Analysis of the contents of pungent compounds in fresh Korean red peppers and in pepper-containing foods

An HPLC method has been developed for the analysis of extracts of fresh peppers containing capsaicinoids and of both capsaicinoids and piperines in pepper-containing foods produced and sold in Korea. The HPLC method was optimized by defining how composition of the mobile phase affected retention times. Both identification and quantification were based on retention times and the following criteria: linearity of the UV response at 280 nm in HPLC, recoveries from spiked samples, and observed individual molecular ions in the mass spectra of the extracts determined by liquid chromatography-mass spectrometry. This method, with a limit of detection of approximately 15-30 ng, was used to quantify the distribution of capsaicinoids in 11 Korean whole peppers and in 12 commercial pepper-containing foods. Total capsaicinoid levels of whole peppers ranged from 1.21 microg/g for the PR Gang ja variety to 121.1 microg/g for the Chung yang variety. The levels in food extracts, four of which also included two piperines, ranged from 11.0 microg/g for radish kimuchi to 3752 microg/g for capsaicin sauce. The results demonstrate (a) the usefulness of the HPLC method for the simultaneous analysis of capsaicinoids derived from red peppers and piperines derived from black and white peppers extracted from complex food matrices and (b) the wide-ranging spread of levels of pungent pepper compounds in fresh peppers and in pepper-containing foods consumed in Korea.     

Pressurized liquid extraction of capsaicinoids from peppers

A method has been developed for the extraction of capsaicinoids from peppers by pressurized liquid extraction (PLE); these compounds are determined by reverse phase high-performance liquid chromatography (HPLC), with detection by fluorescence spectrophotometry and mass spectrometry (MS). The stability of capsaicin and dihydrocapsaicin has been studied at different temperatures (50-200 degrees C), and several extraction variables have been assayed: solvent (methanol, ethanol, and water), different percentages of water in the methanol (0-20%) and in the ethanol (0-20%), and the number of extraction cycles. The study has evaluated the repeatability (RSD < 7%) and the reproducibility (RSD < 7%) of the method. Finally, the PLE method developed has been applied to quantify the capsaicinoids present in three varieties of hot peppers cultivated in Spain, quantifying five capsaicinoids: nordihydrocapsaicin, capsaicin, dihydrocapsaicin, an isomer of dihydrocapsaicin, and homodihydrocapsaicin.     

Determination of capsaicin and dihydrocapsaicin in capsicum fruits by liquid chromatography-electrospray/time-of-flight mass spectrometry

A simple, highly selective, sensitive, and reproducible liquid chromatography-electrospray ionization/time-of-flight mass spectrometry method has been developed for the direct and simultaneous determination of capsaicin and dihydrocapsaicin in Capsicum fruit extracts. Capsaicin and dihydrocapsaicin are the two major members of the so-called capsaicinoid family, which includes other minor analogues, and usually account for at least 90% of the pungency trait in Capsicum fruits. Chromatographic separation of capsaicin and dihydrocapsaicin was achieved with a reversed-phase chromatography column, using a gradient of methanol and water. Quantification was done using as an internal standard (4,5-dimethoxybenzyl)-4-methyloctamide, a synthetic capsaicin analogue not found in nature. Analytes were base-peak resolved in less than 16 min, and limits of detection were 20 pmol for capsaicin and 4 pmol for dihydrocapsaicin. The intraday repeatability values were lower than 0.5 and 12% for retention time and peak area, respectively, whereas the interday repeatability values were lower than 0.6 and 14% for retention time and peak area, respectively. Analyte recoveries found were 86 and 93% for capsaicin and dihydrocapsaicin, respectively. The method developed has been applied to the identification and quantification of capsaicin and dihydrocapsaicin in fruit extracts from different Capsicum genotypes, and concentrations found ranged from 2 to 6639 mg kg(-1).     

