Capsaicinoid Contents in Peppers and Pepper-Related Spicy Foods

Abstract

The quantification of the major capsaicinoids, namely nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin, present in spicy foods made from peppers has been performed. The capsaicin content is directly related to the pungency properties of foods that contain hot peppers. The samples studied included 10 different dried hot peppers, 19 hot sauces, 4 kinds of paprika, and 4 different ketchups. The range of concentrations of capsaicinoids found were as follows: dried hot peppers (554.1–1705.9 mol kg⁻¹), paprikas (582.0–665.0 mol kg⁻¹), spicy ketchups (4.0–12.4 mol kg⁻¹), and hot sauces (4.6–843.8 mol kg⁻¹). Variability in the capsaicinoid content was found, with capsaicin and dihydrocapsaicin present at the highest levels. The capsaicin content was generally higher than the dihydrocapsaicin content.

Keywords:

• Capsaicinoids
• Spicy Food
• Sauces
• Pepper
• Paprika

INTRODUCTION

Hot peppers are spicy or savory food additives that are widely used in numerous countries around the world. These peppers have high value due to their attributes of color, flavor, and pungency. Capsaicinoids are the pungent compounds that are responsible for the characteristic taste of hot peppers. Among these compounds are two major capsaicinoids, namely capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) and dihydrocapsaicin (DHC; 8-methyl-N-vanillylnonanamide), and these represent around 80–90% of the total capsaicinoids present in most species of hot peppers.^[1] In addition to these two major compounds, other capsaicinoids have been found in peppers and these include nordihydrocapsaicin (n-DHC), homocapsaicin (h-C), homodihydrocapsaicin (h-DHC), and nonivamide amongst others. In total, more than 20 capsaicinoids have been identified in different pepper species.^[2−4]

Capsaicinoids are responsible for the spiciness of peppers. It should be noted that the Scoville scale is usually used to measure the pungency of chili peppers. Units for the Scoville scale are directly related to the levels of capsaicin derivatives and 1 mg of capsaicin per kg of chili pepper is represented by 15 Scoville units. Therefore, the determination of capsaicin in foods allows the level of pungency in samples to be ascertained. It has also been demonstrated that capsaicins have several biological properties and health benefits.^[5] These include anticancer activity,^[6,7] antioxidant properties,^[8,9] action on the metabolism of fats,^[10] anti-inflammatory properties,^[11] and gastric mucosal^[12] and antimicrobial properties.^[13] These properties make it of interest to determine the levels of these compounds in common foods in the diet.

Capsaicinoids are usually incorporated into semisolid or solid foods and, as a consequence, it is necessary to extract these compounds from the matrix in which they are present prior to analysis and quantification by chromatographic techniques. The ultrasound-assisted extraction technique has been widely used for the extraction of capsaicinoids from peppers.^[14−16] Barbero et al. used this technique to develop a quantitative and reproducible (RSD <3%) extraction method for capsaicinoids that requires only a short time (10 min) and involves the use of methanol as the extraction solvent.

After extraction, thin layer chromatography,^[17] gas chromatography,^[18] and high performance liquid chromatography (HPLC)^[19] were used to analyze and quantify the components. HPLC is the most commonly used technique in this respect. The aim of the work described here was to survey the five major capsaicinoids present in hot dried pepper samples and also in different foods made from peppers, e.g., paprika, spicy ketchups, and spicy sauces. Ultrasound-assisted extraction^[14] was used to extract the components prior to quantification by reversed phase HPLC using a monolithic column with fluorescence detection.^[20]

MATERIALS AND METHODS

Chemicals

The reference standards for capsaicin (97%) and DHC (90%) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Water was obtained from a Milli-Q water deionization system (Millipore, Bedford, MA, USA). HPLC grade methanol and acetic acid were obtained from Panreac (Barcelona, Spain).

Spicy Foods

The different foods to be analyzed were obtained from different supermarkets in Spain; many of these foods were grown or produced in Spain but others originated from other countries. The samples analyzed were as follows: 19 hot sauces, 10 hot dried peppers, 4 hot paprika samples, and 4 hot ketchups. The dried peppers were milled in a conventional grinder prior to extraction.

