Chemistry and Diversity of Nitrogen-Containing Metabolites in Heliotropium procumbens: A Genus-Wide Comparative Profile

Abstract

Heliotropium procumbens, a Boraginaceae species native to Panama, has remained largely unexplored regarding its nitrogen-containing metabolites, including pyrrolizidine alkaloids (PAs). In the current study, a comprehensive phytochemical investigation of its aerial parts is presented using HPLC-DAD-IT-MS, UHPLC–HRMS, and GC-MS primarily to profile its PA composition. A total of twelve PAs and N-oxides (PANOs) were identified, along with two phenolamides—including N¹, N¹⁰-diferuloylspermidine, which is biosynthetically related to PAs—and the distinctive metabolite heliotropamide. The detected PAs included unsaturated necines, primarily monoesters of retronecine and heliotridine, as well as saturated PAs such as a platynecine-type PA and the less commonly encountered triol necines and their N-oxides. Among these, helifoline-N-oxide was isolated and structurally elucidated by NMR spectroscopy for the first time as a natural product. Comparison with the chemodiversity of PAs within the Heliotropium genus revealed a high degree of diversity in H. procumbens, which can be attributed both to the species’ inherent biosynthetic capacity for chemical variation and to the more comprehensive and extensive studies conducted on it, which naturally enrich the apparent diversity observed. This work expands the phytochemical knowledge of H. procumbens and contributes to a broader understanding of PA diversity in the genus, offering new insights into their potential ecological and toxicological significance.

1. Introduction

The genus Heliotropium contributes to the Boraginaceae family and includes approximately 345 species [1]. This cosmopolitan genus occurs on all continents, with four main centers of diversity located in Central and Southwest Asia, Central and South America, East and Southern Africa, the Southern Arabian Peninsula, and Australia [2]. Several species hold economic value in their native areas due to their use in folk medicine and traditional practices [3]. Some Heliotropium species are recognized for their pharmacological activities, including antimicrobial, anti-inflammatory, antiplatelet, wound healing, cardiotonic, and contraceptive effects [3,4,5,6], while others are known for their toxic effects due to the presence of pyrrolizidine alkaloids (PAs) [7]. There are numerous reports in the literature linking various liver diseases, primarily veno-occlusive disease (VOD), with poisoning caused by PAs. Cases of human intoxication have been associated with herbal tea preparations contaminated with Heliotropium lasiocarpum and other members of the Boraginaceae family. However, in most instances, a direct causal relationship has been difficult to establish as the onset of liver disease often occurs long after the ingestion of PA-containing materials [7].

PAs are a prominent class of secondary metabolites produced by various plant species, especially members of the Boraginaceae, Asteraceae, and Fabaceae families [8]. Although toxic to humans and livestock, their primary biological role is to serve as a chemical defense mainly against herbivores [9]. Structurally, PAs consist of a necine base that can be esterified either at a single position (C-7 or C-9), at both positions simultaneously (forming diesters), or as macrocyclic lactone diesters linking C-7 and C-9 (Figure 1). The combination of various necine bases with a diverse array of necic acids generates a wide variety of PA structures. This chemical diversity is further increased by modifications such as N-oxidation of the necine base’s tertiary nitrogen (producing PAs N-oxides, or PANOs), hydroxylation of either the necine base or necic acid, and acetylation of hydroxyl groups on the acid moiety. Consequently, hundreds of distinct PAs have been characterized to date, with new variants continually being discovered [9].

The structural diversity that PAs exhibit also has a toxicological significance. Toxicity studies have established a clear relationship between the nature of PAs and their harmful effects. In particular, 1,2-unsaturated necines and specific esterification patterns—such as cyclic diesters—are associated with toxicity since PAs exhibiting these structural features require metabolic activation to form reactive pyrrolic metabolites [8,10]. This bioactivation primarily occurs in the liver, making it the main target organ of damage, while other types of toxicity—such as pneumotoxicity, genotoxicity, and neurotoxicity—have also been associated with cases of acute or chronic exposure [11].