Capsaicinoids in vegetative organs of Capsicum annuum L. in relation to fruiting

Pepper (cv. Padrón) shows a spatial gradient in the content of phenolic compounds, and particularly of capsaicinoids, along the stem. These compounds were consistently more abundant in apical fruits than in fruits belonging to middle and basal segments. Analysis of the two principal capsaicinoids in fruits showed that the proportion of capsaicin was always higher than that of dihydrocapsaicin. Capsaicinoids were also found to be present in vegetative organs, such as stem and leaves. In this case, the proportion of individual capsaicinoids was different than that in fruits, and dihydrocapsaicin was found to be more abundant. To find out whether the capsaicinoids in vegetative organs came from the fruits, the floral buds were removed and fruit formation was prevented. Capsaicinoids were not detected in the stem and leaves of floral bud-deprived plants, suggesting that they did originate from the fruit.     

Ground red peppers: capsaicinoids content,Scoville scores,and discrimination by an electronic nose

High-pressure liquid chromatography (HPLC) was used to determine the capsaicin, dihydrocapsaicin, and total capsaicinoids levels of different ground red pepper samples obtained from local retail markets in Izmir, Turkey. Scoville scores were determined using sensory tests. An electronic nose (EN) was used to discriminate ground red pepper samples by headspace volatiles. EN data were analyzed using discriminant function analysis (DFA). An overall correct classification rate of pepper varieties by EN of 91% was obtained. A linear correlation between capsaicin, dihydrocapsaicin, and total capsaicinoids and Scoville scores was also observed, and R (2) values of 0.89, 0.85, and 0.91 were obtained, respectively.     

Antioxidant activity of fresh and processed Jalapeño and Serrano peppers

In this research, total phenols, flavonoids, capsaicinoids, ascorbic acid, and antioxidant activity (ORAC, hydroxyl radical, DPPH, and TEAC assays) of fresh and processed (pickled and chipotle canned) Jalapen?o and Serrano peppers were determined. All fresh and processed peppers contained capsaicin, dihydrocapsaicin, and nordihydrocapsaicin, even though the latter could be quantified only in fresh peppers. Processed peppers contained lower amounts of phytochemicals and had lower antioxidant activity, compared to fresh peppers. Good correlations between total phenols and ascorbic acid with antioxidant activity were observed. Elimination of chlorophylls by silicic acid chromatography reduced the DPPH scavenging activity of the extracts, compared to crude extracts, confirming the antioxidant activity of chlorophylls present in Jalapen?o and Serrano peppers.     

Antioxidant activity of capsinoids

Hot peppers are a good source of dietary antioxidants, encompassing, apart from widespread compounds (flavonoids, phenolic acids, carotenoids, vitamin A, ascorbic acid, tocopherols), also specific constituents such as the pungent capsaicinoids (capsaicin, dihydrocapsaicin, and related analogues). We have shown that capsinoids also show remarkable antioxidant activity. These benign analogues of capsaicin could protect linoleic acid against free radical attack in simple in vitro systems, inhibiting both its autoxidation and its iron- or EDTA-mediated oxidation. These properties were retained in some simple synthetic analogues (vanillyl nonanoate and its dimerization products). Capsiate, dihydrocapsiate, and their analogues were devoid of pro-oxidant activity and showed a highly significant antioxidant activity in all systems investigated. Vanillyl nonanoate, a simple capsinoid mimic, was also tested on cell cultures for cytotoxic activity and the capacity to inhibit FeCl(3)-induced oxidation.     

Purification and structure determination of glucosides of capsaicin and dihydrocapsaicin from various Capsicum fruits

Two new glucosides, capsaicin-beta-D-glucopyranoside (1) and dihydrocapsaicin-beta-D-glucopyranoside (2), were discovered in the fruit of the Capsicum annuum cultivar 'High Heat'. They were sequentially purified by acetone extraction, n-hexane extraction, and acetonitrile extraction, followed by medium-pressure liquid chromatography and high-performance liquid chromatography performed on an octadecylsilane column. Their chemical structures were elucidated by proton nuclear magnetic resonance, carbon nuclear magnetic resonance, and hydrolysis with alpha- and beta-glucosidases. The glucosides were also detected in various pungent cultivars of C. annuum, Capsicum frutescens, and Capsicum chinense by liquid chromatography-mass spectrometry. However, they were not detected in nonpungent cultivars of C. annuum. Furthermore, a positive correlation was observed between the quantity of the capsaicinoids, capsaicin, and dihydrocapsaicin and their glucosides.     