Extraction Procedures

A previously developed ultrasound-assisted extraction method^[14] was applied to extract the relevant components. The following conditions were applied: extraction solvent, methanol; temperature, 50°C; power, 360 W; volume of solvent, 25 mL; extraction time, 15 min; amount of sample, 0.2 to 2 g (depending on the percentage of water in the food to be analyzed). A volume of 0.5 mL of internal standard (2,5-dihydroxybenzaldehyde, 1300 mg L⁻¹) was added to each extract. The extracts were filtered through a 0.45 μm nylon syringe filter (Millex-HN, Millipore) prior to chromatographic analysis. The extractions were performed in an ultrasonic bath (360 W, J.P. Selecta, Barcelona, Spain) coupled to a temperature controller, which allowed the water in the bath to be recirculated.

HPLC-Fluorescence Analysis

HPLC-fluorescence analysis was carried out on a Dionex chromatographic system (Sunnyvale, CA, USA), which consisted of an automated sample injector (ASI-100), a pump (P680), a thermostated column compartment (TCC-100), a photodiode array detector (PDA-100), a fluorescence detector (RF 2000), and a universal chromatography interface (UCI-50). The system was controlled with the Chromeleon 6.60 software package. Capsaicinoids were separated using a Chromolith TH Performance RP-18e (100 mm × 4.6 mm) monolithic column (Merck).

The chromatographic method was developed previously.^[20] This method was run with a gradient of two solvents, acidified water (0.1% acetic acid, solvent A) and acidified methanol (0.1% acetic acid, solvent B), at a flow rate of 6 mL min⁻¹. The gradient used for chromatographic separation of the capsaicinoids (time, solvent B) was: 0 min, 10%; 2 min, 50%; 4 min, 50%; 4.5 min, 55%; 5.5 min, 55%; 6 min, 60%; 7 min, 60%; 9 min, 70%; 10 min, 100%; 15 min, 100%. The temperature of the column was kept constant at 30°C. Fluorescence detection was carried out at 278 nm (excitation) and 310 nm (emission).

Identification of Capsaicinoids by Liquid Chromatography Coupled to Mass Spectrometry

Five major capsaicinoids found in peppers were identified using a High Performance Liquid Chromatography – Mass Spectrometry (HPLC-MS) system. The HPLC-MS analyses of hot pepper extracts were carried out on a Finnigan LCQ-coupled LC-MS system (Thermo Electron Co., San José, CA, USA). This equipment was fitted with a Spectra SYSTEM 2000 model gradient pump (Thermo Separation Products, Fremont, USA) and a mass detector (model LCQ), which consisted of an electrospray interface and an ion trap mass analyzer. Xcalibur version 1.2 software was used to control the equipment and to acquire and treat the data. The sample injection volume was 25 μL. The interface conditions were as follows: positive ionization, capillary temperature 220°C, spray voltage 20 kV, capillary voltage—5 V, focus gas flow 80 (arbitrary units), and auxiliary gas flow 10 (arbitrary units). API-MS data were acquired in the m/z range 50–400.

A gradient method with acidified water (0.1% acetic acid, solvent A) and acidified methanol (0.1% acetic acid, solvent B) at a flow rate of 0.2 mL min⁻¹ was employed for the chromatographic separation. The gradient employed was as follows (time, solvent B): 0 min, 0%; 1 min, 0%; 5 min, 30%; 8 min, 50%; 16 min, 70%; 20 min, 70%; 28 min, 90%; 30 min, 90%; 32 min, 100%; 42 min, 100%. A C-18 column (Luna 5 μm, 150 × 3 mm, Phenomenex) was used to achieve the separation. The capsaicinoids identified in the extracts were n-DHC, capsaicin, DHC, h-C, and h-DHC.

