Untargeted Metabolomic Profiling and Bioactivity Insights into Alkanna corcyrensis

Abstract

This study aimed to characterize the chemical composition and evaluate the biological activities of the aerial parts of Alkanna corcyrensis Hayek (AC), an endemic Greek species not previously studied. Phytochemical analysis of the methanolic extract was performed using UHPLC-ESI-Q-TOF–MS/MS combined with molecular networking analysis. Additionally, the total phenolic content (TPC) and total flavonoid content (TFC) were determined. Chromatographic separations were carried out to isolate major compounds, and the antioxidant capacity, along with enzyme inhibitory activity, was assessed. The analysis led to the tentative identification of 86 compounds, including 67 phenolic compounds (mainly caffeic acid derivatives and flavonoid glycosides), 10 pyrrolizidine alkaloids of trachelanthamidine, platynecine, and retronecine types, and nine organic and fatty acid derivatives. Among these, one flavonol glycoside (kaempferol-O-malonyl methyl ester hexoside) and three pyrrolizidine alkaloids (9-sarracinoyl-trachelanthamidine/isoretronecanol, retronecine-pentoside, and trachelanthamidine/isoretronecanol-hexoside) were reported for the first time. The extract exhibited high TPC (74.45 mg GAE/g extract) and TFC (46.66 mg GAE/g extract). Chromatographic separations resulted in the isolation of five major metabolites, namely rosmarinic acid, danshensu, kaempferol-3-O-glucoside, kaempferol-3-O-galactoside, and quercetin-3-O-glycoside. Biological evaluation revealed considerable antioxidant activity and inhibitory effects against α-glucosidase (6.65 mmol ACAE/g extract). Overall, this study highlights the remarkable phytochemical diversity and richness of AC among alkanet species and demonstrates its promising antioxidant potential, laying the foundation for further investigations towards its future exploitation.

1. Introduction

The genus Alkanna (alkanet) of the family Boraginaceae includes 66 species native to southern and east-central Europe, western Asia, and North Africa [1]. It is represented by a significant number of local and regional endemic species [2]. Alkanna corcyrensis Hayek (AC) is a perennial herb endemic to Greece [3]. Its name is derived from its geographical origin, Corfu Island, which is known as “Kerkyra-Corcyra” in Greek, where it is extensively distributed as well as in other Ionian Islands and seaside areas in northwest Greece [4]. The genus has been recognized for its medicinal properties since ancient times, especially for wound healing and further cosmeceutical uses attributed to the hydroxynaphthoquinones present in its roots [5]. As a result, research efforts have largely focused on alkanets’ root extracts, particularly in Alkanna tinctoria [6,7,8].

Due to the endemic nature of AC, there are no documented traditional uses reported for this species. However, AC root hexane extract has been chemically characterized, revealing the presence of alkannin derivatives including acetyl alkannin, isobutyl alkannin, angelic alkannin, β,β-dimethylacryl alkannin, and isovaleryl-α-methyl-n-butyl alkannin [9], while a later study quantified its content of alkannin/shikonin (A/S) derivatives [10]. More recently, AC seeds were found to be rich in fatty acids—especially gamma-linolenic acid. The seeds’ water-methanolic extract also showed strong antioxidant activity (2.56 mmol TE/100 g by DPPH, 1.24 mmol TE/100 g by ABTS) and high phenolic (525.3 mg GAE/100 g) and flavonoid content (365.3 mg QE/100 g), with a notable concentration of rosmarinic acid (433.7 mg/100 g), alongside phenolic acids like 4-hydroxybenzoic acid and trans-p-coumaric acid, as well as flavonoids such as rutin and quercetin-3-O-glucoside [11].

Few studies have focused on the aerial parts of alkanet species, where various phenolic compounds (PCs) have been identified, including hydroxycinnamic acid derivatives and flavonoids. Specifically, glycosides of quercetin and kaempferol, as well as derivatives of caffeic acid, such as rosmarinic acid, salvianolic acids, lithospermic acid, and rabdosiin, have been reported in aerial parts of Alkanna plants so far [12,13]. Furthermore, similarly with other genera among Boraginaceae plants, Alkanna is characterized by the presence of pyrrolizidine alkaloids (PAs), which are notable for their complex structural diversity while posing health risks due to their high hepatotoxicity [14]. The most studied species in this context are A. tinctoria and A. orientalis, which have been found to contain a significant number of PAs, mainly of retronecine type (e.g., 7-angeloyl-retronecine, 7- and 9-tigloyl-retronecine, triangularine, dihydroxy-triangularine) [15]. Recent research focusing on the alkaloid content of rare and endemic species from the Balkan region has led to the identification of new alkaloid derivatives, including glycoside derivatives of PAs [16,17].

Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) offers a powerful tool for detecting metabolites directly from complex plant matrices without requiring extensive purification steps. This approach provides rapid insights into phytochemical profiles, making it particularly advantageous when dealing with novel or low-abundance compounds [18]. Especially in the case of PAs, LC–MS/MS is recognized as an efficient analytical tool, offering the advantage of detecting N-oxides (PANOs, Pyrrolizidine Alkaloid N-Oxides) without requiring additional reduction steps [14,18]. Direct analysis of PANOs is important because they are naturally more abundant in plants than their respective tertiary nitrogen, parent PAs [19]. In fact, LC–MS/MS is considered a standard method for detecting alkaloids in food products under regulatory compliance guidelines, such as those outlined by the European Pharmacopoeia [20].

Untargeted metabolomics simultaneously analyzes hundreds to thousands of metabolites by processing all mass chromatogram signals without bias, offering a comprehensive view of the metabolome [21]. Identification typically relies on annotation strategies combining exact mass, isotope patterns, retention times, and structural information derived from MS/MS fragmentation data [22]. However, this approach also leads to much more complex data processing and subsequent analysis steps [21]. Computational tools such as the Global Natural Products Social Molecular Networking (GNPS) platform can assist by clustering structurally related molecules based on shared fragment patterns and matching them to reference spectra from curated databases [23,24]. However, interpreting large-scale MS/MS data remains difficult due to vast chemical diversity and incomplete spectral libraries, which are continuously updated as new compounds are characterized [25]. In general, no single approach has been established for comprehensive metabolite characterization, as metabolites exhibit a wide range of diverse physicochemical properties [21,22]. Recognizing chemical groups of interest helps to narrow down the scope of analysis, while having a broader understanding of the biosynthetic capacities of related species is particularly valuable when studying previously uncharacterized plants.

