Determination of the antioxidant activity of capsaicinoids and polyphenols extracted from wild chili pepper

Determinação da atividade antioxidante de capsaicinoides e polifenóis extraídos de pimenta malagueta selvagem

ABSTRACT

Wild chilis are considered a plant-genetic resource native to Mexico; however, the lack of studies on their phytochemical composition limits their sustainable commercial use. This study aimed to evaluate the concentration of capsaicinoids and polyphenols in three varieties of wild chilis from Tezonapa in the high mountains of Veracruz, Mexico, and their correlation with their antioxidant capacity. Determining antioxidant activity and quantifying total polyphenols were performed using spectrophotometric methods, while capsaicinoids were quantified by liquid chromatography. The chili variety factor significantly affected (p≤0.05) antioxidant activity determined by 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ABTS and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) methods and capsaicinoid concentration. The community factor significantly affected (p≤0.05) the variables mentioned above, in addition to the concentration of total polyphenols. Chiltepin and Mirasol varieties from the community of Manzanares had the highest concentration of total capsaicinoids (246.58 ± 14.93 and 256.75 ± 7.15 mg/100 g). In the case of Tabaquero, those from the community of Cerro Azul presented the highest concentration of capsaicinoids (345.93 ± 14.25 mg/100 g). The three Cerro Azul chili varieties had the highest total polyphenols concentration. The total polyphenols concentration was positively correlated with the antioxidant activity of wild chilis, indicating that these are the main compounds responsible for the antioxidant characteristics in these chili varieties. The knowledge generated in this research will contribute to the conservation of these chili species and benefit the communities of Tezonapa by exploiting these fruits for the agro-food sectors focused on functional foods.

Keywords:
Capsicum annuum L. glabriusculum; Chiltepin; Tabaquero; Mirasol.

RESUMO

Algumas pimentas silvestres fazem parte dos recursos genéticos vegetais nativos do México; no entanto, a falta de estudos sobre sua composição fitoquímica limita seu uso comercial sustentável. Este estudo teve como objetivo avaliar a concentração de capsaicinoides e polifenóis em três variedades de pimentas silvestres de Tezonapa, nas altas montanhas de Veracruz, México, e a correlação com sua capacidade antioxidante. A determinação da atividade antioxidante e quantificação dos polifenóis totais foram realizados utilizando métodos espectrofotométricos, enquanto os capsaicinoides foram quantificados por cromatografia líquida. O fator “variedade de pimenta” afetou significativamente (p≤0.05) a atividade antioxidante determinada pelos métodos 2,2'-azino-bis(3-etilbenzotiazoline-6-sulfonic acid) ABTS e 2,2-difenil-1-picrilhidrazilo (DPPH) e a concentração de capsaicinoide. O fator comunidade afetou significativamente (p≤0.05) as variáveis mencionadas acima, além da concentração de polifenóis totais. As variedades Chiltepin e Mirasol da comunidade de Manzanares apresentaram a maior concentração de capsaicinoides totais (246.58 ± 14.93 e 256.75 ± 7.15 mg/100 g). No caso do Tabaquero, os da comunidade de Cerro Azul apresentaram a maior concentração de capsaicinoides (345.93 ± 14.25 mg/100 g). As três variedades de pimenta do Cerro Azul apresentaram a maior concentração total de polifenóis (867.55 ± 17.62 mg/100 g). A concentração de polifenóis totais foi positivamente correlacionada com a atividade antioxidante das pimentas silvestres, indicando que estes são os principais compostos responsáveis pelas características antioxidantes dessas variedades. O conhecimento gerado nesta pesquisa contribuirá para a conservação dessas espécies de pimenta e beneficiará as comunidades de Tezonapa ao explorar esses frutos para os setores agroalimentares voltados para alimentos funcionais.

Palavras-chave:
Capsicum annuum L. variedade glabriusculum; Chiltepin; Tabaquero; Mirasol

Mexico is considered one of the countries with the greatest variety of species of the genus Capsicum distributed throughout the national territory (Aguilar-Meléndez et al., 2021). These fruits have great economic, social, and cultural importance, making them the second most important crop in the country (Luna-Garcia et al., 2023; Sánchez Toledano et al., 2023). Despite this, some species have not been subjected to a process of domestication for commercial use, which is called wild chili (Barboza et al., 2022).