HPLC Calibration

The HPLC method was used to prepare calibration curves for capsaicin and DHC (y = 112.901x + 187 for capsaicin and y = 151.770x + 4589 for DHC), which are the two commercially available capsaicinoid standards. Regression equations and the correlation coefficient (r²; 0.9995 for capsaicin and 0.9995 for DHC), limits of detection (0.008 mg L⁻¹ for capsaicin and 0.011 mg L⁻¹ for DHC), and limits of quantification (0.028 mg L⁻¹ for capsaicin and 0.036 mg L⁻¹ for DHC) were calculated using the ALAMIN software package.^[21]

Quantification of the Capsaicinoids Present in Different Spicy Foods

The five major capsaicinoids (n-DHC, capsaicin, DHC, h-C, and h-DHC) present in the samples were quantified. Capsaicin and DHC were quantified from the calibration curves obtained from the standard solutions. Commercial standards for n-DHC, h-C, and h-DHC were not available and these compounds were quantified from the calibration curves of DHC (for n-DHC and h-DHC) and capsaicin (for h-C) given the structural similarities between these molecules and taking into account their molecular weights. All analyses were carried out in triplicate.

RESULTS AND DISCUSSION

Quantification of Capsaicinoids in Dried Peppers

Dried hot peppers are widely used in Spanish cooking and in cuisine from around the world to provide flavor, color, and taste and to produce spicy dishes. This kind of product can be found in many markets around the world because peppers are very easy to preserve for long time periods.

In the study described here ten different brands of ground dried hot peppers sold in Spain were analyzed, one each from the species Capsicum annuum (sample 1) and Capsicum baccatum (sample 2) and eight from the species Capsicum frutescens (samples 3–10). In Spanish markets a variety of dried ground pepper is usually prepared from cayenne pepper (Capsicum frutescens) and, for this reason, this particular sample is discussed in greater detail.

The amounts of capsaicinoids found in all of these peppers are represented in Fig. 1a.

FIGURE 1 Average capsaicinoid contents found in different samples of dried hot peppers (A), paprika (B) and ketchup (C) (μmol kg^--1 of sample, N = 3).

The capsaicinoid contents determined for dried peppers varied greatly, with some of these peppers showing relatively low total concentrations (554.15 mol kg⁻¹) whilst others showed exceptionally high levels (17050.87 mol kg⁻¹). Capsaicin (C) was present at the highest level in all samples, with percentages in the range between 49 and 68% of total capsaicinoids. The second most common capsaicinoid in the samples was DHC), which was present at percentages between 22 and 34%. These two major capsaicinoids represent between 80 and 95% of the total capsaicinoid contents of the samples analyzed. The C/DHC ratios in these samples of ground, dried hot peppers were in the range between 3.06 and 1.47, i.e., the value was greater than 1 in all cases.

The third most abundant capsaicinoid was h-C, with the exception of sample 3, and this was present in amounts ranging from 3.8 to 8.7% of total capsaicinoids. The next capsaicinoid in terms of abundance in the samples was n-DHC, again with the exception of sample 3. This compound was present at 1.3 to 8.6% as a percentage of total capsaicinoid content. Finally, of the five studied capsaicinoids, the least abundant in all cases was h-DHC, which was only present at levels between 0.3 and 5.0%. The relative values of C and DHC do not provide evidence of a clear difference in behavior between the samples from the different species tested (C. frutescens, C. annuum, and C. baccatum) and, as a consequence, this parameter cannot be used as a criterion for varietal discrimination.

Quantification of Capsaicinoids in Paprika

Paprika is the product obtained by grinding red peppers that are harvested when fully mature, healthy, clean, dry, and completely free of attack by pests or diseases. The preparation of the peppers involves three stages: The first stage is the care of the plantation, the second phase consists of drying the fruit, either by exposure to sun or drying in a hot air stream, and the third stage is the grinding process, in which the sample is passed through a mill several times to produce a fine bright red powder. All of these stages can affect the final capsaicinoid content in the paprika. Paprika powder, as well as being a flavoring additive, is a coloring agent that is used in household cooking and in the food industry. In this study four different brands of paprika sold in Spain were analyzed and all were obtained from the species C. annuum. The paprika that is traditionally produced and marketed in Spain usually comes from different varieties of peppers of this species.