In order to map the chemodiversity of metabolites in complex natural mixtures, analytical techniques such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) coupled with mass spectrometry (MS) are widely used [12,13]. For screening, identifying, and quantifying PAs, HPLC-MS-based methods have become the preferred approach due to their robustness and reproducibility, and they are currently recognized by the European Pharmacopoeia for their analysis of these compounds in herbal products [13]. However, the limited availability of commercial reference standards for many rare PAs restricts unambiguous identification and accurate quantification, especially for minor or novel alkaloids [14]. GC-MS remains a standard approach, offering well-established fragmentation patterns for free base PAs; however, it requires prior chemical reduction of non-volatile N-oxides and more extensive sample preparation [15]. Additionally, PANOs are less studied and lack comprehensive bibliographic data, which complicates their detection and structural elucidation because they require non-reductive analytical methods [11,13]. Nuclear magnetic resonance (NMR) spectroscopy is essential for determining necine base types, identifying ester attachment sites, and elucidating the stereochemistry of necic acids [16]. Therefore, using complementary analytical techniques helps overcome these challenges and enhances the characterization of PAs in natural products.

Heliotropium procumbens Mill. (syn. Euploca procumbens (Mill.) Diane & Hilger) [17], native to America and the East and West Indies, has been locally used as animal feed due to its high nutritional value, particularly its notably high protein content [18]. Regarding its alkaloid content, only a few samples from Mexico have been analyzed, revealing the presence of lindelofidine, retronecine, supinidine, and trachelanthamidine [19].

Given its local use and limited detailed profiling of its alkaloid composition, this study continues our research on Boraginaceae plants [20,21,22,23], specifically the phytochemistry of H. procumbens [24], by analyzing its PA composition. The findings from this study not only enhance the understanding of the PA profile in H. procumbens but also place it within the broader chemodiversity of the Heliotropium genus. This highlights how more comprehensive and advanced analytical techniques reveal greater chemical complexity and diversity across species.

2. Materials and Methods

2.1. Plant Material and Methanolic Extract Preparation

The aerial parts of H. procumbens were collected from Rio Grande (area near the Puente) in April 2004 by Prof. Mahabir Gupta. The plant material was botanically identified by Prof. M.D. Correa and deposited at the Herbarium of Panama University (voucher number 6476). The plant samples were air-dried in a shaded, well-ventilated environment, subsequently ground using a laboratory mill, and stored in a dark environment at room temperature. An aliquot of 900 g of dried plant material was successively extracted with methanol by immersion in the solvent 3 × 24 h at room temperature. The obtained methanolic extract (14.820 g) was evaporated under reduced pressure to dryness.

2.2. UHPLC–HRMS Analysis

The methanolic extract underwent UHPLC–HRMS analysis using a Vanquish UHPLC platform (Thermo Scientific, Bremen, Germany) featuring a binary pump, autosampler, in-line degasser, and thermostatted column chamber. Separation occurred on an Accucore Vanquish UPLC C18 column (2.1 × 50 mm, 1.5 μm) with detection by an Orbitrap Exactive Plus high-resolution mass spectrometer (Thermo Scientific, Bremen, Germany). Samples (100 ppm in 50:50 MeOH/H₂O) were injected at a 5 μL volume. The mobile phase comprised (A) 0.1% (v/v) formic acid in water and (B) acetonitrile, with the following optimized gradient: 5% B (0–3 min), linear increase to 95% B (21 min), hold (26 min), return to 5% B (26.1 min), and re-equilibration (30 min). Analysis parameters included a 0.3 mL/min flow rate, 40 °C column temperature, and 10 °C sample tray temperature. LC-MS grade solvents (MeOH, ACN, FA from Fisher Optima, Loughborough, UK) and purified water (Barnstead MicroPure system, Thermo Scientific, Niederelbert, Germany) were used throughout. Ionization was conducted using a heated electrospray ionization (HESI) source in both positive and negative modes. High-resolution mass spectrometry parameters for both ionization modes were configured as follows: capillary temperature at 320 °C, spray voltage of 2.7 kV, S-lens RF level at 50 V, sheath gas flow rate of 40 arbitrary units, auxiliary gas flow at 8 arbitrary units, and auxiliary gas heater temperature maintained at 50 °C. Analyses employed Fourier transform mass spectrometry (FTMS) in full-scan mode with a resolution of 70,000, with all mass spectra acquired in centroid mode. Data-dependent acquisition (DDA) was implemented at 35,000 resolution in centroided mode, triggering MS/MS fragmentation for the three most intense ions per peak exceeding a predefined intensity threshold, using a 10 s dynamic exclusion window. Normalized collision energy was fixed at 35%. Data acquisition and processing utilized Xcalibur 2.1 software.