Determination of capsaicinoids in habanero peppers by chemometric analysis of UV spectral data

A novel spectrophotometric method for the determination of capsaicinoids in habanero pepper extracts is described that does not require prior analyte separation. The method uses partial-least-squares (PLS-1) multivariate regression modeling techniques in conjunction with ordinary UV absorption spectral data obtained on alcoholic extracts of habanero peppers (Capsicum chinese). The PLS-1 regression models were developed by correlating the known total concentration of the two major capsaicinoids (capsaicin and dihydrocapsaicin) in the extracts as determined by high-performance liquid chromatography with the spectral data. The regression models were subsequently validated with laboratory-prepared test sets. The validation studies revealed that the root-mean-square error of prediction varied from 4 to 8 ppm, based on the results obtained from models prepared from nine test sets. Once a regression model has been developed and validated, analyses of the extracts can be accomplished rapidly by ordinary spectrophotometric procedures without any prior separation steps.     

Direct connection of supercritical fluid extraction and supercritical fluid chromatography as a rapid quantitative method for capsaicinoids in placentas of Capsicum.

The fruits of Capsicum annuum L. are used worldwide as chili peppers and in folk medicines. The pungent components of C. annuum, which are irritants, are called capsaicinoids (CAPS), and the most abundant components are capsaicin, dihydrocapsaicin, and nordihydrocapsaicin. To analyze CAPS in the placentas of Capsicum fruits rapidly and safely, we used a directly connected system of supercritical fluid extraction and supercritical fluid chromatography (SFE/SFC). As a column for SFE/SFC, only a silica-type column was found to be suitable. The CAPS contents in placentas of C. annuum cv. Jalapeno (hot type) and C. annuum cv. Shishitoh (less-hot type) determined by the SFE/SFC method agreed well with those in the range of 0-13.81 mg g(-1) fr. wt determined by the usual extraction-HPLC method. The SFE/SFC method has the advantages of no need for pretreatment and no (or minimal) need for organic solvents. We conclude that this method is useful as a rapid (20 min) and safe screening test for the pungency of various Capsicum fruits.     

Identification and quantification of carotenoids including geometrical isomers in fruit and vegetable juices by liquid chromatography with ultraviolet-diode array detection

A method was established for the identification and quantification of carotenoids including geometrical isomers in fruit and vegetable juices by liquid chromatography with an ultraviolet-diode array detector, using a C(18) Vydac 201TP54 column. The mobile phase used was the ternary methanol mixture (0.1 M ammonium acetate), tert-butyl methyl ether and water, in a concentration gradient, and a temperature gradient was applied. Retinol palmitate was added as an internal standard. An extraction process (ethanol/hexane, 4:3, v/v) was performed, followed by saponification with diethyl ether/methanolic KOH (0.1%, w/v, BHT) (1:1, v/v) for 0.5 h at room temperature. Seventeen different (cis and trans) carotenoids were identified by UV-vis spectra and retention times in HPLC in the juices analyzed. The analytic parameters show that the method proposed is sensitive, reliable, accurate, and reproducible.     

