A New Pyrrole Alkaloid from Capsicum annuum L. var. palmera Grown in La Palma (Canary Islands, Spain)

Abstract

Capsicum annuum, commonly known as pepper, is a widely cultivated crop valued for its nutritional and economic significance. Among its varieties, the small red bell pepper, locally known as “pimienta palmera”, is a unique cultivar from La Palma in the Canary Islands, renowned for its distinctive flavor and use in traditional dishes. Despite its cultural importance, limited research exists on the chemical composition of this pepper variety. This study investigates the chemical constituents of dichloromethane and n-butanol fractions extracted from the pericarp of Capsicum annuum var. palmera. Isolation and structural elucidation of compounds were achieved using high-resolution mass spectrometry (HREIMS), nuclear magnetic resonance spectroscopy (NMR), and chromatographic techniques. A previously unreported pyrrolo-lactone compound, 2-(6-formyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazin-4-yl)acetic acid, was identified alongside other metabolites, including α-tocopherol, β-sitosterol, and oleic acid. This alkaloid adds to the chemical diversity of Capsicum species and highlights the unique bioactive potential of this regional pepper. These findings enhance the understanding of pimienta palmera’s composition and suggest potential applications in nutraceuticals and pharmacology due to the biological activity associated with pyrrole alkaloids. The characterization of this compound underscores the need to further explore the phytochemical richness of traditional crops with limited prior research.

1. Introduction

Pepper, specifically Capsicum annuum, is the general name for plants from the Capsicum species of the Solanaceae family and is native to southern North America and northern South America. It is an important agricultural crop not only because of its economic significance but also due to the nutritional value of its fruits as they are rich in antioxidant compounds and natural pigments that contribute to health benefits [1,2]. Antioxidant nutrients play an important role in health maintenance by neutralizing harmful free radicals that cause cellular damage. These nutrients have been strongly linked to protection against numerous diseases, including heart disease, cancer, and immune dysfunction. Red pepper, in particular, is one of the oldest, most popular, and economically important natural coloring and flavoring agents, with an average annual production of 2.5 million tons [3]. The diversity of carotenoids in red pepper pods depends largely on the variety and geoclimatic conditions of cultivation.

On the island of La Palma, the small red bell pepper known locally as “pimienta palmera” (Figure 1) is an essential ingredient in traditional dishes, especially “mojos” served with wrinkled potatoes. Its distinctive flavor is likely influenced by the unique climate, careful seed selection, and cultivation practices used. While peppers are generally considered a balanced source of essential nutrients, including proteins, minerals, vitamin C, and natural pigments, there is no information available in the literature, as far as we are aware, regarding the chemical composition of this pepper variety.

Natural products often originate from biosynthetic pathways involving the modification of primary metabolites such as amino acids, fatty acids, or terpenoids. These biochemical processes yield a diverse array of structures with functional and ecological significance, ranging from chemical defense agents to signaling molecules in plants. The case of Capsicum annuum var. palmera is particularly intriguing, given the unique environmental and cultivation conditions in La Palma that may influence the biosynthesis of specialized metabolites. Previous studies on other Capsicum annuum strains have reported significant variability in the chemical composition of their extracts, influenced by factors such as maturity stage, genotype, and environmental conditions [1,2,3]. For example, Conforti et al. [1] and Deepa et al. [2] highlighted the influence of maturity on the antioxidant content of hot and sweet peppers, respectively, while Ranjith et al. [3] provided insights into the diversity of carotenoids in red pepper varieties. These findings underscore the importance of studying the unique conditions under which Capsicum annuum var. palmera is cultivated, which may contribute to its distinctive phytochemical profile.

Peppers are a rich source of bioactive compounds, including alkaloids, carotenoids, and antioxidants, which have been linked to significant health benefits such as antioxidant, anti-inflammatory, and lipid-lowering effects. These properties make them valuable for applications in nutraceuticals and pharmacology. The distinctive cultivation practices and geoclimatic conditions of La Palma, coupled with the traditional use of Capsicum annuum var. palmera in local cuisine, suggest that this variety may possess a unique chemical profile with potential bioactive properties. Investigating the composition of this pepper is essential not only to expand our understanding of its phytochemical diversity but also to explore its potential contributions to human health and nutrition.