2.3. HPLC-ESI-DAD-IT MS

2.3.1. Sample Preparation for HPLC-ESI-DAD-IT MS Analysis

For HPLC-ESI-DAD-IT MS analysis, plant material was subjected to an extraction process following the standard BfR protocol, using the dried aerial parts of the plant [25]. This protocol, developed by the German Federal Institute for Risk Assessment (BfR), is widely recognized for its scientific rigor and regulatory acceptance in the reliable and reproducible extraction and analysis of PAs from plant materials. A total of 2 g of powdered material was extracted with 20 mL of 0.05 M H₂SO₄ for 15 min in an ultrasonic bath. Following extraction, the mixture was centrifuged for 10 min at 3800 rpm, and the supernatant was collected through decantation. The extraction procedure was repeated with an additional 20 mL of the same solvent, and the resulting supernatant was combined with the first. The total extract was then neutralized with 25% aqueous ammonia solution (to pH = 7). The neutralized extract was filtered using a filter paper, and the solid phase extraction method (SPE) was performed in a special vacuum chamber using C-18 cartridges (C18-E, 55 μm, 70 Å, 500 mg/6 mL, Phenomenex Strata^®, Phenomenex Inc., Torrance, CA, USA). The SPE procedure involved two conditioning steps (5 mL MeOH followed by 5 mL dd H₂O), sample loading (10 mL of neutralized extract), washing (2 × 5 mL dd H₂O), drying of cartridges under low vacuum for 5–10 min, and elution of pyrrolizidine alkaloids with 2 × 5 mL MeOH.

2.3.2. HPLC-ESI-DAD-IT MS Analysis

For the analysis of the methanolic extract obtained from the SPE extraction, HPLC coupled with ESI-DAD-IT MS was employed. The system utilized a mass detector HPLC–LCQ with an ion trap detector MSn (Finningan LCQ). Agilent|1100 Series LC/MS 0.1 mm, dp = 3 μm) (Waters Milford, MA, USA) was utilized. The chromatograph used a diode array detector autosampler, dual grading pump, and column heater equipped with dual spray source: electrospray (ESI) for sample and reference masses, connected to N₂ generator (Parker Hannifin Corporation, Haverhill, MA, USA; generating N₂ at purities > 99%), compressed air generator (Jun-Air, Oxymed, Łódź, Poland), and compressed air container. The analytical column was a 5 μm particle size, XTerra C18 (Waters, Milford, MA, USA), 150 × 4.6 mm (i.d). The column was held at 25 °C, and the mobile phase flow rate was 1.0 mL/min. As mobile phase, solvent A 15 mM NH₃ and solvent Β 100% ACN were used for gradient elution as follows: 0–20 min—linear gradient: 5–50% of solvent B; 20–25 min—isocratic run: 50% of solvent B; 25–28 min—linear gradient elution of solvent B from 50–100%; the 28–33 min—isocratic run of 100% B; and at the end, 33–36 min—linear gradient elution 100–5% B. Total analysis time was 36 min. The injection volume was 20 μL (triple repeat). Analysis was performed in positive ionization mode with different fragmentation voltages (140 V, 200 V, 250 V). Mass Hunter 2.2.1 LC/MS spectra analysis software was used for data acquisition and analysis thereof.

2.4. GC-MS

2.4.1. Sample Preparation for GC-MS Analysis

For the GC-MS analysis, a portion of the methanolic extract was subjected to reduction using zinc dust (Riedel-de Haën, Seelze, Germany) in 3 mL of 2 M HCl solution (Merck™, Darmstadt, Germany) to convert alkaloid N-oxides into their corresponding free bases. The mixture was then alkalized with 25% ammonia solution (NH₃, reagent grade, Panreac Química SA, Barcelona, Spain) to a pH of 10.5 and extracted three times with 50 mL portions of dichloromethane (HPLC grade, Fisher Scientific, Loughborough, Leics, UK) to remove nonalkaloidal compounds. The combined organic layers were dried over anhydrous sodium sulfate (Na₂SO₄, Lach-Ner, s.r.o., Neratovice, Czech Republic) and evaporated under reduced pressure to yield the PAs fraction, which represented the total alkaloid content, including both tertiary alkaloid bases and their N-oxide derivatives [20].