In the present study, as part of our ongoing research on Boraginaceae plants [12,17,26,27,28], a comprehensive UHPLC-ESI-Q-TOF–MS/MS analysis of AC’s chemical profile was performed, particularly focused on pharmacologically and toxicologically important chemical classes of secondary metabolites (PCs and PAs). The analysis is complemented by biological activity tests that assess antioxidant capacity and enzyme inhibitory effects against cholinesterase, α-amylase, and α-glucosidase enzymes. To our knowledge, the endemic species A. corcyrensis has never been studied before.

2. Materials and Methods

2.1. Plant Material and Extraction

The aerial parts of AC were collected from Preveza (Epirus, North-West Greece) during the flowering season and were botanically identified by Dr. E. Kalpoutzakis (Dept. of Pharmacy, NKUA). The plant material was deposited at the Herbarium of the Laboratory of Pharmacognosy Department of Pharmacy at the National and Kapodistrian University of Athens (NKUA) (voucher number IC238).

The aerial parts of the plant were naturally dried at room temperature in a shaded and well-ventilated environment. The air-dried material was then ground using a laboratory mill. A total of 9 g of powdered plant material underwent maceration with methanol (200 mL) at room temperature for 24 h. The extraction procedure was repeated three times. The combined extracts were filtered through white filter paper and evaporated under vacuum at a maximum temperature of 40 °C using a rotary evaporator, yielding 1.1 mg of methanolic extract.

2.2. Isolation of Compounds

A total of 0.67 g of the methanolic extract was subjected to column chromatography on a Sephadex LH-20 (25–100 μm, Pharmacia), using a gradient elution of dichloromethane: methanol (DCM: MeOH, 10:90 → 0:100), yielding 23 fractions (AC1–AC23). Fractions AC11 (5.7 mg) and AC17 (8.5 mg) yielded danshensu (salvianic acid A) and rosmarinic acid, respectively. Fraction AC19 (21.4 mg) was further purified by preparative thin-layer chromatography (prep. TLC) on reversed-phase silica gel RP-18 (Merck™, Darmstadt, Germany), using water: methanol (50:50) as the mobile phase. The resolved bands were scraped from the TLC plate and extracted with methanol, affording three flavonoid glycosides identified as kaempferol-3-O-β-glucoside, kaempferol-3-O-β-galactoside, and quercetin-3-O-β-glucoside. All isolated compounds were structurally characterized by NMR. ¹H-NMR spectra were recorded on a Bruker DRX 400 spectrometer (Bruker Corporation, Billerica, MA, USA), operating at 400 MHz and using methanol-d₄ (Euriso-Top, Cambridge Isotope Laboratories, Tewksbury, MA, USA) as the solvent. The chemical shift of methanol-d₄ (3.31 ppm) was used as an internal standard. Compound identification was confirmed by comparison with data in the literature [28,29]. All solvents used for chromatographic separations were of HPLC grade and purchased from Fisher Chemical (Fisher Scientific, Loughborough, Leics, UK).

2.3. Solid-Phase Extraction (SPE) for PAs/PANOs Enrichment

For the PA extraction, the dried and powdered aerial parts of the plant were processed following the standard BfR protocol [30]. Specifically, 2 g of the powdered sample was treated with 20 mL of 0.05 M sulfuric acid (H₂SO₄) and sonicated for 15 min in an ultrasonic bath. After extraction, the mixture was centrifuged at 3800 rpm for 10 min, and the clear supernatant was carefully separated by decantation. This extraction process was repeated with an additional 20 mL of the same acidic solution, and both supernatants were combined. The resulting total extract was then neutralized to a pH of 7 by adding 25% aqueous ammonia. The neutralized solution was filtered using filter paper, and solid phase extraction (SPE) was performed using C18 cartridges (C18-E, 55 μm, 70 Å, 500 mg/6 mL, Phenomenex Strata^®, Phenomenex Inc., Torrance, CA, USA) in a vacuum setup. The SPE protocol involved conditioning the cartridges first with 5 mL methanol, then with 5 mL deionized water, loading 10 mL of the neutralized extract, washing twice with 5 mL deionized water each time, drying the cartridges under low vacuum for 5 to 10 min, and finally eluting the PAs with two 5 mL portions of methanol.

2.4. UHPLC-ESI-Q-TOF–MS and MS/MS Analysis

UHPLC-ESI-Q-TOF–MS/MS analysis was conducted using an Agilent Technologies (Santa Clara, CA, USA) 6530 B system including an ESI-Jet Stream^® ion source, a quadrupole time-of-flight (Q-TOF) mass analyzer, a gradient pump, a diode array detector (DAD), an autosampler, and a column oven.

For the chromatographic analysis of AC extract, a Zorbax Stable Bond RP-18 column (250 × 2.1 mm, particle size = 5 μm) was utilized. The mobile phase consisted of solvent A: 1% acetonitrile in H₂O with 0.1% (v/v) formic acid and solvent B: 95% acetonitrile in H₂O with 0.1% (v/v) FA. A method with a total run time of 45 min was employed. Τhe gradient procedure was as follows: 0–5 min, 95% A/5% B (isocratic); 5–35 min, linear gradient from 95% A/5% B to 5% A/95% B; 35–40 min, linear gradient from 5% A/95% B back to 95% A/5% B. The column temperature was maintained at 25 °C, with a flow rate of 0.2 mL/min and an injection volume of 10 µL. Analysis was performed according to the following parameters of the ion source: dual spray jet stream electrospray (ESI) in negative ion mode; nitrogen flow rate: 10 L/min; nebulizer pressure: 35 psi, gas temp.: 350 °C; sheath gas temperature: 325 °C; fragmentor voltage: 140 V, 200 V, and 250 V; m/z range from 100 to 1000 units in Auto MS/MS acquisition mode.

For the chromatographic analysis of the PA/PANOs fraction, an Atlantis HILIC column was used (3.5 μm, 150 mm × 2.1 mm) (Waters, Milford, MA, USA). The mobile phase consisted of solvent A: acetonitrile (95%) with 10 mM ammonium formate (0.2%) and solvent B: acetonitrile (50%) with 10 mM ammonium formate (0.2%). Τhe gradient procedure was as follows: 0–10 min (isocratic), 100% (A)–0% (B); 10–30 min (linear gradient), 100% (A) to 92% (A); 30–40 min, 92% (A) to 64% (A), 40–45 min (linear gradient), 64% (A) to 100% (A). The total analysis time was 45 min with a flow rate of 0.25 mL/min. The injection volume was set at 5 μL. Analysis was performed according to the following parameters of the ion source: dual spray jet stream ESI, in positive ion mode; nitrogen flow rate: 10 L/min; nebulizer pressure: 35 psi; gas temp.: 350 °C; sheath gas temp.: 325 °C; fragmentor voltage: 120 V; m/z range from 50 to 700 units in Auto MS/MS acquisition mode.

For both chromatographic analyses, data acquisition was performed using Mass Hunter version 2.2.1 (Agilent Technologies).