Wild chilis (Capsicum annuum L. variety glabriusculum) have growing conditions that make them stand out. They have become a subject of study because they are considered a genetic resource of cultural importance for various rural communities. Therefore, their care and preservation are paramount (SIAP, 2023). These chilis are compared to domestic grow in conditions where no controlled human agricultural practice interferes with irrigation, fertilization, or pest management that could attack the plant (Serrano-Mejía et al., 2022). This affects morphological characteristics, such as weight, size, and number of seeds (Hernández-Verdugo et al., 2012).

One of the regions of Mexico where we can find a variety of wild chilis is “Las altas montañas” in Veracruz, Mexico (Hernández-Salinas et al., 2021). Within this area, there are microclimates caused by the geographical differences between the communities that make up this region, which can affect the morphological characteristics and antioxidant activity of chilis (Hernández-Salinas et al., 2024). This is because environmental factors have been reported to significantly affect the metabolomics associated with producing phytochemicals that endow chilis with their characteristic antioxidant activity.

The effect of these factors on the concentration of secondary metabolites in wild chilis is of great importance because these fruits are considered rich in capsaicinoids, vitamins, polyphenols, and carotenoids (Batiha et al., 2020). The most representative of these are capsaicinoids, alkaloids that give these fruits their pungency. They are essential for the pharmaceutical industry due to their anti-inflammatory, analgesic, antidiabetic, and antimicrobial effects (Yasin et al., 2023). Among the bioactive compounds present in peppers, polyphenols are another one of the most outstanding due to their high antioxidant capacity and their potential in the prevention and treatment of chronic diseases such as cardiovascular disease, neurodegenerative diseases, and cancer (Güneş Bayir et al., 2019; Jayaprakasha et al., 2012).

Because of the above, this study aims to evaluate the concentration of capsaicinoids and polyphenols in three varieties of wild chilis (Chiltepin, Tabaquero, and Mirasol) and their correlation with their antioxidant capacity. These results will allow us to know how the area of cultivation affects the composition of phytochemical compounds present in these varieties of wild chili and how they affect antioxidant activity. But also contribute to the conservation of these wild chilis, highlighting their antioxidant and phytochemical characteristics that interest the agro-food sectors focused on producing functional foods.

MATERIAL AND METHODS

Sample preparation

For the present research work, three varieties of wild chilis were harvested: (1) Chiltepin, (2) Tabaquero, and (3) Mirasol. These were obtained from 6 communities in “Las altas montañas” region of Tezonapa, Veracruz, Mexico. The data of the communities from which the chilis were harvested are shown in Table 1. The fruits were randomly selected, free from physical and pathogenic damage. The chilis of the four varieties were harvested in an unripe grade of ripeness. These were dried in a convection oven for 48 hours at a temperature of 45°C. Subsequently, samples were macerated with an electric mill (Krups GX4100®, Mexico) and stored at -20°C until analysis.

Ultrasound extraction for capsaicin analysis

This procedure was carried out using as reference the previous work by Morozova et al. (2019) with some modifications. For extraction, 50 mg of chili powder was mixed with 4 mL of MeOH:H₂O (80:20) solvent. The samples were then sonicated in an ultrasonic bath for 30 minutes, followed by centrifugation at 3500 rpm for 10 minutes. The supernatant was filtered through 0.45 µm nylon filters.

Table 1
Communities in “Las altas montañas”, a region of Veracruz, Mexico, where three varieties of wild chili were collected. Zongolica, Veracruz, México, Instituto Tecnologico Superior de Zongolica, 2024.