Antioxidant activity of the main phenolic compounds isolated from hot pepper fruit (Capsicum annuum L)

Four cultivars (Bronowicka Ostra, Cyklon, Tornado, and Tajfun) of pepper fruit Capsicum annuum L. were studied for phenolics contents and antioxidant activity. Two fractions of phenolics, flavonoids (with phenolic acids) and capsaicinoids, were isolated from the pericarp of pepper fruit at two growth stages (green and red) and were studied for their antioxidant capacity. Both fractions from red fruits had higher activities than those from green fruits. A comparison of the capsaicinoid fraction with the flavonoid and phenolic acid fraction from red fruit with respect to their antioxidant activity gave similar results. Phenolic compounds were separated and quantified by LC and HPLC. Contents of nine compounds were determined in the flavonoid and phenolic acid fraction: trans-p-feruloyl-beta-d-glucopyranoside, trans-p-sinapoyl-beta-d-glucopyranoside, quercetin 3-O-alpha-l-rhamnopyranoside-7-O-beta-d-glucopyranoside, trans-p-ferulyl alcohol-4-O-[6-(2-methyl-3-hydroxypropionyl] glucopyranoside, luteolin 6-C-beta-d-glucopyranoside-8-C-alpha-l-arabinopyranoside, apigenin 6-C-beta-d-glucopyranoside-8-C-alpha-l-arabinopyranoside, lutoeolin 7-O-[2-(beta-d-apiofuranosyl)-beta-d-glucopyranoside], quercetin 3-O-alpha-l-rhamnopyranoside, and luteolin 7-O-[2-(beta-d-apiofuranosyl)-4-(beta-d-glucopyranosyl)-6-malonyl]-beta-d-glucopyranoside. The main compounds of this fraction isolated from red pepper were sinapoyl and feruloyl glycosides, and the main compound from green pepper was quercetin-3-O-l-rhamnoside. Capsaicin and dihydrocapsaicin were the main components of the capsaicinoid fraction. A high correlation was found between the content of these compounds and the antioxidant activity of both fractions. Their antioxidant activities were elucidated by heat-induced oxidation in the beta-carotene-linoleic acid system and the antiradical activity by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) decoloration test. The highest antioxidant activity in the beta-carotene-linoleic acid system was found for trans-p-sinapoyl-beta-d-glucopyranoside, which was lower than the activity of free sinapic acid. Quercetin 3-O-alpha-l-rhamnopyranoside had the highest antiradical activity in the DPPH system, which was comparable to the activity of quercetin. The activities of capsaicin and dihydrocapsaicin were similar to that of trans-p-feruloyl-beta-d-glucopyranoside in the DPPH model system.     

Glycoalkaloid and calystegine contents of eight potato cultivars

Diverse procedures have been reported for the separation and analysis by HPLC of the two major glycoalkaloids present in potatoes, alpha-chaconine and alpha-solanine. To further improve the usefulness of the HPLC method, studies were carried out on the influence of several salient parameters on the analysis of the two potato glycoalkaloids. Effects on retention (elution, separation) times of the (a) composition and pH of the mobile phase (acetonitrile and phosphate buffer), (b) concentration of the phosphate buffer, (c) capacity values of column packing of four commercial HPLC amino columns, (d) column temperature were studied. Except for pH, all of the variables significantly influenced the retention times. The results make it possible to select analysis conditions that produce well-separated as well as symmetrical peaks of the two glycoalkaloids. This improved HPLC method (limit of detection of approximately 150 ng) was evaluated with extracts from the cortex of one whole potato variety (May Queen) grown in Japan and the freeze-dried peel and flesh from the following eight cultivars grown in the United States: Atlantic, Dark Red Norland, Ranger Russet, Red Lasoda, Russet Burbank, Russet Norkota, Shepody, and Snowden. In addition, the same samples were analyzed by GC-MS for the presence of two water-soluble nortropane alkaloids, calystegine A(3) and calystegine B(2), reported to be potent glycosidase inhibitors. The following ranges for the eight varieties of total glycoalkaloid and calystegine levels were observed: dry flesh, 5-592 and 6-316 mg/kg; dry peel, 84-2226 and 218-2581 mg/kg; dry whole potatoes, 40-883 and 34-326 mg/kg; wet flesh, 1-148 and 1-68 mg/kg; wet peel, 12-429 and 35-467 mg/kg; wet whole potatoes, 7-187 and 5-68 mg/kg. The possible significance of the results to plant and food sciences is discussed.     