2.5. Data Processing

Data processing was carried out using MZMine 4.2.0/MZIO software. The raw UHPLC-HRMS files from Agilent (*.D) were first converted to the *.mzML format using the MSConvert tool from the ProteoWizard suite [31,32]. For data processing in the MZMine environment and the corresponding parameters, consult the Supplementary Information (Supplementary Materials Table S1). The tentative identification of compounds was performed according to the accurate mass, the potential adducts and isotopes in correlation with MS/MS fragmentation spectra, and by comparison with commercial and noncommercial databases and spectral libraries (e.g., Dictionary of Natural Products, library mzCloud), as well as data from literature.

2.6. Molecular Networking and Chemical Dereplication

The processing output (MS1, MS2 spectral data, and sample metadata) was subsequently used for the construction of a feature-based molecular network (FBMN) using the comprehensive Global Natural Products Social Molecular Networking (GNPS2) platform [33,34]. The parametrization within the GNPS2 environment included the following general parameters: precursor ion tolerance set to 0.02 Da; fragment ion tolerance set to 0.02. For the networking parameters, the following were applied: minimum cosine score of 0.7; minimum matched peaks of 4. The data from the MZMine were also imported into the robust SIRIUS computational environment [35] to predict the identity and chemical classes of compounds using the built-in tools and algorithms CSI: FingerID [36], CANOPUS [37], and ClassyFire [38]. The outputs from the GNPS2 and SIRIUS analyses were integrated into the Cytoscape environment (version 3.10.2, Cytoscape Consortium, San Diego, CA, USA) [39]. The FBMN output and parametrization of the corresponding workflow can be found on the GNPS2 repository: https://gnps2.org/status?task=44576daacb844287930eb5655fb5f4ad (accessed on 27 January 2025). Detailed networking parameters can be found in Table S1.

2.7. Total Phenolic Content (TPC) and Flavonoid Content (TFC) Assays

The TPC and TFC values of the extract were determined by colorimetric assays, using well-established procedures such as the Folin–Ciocalteu method for TPC and the aluminum trichloride (AlCl₃) method for TFC [28,40]. In brief, for the evaluation of TPC, 0.25 mL of the sample solution (2 mg/mL in methanol) was combined with 1 mL of diluted Folin–Ciocalteu reagent (1:9). After incubating for 3 min, 0.75 mL of a 1% Na₂CO₃ solution was added. The mixture was incubated in darkness at room temperature for 2 h, and its absorbance was measured at 760 nm. TPC results were expressed as milligrams of gallic acid equivalents per gram of extract (mg GAE/g of extract), and the calibration curve for standard gallic acid at different concentrations (0–5 µg) was y = 0.2163x, R²: 0.995.

For the evaluation of TFC, 1 mL of the sample solution (2 mg/mL in methanol) was mixed with 1 mL of 2% AlCl₃ in methanol. To prepare the blank of the sample, 1 mL of the sample solution was mixed with 1 mL of methanol (without AlCl₃). After incubating for 10 min at room temperature, the absorbances were measured at 415 nm (Shimadzu UV-1800). TFC results were expressed as milligrams of rutin equivalents per gram of extract (mg RE/g of extract), and the calibration curve for standard rutin at different concentrations (0–20 µg) was y = 0.1301x + 0.05, R²: 0.994.

Both quantification assays were conducted in triplicate, and the results were expressed as mean values and standard deviation (SD).

2.8. Biological Activity Assays

2.8.1. Antioxidant Assays

The antioxidant properties of AC extract were evaluated using established antioxidant assays, including metal chelating, phosphomolybdenum, FRAP (Ferric Reducing Antioxidant Power), CUPRAC (Cupric Reducing Antioxidant Capacity), ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays [28,40]. All antioxidant assays were performed in triplicate, and the results were expressed as mean values and standard deviation (SD).

In the DPPH assay, 1 mL of the sample solution (2 mg/mL in methanol) was mixed with 4 mL of a 0.004% DPPH methanol solution. The mixture was incubated for 30 min in the dark at room temperature, and its absorbance was measured at 517 nm. The result was expressed as milligrams of Trolox equivalent per gram of dry extract (mg TE/g), and the calibration curve for standard Trolox at different concentrations (0–5 µg) was y = 14.058x + 1.932, R²: 0.995.

For the ABTS assay, the radical cation was produced by incubating a 7 mM solution of ABTS•+ with 2.45 mM potassium persulfate in the dark at room temperature. Subsequently, 1 mL of the sample solution (2 mg/mL in methanol) was mixed with 2 mL of the ABTS•+ solution, and the absorbance was measured at 734 nm, following a 30 min incubation at room temperature. The result was expressed as the Trolox equivalent (mg TE/g), and the calibration curve for standard Trolox at different concentrations (0–2.5 µg) was y = 12.054x − 2.7398, R²: 0.967.

In the FRAP assay, 0.1 mL of the sample solution (2 mg/mL in methanol) was mixed with 2 mL of the reagent solution comprising 10 mM 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl, acetate buffer (0.3 M, pH 3.6), and 20 mM ferric chloride in a volume ratio of 1:10:1 (v/v/v). After incubating for 30 min at room temperature, the absorbance was measured at 593 nm. The result was expressed as the Trolox equivalent (mg TE/g), and the calibration curve for standard Trolox at different concentrations (0–2.5 µg) was y = 0.3178x + 0.0053, R²: 0.999.

For the CUPRAC assay, 0.5 mL of the sample solution (2 mg/mL in methanol) was combined with a mixture of 1 mL of 10 mM CuCl₂, 1 mL of 7.5 mM neocuproine, and 1 mL of ammonium acetate buffer (1 M, pH 7.0). Absorbance was recorded at 450 nm after a 30 min incubation at room temperature. The result was expressed as the Trolox equivalent (mg TE/g), and the calibration curve for standard Trolox at different concentrations (0–2.5 µg) was y = 0.1317x + 0.0068, R²: 0.996).

In the phosphomolybdenum assay, 0.3 mL of the sample solution (2 mg/mL in methanol) was mixed with 3 mL of the reagent solution comprising 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. The mixture was incubated at 95 °C for 90 min, and absorbance was measured at 695 nm. The result was expressed as the Trolox equivalent (mmol TE/g), and the calibration curve for standard Trolox at different concentrations (0–100 µg) was y = 0.0061x + 0.0056, R²: 0.999.

For the metal chelating activity assay, a 2 mL portion of the sample solution (2 mg/mL in methanol) was mixed with 0.05 mL of a 2 mM FeCl₂ solution. Then, 0.2 mL of a 5 mM ferrozine solution was added. The absorbance was measured at 562 nm after incubating the mixture for 10 min at room temperature. The result was expressed as the EDTA equivalent (mg EDTAE/g), and the calibration curve for standard EDTA at different concentrations (0–4 µg) was y = 13.314x − 3.5881, R²: 0.978).