Variety	Community	Latitude	Longitude	Altitude (m)	Temperature (°C)	Precipitation (mm)	Code
Chiltepin	Cerro Azul	18° 36’ 30’’	96° 42’ 10’’	655	27	230	C1
	Presidio	19° 04’ 10’’	96° 58’ 16’’	1330	17	171	C2
	Manzanares	18° 31’ 52’’	96° 45’ 47’’	139	21	126	C3
	Ixtacapa	18° 24’ 45’’	96° 43’ 43’’	103	21	138	C4
Tabaquero	Villanueva	18° 37' 32"	96° 49' 21"	921	21	113	T1
	Presidio	19° 04’ 10’’	96° 58’ 16’’	1330	17	171	T2
	Cerro Azul	18° 36’ 30’’	96° 42’ 10’’	655	27	230	T3
Mirasol	Josefinas	18° 29’ 26’’	96° 41’ 35’’	121	21	128	M1
	Vázquez Vela	18° 39’ 45’’	96° 47’ 20’’	547	21	113	M2
	Cerro Azul	18° 36’ 30’’	96° 42’ 10’’	655	27	230	M3
	Manzanares	18° 31’ 52’’	96° 45’ 47’’	139	21	126	M4
	Villanueva	18° 37' 32"	96° 49' 21"	921	21	113	M5

Quantification of total capsaicinoids by HPLC

The chromatographic conditions reported by Oney-Montalvo et al. (2021) were used as reference with some modifications. Capsaicinoids were quantified using an Ultimate 3000 HPLC (Thermo Fisher Scientific) system equipped with a four-channel photodiode detector (PDA). The column was a Hypersil GOLD™ C18 (4.6 mm x 100 mm, and particle size of 3 µm) Thermo Scientific^®. Meanwhile, the mobile phase was water: ACN:H20 (50:50) solution with 0.2% formic acid. The detection was carried out at 280 nm wavelength, a flow rate of 1 mL/min, with 15 min analysis time. External calibration was performed with working solutions prepared from the capsaicin reference standard for the detection.

Ultrasound extraction for polyphenols and antioxidant analysis

The extraction of polyphenols and antioxidant compounds was performed using the methodology reported by Liu et al. (2020) with some modifications. For extraction, 250 mg of each sample was weighed in 15 mL Falcon tubes, and then 4 mL of ethanol:water mixture (50:50) was added and homogenized. The extracts were sonicated for 30 minutes and centrifuged at 3500 rpm and 4°C for 15 minutes. Finally, the supernatant was filtered with a nylon membrane of 0.45 μm pore size.

Quantification of total polyphenols

Total polyphenols were quantified using the Folin Ciocalteu methodology (Blainski et al., 2013). To this, 50 μL of the sample was taken and placed in 15 mL Falcon tubes, 3 mL of distilled water, and 250 μL of Folin-Ciocalteau reagent added. The contents are then homogenized and left to stand for 8 minutes in darkness. After this time, 750 μL of 20% sodium carbonate (NaCO₃) and 950 μL of distilled water was added. The sample was homogenized again, kept in darkness at room temperature for 2 hours, and measured at 765 nm. External calibration was performed using gallic acid as a polyphenol reference at concentrations of 50, 100, 150, 200, 250, 300 and 400 mg/L (Figure 1).

Determination of antioxidant capacity by ABTS

The determination of antioxidant capacity by ABTS methodology was made using as reference the work of Nenadis et al. (2004) as reference, with some modifications. In a 15 mL Falcon tube, 2970 μL of the ABTS solution was added, followed by 30 μL of antioxidant extract and homogenization using a vortex. Let stand for 6 minutes, and the absorbances were read on the spectrophotometer at 734 nm to calculate the % ABTS inhibition for each sample. The calibration curve was prepared using different Trolox as antioxidant reference.

Figure 1
Calibration curve for the determination of total polyphenols using gallic acid as standard at different concentrations (50, 100, 150, 200, 250, 300 and 400 mg/L). Zongolica, Veracruz, México, Instituto Tecnologico Superior de Zongolica, 2024.

Determination of antioxidant capacity by DPPH

The antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazil (DPPH) radical removal method following the methodology described by Brand-Williams et al. (1995), with some modifications. In 15 mL Falcon tubes, 3800 μL of the DPPH solution (0.1 mM) was added, followed by 200 μL of the extract and homogenization using a vortex. The extracts shall be left to stand for 30 minutes to react with the radical. Absorbance shall then be measured at 515 nm using a UV-Vis spectrophotometer, and the % inhibition of DPPH shall be calculated for each sample. The calibration curve uses Trolox at different concentrations as a reference standard.

