method previously described by Pulido et al. [27]. Briefly, the FRAP reagent was prepared by mixing 25 mL of 0.3 mM acetate buffer (pH 3.6); 2.5 mL of 10 mM of TPTZ in 40 mM HCl solution, and 2.5 mL of 20 mM FeCl 3 ·6H 2 O solution. The analyses were performed by adding 90 µL of water and 0.9 mL of FRAP reagent to 30 µL of the extracts and measuring the absorbance after incubation at 37 • C for 30 min at 595 nm.

Determination of Phenolic Compounds in Pepper Extracts by HPLC-ESI-TOF-MS Analysis
Polar compounds present in the pepper extracts, obtained with the optimized conditions, were analyzed using an Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters Corporation, Milford, MA, USA) coupled to an electrospray ionization (ESI) source operating in the negative mode and a mass detector time of flight (TOF) micro mass spectrometer (Waters). The compounds of interest were separated on an ACQUITY UPLC BEH Shield RP18 column (1.7 µm, 2.1 × 100 mm; Waters Corporation, Milford, MA, USA) at 40 • C using a gradient previously used by Verni et al. [28]. H 2 O acidified with 1% of acetic acid and acetonitrile were used as phase A and B, respectively. MassLynx 4.1 software (Waters Corporation, Milford, MA, USA) was used for elaborating the data. An indicative base peak total ion chromatogram of the pepper samples analyzed by HPLC-MS is attached as Figure S1. To quantify the phenolic compounds identified in pepper samples, five calibration curves were made: vanillic acid, chlorogenic acid, ferulic acid, quercetin, and rutin. Table 2 contains the standards used, with their calibration ranges and curves, the regression coefficients, and the limits of detection (LOD) and quantification (LOQ). The calibration curves were elaborated by using the peak areas of each standard measured by HPLC at different concentrations. Calibration ranges were determined previously according to the LOQ values. The regression coefficients were >0.99 in all cases which means that all calibration curves had good linearity. LOD ranged between 0.17 and 2.76 µg/mL, and LOQ between 0.57 and 9.20 µg/mL.

Carotenoid Analysis by HPLC-MS
Carotenoids were extracted according to the protocol described by Castro-Puyana et al. 2013 [29] with some modifications. Briefly, 10 mg of lyophilized and powdered pepper was added to 1.5 mL of ethanol containing 0.1% (v/w) of butylated hydroxytoluene.
The mixture was centrifuged for 10 min at 10,000 (4 • C). The extracts were collected and filtered through 0.2 µm nylon syringe filters and stored at −18 • C until the analysis. Pepper extracts were analyzed by an Acquity UPLC coupled to a XEVO-TQ-S triple quadrupole mass spectrometry (Waters Corporation, Milford, MA, USA). Carotenoids were separated on a YMC-C30 column (250 × 4.6 mm 3 µm). The mobile phases consisted of methanol with 5% water and 0.1% formic acid as mobile phase A and methyl tert-butyl ether as mobile phase B. The conditions of the solvent gradient were 60% A to 0% A in 30 min with a flow rate of 1 mL min −1 . MRM transitions of each carotenoid were obtained by infusion of the analytical standards in positive ionization mode. Additional mass spectrometric parameters were as follows: source temperature: 150 • C, desolvation temperature: 500 • C, cone gas flow: 150 • C, source offset: 30 V, desolvation gas flow: 1000 L/h, collision gas flow: 0.15 mL min −1 , and collision gas: argon. The data were acquired using MassLynx version 4.1 (Waters, San Jose, CA, USA). Carotenoids were quantified by standards of violaxanthin, neoxanthin, meso-zeaxanthin, zeaxanthin, lutein, lycopene, and β-carotene ( Table 3). The calibration curves were prepared from the limit of quantification (LOQ) to 500-625 µg/L. All calibration curves revealed a good linearity among different concentrations, and the determination coefficients of the linear regression were higher than 0.99 in all cases. The method used for analysis showed a limit of detection (LOD) within the range 0.02-1.18 µg/L and the LOQ was within 0.08-3.96 µg/L. Table 3. Standard analytes used for elaborating the calibration curves with the range used, equations, R 2 , LOD, and LOQ of each compound for the carotenoid analysis.

Chlorophylls Analysis
The content of chlorophyll pigments a and b was determined following the procedure described by Porra et al. (1989) [30]. Briefly, the samples were extracted by acetone/water 80% in an ultrasound bath for 15 min. Next, the samples were centrifuged at 9000 rpm for 10 min. The supernatant was collected and filtered through 0.2 µm filters. Then, the absorbance of the extract samples, appropriately diluted, was measured at 647, 664, and 750 nm on a UV-Vis spectrophotometer (Perkin-Elmer Lambda 25). The results, expressed as µg/mL, were calculated using the equations: where A is the absorbance at the established wavelengths. All absorbance measurements must have the absorbance at 750 nm (A 750 ) subtracted.