2.8.2. Enzyme Inhibition Assays

Τhe enzyme inhibitory activity of AC extract was assessed against key enzymes such as cholinesterases (AChE and BChE), α-amylase, and α-glucosidase [28,40]. All enzyme inhibition assays were performed in triplicate, and the results were expressed as mean values and standard deviation (SD).

For the cholinesterases inhibition assays, 50 μL of the sample solution (2 mg/mL in methanol) was combined with 125 μL of DTNB (5,5′-dithiobis (2-nitrobenzoic acid), 0.3 mM), and 25 μL of AChE or BChE solution (0.03 unit/mL) in Tris-HCl buffer (50 mM, pH 8.0). The mixtures were incubated at room temperature for 15 min. The reactions were then initiated by adding 25 μL of acetylthiocholine iodide (1.5 mM) or butyrylthiocholine chloride (1.5 mM). After a further 10 min incubation at room temperature, the absorbances of the mixtures were measured at 405 nm. Similarly, a blank was prepared by adding the sample solution to all reaction reagents without the enzyme (AChE or BChE) solution. The results were expressed in milligrams of galantamine equivalents per gram of extract (mg GALAE/g of extract), and the calibration curve for standard galantamine at different concentrations (0–2 µg) was y = 185.44x + 0.5502, R²: 0.996.

In the α-amylase inhibitory assay, 50 μL of the enzyme solution (10 units/mL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) was added to 25 μL of the sample solution (2 mg/mL in methanol). The mixture was incubated at 37 °C for 10 min. Subsequently, 50 μL of a 0.05% starch solution was added, and the mixture was incubated for another 10 min at 37 °C. Similarly, a blank was prepared by adding the sample solution to all reaction reagents without the enzyme (α-amylase) solution. The reaction was stopped by adding 25 μL of 1 M hydrochloric acid (HCl), followed by the addition of 100 μL of an iodine-potassium iodide solution. The absorbance of the mixture was measured at 630 nm. Results were expressed in millimoles of acarbose equivalents per gram of extract (mmol ACAE/g of extract), and the calibration curve for standard acarbose was y = −0.0312x² + 3.0426x + 0.4457, R²: 0.993, concentration range: 0–10 µg.

In the α-glucosidase inhibitory assay, 50 μL of the sample solution (2 mg/mL in methanol) was mixed with 50 μL of glutathione (0.5 mg/mL), 50 μL of α-glucosidase solution (0.2 unit/mL) in phosphate buffer (pH 6.8, 0.1 M phosphate buffer), and 50 μL of p-nitrophenyl-α-D-glucopyranoside (10 mM, PNPG). The mixture was incubated at 37 °C for 15 min. Similarly, a blank was prepared by adding the sample solution to all reaction reagents without the enzyme (α-glucosidase) solution. To terminate the reaction, 50 μL of 0.2 M sodium carbonate was added. The absorbances were measured at 400 nm. Results were expressed in millimoles of acarbose equivalents per gram of extract (mmol ACAE/g of extract), and the calibration curve for standard acarbose was y = −0.0013x² + 0.686x + 1.1301, R²: 0.994, concentration range: 0–300 µg).

3. Results

3.1. Analysis of AC Extract

UHPLC-ESI-Q-TOF–MS analysis of AC extract led to the tentative identification of 76 metabolites in negative ionization mode. Compound annotation was performed by interpreting the MS and MS/MS fragmentation data and by comparison with the data in the literature. The annotated compounds, along with their major detected fragments, are presented in

Table 1. UHPLC-ESI-Q-TOF–MS/MS analysis of Alkanna corcyrensis methanolic extract.