Statistical analysis

Analysis of variance (ANOVA) was used to α = 0.5% to analyze all data. The statistical test used to separate means was the last significant difference test with a 95% confidence level. A Person correlation analysis was used to assess the possible correlations between capsaicinoids and total polyphenols with antioxidant activity (DPPH and ABTS). All statistical analyses were performed with the software Statgraphics Centurion XVII.II-X64 (Statgraphics Technologies Inc., the Plains, VA, USA).

RESULTS AND DISCUSSION

Analysis of variance

Table 2 shows the results of the multifactorial variance analysis (ANOVA), showing that the chili variety factor significantly affected antioxidant activity determined by the ABTS and DPPH methods and capsaicinoid concentration. The community factor significantly affected the above mentioned variables and the total polyphenols concentration.

The variety of chili is a factor affecting the concentration of bioactive compounds, with variations in capsaicinoids and other metabolites responsible for antioxidant activity in these fruits, which enable them to stand out in the functional food sector (Lidiková et al., 2021). These metabolomic characteristics of the chili varieties are associated with their genetic differences, which lead to the production of some secondary metabolites over others, affecting their antioxidant capacity (Carvalho et al., 2015; Kim et al., 2021).

On the other hand, the effect of community is due to environmental characteristics such as microclimates and the physicochemical composition of the soils of each region, which affect the biosynthesis of antioxidant phytochemicals. The work previously done by Oney-Montalvo et al. (2021) shows that these factors can decrease or increase the biosynthesis of capsaicinoids, one of the main antioxidant compounds present in chili and responsible for the spiciness that characterizes them. Aguirre et al. (2017) have evaluated the effect of different factors on the concentration of capsaicin and dihydrocapsaicin in wild chilis, demonstrating that altitude has a positive effect on the compounds responsible for pungency together with other factors such as temperature and light availability. This is corroborated by the work done by Gurung et al. (2011), in which the chilis that were grown in higher areas also had a higher concentration of capsaicinoids, although it fluctuated for low-pungency varieties.

But not only the capsaicinoids are the antioxidant compounds that will be affected by environmental factors, since the biosynthesis of polyphenols is also influenced by them, reporting that the soil where the crop develops is related to the concentration and profile of polyphenols in chilis (Oney-Montalvo et al., 2020). It has been observed in the work performed by Meckelmann et al. (2015) as the altitude of the locality where the crop is developed, the temperature, the precipitation and the sunlight also affect the concentration of total polyphenols and the antioxidant capacity.

Table 2
p values of factors variety and community in the concentration of capsaicinoids, polyphenols, and antioxidant activity (DPPH and ABTS). Zongolica, Veracruz, México, Instituto Tecnologico Superior de Zongolica, 2024.

	Variety	Community
DPPH	0.0005*	<0.0001*
ABTS	<0.0001*	<0.0001*
Polyphenols	0.1016	<0.0001*
Capsaicinoids	<0.0001*	<0.0001*

• * = Significant effect.

Capsaicinoids content

Chilis of the chiltepin species from the community of Manzanares had the highest concentration of total capsaicinoids (246.58 ± 14.93 mg/100 g), followed by those collected from Cerro Azul (205.86 ± 18.6 4 mg/100 g) and Ixtacapa (194.36 ± 15.18 mg/100 g), with the community of Presidio having the lowest concentration of these (59.88 ± 5.15 mg/100 g). In the case of chilis Tabaquero, those from the community of Cerro Azul presented the highest concentration of capsaicinoids (345.93 ± 14.26 mg/100 g), while those from Villanueva had the lowest (63.45 ± 8.21 mg/100 g). Mirasol chili from the Manzanares community had the highest capsaicinoid concentration (256.75 ± 18.03 mg/100 g), and those from the Josefinas community had the lowest concentration (143.64 ± 5.63 mg/100 g).

These results confirm that the community from which all three varieties of chili come affects the production of capsaicinoids. There are reported biotic and abiotic factors that could be causing these variations in the concentration of capsaicinoids; among the main ones are temperature, the presence of exogenous substances, the availability of water, and the nutritional characteristics of the soil (Yang et al., 2024). Although the communities evaluated in this work belong to “Las altas montañas”, the region of Veracruz, Mexico, the different altitudes (Table 1) at which they are located cause different microclimates that influence the synthesis of capsaicinoids (Figure 2).