Rapid electrochemical method for the evaluation of the antioxidant power of some lipophilic food extracts.

In this paper, a novel electrochemical method to evaluate the antioxidant power of lipophilic compounds present in vegetables, such as carotenoids, chlorophylls, tocopherols, and capsaicin, is reported. The method is based on a flow injection system with an electrochemical detector equipped with a glassy carbon working electrode operating amperometrically at a potential of + 0.5 V (vs Ag/AgCl). The proposed method is selective for lipophilic compounds having antioxidant power. When applied to pure compounds, the order of antioxidant power resulted as follows: lycopene > beta-carotene > zeaxanthin > alpha-carotene > beta-cryptoxanthin > lutein > alpha-tocopherol > capsaicin > chlorophyll a > chlorophyll b > astaxanthin > canthaxanthin. Results obtained on five vegetable and two fruit extracts were compared to those obtained by the 2,2'-azinobis(3-ethylbenz-thiazoline-6-sulfonic) acid (ABTS) radical cation decolorization assay, one of the most used methods to evaluate the total antioxidant capacity of foods. A good correlation between the two methods was found, except for spinach, because of the different antioxidant powers assigned by the two methods to chlorophylls. In conclusion, results suggest that the proposed electrochemical method can be successfully employed for the direct, rapid, and reliable monitoring of the antioxidant power of lipophilic food extracts.     

Inheritance of capsaicin and dihydrocapsaicin, determined by HPLC-ESI/MS, in an intraspecific cross of Capsicum annuum L

The quantitative inheritance of capsaicin and dihydrocapsaicin contents in fruits has been studied in an intraspecific cross of Capsicum annuum L. across two different environments, namely, fruits developed in spring and summer. A liquid chromatography-electrospray ionization/time-of-flight mass spectrometry [HPLC-ESI/MS(TOF)] method was used to identify and quantify capsaicin and dihydrocapsaicin in extracts of pepper fruits. The analytical method used was able to determine the pungency of genotypes that, using other methods, would have been classified as non-pungent. Capsaicin and dihydrocapsaicin contents varied largely among families, and families did not respond similarly in producing these capsaicinoids when their fruits were grown in spring and summer, with some families showing no increase, whereas in others, the increase was more than 2-fold. Heterosis for the pungency trait, assessed by the capsaicin and dihydrocapsaicin contents in fruits, was found, indicating the existence of epistasis, over-dominance, or dominance complementation. Non-pungent parent alleles contributed to the capsaicin and dihydrocapsaicin contents since transgressive segregation did occur. Furthermore, the type of gene action varied between capsaicin and dihydrocapsaicin, and a seasonal effect during fruit development could affect gene action.     

Pungency in paprika (Capsicum annuum). 2. Heterogeneity of capsaicinoid content in individual fruits from one plant

The capsaicinoid content of individual fruits from a single plant harvested at the same time after flowering exhibits a wide range of values with a rather uniform pattern for the ratio of capsaicin, dihydrocapsaicin, and nordihydrocapsaicin. This observation is confirmed by the analysis of fruits from a second and third plant and for several harvest times at different stages of maturity. Competition with lignin-like material, environmental influences, and subcellular distribution may play a role in the synthesis and transformation of capsaicinoids.     