Fitting the Model
A Box-Behnken design combined with RSM was used to establish the optimal conditions of time (X 1 ), ethanol/water ratio (X 2 ), and solvent/sample ratio (X 3 ) in ultrasound bath technology to extract phenolic compounds from pepper edible parts. The experimental values of the total phenolic content (TPC) for each run are presented in Table 4. The observed results ranged from 2.5 to 11.6 mg GAE/g d.w., which correspond to the extraction conditions 5 min and 100% ethanol, and 45 min and 50% ethanol, respectively, both at a solvent/sample ratio of 105 v/w. In fact, all the runs using 100% ethanol as solvent reported lower recovery of TPC. Table 4. Box-Behnken design with natural and coded values (parenthesis) of the conditions of extraction and the experimental results obtained for total phenolic content (TPC) expressed with the average and the standard deviation.

Run
Independent Factors Response The experimental data were analyzed adjusting it to a second-order polynomial equation, a regression model that provides the lowest residual value using the least-squares method. All regression coefficients are shown in Table 5. With a significance level of p < 0.05, the lineal terms of time (β 1 ) and ratio ethanol/water (β 2 ), the crossed between them (β 12 ), and all the quadratic terms (β 11 , β 22 , β 33 ) had a significant effect. After discarding the non-significant terms, the model was recalculated and tested by ANOVA. As can be seen in Table 5, the model revealed a high correlation between the response variable and the factors (R 2 = 0.9173), a good fit to the regression model (p < 0.05), and an insignificant lack of fit (p > 0.05); therefore, as reported by Bezerra et al. (2008) [31], the adequacy of the model is confirmed. The selection of the optimal conditions was carried out by using RSM among the threedimensional graphs as shown in Figure 2. Thus, a compromise between the minimum possible values of each independent factor was made. As can be seen, when increasing the percentage of ethanol higher than 40% combined with the effect of time or solvent/sample ratio, lower amounts of phenolic compounds were achieved. Regarding the combined effect of time with ratio solvent/sample intermedium, values of those two parameters allowed the highest recovery of TPC. Therefore, these observations made it possible to establish the best conditions as: 30 min, 20% ethanol/water, and ratio solvent/sample 120 v/w, which provided a predicted value of 11.18 ± 2.03 mg GAE/g d.w for TPC. Furthermore, the model was verified and did not report significant differences between the observed (11.26 ± 1.71 mg GAE/g d.w.) and predicted value (p < 0.05).  [33] reported methanol as the better solvent for extracting phenolic compounds from Capsicum Chinese pepper leaves compared to acetone and hexane. However, those solvents are hazardous for human health and could not be used for nutraceuticals. In contrast, Rybak et al. (2020) [34], using water as a solvent in a ratio solvent/sample (v/w) of 4 for a total of 90 min at 20 • C, reported a value of 25.5 mg/g d.w. by ultrasound technology in red bell pepper, which demonstrated that water is a food-grade safe solvent that allowed the recovery of phenolic compounds from pepper. In the same way,   [35] reported the maximum flavonoid recovery from pepper when using ethanol instead of methanol or N-N-dimethylformamide. Additionally, Dias et al. (2017) [36] confirmed that ethanol allowed the extraction of more antioxidant compounds than methanol by ultrasound bath technology from dedo de moça pepper (Capsicum baccatum L.).  [40] reported a total phenols content from 0.7 to 1.11 mg/g d.w. in C. annuum var. Orangela/Terrano. In this context, the optimized ultrasound conditions allowed the achievement of a high recovery of phenols from pepper by using a food-grade and non-hazardous solvent, ethanol/water 20:80, avoiding thermal degradation as no temperature was used during the process, and which is less time consuming than other conventional methods.