In this study, we analyzed the dichloromethane and n-butanol fractions of the pericarp of Capsicum annuum L. var. palmera grown in Fuencaliente (southwest La Palma). Using advanced spectroscopic techniques and chromatographic separations, we identified a new pyrrolo-lactone compound 8 alongside several known metabolites, providing the first detailed compositional profile of this regional pepper variety.

2. Results and Discussion

Figure 2 shows the chemical structures of compounds 1–8 that were isolated as described in the experimental section from the dichloromethane and n-butanol fractions of the pericarp of the above-mentioned Capsicum annuum L. var. palmera.

The compounds isolated in this study belong to well-characterized classes of natural products known for their broad occurrence and significant roles in biological systems. For instance, α-tocopherol (compound 1) is a lipophilic antioxidant crucial for preventing lipid peroxidation in cellular membranes, while β-sitosterol (compound 5) is a phytosterol associated with cholesterol-lowering and anti-inflammatory effects. Other triterpenoids such as α-amyrin and lupeol exhibit diverse pharmacological activities, including anti-inflammatory and hepatoprotective properties. These findings align with previous studies on Capsicum annuum. Conforti et al. [1] reported variability in these compounds across different maturity stages of hot peppers, while Deepa et al. [2] observed similar trends in sweet peppers. The carotenoid diversity reported by Ranjith et al. [3] complements the current findings, highlighting that the chemical composition of peppers can be strongly influenced by genotype, geographical origin, and cultivation conditions. The identification of these metabolites in Capsicum annuum var. palmera underscores the pepper’s rich phytochemical profile and its potential health benefits, which may be further enhanced by the unique environmental and agricultural practices of La Palma.

Fractions 14-15D

Fractions 14-15D were eluted with hexanes-EtOAc 8:2. TLC analysis under UV light showed the presence of a single substance that, once concentrated by elimination of the solvent under vacuum, yielded 7.8 mg of compound 1. It has a molecular formula of C₂₉H₅₀O₂, as determined by high-resolution mass spectrometry (HREIMS), where the molecular ion is observed at m/z 430.3827 [M]⁺ (100%) (Calcd. for C₂₉H₅₀O₂ m/z 414.3811). The ¹H NMR spectrum in deuterated chloroform shows signals from hydroxyl groups at δ_H 4.19 (1H, s) for benzylic protons at δ_H 2.61 (2H, t, J = 6.9 Hz) and three methyl groups on the aromatic ring at δ_H 2.17 (3H, s) and δ_H (2.12, 6H, s). Other clearly discernible signals in this spectrum are those due to protons H-3a and H-3b at δ_H 1.83 and 1.77 ppm (each 1H, ddd, J = 7.1, 6.8 and 6.8 Hz), and the HSQC peak at δ_C 31.7 ppm. One methyl group at δ_H 1.23 ppm (3H, s) and four aliphatic methyl groups at δ_H 0.88 (6H, d, J = 6.6 Hz) and δ_H 0.87 (3H, d, J = 6.6 Hz) and δ_H 0.86 (3H, d, J = 6.4 Hz) were detected. The remaining observed signals between δ_H 1.59 and 0.83 ppm were assigned on the basis of a thorough two-dimensional correlation analysis and by comparison with data given in the literature [5] for vitamin E (α-tocopherol, 1), which is ubiquitous in biomembranes and is believed to be the most important lipophilic antioxidant that inhibits lipid peroxidation via a chain-breaking radical scavenging mechanism [6,7,8].