No.	Identified Compounds	Rt (min)	Relative Abundance (%)	Precursor Ion	Molecular Formula	Error (ppm)	MS/MS	Reference
HYDROXYCINNAMIC ACIDS
Coumaroyl derivatives
1.	Coumaric acid *	7.79	0.09	163.0387	C9H8O3	−5.02	119.05	[41]
2.	Coumaroyl-glyceric acid	7.34	0.14	251.0544	C12H12O6	−4.63	163.04/145.03/119.05/105.02	[42]
3.	Coumaroyl- threonic/erythronic acid	6.92	0.18	281.0649	C13H14O7	−4.72	163.04/135.03/119.05/75.01	[43]
4.	Coumaroyl malic acid methyl ester	9.45	0.15	293.0655	C14H14O7	−2.14	163.04/145.03/133.05/119.04	[44]
5.	Coumaroyl-threonic/erythronic acid methyl ester	6.47	0.09	295.0805	C14H16O7	−4.33	193.04/163.04/149.03/133.05/119.05	[45]
6.	Coumaric acid hexoside	4.89	0.05	325.0911	C15H18O8	−3.82	179.05/163.04/145.02/135.04/119.05	[46]
Feruloyl derivatives
7.	Feruloyl malic acid methyl ester	10.7	0.21	323.0758	C15H16O8	−2.76	193.04/175.04/149.06/134.04	[47]
Caffeoyl derivatives
Monomers
8.	Caffeic acid *	4.23	0.57	179.0332	C9H8O4	−6.89	135.05	[41]
9.	Methyl caffeate	15.4	0.19	193.0493	C10H10O4	−4.06	161.02/133.03	[48]
10.	Danshensu *	2.27	0.42	197.0460	C9H10O5	5.08	179.03/135.04/123.05/93.03/72.99	[49]
11.	Caffeoyl-glyceric acid	4.5	2.28	267.0484	C12H12O7	−7.78	179.04/161.02/135.04/105.02/75.01	[50]
12.	Caffeoyl-threonic/erythronic acid	2.95	0.24	297.0628	C13H14O8	6.25	179.03/161.02/135.03/75.01	[50]
13.	Caffeoyl malic acid methyl ester	4.93	0.45	309.0605	C14H14O8	−1.75	179.03/161.02/135.04/119.05	[51]
14.	Caffeoyl-methyl threonic/erythronic acid	3.89	0.29	311.0768	C14H16O8	0.35	179.03/163.04/149.05/119.05	[52]
Dimers
15.	Nepetoidin B (Isomer I)	32.62	0.33	313.0710	C17H14O6	−0.68	161.02/151.04/133.03/123.04	[53]
16.	Nepetoidin B (Isomer II)	31.84	0.20	313.0717	C17H14O6	1.56	161.02/151.04/133.03/123.04	[53]
17.	Caffeoyl-4-hydroxyphenyllactic acid	23.98	0.19	343.0823	C18H16O7	1.52	197.04/179.03/161.02/145.03/135.04/72.99	[54]
18.	Rosmarinic acid *	18.52	14.3	359.0778	C18H15O8	3.08	197.05/179.03/161.02	[41]
19.	Rosmarinic acid methyl ester	25.63	0.21	373.0928	C19H18O8	1.23	197.04/175.04/135.04	[50]
20.	Danshen suan C (Salvianic acid C)	4.5	0.16	377.0857	C18H18O9	−4.13	198.05/179.03/161.02/135.04	[55]
21.	Rosmarinic acid hexoside Ι	12.77	0.15	521.1277	C24H26O13	−3.48	359.09/197.04/161.03	[54]
22.	Rosmarinic acid hexoside ΙΙ	14.08	0.18	521.1280	C24H26O13	−2.91	359.09/197.05/179.03/135.04	[54]
Trimers
23.	Salvianolic acid C *	28.52	0.85	491.0973	C26H20O10	−1.06	311.06/295.06/185.02/135.04	[49]
24.	Salvianolic acid A *	21.73	1.09	493.1117	C26H22O10	−3.59	295.06/185.02/109.03	[54]
25.	Yunnaneic acid D	9.21	0.71	539.1171	C27H24O12	−3.43	359.07/297.07/197.04/161.02/135.04	[49]
26.	Yunnaneic acid I	19.42	0.18	541.1339	C27H26O12	−1.30	343.08/329.07/299.09/267.06/197.04/135.04	[56]
27.	Monomethyl lithospermate	29.12	0.10	551.1195	C28H24O12	1.00	519.08/353.07/339.05/321.04/295.05/197.04	[57]
28.	Salvianolic acid K	11.54	0.14	555.1151	C27H24O13	2.22	467.13/359.07/313.07/269.08/197.04/135.04	[58]
29.	Yunnaneic acid E *	11.5	0.74	571.1079	C27H24O14	−1.54	527.12/439.14/285.07/241.09/197.04	[49]
30.	Yunnaneic acid F *	11.22	0.32	597.1224	C29H26O14	−3.40	491.14/329.10/311.09/267.10/197.04/179.03/135.04	[49]
Tetramers
31.	Sebesteniod E	27.66	0.18	671.1394	C35H28O14	−1.01	520.08/473.08/321.03/169.91	[56]
32.	Lithospermic acid B *	27.75	0.30	717.1428	C36H30O16	−3.85	519.09/321.04	[50]
33.	Trigonotin B/Rupestrin A	19.11	0.19	733.1950	C34H38O18	−4.08	689.21/367.07/323.09/193.01/161.04/125.02	[59]
34.	Sodium rabdosiin *	27.62	0.28	739.1259	C36H29NaO16	−2.17	559.08/515.10/335.06/291.06	[12]
35.	Sodium lithospermate B	18.89	0.23	741.1400	C36H31O16Na	−4.26	579.10/381.06/219.02/197.04/179.03/135.04	[60]
36.	Sodium salvianolic acid E	17.6	0.27	741.1428	C36H31O16Na	−0.48	561.10/381.06/161.02	[60]
37.	Trigonotin A/Rupestrin C	13.64	0.32	763.2083	C35H40O19	−0.33	633.03/500.10/369.10	[59]
38.	Pulmonarioside C/Echiumin E	15.44	0.22	983.2814	C47H52O23	−0.73	837.23/793.23/629.18/469.13/367.08/145.03	[45]
39.	Pulmonarioside C/Echiumin E	20.41	0.13	983.2816	C47H52O23	−0.52	837.22/793.21/469.13/367.08	[45]
FLAVONOIDS AND FLAVONOID GLYCOSIDES
Kaempferol derivatives
40.	Kaempferol	32.73	0.04	285.0396	C15H10O6	−1.10	285.04/255.03/227.03/151.00	[61]
41.	Kaempferol-methylether *	33.24	0.05	299.0548	C16H12O6	−2.55	284.03/255.03/227.03	[62]
42.	Kaempferol-O-hexoside I *	14.99	0.94	447.0929	C21H20O11	0.37	447.09/284.03/151.00	[61]
43.	Kaempferol-O-hexoside II *	16.2	7.71	447.0930	C21H20O11	0.59	447.09/284.03/151.00	[61]
44.	Kaempferol-O-acetyl-hexoside I *	22.74	0.58	489.1037	C23H22O12	0.82	285.04/284.03/227.03	[63]
45.	Kaempferol-O-acetyl-hexoside II *	24.91	0.08	489.1046	C23H22O12	2.66	285.04/255.03	[63]
46.	Kaempferol diacetyl-deoxyhexoside	28.87	0.15	515.1177	C25H24O12	−2.43	285.04/255.02/179.03	[64]
47.	Kaempferol-O-malonyl hexoside	20.56	0.08	533.0938	C24H22O14	1.26	489.10/285.04	[50]
48.	Kaempferol-O- (malonyl methyl ester)-hexoside	24.79	0.09	547.1107	C25H24O14	−2.91	515.09/489.09/471.09/447.09/309.04/285.04
49.	Kaempferol-triacetyl hexoside	22.96	0.09	573.1255	C27H26O14	1.87	489.10/368.99/285.04/125.02
50.	Kaempferol-O-hexose-deoxyhexose I *	13.09	0.09	593.1490	C27H30O15	−2.77	431.10/285.04/284.03/255.03	[41]
51.	Kaempferol-O- hexose-deoxyhexose II *	14.95	0.34	593.1500	C27H30O15	−1.09	447.08/285.04/284.04/255.03/227.03	[41]
52.	Kaempferol-O-caffeoyl-hexoside	25.51	0.09	609.1264	C30H26O14	3.23	447.09/323.08/285.04/179.03	[65]
Quercetin derivatives
53.	Quercetin *	28.66	0.07	301.0346	C15H10O7	−0.76	178.99/151.00/107.02	[66]
54.	Quercetin-O-hexoside *	12.18	6.96	463.0863	C21H20O12	−2.92	300.03/301.03/151.00	[61]
55.	Quercetin-O-acetyl-hexoside I *	16.66	3.16	505.0996	C23H22O13	3.83	300.03	[63]
56.	Quercetin-O-acetyl-hexoside II *	18.48	0.11	505.1005	C23H22O13	2.74	446.09/360.08/300.03/151.00	[63]
57.	Quercetin-O-malonyl-hexoside	16.7	0.30	549.0902	C24H22O15	3.92	505.10/463.10/301.03	[63]
58.	Rutin *	9.85	0.07	609.1438	C27H30O16	−2.89	300.03	[66]
Other flavonoid derivatives
59.	Dimethoxy-dihydroxy-flavone *	36.68	0.12	313.0717	C17H14O6	1.56	298.05/283.02	[67]
60.	Isorhamnetin	30.08	0.18	315.0508	C16H12O7	1.02	300.03/271.03/255.03	[68]
61.	Dimethoxy-trihydroxy-flavone *	34.87	0.15	329.0654	C17H14O7	−2.21	314.04/299.02/285.03/271.02	[67]
62.	Trimethoxy-dihydroxy-flavone *	35.2	0.13	343.0818	C18H16O7	0.06	328.06/313.03/298.01/285.03/270.01	[67]
63.	Dimethoxy-tetrahydroxyflavone	29.78	0.06	345.0605	C17H14O8	−1.57	330.04/315.01/287.01	[67]
64.	Myricetin-O-hexoside	6.39	0.22	479.0810	C21H20O13	−3.27	317.02/316.02/271.03/178.99/151.01	[69]
65.	Dimethoxy-trihydroxy-flavone-O-hexoside	31.1	0.08	491.1165	C23H24O12	−4.99	429.11/329.06/314.04/135.04	[70]
66.	Trihydroxy-trimethoxyflavone-O-hexoside *	27.83	0.29	521.1287	C24H26O13	−1.57	359.07/225.86	[65]
COUMARINS
67.	Hydroxy coumarin	18.52	0.62	161.0243	C9H6O3	2.68	133.03	[54]
ORGANIC ACIDS
68.	malic acid *	1.8	0.25	133.0150	C4H6O5	9.79	115.01/73.00/71.02	[71]
69.	Azelaic acid	13.84	0.10	187.0962	C9H16O4	−4.46	125.10/123.08/98.08	[72]
70.	Citric acid	2.13	0.57	191.0202	C6H8O7	5.35	129.02/111.01/87.01/85.03	[71]
71.	Gluconic/galactonic acid *	1.8	0.25	195.0507	C6H12O7	1.14	162.03/105.02/99.01/87.00/75.01/71.01	[72]
FATTY ACIDS
72.	Trihydroxy-octadecenoic acid	32.38	0.15	329.2319	C18H34O5	−2.73	331.25/229.14/211.13/171.10	[57]
73.	Hydroxy-octadecadienoic acid	38.84	0.14	295.2267	C18H32O3	−2.10	183.14	[73]
74.	Trihydroxy-octadecadienoic acid	31.02	0.13	327.2167	C18H32O5	−1.37	327.21/229.14/211.13/171.10	[57]
75.	Hydroxy-octadecatrienoic acid	38.04	0.12	293.2110	C18H30O3	−2.28	275.20/235.17/223.17/183.10	[73]
76.	Hydroxy-octadecenoic acid *	39.65	0.08	297.2432	C18H34O3	0.77	297.24/279.23/171.10	[40]