HPLC Analysis of Polar Compounds in Pepper Extracts
A total of 38 polar compounds have been tentatively identified in the pepper optimal extracts. Between them, there are organic acids, amino acids, terpenoids, lignan derivates, phenolic acids, flavonoids, and other metabolites. Table 6 shows an overview of all proposed compounds with their retention time (min), molecular formula, experimental and calculated m/z, and m/z fragments. Furthermore, all metabolites showed a score higher than 90% and an error (ppm) lower than 5. These parameters were given by the software MassLynx 4.1. To identify the compounds, the generated molecular formulas and some in-source fragments were checked, studied, and compared with different databases such as PubChem, Mass bank, Phenol-Explorer, and the literature.
Three organic acids were identified. Peak 2, at 0.49 min and m/z 191, was recognized as quinic acid; peak 3, at 0.59 min and m/z 175, was identified as ascorbic acid; and peak 4, at 0.63 min and m/z 191, was identified as citric acid. At peak 5 and 7 with m/z 164 and 203, two amino acids were identified, DL-phenylalanine and tryptophan, respectively. Pantothenic acid (vitamin B5) was found at 1.21 min corresponding to peak 6. Furthermore, a lignan derivate was identified with an m/z of 521 (peak 29) named lariciresinol glucopyranoside. The results were in concordance with the previous literature [33,41,42].
Flavonoids are the more numerous group of phenolic compounds found in the pepper samples analyzed. Corresponding with peaks 16 [46]. At 11.15 min (peak 37), a kaempferol derivate with the kaempferol m/z fragment 285 was detected that, according to the PubChem database, was named kaempferol 3-(3"-acetylalpha-L-arabinopyranosyl)-glucoside in concordance with other authors that reported similar compounds in pepper leaves [32]. Moreover, the compound with an m/z of 665 (peak 38) was luteolin-7-(2-O-apiosyl-6-O-malonyl) glucoside as reported previously in Capsicum chinense pepper leaves [33] and sweet red peppers [46].      Finally, two terpenoids have been found and reported here for the first time. Corresponding with peaks 15 and 18, two isomers of a monoterpene iridoid named deoxyloganic acid were detected according to the m/z fragment 197 according to the MassBank record: PR309193 by Tsugawa et al. (2019) [48] and the PubChem database. Similarly, the compound found at 9.63 min (peak 33) with the m/z fragment 155 corresponding to menthol was named menthol glucuronide, previously described as a tea metabolite [47].

Quantification of Phenolic Compounds in Pepper Extracts by HPLC-MS
A total of 9 phenolic acids and 19 flavonoids were quantified in WP and EP extracts obtained with the optimized conditions ( Table 7). The analyses were made in triplicate.   As can be seen in Table 7, WP samples had a higher content of total phenolic compounds than EP samples; however, some differences between them should be highlighted. In general, phenolic acids were the most representative polyphenols accounting for 66-93% of the total phenolic content. WP showed a higher content of total phenolic acids with feruloyl glucose being the main compound followed by sinapic acid-O-hexoside and vanillic acid 1-O-β-D-glucopyranosylester. In the EP, the major phenolic acid was sinapic acid-O-hexoside followed by galloyl-1,4-galactarolactone and feruloyl glucose. Caffeic acid derivatives also had a significant impact on the phenolic acid content in both types of samples. No significant differences were found between samples 1 and 2 in WP nor in EP. In contrast, EP samples showed a higher content of total flavonoids than WP. The major flavonoid in all cases was kaempferol-3-(3"-acetyl-alpha-L-arabinopyranosyl)-glucoside. Moreover, high amounts of quercetin, luteolin, and apigenin derivatives were found. Furthermore, some differences were found between samples 1 and 2. Samples coded as 2 had around half of the total flavonoid content of 1. This is mainly attributed to the slight differences in the environmental conditions due to the different locations of the culture that can affect the stress or non-stress conditions of the plant and consequently promote the production of different metabolites. Chel-guerrero et al. (2021) [49] compared two habanero peppers grown in black and red soils of Yucatán, México, and obtained values of total phenolic compounds ranging from 0.56 to 1.54 mg/g d.w. Dos Anjos et al. (2021) [32], in C. frutescens peppers from Brazil, reported a phenolic content from 15 to 17 mg/g d.w. and total flavonoids content from 6.1 to 7.8 mg/g d.w. Guclu et al. (2021) [43] found a total phenolic content of 368.77-496.07 µg/g in sweet red pepper samples. The obtained results are in the same range of magnitude as those previously reported by other authors in other pepper samples, and it can be confirmed that the origin and variety significantly affected the content in phenolic compounds.

Antioxidant Activity of Pepper Extracts by DPPH, ABTS and FRAP Assays
The antioxidant activity of the extracts obtained with the ultrasound optimal conditions was measured by three different methods and the results are summarized in Table 8. The values ranged from 11. 58-12.44, 9.66-11.88, and 14.96-19.55 mg TE/g d.w. for the DPPH, ABTS, and FRAP, respectively. As reported by Ou et al. (2002) [50], polyphenols are chain-breaking antioxidants. Pepper extracts are rich in phenolic acids and flavonoids that are responsible for the antioxidant activity reported here. No significant differences were found between EP and WP samples for DPPH and ABTS assays, but there were for FRAP. In this case, WP presented higher radical scavenging, reducing ferric anion. However, this lack of differences between the pepper without peel and the whole pepper may be due to its composition. EP has been found to be richer in flavonoids; meanwhile, WP was richer in the major phenolic component, phenolic acids. Therefore, these differences seem to compensate and give the sample the same antioxidant activity for the DPPH and ABTS assays. However, in contrast, for the FRAP assay, phenolic acids seemed to have the strongest influence. Compared with other authors, the results are in the range reported in other bell pepper varieties [3,40,51,52].