The amounts of capsaicinoids present in all of the paprika samples investigated are shown in Fig. 1b. The composition in terms of individual capsaicinoids was very similar in most cases and the most marked differences were found in the minor capsaicinoids, i.e., n-DHC, h-C, and h-DHC. The differences in the major compounds were significantly lower. For example a difference of 8% was found in the case of C but the difference was 34% in the case of DHC. The levels of capsaicinoids ranged from 581 to 665 mmol kg⁻¹. These levels are significantly lower than those found in the dried hot peppers analyzed previously (Fig. 1a). This difference is due to the species of pepper used to prepare the products. It can be observed that the major capsaicinoid was C in all of the peppers studied and this was present at levels between 55 and 59% of total capsaicinoids. Once again, the second most common capsaicinoid was DHC and this was present in the range from 24 to 34% of total capsaicinoids depending on the sample. The sum of these two major capsaicinoids represents between 82 and 92% of the total capsaicinoids. The C/DHC ratio in the samples was between 2.5 and 1.6.

The variability in the minor capsaicinoids in the paprika samples was more pronounced than for the major capsaicinoids. In this case, the third most abundant capsaicinoids were h-DHC for samples 3 and 4, h-C for sample 2, and n-DHC for sample 1. In these samples, the percentage of n-DHC ranged between 3.4 and 5.4%, whereas h-C ranged between 2.3 and 10.7% and h-DHC between 0.7 and 6.5%. The combined quantities of these minor compounds did not exceed 18% of the total capsaicinoid content.

Quantification of Capsaicinoids in Ketchup

Ketchup is a tomato sauce that is widely used throughout the world. In addition to tomato, vinegar, sugar and salt, various spices are used in the preparation of this type of sauce. Spicy ketchup is prepared using hot peppers and capsaicinoids should be present in samples of this sauce. Four different brands of spicy ketchup that are commercially available in Spain were studied. In Spain, sweet ketchup is more widely consumed than spicy ketchup, but in recent years spicy ketchups have become increasingly available.

The levels of capsaicinoids present in the studied ketchup samples are shown in Fig. 1c. As in the case of hot peppers, only small differences in the total contents of capsaicinoids were found in the different samples. In this case, however, both major and minor components showed only small differences in abundance. Two samples contained levels of n-DHC, h-DHC, and h-C that were below the limits of detection. In any case, the total levels of capsaicinoids in these samples did not exceed 15 μmol per kilogram, i.e., up to two orders of magnitude lower than the levels found in the dried hot peppers. These low contents reflect the intended use of the food and its direct introduction into the diet—in contrast to dried hot peppers or paprika, which are used as condiments.

Regarding the major components, C was not the most abundant capsaicinoid in all cases as DHC was the major component in ketchup sample 3. Capsaicinoid C was present in percentages between 63 and 17%, whereas DHC was present in percentages ranging between 60 and 33%. The sum of these two major capsaicinoids was between 77 and 100% of the total capsaicinoid content.

Regarding minor capsaicinoids, two ketchup samples (1 and 2) contained levels that were below the limit of detection. In the other ketchup samples (samples 3 and 4) the third most abundant capsaicinoid was h-DHC (12–15%), followed by h-C (5–7%), and finally n-DHC (1–3%); the sum of these compounds did not exceed 23% of the total capsaicinoids.

The C/DHC ratios were markedly different between ketchup samples. These values range from 0.28 (DHC/C = 3.51) for sample 3 to 1.67 for sample 1. For the other two ketchup samples the C/DHC ratios were 1.41 (sample 4) and 1.14 (sample 2). The different relative compositions can be attributed to the use of different species of peppers in the preparation of the ketchup samples.