* Previously reported within the Alkanna genus. The most abundant ions are shown in bold. Compounds shown in bold are isolated, with their ¹H-NMR data available in Supplementary Materials.

.

3.2. Molecular Network

The global molecular network constructed by the GNPS2 workflow is presented in Figure 1, showing cluster linking nodes based on their MS/MS spectral/fragmentation similarities, expressed through a cosine similarity coefficient above 0.7, with at least four common ions, along with solitary nodes that are not related to clusters due to differing chemical structures or having fewer than four ions in common with other nodes. The major clusters were highlighted, representing the major chemical classes of secondary metabolites contained in the AC extract.

3.3. LC–MS Analysis of PAs/PANOs Fraction

LC–MS analysis of the obtained PAs/PANOs fraction resulted in the tentative identification of seven PAs and three PANOs (

Table 2. Annotated alkaloids from AC PAs/PANOs fraction analysis by LC–MS.

No.	Identified Compounds	Rt (min)	Relative Abundance (%)	Precursor Ions	Type	Molecular Formula	Δppm	MS/MS	Reference
PA1.	Trachelanthamidine/Isotretronecanol	11.62	5.26	142.1223	TRA	C8H15NO	−2.4	124.11/108.08	[74]
PA2.	Platynecine *	4.6	1.26	158.1166	PLAT	C8H15NO2	−6.04	140.11/122.09/105.07	[17]
PA3.	9-sarracinoyl-trachelanthamidine/isotretronecanol	16.96	0.74	240.1594	TRA	C13H21NO3	−0.08	142.12/124.11/108.08
PA4.	Retronecine-pentoside	3.91	0.57	288.1435	RET (monoester)	C13H21NO6	−0.57	156.10/138.09/120.08/108.08
PA5.	Trachelanthamidine/Isotretronecanol-hexoside	8.95	8.16	304.1758	TRA	C14H25NO6	1.1	142.12
PA6.	Leptanthine	12.41	1.41	316.1751	RET (monoester)	C15H25NO6	−1.15	298.16/254.14/156.10/139.10/138.09/122.09/120.08/108.08	[17]
PA7.	Leptanthine-N-oxide	4.9	1.70	332.1709	RETNO (monoester)	C15H25NO7	1.57	172.09/155.09/138.08/111.06	[17]
PA8.	Dihydroxy triangularine *	13.4	7.41	370.1859	RET (diester)	C18H27NO7	−0.35	341.12/326.16/288.14/238.14/220.13/138.09/120.08	[17]
PA9.	Dihydroxy triangularine N-oxide *	13.1	6.45	386.1796	RETNO (diester)	C18H27NO8	−3.48	254.14/220.13/154.08/136.07	[17]
PA10.	7-senecioyl-9-(3-acetoxy-2-hydroxy-2-methylbutyryl) retronecine N-oxide *	14.29	1.11	412.196	RETNO (diester)	C20H29NO8	−1.44	238.14/138.09/120.08/103.05	[17]

* Previously reported within the Alkanna genus.

). Compounds were further classified according to their necine base moiety as trachelanthamidine type (TRA), platynecine type (PLAT), retronecine type (RET), and the respective N-oxides (RETNO) type.

3.4. TPC and TFC

The methanolic extract of AC was assessed for its total phenolic and total flavonoid content, and the results are presented in

Table 3. Total phenolic and total flavonoid contents of AC methanolic extract.

Phytochemical Assays	AC *
TPC (mg GAE/g of extract)	74.45 ± 4.37
TFC (mg RE/g of extract)	46.66 ± 0.11

* Values expressed are means ± S.D. of three parallel measurements. GAE: gallic acid equivalents; RE: rutin equivalents.

.

3.5. Biological Activity

3.5.1. Antioxidant Activity Assays

To evaluate the antioxidant potential of the AC methanolic extract, six different assays were conducted. The DPPH and ABTS assays were used to assess radical scavenging activity. The phosphomolybdenum, CUPRAC, and FRAP assays were employed to determine the extract’s reducing power. Furthermore, a metal chelating assay was performed to evaluate the extract’s metal ion binding capacity. The results of all antioxidant assays are summarized in

Table 4. Antioxidant properties of AC methanolic extract.