Identification and quantification of polyphenolic compounds in Longan (Euphoria longana Lam.) fruit

Regular consumption of fruit and vegetables is associated with a lower risk of some chronic diseases including various forms of cancer and cardiovascular diseases. The health-promoting potential of these foods may be due, in part, to the phytochemical bioactive compounds present in the plants. Fruit of Euphoria longana Lam. (longan) are consumed throughout Asia and are a major crop in Thailand. In the present study phytochemicals were extracted with 70% methanol from peel, pulp, and seed tissues of longan fruit, and the major components were identified as gallic acid, corilagin (an ellagitannin), and ellagic acid. A high-through-put reversed phase HPLC method was developed to determine the content of these three compounds in different parts of the longan fruit and among different cultivars. The analyses showed that there was a large variation in the contents of gallic acid, corilagin, and ellagic acid in different plant tissues and cultivars. Seed contained the highest levels of the three phenolics, and pulp contained the lowest. Among commercial cultivars, Biewkiew and Edor contained the highest levels of gallic and ellagic acid while Srichompoo contained the highest content of corilagin. These three cultivars may be used in directed breeding and cultivation programs and to develop concentrated longan seed extracts to promote good health. Utilization of this byproduct material will support the use of thousands of tons of waste longan seeds after the production of canned longan pulp.     

Quantitation of clovamide-type phenylpropenoic acid amides in cells and plasma using high-performance liquid chromatography with a coulometric electrochemical detector

A high-performance liquid chromatography (HPLC) method was developed for measuring the concentrations of clovamide-type phenylpropenoic acid amides (N-caffeoyldopamine and N-caffeoyltyramine) in cell and plasma samples. The separation was performed on a Nova-Pak C18 column using an isocratic buffer with a coulometric electrochemical detector with four electrode channels. Using the HPLC method, N-caffeoyldopamine and N-caffeoyltyramine could be detected with good peak resolutions at respective retention times (4 and 6.4 min). The calibration curves were linear over the ranges (0.1 and 100 microM), and their lower limit of detection was as little as 100 fmol. For quantifying N-caffeoyldopamine and N-caffeoyltyramine in cell and plasma samples, the samples were extracted by extraction methods with more than 95% recoveries. After extraction, the amides were detected with the same sensitivity, peak resolutions, and retention times. Using this method, plasma concentrations of N-caffeoyltyramine were determined in blood samples collected at 12, 24, 30, 36, 48, 60, and 75 min after the oral administrations of N-caffeoyltyramine (0.5 mg and 2 mg/30 g body weight). This HPLC method with an electrochemical detector is the first reported method able to quantify N-caffeoyldopamine and N-caffeoyltyramine in biological samples with excellent detection limits, peak resolutions, discrete retention times, and consistent reproducibility.     

Rapid screening of ascorbic acid, glycoalkaloids, and phenolics in potato using high-performance liquid chromatography

Evaluation of phenolic metabolism in potato tubers (Solanum tuberosum) would be facilitated by faster analytical methods. A high-throughput HPLC method was developed for the qualitative and quantitative determination in potato of numerous phenolic compounds, the sum of the glycoalkaloids chaconine and solanine, plus ascorbic acid. Following a fast extraction, HPLC run times of 12 min were achieved with the use of a monolithic RP C18 column. UV and MS analyses were used to characterize the phenolic complement in extracts from two white-fleshed varieties. Over 30 compounds were identified, some of which are thought to possess either nutritional value or are involved in plant disease resistance. This method is expected to be useful for germplasm mining and for varietal development programs in which large numbers of lines are generated.     

Quantitative characterization of flavonoid compounds in Rooibos tea (Aspalathus linearis) by LC-UV/DAD

Rooibos tea originates from the leaves and stems of the indigenous South African plant Aspalathus linearis. It has gained much attention for clinical purposes in the case of nervous tension, allergies (dermatitis), and various indigestive problems. Recently, antioxidative activity was also attributed to the tea on the basis of its flavonoid content. Therefore, an HPLC method using a C(18) reversed phase column was developed for the assay of 10 flavonoids in aqueous and methanolic infusions. Main compounds determined were the dihydrochalcone aspalthin, rutin, and orientin, and their content was in the range of 1.0 to 1.3 mg/g. The identity of detected flavonoids was confirmed by comparing their retention times and UV and MS spectra with those of corresponding standards. In addition, the MS analysis showed evidence of the presence of other compounds such as nothofagin, dihydroisoorientin, and dihydroorientin.