2.4.2. GC-MS Analysis

The analysis of the PAs fraction was performed using an Agilent Technologies 7820A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent Technologies 5977B (Agilent Technologies, Santa Clara, CA, USA)mass spectrometer, operated in both split and splitless modes. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. Electron ionization (EI) mass spectra were acquired under the following conditions: electron energy of 70 eV and ion source temperature set at 205 °C. Separation was achieved on an HP-5MS capillary column (Agilent Technologies, Santa Clara, CA, USA, 30 m length, 0.25 mm internal diameter, 0.25 μm film thickness). The oven temperature program was initiated at 120 °C with a hold time of 3 min, followed by a ramp to 230 °C at 20 °C/min with a 10 min hold, and then increased to 290 °C at 10 °C/min with a final hold of 2 min.

2.5. Isolation and Identification of Helifoline-N-oxide

A portion of the dried methanol extract (10.0 g) was subjected to column chromatography using microcrystalline cellulose (20–160 μm, Merck, Darmstadt, Germanyas the stationary phase and a solvent mixture of cyclohexane (A)/ethyl acetate (B)/methanol (C)/water (D) as the eluting system. A gradient elution was applied as follows: 100:0:0:0 → 0:100:0:0 → 0:80:20:0 → 0:0:50:50, yielding 46 fractions (HC1–HC46). Fraction HC9 (500 mg) was further purified by molecular weight chromatography on Sephadex LH-20 (25–100 μm, Pharmacia, Uppsala, Sweden) using methanol as the mobile phase, resulting in 15 fractions (S2HC1–S2HC15). For monitoring the elution of PAs and PANOs, silica gel TLC plates (Merck™, Darmstadt, Germany) were developed with DCM:MeOH:[NH₃OH]- (85:14:1) or (75:25:1) as mobile phases and visualized using sulfuric vanillin reagent. Upon heating, PAs typically produced blue or purple spots, while PANOs appeared as pink to reddish or brownish spots [26]. Mattocks-Molyneux reagent was also used for the detection of PANOs containing a 1,2-unsaturated necine moiety, which characteristically produce purple spots [27]. Fraction S2HC4 (29.1 mg), which gave a positive reaction in the PA detection assay, was further purified by preparative-TLC on silica gel 60 F254 aluminum plates (0.2 mm layer thickness, Merck™, Darmstadt, Germany) using DCM:MeOH:[NH₃OH]^- (75:25:1) as the eluent. The resolved zones were extracted with a mixture of ethyl acetate:methanol (80:20), leading to the successful isolation of helifoline-N-oxide, which was identified by NMR spectroscopy using CD₃OD (Euriso-Top, Cambridge Isotope Laboratories, Tewksbury, MA, USA). All solvents used for chromatographic separations were of HPLC grade and purchased from Fisher Chemical (Fisher Scientific, Loughborough, Leics, UK). Ammonium hydroxide (NH₄OH) was obtained from Vioryl (Afidnes, Greece), and sulfuric acid (H₂SO₄) from Merck™ (Darmstadt, Germany). ¹H-NMR and 2D-NMR experiments (COSY, HMBC) were recorded on a Bruker DRX400 spectrometer (Bruker Corporation, Billerica, MA, USA) operating at 400 MHz.

2.6. Data Collection and Visualization of PAs Chemodiversity in the Heliotropium Genus

To gather chemical information on PAs within the Heliotropium genus, data were compiled from existing reviews on PAs, including those covering the Heliotropium genus [3,16]. This dataset was further supplemented by recent PA analyses of Heliotropium species identified through targeted literature searches on platforms such as PubMed, Google Scholar, and Scopus [28,29,30,31,32,33,34,35]. The species list with corresponding citations is provided in Supplementary Materials Section S1.1. For visualization, RAWgraphs (rawgraphs.io) was used to create an alluvial diagram illustrating the distribution and diversity of PA types across species [36].

3. Results

3.1. Phytochemical Profiling of H. procumbens Extracts

The HPLC-ESI-DAD-IT-MS profiling of H. procumbens extracts revealed a diverse array of 12 PAs and PANOs (

Table 1. Data of compounds detected in H. procumbens extracts by HPLC-ESI-DAD-IT-MS and UHPLC–HRMS.