According to Zamljen et al. (2020), the insufficiency of irrigation is associated with increased capsaicinoid production in chilis due to the stress on the plant that activates the synthesis of these secondary metabolites. On the other hand, the effect of temperature has been studied by Oh & Koh (2019), in which it was observed that the content of capsaicinoids increases over a temperature range of 15 to 30°C, having the highest concentration of these compounds at 30°C. These results indicated how these factors affect the synthesis of the compounds responsible for the pungency and that the different microclimates of the communities evaluated in this research (Table 1) end up being a determining factor in the pungency of wild chilis.

Polyphenols content

Figure 3 shows the results of determining total polyphenols in all three varieties of wild chili. Chiltepin peppers from the community of Cerro Azul had the highest concentration of total polyphenols (867.55 ± 17.62 mg/100 g). The same behavior was observed in the Tabaquero and Mirasol chili, where those collected at Cerro Azul also had a higher concentration of these secondary metabolites (574.16 ± 11.17 and 767.53 ± 11.72 mg/100 g, respectively).

Figure 2
Results of the determination of total capsaicinoids in three varieties of wild chilis from different communities in the high mountains region of Veracruz, Mexico. C: Chiltepin (1) Cerro Azul, (2) Presidio, (3) Manzanares, (4) Ixtacapa. T: Tabaquero (1) Villanueva, (2) Presidio, (3) Cerro Azul. M: Mirasol (1) Josefinas, (2) Vázquez Vela, (3) Cerro Azul, (4) Manzanares, (5) Villanueva. Zongolica, Veracruz, México, Instituto Tecnologico Superior de Zongolica, 2024.

The fact that all three varieties of wild chili from the Cerro Azul community had the highest concentration of polyphenols indicate that the community's environmental characteristics contribute to the biosynthesis of these compounds, this community reported the highest temperature (27°C) and precipitation (230 mm) of the communities studied. Geographical location is considered to have an effect related to altitude because high elevation is generally associated with high polyphenol production (Ahmed et al., 2019).

Other factors related to the possible microclimates of communities and polyphenol production are water availability, which could cause water stress in the plant and change metabolic processes. Zamljen et al. (2020) showed that irrigation is one of the main factors affecting the production of polyphenols in chilis, since when plants are subjected to a process of irrigation deficiency, a significant decrease in these secondary metabolites is observed, If the plant has a suitable irrigation system, the polyphenol concentration increases. Additionally, different biotic and abiotic factors that could also generate stress on the plant and affect the polyphenolic composition of chilis are the physicochemical characteristics of the soil, temperature, exposure to solar radiation, and pathogens (Bruni & Sacchetti, 2009).

Antioxidant activity

Table 3 shows the results of determining antioxidant activity. Chiltepin chili from the Cerro Azul community had the highest antioxidant activity of the three varieties evaluated in this study, 92.45 ± 2.02 (DPPH)and 1053.84 ± 16.68mg of trolox/100 g (ABTS) of chili. In the case of Tabaquero chili, the Villanueva community showed more antioxidant activity using the DPPH technique (79.73 ± 1.27 mg of trolox/100 g of chili), while the ABTS technique showed higher values for the Presidio community (981.35 ± 17.85 mg of trolox/100 g of chili). Meanwhile, the Mirasol chilis from the Cerro Azul and Villanueva communities showed the highest antioxidant activity for this variety of chili, 87.21 ± 2.40 and 1101.87 ± 17.57 mg of trolox/100 g of chili.

Figure 3
Results of the determination of total polyphenols in three varieties of wild chilis from different communities in the high mountains region of Veracruz, Mexico. C: Chiltepin (1) Cerro Azul, (2) Presidio, (3) Manzanares, (4) Ixtacapa. T: Tabaquero (1) Villanueva, (2) Presidio, (3) Cerro Azul. M: Mirasol (1) Josefinas, (2) Vázquez Vela, (3) Cerro Azul, (4) Manzanares, (5) Villanueva. Zongolica, Veracruz, México, Instituto Tecnologico Superior de Zongolica, 2024.

The Precedent chilis of Cerro Azul and V. Nueva communities stood out as those with the greatest antioxidant activity compared to the chilis from the other communities. This shows that these three communities' environmental and soil characteristics could contribute to wild chilis producing more antioxidant compounds.