Fractions 27-30D

Fractions 27-30D eluted with hexane-EtOAc 8:2 yielded 15.5 mg of a colorless needle crystalline solid with a melting point of 140–154 °C. It has a molecular formula of C₃₀H₅₀O, as determined by high-resolution mass spectrometry (HREIMS), where the molecular ion is observed at m/z 426.3848 [M]⁺ (13.5%)] (Calcd. for C₃₀H₅₀O m/z 426.3848). The ¹H NMR spectrum shows signals of olefinic protons at δ_H 5.19 (t, J = 3.5 Hz) and 5.13 (t, J = 3.6 Hz) and two olefinic protons at δ_H 4.69 and 4.57 brs, characteristic of an exocyclic double bond. Three signals are also observed at δ_H 3.234 dd (J = 11.1 and 4.6 Hz), δ_H 3.231 dd (J = 11.1, 4.6 Hz) and at δ_H 3.19 dd (J = 11.3 and 4.8 Hz), and these signals are typical of proton geminal to hydroxyl groups. Inspection of the integrals corresponding to these signals reveals that we are actually dealing with a mixture of at least three different products at a ratio of 5:3:2, where we can tentatively deduce that we are dealing with a mixture of α-amyrin, a major component, β-amyrin, an intermediate, and lupeol, a minor component. The ¹³C NMR spectrum shows typical carbon signals for a terpenoid skeleton, including carbons attached to OH groups at the C-3 position at δ_C 79.41, 79.39 and 79.36 ppm. The olefinic carbons of the exocyclic double bond appeared at 151.16 (quaternary C) and 109.54 ppm (methylene C), which are assigned to the C-20 and C-29 double bonds of the minor compound lupeol. The C-12 and C-13 carbons of α-amyrin are observed at δ_C 124.88 (C-H) and δ_C 140.00 (C-quaternary), and for β-amyrin, they appear at δ_C 122.17 (C-H) and δ_C 145.5 (C-quaternary). RP-18 chromatography with acetonitrile-MeOH (95:5) as the eluent at a flow rate of 1.0 mL/min revealed the presence of two major constituents with retention times of 17.3 and 28.3 min, which were identified by ESI-TOF mass spectrometry and ¹H and ¹³C NMR spectra as lupeol and α-amyrin, respectively. β-Amyrin was not detected. It should be considered that the HPLC system used in this study relies on a UV detector, which primarily detects compounds with significant UV absorption, such as those containing double bonds or carbonyl groups. As a result, β-amyrin, due to its structural characteristics and concentration, is not detected under these conditions. However, the presence of β-amyrin in the mixture is confirmed by NMR data, which provides complementary evidence to support the composition of the fraction.

Fractions 37-41D

Fractions 37-41D, which were eluted with a 7:3 hexane-EtOAc mixture, yielded 9.2 mg of colorless crystals with a melting point of 125 °C in acetone (lit. m.p. 130 °C) [9]. These present a molecular formula of C₂₉H₅₀O, determined by high-resolution mass spectrometry (HREIMS), where the molecular ion is observed at m/z 414.3831 [M]⁺ (Calcd. for C₂₉H₅₀O m/z 414.3862) (82.08%)]. The ¹H NMR spectrum shows signals from an olefinic proton at δ_H 5.36 broad singlet, a geminal proton to a hydroxyl group at the δ_H 3.53 triplet of triplet (J = 11.0 and 4.7 Hz). In addition, this spectrum shows two singlets with three protons each at δ_H 0.68 and 1.01 ppm, indicative of C-18 and C-19 angular methyl groups, respectively. The signals at δ_H 0.80 (3H, d, J = 6.85 Hz), 0.83 (3H, d, J = 7.5 Hz), 0.82 (3H, d, J = 6.85 Hz) and 0.91 (3H, d, J = 6.6 Hz) were assigned to C-27, C-29, C-26 and C-21, respectively [10].

The peaks observed in the mass spectrum at m/z 396 [M-H₂O]⁺ due to water loss are indicative of the presence of OH groups in the molecule. The formation of fragments at m/z 303 [M-C₇H₁₁O]⁺ and 329 [M-C₇H₁₁O+26]⁺ is the most characteristic indication of the presence of a double bond between the C-5 and C-6 atoms of a steroid [11,12,13]. The above data are in agreement with the assignment of the structure of β-sitosterol for our compound 5.