No	Rt (min)	[M+H]+	Molecular Formula	Error Δppm	MS/MS	Annotated Compounds	Ref.
1	0.41	142.1228	C8H15NO	1.12	124	Trachelanthamidine/Lindelofidine (S)	[16,37]
2	0.39	174.1127	C8H15NO3	1.32	156, 138, 112	Helibracteatinecine/Helibractinecine/Croalbinecine (S)
3	0.41	190.1075	C8H15NO4	0.61	172, 155, 129, 98	Helibracteatinecine/Helibractinecine/Helifolinecine-N-oxide (S)
4	2.42	238.1437	C13H19NO3	−0.29	156, 138, 120, 94, 83	9- angeloylretronecine * (US)	[16,38]
5	2.13	238.1436	C13H19NO3	−0.71	156, 138, 120, 94, 83	9- angeloylheliotridine * (US)	[37]
6	2.81	240.1594	C13H21NO3	−0.08	208, 178, 158, 140, 122, 83	7-angeloylplatynecine * (S)	[39,40,41]
7	4.19	254.1387	C13H19NO4	0.06	247, 172, 154, 136, 112, 83	9- angeloylheliotridine * -N-oxide (US)	[37]
8	1.95	254.1388	C13H19NO4	0.45	247, 174, 137, 111, 106, 83	7- angeloylheliotridine *-N-oxide (US)	[16]
9	1.45	256.1544	C13H21NO4	0.26	238, 174, 156, 138, 120, 83	Helifoline * (S)	[42]
10	1.84	256.1544	C13H21NO4	0.26	174, 156, 106, 83	Heliscabine or isomer * (S)	[16]
11	2.10	272.1492	C13H21NO5	−0.18	190, 172, 155, 129, 98, 83	Heliscabine or isomer *-N-oxide (S)
12	2.98	272.1493	C13H21NO5	0.19	190, 172, 155, 129, 98, 83	Helifoline *-N-oxide (S)
13	6.53	498.2604	C27H35N3O6	1.08	322, 234, 177, 145	N1, N10-Diferuloyl spermidine	[43]
14	8.71	625.2552	C36H36N2O8	1.21	417, 325, 272, 301, 137, 121	Heliotropamide

Retention times (Rt) and observed [M+H]⁺ m/z values correspond to UHPLC–HRMS analysis. Compounds marked with “*” were also detected by GC-MS analysis (see Supplementary Materials Section S1 Figure S2, Table S1). Saturated PAs are indicated as (S), and unsaturated PAs as (US). MS/MS spectra obtained from UHPLC-HRMS of compounds 2, 3, 11, and 14 can be found in Supplementary Materials Section S1, Figures S3–S6.

), tentatively identified based on MS/MS fragmentation patterns (see Supplementary Materials Section S1 Figure S1). These findings were further confirmed by UHPLC–HRMS analysis, which validated molecular formulas and provided high-resolution fragmentation data. Complementary GC-MS analysis of the reduced extract offered additional confirmation, leveraging its frequent application in alkaloid detection, particularly within Heliotropium species [15].

3.2. Structure Elucidation of Helifoline-N-oxide

Helifoline-N-oxide (Figure 2) was obtained as a white, amorphous powder. Its presence in the selected fraction was detected on silica gel TLC plates using a mobile phase of DCM:MeOH:NH₄OH (85:14:1) and visualized with sulfuric vanillin reagent, producing a vivid red-pink spot upon heating. The [M+H]⁺ m/z 272.1493 established its elemental formula as C₁₃H₂₁NO₅ (Δppm 0.19). The proposed fragmentation pattern of helifoline-N-oxide can be found in Supplementary Materials Section S1 (Figure S7).

The ¹H and ¹³C NMR spectra (

Table 2. NMR data (400 MHz, methanol-d4) for helifoline-N-oxide (compound 12).

Position	δC	δH (J in Hz)	COSY	HMBC
1	43.6	2.76 m	2, 8, 9a, 9b	-
2	71.8	4.64 m	1, 3a, 3b	C-9
3a	71.1	3.75 dd (11.07, 6.12)	2, 3b	C-1, C-8
3b		3.39 dd (11.07, 9.15)	2, 3a	C-2, C-5
5a	68.7	3.57 m	5b, 6a, 6b
5b		3.91 m	5a, 6a, 6b	C-6, C-7
6a	34.3	2.61 m	6b, 5a, 5b, 7	C-5
6b		2.12 m	6a, 5a, 5b, 7	-
7	73.7	4.50 m	8, 6a, 6b	-
8	90.4	3.85 dd (8.25, 5.61)	7, 1	C-1, C-9
9a	62.9	4.44 dd (11.23, 4.69)	9b, 1	C-1, C-2, C-1′, C-8
9b		4.29 dd (11.23, 6.98)	9a, 1	C-1, C-2, C-1′, C-8
1′	167.8	-	-	-
2′	127.5	-	-	-
3′	138.5	6.15 dq (7.20/1.40)	4′	-
4′	15.8	1.99 dd (7.20/1.53)	3′	C-3′, C-2′
5′	20.9	1.92 s	-	C-3′, C-2′, C-1′