Table 3
Results obtained from the determination of antioxidant activity in three varieties of wild chilis from high mountains in Veracruz, Mexico. Zongolica, Veracruz, México, Instituto Tecnologico Superior de Zongolica, 2024.

Code	Variety	Community	DPPH Inhibition mg oftrolox/100g of chili	ABTS Inhibition mg oftrolox/100g of chili
C1	Chitepin	Cerro Azul	92.45 ± 2.02b	1053.84 ± 16.68b
C2	Chitepin	Presidio	73.21 ± 1.45e	956.69 ± 18.39c
C3	Chitepin	Manzanares	80.16 ± 4.61d	883.21 ± 17.48d
C4	Chitepin	Ixtacapa	64.85 ± 3.12f	972.56 ± 21.18c
T1	Tabaquero	Villanueva	79.73 ± 1.27d	720.39 ± 9.92f
T2	Tabaquero	Presidio	72.25 ± 1.92e	981.35 ± 17.85c
T3	Tabaquero	Cerro Azul	58.33 ± 1.67g	890.35 ± 16.68d
M1	Mirasol	Josefinas	56.46 ± 2.50g	786.11 ± 27.69e
M2	Mirasol	Vázquez Vela	84.64 ± 1.56c	985.85 ± 28.00c
M3	Mirasol	Cerro Azul	87.21 ± 2.40c	1101.87 ± 17.57a
M4	Mirasol	Manzanares	71.64 ± 3.83e	914.44 ± 33.55d
M5	Mirasol	Villanueva	105.93 ± 3.01a	1060.87 ± 11.47b

• Values presented are the means ± standard deviation; a, b, c means groups of responses. Values followed by different letters in the same column indicate statistically significant differences using the least significant difference (LSD) test at p≤0.05. The antioxidant activity of DPPH and ABTS was performed by inhibition curve.

Correlation analysis

Figure 4 shows the results of the Pearson correlation analysis. Capsaicinoids did not have a high correlation with the antioxidant activity of the analyzed chilis. On the other hand, total polyphenols had a correlation of 0.52 and 0.66 for antioxidant activity determined by the DPPH and ABTS methods, respectively. On the other hand, of the environmental factors evaluated, temperature was the one that showed the greatest positive and significant effect in the production of both capsaicinoids and polyphenols. Followed by precipitation, which also showed an effect on the production of these compounds, in addition to contributing to the production of antioxidants by the ABTS technique. Altitude alone did not have a significant effect on the production of the compounds evaluated in this research.

These results confirm that although capsaicinoids are one of the predominant compounds in wild chilis, their antioxidant activity is mainly due to the fruit's polyphenols. This may be because polyphenols are the metabolites with the highest reported antioxidant activity, so they are widely studied and constantly sought for their production and use by the pharmaceutical and food industry. It also demonstrates the effect of environmental factors such as temperature and precipitation on the production of capsaicinoids, polyphenols, and antioxidant compounds.

Figure 4
Analysis of the Pearson correlation of the variables: antioxidant activity (DPPH and ABTS), total polyphenols, capsaicinoids, altitude, temperature, and precipitation. Zongolica, Veracruz, México, Instituto Tecnologico Superior de Zongolica, 2024.

Significant differences (p≤0.05) were observed in the effect of communities and variety of wild chilis on the concentration of total polyphenols, capsaicinoids, and antioxidant activity; these factors are shown to influence the biosynthesis of these compounds. The total polyphenols concentration was positively correlated with the antioxidant activity of wild chilis, indicating that these are the main compounds responsible for the antioxidant characteristics in these chili varieties. This highlights the community of Cerro Azul, which obtained the highest concentration of total polyphenols in the three varieties of wild chili evaluated (Figure 3). This community was characterized by having the highest temperature (27°C) and the highest precipitation (230 mm), showing that these factors influence the polyphenol synthesis in wild chilis. This knowledge will contribute to the conservation of these crops of great cultural and social importance in the high mountain region by their exploitation by agro-industrial sectors focused on functional foods.

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Publication Dates

• Publication in this collection
  24 Mar 2025
• Date of issue
  2025

History

• Received
  09 Dec 2024
• Accepted
  02 Feb 2025