Antioxidant Assays	AC *
DPPH• (mg TE/g of extract)	227.01 ± 2.15
ABTS•+ (mg TE/g of extract)	450.91 ± 11.42
FRAP (mg TE/g of extract)	365.59 ± 0.61
CUPRAC (mg TE/g of extract)	462.42 ± 7.95
Phosphomolybdenum (mmol TE/g of extract)	2.11 ± 0.02
Metal chelating (mg EDTAE/g of extract)	7.91 ± 0.53

* Values expressed are means ± S.D. of three parallel measurements. TE: trolox equivalents; EDTAE: EDTA equivalents.

.

3.5.2. Enzyme Inhibitory Activity Assays

The enzyme inhibitory effects of AC were assayed against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-amylase, and α-glucosidase. The results are presented in

Table 5. Enzyme inhibitory activities of AC methanolic extract.

Enzyme Inhibitory Assays	AC *
AChE inhibition (mg GALAE/g of extract)	1.43 ± 0.10
BChE inhibition (mg GALAE/g of extract)	0.38 ± 0.14
α-amylase inhibition (mmol ACAE/g of extract)	0.47 ± 0.01
α-glucosidase inhibiton (mmol ACAE/g of extract)	6.65 ± 0.18

* Values expressed are means ± S.D. of three parallel measurements. AChE: acetylcholinesterase; BChE: Butyrylcholinesterase; GALAE: galanthamine equivalents; ACAE: acarbose equivalents.

.

4. Discussion

For the analysis of AC, the methodology followed was consistent with previously described approaches for the aerial parts of Alkanna species, such as A. sfikasiana, A. kotschyana, A. orientalis, and A. tinctoria [12,26]. Other solvents and extraction techniques have also been tested for the analysis of Alkanna’s aerial parts [40,75]. For example, different extraction solvents tested in A. tubulosa showed qualitative and quantitative variations in the chemical profile, as well as differences in the biological activities exhibited by the extracts [40].

There is a tendency toward using solvents that are safe for human consumption, reflecting their relevance in potential therapeutic applications. However, several Alkanna species contain PAs [76], which pose significant health risks if not properly controlled. The characterization of the PA profile in AC extract was conducted using the BfR protocol based on German Federal Institute for Risk Assessment (BfR) guidelines. The method involves extracting PAs from plant material using an acidic aqueous solution, and purifying the obtained extract by solid-phase extraction [30]. Acidified extraction methods have previously been applied in Alkanna species for the recovery of PAs [14,17]. However, PAs have also been extracted using other solvents such as water, ethanol, or their mixtures, as reported in A. tubulosa, and maceration with methanol has also been employed [14,40].

For annotation, it is ideal to have informative fragmentation patterns, since the detection of prominent fragment ions helps the study of the fragmentation behaviors of the representative components. PCs generally fragment in more predictable patterns when the negative ionization mode is used [77]. The acidic functional groups—such as the multiple hydroxyl groups present in flavonoids and caffeic acid derivatives—readily undergo deprotonation, forming stable negative ions and improving ionization efficiency in this mode. In contrast, PAs require a positive ionization mode because of their basic properties. The fragmentation behavior of various PA structural types has been extensively studied due to the importance of risk assessment and the establishment of legislation limiting PA levels in food products [20,78]. The characteristic and predictable fragmentation patterns of PAs depend on factors such as the necine base, necic acid, ester type, and the N-oxidation status of the bicyclic ring system [78].

4.1. PCs Analysis

4.1.1. Hydroxycinnamic Acid Derivatives

Among the PCs detected, a total of 39 hydroxycinnamic acid derivatives were identified in AC, representing the largest category of PCs found in the methanolic extract. A more detailed classification revealed that they were primarily composed of coumaroyl (1–6) and caffeoyl moieties (8–39).

Coumaroyl Derivatives

Coumaric acid (1), along with five derivatives (2–6), was tentatively identified in AC (Table 1). Although compound 1 has been reported in A. tubulosa and A. trichophila [40,75], no other coumaroyl derivatives have been found in Alkanna species. Coumaric acid (1), with a precursor ion at m/z 163.0387, exhibited a characteristic fragment ion at m/z 119 ([M-H-44]⁻), corresponding to the loss of CO₂ [66]. Fragment ions at m/z 163 and m/z 119 were also observed in MS/MS spectra of 2–6, indicating the presence of a coumaroyl moiety. Compounds 3 and 5 were tentatively annotated as conjugates of coumaric with threonic/erythronic acid and its methyl ester. In addition to the common fragment ions corresponding to coumaric acid, the MS/MS fragments at m/z 135 (C₄H₇O₅⁻) and m/z 149 (C₅H₉O₅⁻) corresponded to the deprotonated ions of threonic/erythronic acid and its methyl ester, respectively. Notably, coumaroyl-threonic acid methyl ester has been previously isolated from Onosma bracteatum from the Boraginaceae family, too [45].

Caffeoyl Derivatives

Caffeic acid and its derivatives emerged as the main class of hydroxycinnamates, with a total of 31 metabolites identified. They are categorized into monomeric, dimeric, trimeric, and tetrameric assemblies of caffeoyl units. The findings align with Boraginaceae’s phytochemical profile, where caffeic acid and its derivatives are commonly found, ranging from monomers and dimers (e.g., rosmarinic acid) to trimers (e.g., lithospermic acid) and tetramers like rabdosiin and lithospermic acid B [79].

• Monomers

Caffeic acid (8), with a precursor ion at m/z 179.0332, exhibited a characteristic MS/MS fragment ion at m/z 135 ([M-H-44]⁻), corresponding to the loss of CO₂ [66]. Compound 10, already described in A. tubulosa, has been identified as danshensu (salvianic acid A) and isolated from AC extract. Caffeic acid conjugates with organic acids, such as glyceric, threonic/erythronic, and malic acid, were also detected (11–14). For instance, compound 12 was putatively identified as the threonic/erythronic ester of caffeic acid based on its MS/MS spectra with the characteristic product ion at m/z 179 ([M-H-C₄H₆O₄]⁻), corresponding to the loss of the dehydrated threonic/erythronic acid moiety. Additional product ions at m/z 161 and m/z 135 confirmed the caffeoyl moiety [66]. Although the presence of 3-O-(E)-caffeoyl-threonic acid and 2-O-(E)-caffeoyl-threonic acid has been previously reported from Pulmonaria officinalis in Boraginaceae [50], caffeoyl conjugates with organic acids are novel to the Alkanna genus.