) showed the presence of a saturated triol necine base. More specifically, the characteristic proton signals at δ_H 2.76 (H-1), 4.64 (H-2), and 4.50 (H-7), together with the carbon signals at δ_C 43.6 (C-1), 71.8 (C-2), and 73.7 (C-7), are indicative of a saturated triol necine. The presence of a proton signal at δ_H 2.76, along with proton and carbon resonances significantly deshielded by an electronegative substituent, supports this assignment. Key correlations observed in the COSY spectrum—namely, between H-2 and H-1, as well as between H-7 and H-6a/H-6b (the most shielded among the protons of helifoline’s necine base) and H-8 (δH 3.85, dd J = 8.25/5.61)—were decisive for the assignment of these protons. Key HMBC correlations between the carbonyl carbon at δ_C 167.8 (C-1′) and H-9a/H-9b indicated the presence of a necic acid unit esterified at O-9. Indeed, the signals at δ_H 6.15 (H-1′, dq, J = 7.2/1.4 Hz), 1.99 (3H-4′, dd, J = 7.2/1.53 Hz), and 1.92 (3H-5′, s), along with the signals at δ_C 167.8 (C-1′), 127.5 (C-2′), 138.5 (C-3′), 15.8 (C-4′), and 20.9 (C-5′), were in close agreement with those reported for the angeloyl moiety in helifoline [42]. COSY correlations among H-3′/3H-4′, as well as HMBC correlations of C-1′ to 3H-5′ and C-2′ and C-3′ to 3H-4′ and 3H-5′, further supported the proposed structure, although no correlation signals were observed, including allylic H-3′. The ¹H and ¹³C NMR spectra can be found in Supplementary Materials Section S1 (Figures S8–S10).

4. Discussion

4.1. Profiling of PAs and Phenolamides

The comprehensive profiling of H. procumbens extracts in this study uncovered a variety of PAs and PANOs, whose tentative identification through multiple mass spectrometric techniques provided a strong foundation for further structural characterization. Extensive mass spectral data on PAs are documented, providing valuable insights into the structure of necine bases and their substituents. For instance, 1,2-unsaturated necine bases esterified at C-9 and bearing a free hydroxyl group at C-7 (such as retronecine) typically exhibit characteristic fragment ions at m/z 156, 138, and 120, whereas fragment ions at m/z 172 indicate their corresponding N-oxides. In contrast, 1,2-saturated necines (such as platynecine) generate fragment ions at m/z 140 and 122 [44]. Necines of the same saturated type but lacking a hydroxyl group at C-7 (such as trachelanthamidine and lindelofidine) show a diagnostic fragment ion at m/z 124 [44].

The detected compounds include both saturated and unsaturated necine bases, while the chemical profile shows a predominance of saturated PAs, suggesting a biosynthetic preference. Compound 1, tentatively assigned to the stereoisomers trachelanthamidine and/or lindelofidine, appeared only in trace amounts. These stereoisomers have been recurrently documented across Heliotropium species, including H. procumbens itself [16], reinforcing their taxonomic and chemotaxonomic significance.

Compound 2, with a parent ion at m/z 174.1127 and molecular formula C₈H₁₅NO₃, was identified as a saturated triol necine, alongside the detection of an N-oxide (3) of the same necine type. Notably, N-oxides of such triol necines have not been previously reported. This type of PAs has been documented in Heliotropium species; for example, the stereoisomeric compounds helibracteatinecine and helibractinecine were isolated from H. bracteatum [45,46], while croalbinecine (or helifolinecine) has been reported as the hydrolysis product of helifoline from H. ovalifolium [42]. Due to the absence of available spectral data under ESI conditions for these metabolites, and their non-detection by GC-MS—likely due to their trace levels in the sample and the comparatively lower sensitivity of the GC-MS system used versus HPLC-ESI-DAD-IT-MS and UHPLC–HRMS—an unambiguous assignment of the chromatographic peak to a single isomer was not achievable. Regarding croalbinecine, although it has not yet been isolated, we cannot exclude the possibility that it may also occur as a free base.