Carotenoid Composition of Pepper by HPLC-MS
The carotenoid content was analyzed in triplicate in each sample, and the obtained results are presented in Table 9. Carotenes are responsible for the yellow, red, and orange colors of fruits and vegetables, and offer beneficial health effects against various diseases such as cardiovascular disease, cancer, and other chronic diseases [40]. According to the results reported by de Azevedo-Meleiro and Rodriguez-Amaya [53], the main carotenoids in the studied pepper were violaxanthin, lutein, and β-carotene. Other minor compounds were zeaxanthin, α-carotene, neoxanthin, lycopene, and meso-zeaxanthin. As seen in Table 9, EP samples showed higher amounts of carotenoids compared with the respective WP samples confirming that the localization of these compounds is in the edible portion of the fruit. Total carotenoid content ranged from 82.8 to 103.6 µg/g d.w. in EP samples and from 63.2 to 78.2 µg/g d.w. in WP samples. These data are in the same order of magnitude as those reported in the literature for yellow peppers [53], but lower than others reported by other authors [54,55].
It should be taken into account that, as reported by Jarén-Galán et al. (1999) [56], up to 30% of the pigments could be destroyed after post-harvesting due to autoxidation and the presence of pepper lipoxygenase enzyme. Moreover, the carotenoid content depends strongly on the variety and pedo-climatic conditions. However, several authors have reported results similar to those obtained here for C. annuum var. Kioto in other cultivars.  [40] reported a β-carotene content of 86.2 µg/g d.w. and lycopene content of 0.03 µg/g d.w. in C. annuum var. Orangela/Terrano. Carvalho et al. (2015) [38] reported values of 1.95-3.12, 0.08-4.6, 0.0-5.16, and 0.0-44.42 µg/g d.w. of lutein, zeaxanthin, alpha-carotene, and beta-carotene, respectively, in other varieties of C. annuum.

Chlorophylls Content of Pepper
The chlorophyll content was evaluated and the results are presented in Table 10. Total chlorophyll content ranged from 5.40 to 6.63 µg/g d.w. for the EP samples and from 25.13 to 27.79 µg/g d.w for the WPs. As can be seen from the results, yellow bell peppers showed a low chlorophyll content, not being their major pigment; however, differences can be appreciated among the samples. Those composed of the whole pepper had higher chlorophyll a and b content and total content in both cases. Besides there is a difference in the chlorophyll a/chlorophyll b ratio. WP samples had a ratio < 1 and EP samples had a > 1 ratio which indicates that chlorophyll a is mainly in the edible portion and chlorophyll b is present in the other parts of the peppers. Overall, these differences can be mainly attributed to the presence of peduncles and seeds in the whole pepper samples. There are few previous references of chlorophylls in yellow bell pepper samples. Ignat et al. (2013) [59] evaluated the effect of the maturity stage in the total chlorophyll content in intact yellow bell peppers and reported values in the same range as those obtained here for the mature ones, which additionally confirms the maturity stage of the analyzed peppers.

Conclusions
The ultrasound-assisted extraction of phenolic compounds from C. annuum var Kioto has been optimized using a Box-Behnken design combined with the RSM methodology. Optimal established conditions were 30 min, 20% ethanol, and a solvent/sample ratio of 120 v/w. The optimal extracts were characterized and a total of 38 polar compounds were identified from which seven were identified in pepper fruit extracts for the first time. Moreover, EP and WP were compared according to their phenolic compounds, antioxidant activity, and pigment content. WP samples presented a higher phenolic acids content; meanwhile, EP samples had a higher flavonoids content. No differences were found in the antioxidant activity except for the FRAP assay where the WP sample showed higher radical scavenging activity. Regarding pigments, it was noticed that carotenoids are much more concentrated in the edible portion of pepper, whereas chlorophylls are in the whole pepper.
Overall, the bell peppers that are usually wasted during the selection process of fruits could be considered a valuable raw material to obtain bioactive compounds such as phenolic compounds and carotenoids that can be used as functional food, nutraceutical, and cosmeceutical ingredients.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11030557/s1, Figure S1: Indicative base peak total ion chromatogram of the pepper samples analysed by HPLC-MS.