Fractions 71-73D

Fractions 71-73D yielded a colorless oil. The high-resolution mass spectrum shows a molecular ion at m/z 283.2640 [M+H]⁺ (100%) (Calcd. for C₁₈H₃₅O₂ m/z 283.2637) according to the empirical formula C₁₈H₃₅O₂ [M+1]⁺. In its ¹H-NMR spectrum, the characteristic signals of an unsaturated fatty acid are as follows: terminal methyl of an aliphatic chain at δ_H 0.88 (3H, t, J = 6.7 Hz) and eleven methylenes at 1.26 ppm, two allylic methylenes at δ_H 1.63 m, and δ_H 2.01 m, a methylene contiguous to an acidic group at δ_H 2.35 (2H, t, J = 7.4 Hz), and a multiple at δ_H 5.35 ppm with an intensity of two vinyl hydrogens. The above data allowed us to identify this substance as oleic acid 6 (Figure S8).

A 4.5 g portion of the n-butanol extract was subjected to column chromatography with Hex-EtOAc mixtures of varying polarity as the eluent on a SiO₂ column (column B). The eluted fractions were collected in 59 portions of 200 mL each.

Fraction 6B

Fraction 6B was eluted with Hex-EtOAc at a 1:1 ratio after high-performance liquid chromatography (HPLC) was performed (SiO₂, EtOAc-Hex 1:2, flow rate of 2 mL × min⁻¹, TR of 14 min), and 18 mg of colorless compound 7 had a syrupy appearance. The high-resolution mass spectrum shows a molecular ion at m/z 126.0322 [M]⁺ (71%) (Calcd. for C₆H₆O₃ m/z 126.0317) according to the empirical formula C₆H₆O₃.

The diversity of metabolites observed in this study reflects the metabolic versatility of Capsicum plants, which adapt their biosynthetic pathways to local environmental conditions. Pyrrolo-lactones are likely derived from amino acid modifications, while tocopherols and triterpenoids are products in the isoprenoid biosynthesis pathway. Environmental factors such as La Palma’s volcanic soil composition, high UV exposure, and oxidative stress may play crucial roles in driving the production of specialized metabolites. Furthermore, the metabolic interactions between primary and secondary pathways could explain the co-occurrence of antioxidant molecules and sterols, which are critical for plant defense and resilience.

The ¹HNMR spectrum of 7 taken in deuterated acetone shows signals for two mutually coupled protons at δ_H 7.37 (1H, d, J = 3.8 Hz) and at δ_H 6.57 (1H, d, J = 3.8 Hz), characteristic of H-3 and H-4 of a disubstituted furanic ring, two oxymethylene protons at δ_H 4.63 (2H, broad singlet) and an aldehyde proton at δ_H 9.58 (1H s). The ¹³C NMR spectrum of 6 shows two olefinic carbons at δ_C 123.4 and 110.4, two sp² quaternary carbons at δ_C 153.9 and δ_H 163.3, and one carbonyl carbon at δ_C 178.2, suggesting a 5-oxymethyl-2-formyl-furan skeleton, 5-hydroxymethylfurfural (HMF). This was corroborated by the correlations observed in the HMBC spectrum between H-7 and C-2 and C-3 and between H-6 and C-4 and C-5; between H-3 and C-2, C-4, and C-5; and between H-4 and C-5 and C-2 (Figura 3a).

Fractions 15-16B

Fractions 15-16 B were eluted with EtOAc, and after HPLC rechromatography (SiO₂, EtOAc, flow 2 mL min⁻¹, TR 12.7 min), 7.4 mg of 8, a colorless oil ([α_D]²⁰ −65.13° (7.4 mg in 1 mL MeOH) with the molecular formula C₁₀H₉NO₅ was determined by HREIMS, where the molecular ion was observed at m/z 223.0493 [M]⁺ (9.0%) (Calcd. for [C₁₀H₉O₅]⁺ m/z 223.0481), in addition to the loss of a proton at m/z 222.0384 [M-H]⁺ (69.0%).