Angeloyl esters of triol necines and their N-oxides were also tentatively identified, reflecting patterns observed in related Heliotropium species, such as H. bracteatum, H. scabrum, and H. ovalifolium, where helibracteatine, heliscabine (10), and helifoline (9) were, respectively, isolated [42,45,46] but without their corresponding N-oxides (11 and 12).

Among saturated PAs, compound 6 was assigned as 7-angeloylplatynecine, supported by characteristic fragment ions at m/z 140 and 122, indicative of a platynecine-type PA. Together with previously reported GC-MS spectral data for this compound [39,40,41], these findings confirm its presence in the extract. Platynecine-type PAs are rare in the Heliotropium genus, with only megalanthonine—an ester of viridifloric acid and platynecine—reported in H. megalanthum [47].

Among the unsaturated compounds, 9-angeloylretronecine (4) was recently isolated from H. sarmentosum [32]. In contrast, its isomer 9-angeloylheliotridine (5), along with its N-oxide (7), has not been previously observed in the genus. The compound 7-angeloylheliotridine has been reported in H. curassavicum [48] and H. supinum, while the N-oxide of 7-angeloylheliotridine (8) isolated from the latter was considered an artifact [49]. In terms of spectral differentiation of closely related necine bases, retronecine is distinguished from heliotridine by the abundance of the fragment ion at m/z 94, which serves as the base peak for retronecine but not for heliotridine [37]. The 7-angeloyl derivative was assigned based on the characteristic fragment ion at m/z 106.

Beyond the pyrrolizidine alkaloids, two additional nitrogenous derivatives were tentatively annotated. Compound 13 was identified as N¹, N¹⁰-diferuloylspermidine based on its characteristic fragmentation pattern in positive ion mode. This phenolamide is a noteworthy metabolite due to its biosynthetic link with PAs via common polyamine precursors, putrescine and spermidine. Biosynthetically, putrescine and spermidine, derived from the amino acid arginine, serve as substrates for homospermidine synthase (HSS) to produce homospermidine, which subsequently undergoes oxidation, cyclization, and reduction steps to form 1-hydroxymethylpyrrolizidine, the core structure of PAs [9] (Figure 3). Although the presence of these polyamines in a free form has been reported in leaves and inflorescences of H. angiospermum and H. indicum plants [50], phenolamides are much less documented in this genus. To date, only one phenolamide derivative—N¹,N¹⁰-dicoumaroylspermidine—has been detected in H. crispum [29]. The detection of diferuloyl spermidine in this study, however, represents the first known occurrence of this compound in any Heliotropium species. Beyond their biosynthetic importance, phenolamides are also notable for their bioactivities, including anti-inflammatory, antidiabetic, and antityrosinase effects, which may have implications for human health [43].

Heliotropamide (compound 14)—a phenolamide featuring an oxopyrrolidine-3-carboxamide core—was first isolated as the major constituent of the dichloromethane extract from the aerial parts of H. ovalifolium [51]. However, it has not been reported in other Heliotropium spp. so far. Regarding its biosynthesis, similar to compound 13, heliotropamide combines the phenylpropanoid with amine biosynthetic pathways. Unlike other phenolamides that typically involve polyamines, its formation specifically involves an aromatic amine, namely, tyramine. Structurally, heliotropamide contains two feruloyl moieties and two tyramine units, reflecting this biosynthetic origin [52,53]. Recently, it has also been isolated from the fruits of Lycium barbarum and tested for inhibition against α-glucosidase, exhibiting significant activity with an IC₅₀ of 33.53 μM, 5 times stronger than acarbose (IC₅₀ = 169.78 μM) [54].

4.2. Isolation of Helifoline-N-oxide

The structural characterization of helifoline-N-oxide (12) by NMR and mass spectral data established this compound as a saturated triol necine monoester N-oxide, providing a novel addition to the known PAs within Heliotropium species. Among the inherent challenges in PAs’ research is the isolation of N-oxide derivatives. Their chemical and physicochemical properties contribute significantly to this difficulty as N-oxides are highly polar, extremely water-soluble, and exhibit salt-like behavior, which complicates their extraction and purification using conventional methods. These characteristics, however, are closely linked to their biological roles since amine N-oxides, in contrast to their tertiary amines, are probably membrane-impermeable, a feature believed to be important for the transport and storage of PAs within plant tissues [9].