Quantification of Capsaicinoids in Hot Sauces

In gastronomy, the function of the sauce is to accompany other foods as a garnish to improve the taste by contrasting or complementing the food in question. For this reason, sauces usually offer relatively sharp sensations on the palate that stimulate the senses of taste and smell, as is the case with spicy sauces. A number of commercially available hot sauces and foods contain spicy peppers as an ingredient. Most hot sauces have only a small proportion of hot peppers in the ingredients (0.42–3.5%). However, there are sauces that are made with high levels of crushed hot peppers and this gives them a very intense degree of pungency.

In this study a total of 19 different brands of hot sauce marketed in Spain were analyzed. These 19 sauces encompassed a variety of types in terms of the percentage of pepper used in the preparation and in the colors, uses and textures. The amounts of capsaicinoids present in these sauces are represented in Figs. 2a and 2b.

FIGURE 2 Average capsaicinoid levels found in different samples of hot sauces containing less than 3.5% of peppers (A) and hot sauces containing more than 3.5% of peppers (B) (μmol kg^--1 of pepper, N = 3).

The variation in the levels of total and individual capsaicinoids was quite high in these samples. A number of the sauces contained very low total contents of capsaicinoids (4–20 μmol kg⁻¹), whereas other sauces had high levels of total capsaicinoids (>400 μmol kg⁻¹), which in some cases were above 800 μmol kg⁻¹.

As observed previously for other samples, the two major capsaicinoids were C and DHC. In most cases the major capsaicinoid is C, but in certain sauce samples (samples 1, 3, 6, 7, and 19) this order is reversed and DHC is the major capsaicinoid. The percentage of C with respect to the total percentage of capsaicinoids was between 76 and 38% for those sauces in which C was the major capsaicinoid, and between 43 and 35% for those sauces in which DHC was the most abundant capsaicinoid. Similarly, DHC was present at percentages between 62 and 41% for those sauces in which DHC was the major capsaicinoid, and between 48 and 15% for sauces in which C was the major capsaicinoid. The sums of the percentages of the two major capsaicinoids (C and DHC) in the analyzed samples were in the range 64–100% and the mean value for the 19 samples was 87%.

In two samples (sauces 2 and 8) n-DHC was the second most abundant capsaicinoid, thus relegating DHC to third position. In these two samples the percentages of n-DHC were 26.5 and 25.8%, respectively. Apart from these two cases, n-DHC was generally found to be the third most abundant capsaicinoid in 11 of the sauces and this was present at levels of up to 16.2%. The third most abundant capsaicinoid in four of the sauces tested was h-C, with levels of up to 13.2%, but h-DHC was the third most abundant capsaicinoid in one of the sauces, with a percentage of 13.0%.

The C/DHC ratios for the samples ranged between 3.55 and 1.03 for sauces in which C was the major capsaicinoid. In cases where the major capsaicinoid was DHC, the C/DHC ratio ranged between 0.36 and 0.95. These differences in the levels of capsaicinoids in the sauces are due to the use of different species of peppers in the preparation of the sauces.

CONCLUSIONS

Differences in the total capsaicinoid contents, as well as in the individual levels of capsaicinoids, were extremely high for processed foods. These variations were found to be significantly smaller for dried hot pepper samples and even less marked in the case of spicy ketchups. In almost all samples, the two major capsaicinoids were C and DHC, with C generally being the most abundant. The combined percentages of C and DHC with respect to the total capsaicinoid contents varied as follows in the different foods studied: 80–95% (dried hot peppers), 82–92% (paprika), 77–100% (spicy ketchup), 64–100% (hot sauce). The results obtained indicate that there is no clear rule to determine the third most prevelant capsaicinoid in the samples—this was h-C for most dried peppers, h-DHC for several ketchup and paprika samples, and n-DHC for many of the hot sauces. As far as the C/DHC ratio is concerned, values of 3.55 were obtained for the samples in which C was the major capsaicinoid. Samples in which DHC was the main capsaicinoid gave a DHC/C ratio of 3.51, i.e., a similar value but with the order reversed.

ORCID

Gerardo Fernández Barbero

http://orcid.org/0000-0001-7302-6605

Miguel Palma

http://orcid.org/0000-0001-8509-4226

Carmelo García Barroso

http://orcid.org/0000-0003-2284-2244