The identification of a previously unreported pyrrolo-lactone compound in Capsicum annuum var. palmera expands the chemical diversity of pyrrole-containing natural products, which are known for their significant biological activities, including antioxidant, anti-inflammatory, and antimicrobial properties. The specific structural features of compound 8, such as the 5-oxymethyl-2-formyl-pyrrole skeleton and fused succinic acid moiety, are rarely encountered in Capsicum species and suggest a potential for bioactivity exploration. Additionally, this compound represents a new entry into the alkaloid diversity of peppers, underscoring the importance of studying regional varieties cultivated under unique environmental conditions, such as La Palma’s volcanic soils and semi-arid climate. These findings open new avenues for investigating the nutraceutical and pharmaceutical applications of this alkaloid family.

A comparison of the ¹H and ¹³C NMR spectra of 7 and 8 shows similarities, with the main difference being the chemical shifts observed for the carbons at δ_C 153.9 ppm, C-2 and δ 163.3 ppm C-5 for compound 7 versus 133.2 ppm C-2 and 145.6 C-5 for compound 8 (see

Table 1. ¹H (600 MHz) and ¹³C (150 MHz) NMR data of HMF 7 and pyrrolo-lactone 8 in acetone-d₆.

	δH (J Values Are Given in Hz)		δC
Atom/Position	7	8	7	8
OH		10.21 s	--
2			153.9	133.5
3	7.37 d (3.8)	7.10 d(3.8)	123.4	126.3
4	6.57 d (3.8)	6.31 d (3.8)	110.4	111.3
5			163.3	145.6
6a	4.63 brs	4.73 d (14.1)	57.91	56.9
6b	4.63 brs	4.73 d (14.1)	57.91	56.9
7	9.58 s	9.41 s	178.2	180.4
1’		5.78 brdd (9.5, 7.0)	--	56.5
2’			--	169.8 C’-2
3’a		3.14 dd (16, 9.5)	--	38.5
3’b		2.80 brdd (16, 7.0)	--	38.5
4’				175.1 C’4

), indicating the presence of a 5-oxymethyl-2-formyl-pyrrole skeleton in 8 instead of the 5-oxymethyl-2-formyl-furan skeleton in 7. In addition, the ¹H and ¹³C NMR spectra of 8 show resonances of a methylene carbon at δ_C 38.5 ppm, correlated with the protons at δ_H 3.14 (1H, dd, J = 16.0 and 9.5 Hz) and δ_H 2.80 (1H, dd J = 16.0 and 7.0 Hz) in the HSQC spectrum. These protons are coupled with the signals at δ_H 5.78 (1H, dd, J = 9.5, 7.0 Hz) and HSQC 56.9 ppm. Two quaternary carbons, δ_C 169.8 and 175.1 ppm, were assigned to the carbonyl carbons of a lactone and an acid, respectively. The correlations observed in the heteronuclear HMBC correlation experiments (Figure 3b) between H’-1 and C-2, C-5, C-3, C’-2 and C’-4 confirmed the binding of a succinic acid moiety fused to a 5-oxymethy1-2-formyl-pyrrole fragment in 8. The methyl ester derivative was synthesized by Sang Ho and coworkers and has the ability to recover glutathione in living cells against noxious oxygen species [14]. The bioactive properties of the identified compounds, particularly the pyrrolo-lactone derivative (8), suggest potential applications in nutraceuticals and pharmacology. Pyrrolo-lactones have been previously associated with cytoprotective and antioxidant effects, while tocopherols are widely recognized for their ability to prevent lipid peroxidation in biological systems. The unique chemical profile of Capsicum annuum var. palmera thus positions it as a valuable resource for further bioactivity screening and functional food development. Future studies may focus on evaluating the biological efficacy of these compounds in vitro and in vivo, which could lead to the development of new natural-product-based therapies.