Within the genus Heliotropium, only a limited number of PANOs have been isolated, the majority of which correspond to the most frequently occurring unsaturated monoester N-oxides characteristic of the genus [28,30,31]. Previously reported triol necine N-oxides include those of rosmarinine, 12-O-acetylrosmarinine, angularine, 12-O-acetylhadiensine, and hadiensine [55] all of which are macrocyclic diesters, while 2-hydroxysarracine N-oxide, an open-chain diester, was detected in Senecio ovatus pollen [56]. To the best of our knowledge, helifoline-N-oxide represents the first monoester N-oxide derived from a triol necine.

4.3. PA Diversity in Heliotropium and H. procumbens

Given the detection of some relatively rare necine types in this species, the analysis was extended to examine the broader PA diversity within the Heliotropium genus. This comparative approach provides context for the chemical profile of H. procumbens and allows for an assessment of how its PA composition aligns with the genus-wide chemodiversity. To facilitate this, PAs were categorized primarily according to their necine base types and esterification patterns (Figure 4). While further subclassification based on the stereochemistry of the necine base or the type of necic acid could give deeper insights, such an exhaustive review falls outside the scope of the present study. The necine type, however, remains a key factor influencing PA toxicity (including saturation or unsaturation and the positions of hydroxylation that influence esterification potential), making it a relevant and practical parameter for comparative analysis.

The visual summary of PA chemodiversity across Heliotropium species reveals a clear predominance of 1,2-unsaturated necine types, of retronecine (RET), and supinidine (SUP). These unsaturated necines are most commonly found as monoesters (M), while diesters (DI) and macrocyclic (MC) esters occur less frequently. Among the saturated necine types, trachelanthamidine (TRA) is the most prevalent, followed by subulacine (SUB). Triol necines, such as helibracteatinecine or croalbinecine (TRIOL), as well as platynecine (PLAT), are encountered much more rarely.

It is important to recognize that the observed chemodiversity reflects not only true chemical variation but also the extent to which each species has been studied; species analyzed using more comprehensive analytical techniques or examined across multiple investigations naturally display greater apparent diversity in their PA profiles. For example, H. indicum has been extensively studied for its alkaloid content, leading to the identification of numerous PAs and PANOs [57], while the PA profiles of H. spatulatum and H. europeum have been expanded through more recent investigations [32,34].

H. procumbens is recognized as one of the more chemodiverse species within the genus, exhibiting a PA profile that encompasses a broad range of necine types. Its profile follows the general Heliotropium pattern, characterized by the presence of 1,2-unsaturated necine monoesters of the retronecine (RET) type, which are among the most common PAs in the genus. However, it also contains less commonly reported saturated necine types—trachelanthamidine (TRA), triol (TRIOL), and platynecine (PLAT)—which contribute to its unique chemical signature. The complementary analytical techniques applied in this study have enriched the previously reported alkaloid content, extending it from simple unesterified necines, such as lindelofidine, retronecine, supinidine, and trachelanthamidine [19], to include monoesters of RET and PLAT types, as well as triol necines and their esterified derivatives.

5. Conclusions

In the current study, the utilization of complementary analytical approaches (HPLC-ESI-DAD-IT-MS, UHPLC–HRMS, and GC-MS) enabled a thorough characterization of the chemical profile of the aerial parts of H. procumbens. Focusing on N-containing metabolites, a diverse array of PAs, along with two phenolamides, were detected. The combined use of these advanced techniques provided a highly efficient and accurate approach for comprehensive metabolite profiling, integrating sensitive detection, precise molecular identification, and structural confirmation.

Among the detected PAs, saturated types predominated over unsaturated ones, with triol necines and platynecine types—rarely reported in Heliotropium species—characteristically detected. This study presents the first report of N-oxides of unesterified triol necine bases and the N-oxide of helifoline, which was isolated as a natural product for the first time. The biosynthetically related phenolamide N¹,N¹⁰-diferuloylspermidine was also reported for the first time in the genus, alongside the detection of heliotropamide.

Comparison of the PA profile of H. procumbens with other species in the genus highlighted its considerable chemical diversity, which likely reflects both its inherent biosynthetic capabilities and the advantages of applying multiple analytical methods. These results encourage further research on less studied Heliotropium species and underscore the value of alkaloid detection for advancing chemical characterization and systematic insights within the genus.