It is worth highlighting that 2-formylpyrrole natural products represent a fascinating class of alkaloids characterized by their unique chemical scaffolds and diverse biological activities. These compounds are widely distributed in nature and are often biosynthesized through enzymatic modifications of amino acids or polyketides. The 2-formylpyrrole motif is commonly associated with bioactivities such as antioxidant, anti-inflammatory, antimicrobial, and cytotoxic properties, making it a subject of considerable interest in natural product chemistry and drug discovery. Recent advancements in understanding their occurrence, biosynthesis, and functional potential have been summarized in the review by Wood, Furkert, and Brimble [15], which highlights their versatility and relevance in natural product research. Structure 8, a pyrrolo-lactone derivative, expands this chemical family and provides insights into the untapped diversity of bioactive metabolites in Capsicum annuum var. palmera. This previously unreported compound likely arises from modifications involving amino acid precursors, similar to other pyrrole-containing alkaloids.

3. Experimental Procedure

3.1. Materials and Methods

Melting points are uncorrected and were measured on a Reichert Thermovar apparatus. Optical rotations were determined via a Perkin Elmer 2H polarimeter with a 1 dm cell. ¹H and ¹³C NMR spectra were measured via Bruker Advance II 500 and Bruker Advance III 600 spectrometers. The spectra were recorded mainly in CDCl₃, C₆D₆, CD₃COCD₃ and C₅D₅N with the solvent used as an internal reference (δ_H 7.26; δ_C 76.7), (δ_H 7.16; δ_C 128.39), (δ_H 2.05, δ_C 29.92), and (δ_H 8.74, 7.58, 7.22; δ_C 150.35, 135.91, 123.87), respectively. ESI-TOF and exact mass measurements were performed via a Micromass LCT Premier XE instrument. Column chromatography was performed over Sephadex LH-20 Pharmacia (ref. 17-0090-01) silica gel (Merck 230–400 mesh), octadecyl-functionalized silica gel (Aldrich 377635-1006) and analytical TLC Merck Kieselgel 60 F254. HPLC separations were carried out on a JASCO Pu-980 series pumping system equipped with a JASCO UV-975 detector and a Waters Kromasil Si 5 mm (10 × 250 mm) column. A Mackerey-Nagel VP 250/10 nucleodur Sphinx RP, 5 µm column was used for HPLC–RP chromatography; TLC samples were visualized under UV light at 255 and 366 nm and/or sprayed with oleum followed by heating. All the solvents were distilled before use.

3.2. Preparation of Plant Extracts

Palmera-type peppers (Capsicum annuum L. var. palmera) were obtained from a commercial plantation in southwest La Palma (Fuencaliente, Spain). The plants were planted in open fields in April and harvested from June to August 2022. Two kgs of air- and sun-dried fruit, free of seeds, were finely crushed via a food crusher. These samples were macerated until exhaustion in a 95:5 ethanol/water mixture. The mixture was subsequently filtered and concentrated under vacuum at 60 °C in a rotary evaporator, resulting in a red–orange mass with a rubbery texture of 181 g. The crude extract was resuspended in water and extracted with different organic solvents. First, three extractions were performed with dichloromethane, and the aqueous fraction from this process was again subjected to three more extractions, this time with n-butanol. Thus, three different fractions were obtained: CH₂Cl₂ (38.7 g), n-butanol (28.8 g) and H₂O (123 g). An aliquot of the dichloromethane residue (3.3 g) was subjected to column chromatography on silica gel (SiO₂ Merck 230–400 mesh). The eluting solvent was a gradient of hexane-EtOAc (0 to 100%), and 109 fractions (150 mL each) were collected. On the basis of TLC analyses, the fractions were pooled to yield four fractions.

4. Conclusions

A new pyrrolo-lactone (i.e., 2-(6-formyl-3-oxo-3,4-dihydro-1H-pyrrolo[2,1-c][1,4]oxazin-4-yl) acetic acid) was isolated from Capsicum annuum L. var. palmera, a unique red pepper cultivated in La Palma (Canary Islands, Spain). Its structure was elucidated by NMR spectroscopy and by comparison with synthetic analogs. This discovery highlights the distinct chemical profile of this regional pepper variety and contributes to the broader understanding of alkaloid diversity in Capsicum species. The characterization of this new pyrrole alkaloid expands the potential applications of Palmera peppers, suggesting promising uses in nutraceuticals and pharmacology due to the known bioactive properties of pyrrole alkaloids.