• Dimers

Among caffeic acid dimers, rosmarinic acid (18) has been previously suggested as a chemotaxonomic marker for plants in the Boraginaceae family [12], and, as a major metabolite, it has been isolated from AC extract. Its fragmentation pattern, well-documented in the literature, showed a precursor ion at m/z 359 and characteristic fragment ions at m/z 197, 179, and 161 [41,54,71]. Additionally, the AC extract contained several of its derivatives, including rosmarinic acid methyl ester (19) and two hexosides of rosmarinic acid (21, 22). Compounds 15 and 16 were characterized as isomers of nepetoidin B previously reported from Boraginaceae species such as Cordia dichotoma [53], while 17, with a deprotonated molecular ion at m/z 343.0823, was identified as caffeoyl-4-hydroxyphenyllactic acid [54]. Notably, the detected caffeoyl dimers, except rosmarinic acid, are reported for the first time in Alkanna species.

• Trimers

Regarding caffeic acid trimers, salvianolic acid C (23) has been previously reported in A. tubulosa, A. orientalis, A. tinctoria, and A. kotschyana [12,40]. It exhibited a precursor ion at m/z 491.0973 along with characteristic fragment ions at m/z 311 ([M-H-C₉H₈O₄]⁻) and m/z 295 ([M-H-C₉H₈O₄-H₂O]⁻), indicating the loss of a caffeic acid unit and of H₂O, respectively. Compound 27 was identified as monomethyl lithospermate, and, although it has been detected in Symphytum anatolicum from Boraginaceae [57], there are no reports of its presence in Alkanna spp.

Four yunnaneic acids (25, 26, 29, 30) have also been identified in AC [49,50]. Yunnaneic acids D, E, and F, first isolated from Salvia yunnanensis, are characterized by their intricate bicyclic structures [80]. Proposed fragmentation patterns of yunnaneic acids D (25) and E (29) can be found in Figure S1. Apart from yunnaneic acids E and F, which have been previously reported in A. tubulosa [40], no other yunnaneic acids or isomers have been mentioned within alkanet plants.

• Tetramers

Three caffeoyl tetramers with a 2-arylbenzofuran type neolignan skeleton were identified (31, 32, 35). Compound 32 was identified as lithospermic acid B and showed a deprotonated ion [M-H]⁻ at m/z 717.1428, and characteristic fragment ions at m/z 519 ([M-H-C₉H₈O₄-H₂O]⁻), corresponding to a neutral loss of a caffeoyl moiety and H₂O, and at m/z 321 ([M-H-C₉H₈O₄-H₂O-C₉H₈O₄-H₂O]⁻), resulting from the subsequent loss of another caffeic acid unit and H₂O. Additionally, two derivatives of lithospermic acid B were detected, including a sodium salt derivative (m/z 741.1400) (35) and sebestenoid E (m/z 671.1394) (31). Lithospermic acid B has been previously detected in several Alkanna species [12], while its derivatives are novel documentations for the genus.

Compound 34, the monosodium salt of rabdosiin (m/z 739.1259), has been previously detected in several Alkanna species [12], while its disodium salt was first isolated by our team from A. sfikasiana [26]. The fragmentation spectra of compound 34 revealed a major product ion at m/z 559 ([M-H-C₉H₈O₄]⁻), corresponding to the loss of a caffeoyl moiety, along with product ions at m/z 515 and at m/z 335, resulting from an additional CO₂ loss, and a subsequent loss of another caffeoyl unit.

Compounds 33, 37–39 were tentatively identified as lignan oligosaccharides characterized by an aryl-dihydro-naphthalene core structure that forms a macrocyclic ring through esterification with fructose [81]. Further substitution with acetylated hexoses (33, 37) or coumaroyl-rhamnoside units (38, 39) yielded these complex glycosylated lignan structures. Compounds 33 and 37 were putatively identified as trigonotin A/rupestrin C (m/z 763.2083) and as trigonotin B/rupestrin A (m/z 733.1950) isomers, respectively. Both trigonotins and rupestrins have been isolated from Boraginaceae species (Trigonotis peduncularis and Eritrichium rupestre, respectively) [59,81]. Compounds 38 and 39 were tentatively characterized as pulmonarioside C/echiumin E isomers, showing precursor ions at m/z 983.2814 and at m/z 983.2816. The fragmentation patterns of these compounds mainly involve losses related to their glycosidic components. Indeed, the fragmentation spectra analysis of compound 38 revealed a fragment at m/z 837 ([M-H-C₆H₁₀O₄]⁻), corresponding to the loss of a rhamnose unit. These metabolites have not been previously detected within the Alkanna genus. Proposed fragmentation patterns of trigonotin B/rupestrin A (33) and pulmonarioside C/echiumin E (38) can be found in Figure S2.

4.1.2. Flavonoids and Flavonoid Glycosides

A total of 27 flavonoids were annotated, including eight flavonols and 19 flavonol O-glycosides. Nearly half of these were kaempferol derivatives, while the remainder included quercetin and other related compounds. Kaempferol, quercetin, and isorhamnetin were reported as the main flavonoids in the Boraginoideae [79], which is consistent with findings in Alkanna species. In particular, kaempferol and quercetin 3-O-glycosides, 3-O-rutinosides, as well as 3-O-acetyl glycosides, were identified [12,40].

Kaempferol Derivatives

Compound 40 exhibited a precursor ion at m/z 285.0396 and fragment ions at m/z 255 and at m/z 227, consistent with kaempferol’s fragmentation pattern [61]. Although various kaempferol glycosides were found in Alkanna, the aglycone of kaempferol is detected for the first time in the genus. However, its methyl ether derivative (compound 41) has been reported in several Alkanna species [12,40], exhibiting a parent ion at m/z 299.0548 and a common MS/MS pattern with compound 40 [61].

Regarding kaempferol-glycosides, all detected compounds were assigned as O-glycosides exhibiting multiple variations. The analysis of the fragmentation spectra for most of these metabolites revealed characteristic fragment ions at m/z 285 and/or 284, corresponding to kaempferol [61] and additional neutral losses of acetyl (−42 Da), hexose (−162 Da), and deoxyhexose (−146 Da) moieties. Accordingly, 42 and 43 were identified as O-hexosides of kaempferol, exhibiting a deprotonated ion [M-H]⁻ at m/z 447 and typical fragment ions at m/z 284 and/or m/z 285, corresponding to the loss of a hexose unit. Their isolation further confirmed both the nature and the position of the sugar substituents as 3-O-β-glucoside and 3-O-β-galactoside, respectively.

Compound 47 was putatively identified as kaempferol-O-malonyl hexoside. Its fragmentation pattern involved the decarboxylation of malonic acid at m/z 489, followed by the loss of the remaining acetylated hexose at m/z 285. Compound 48 was annotated as the methyl ester of 47, kaempferol-O-malonyl-methyl ester-hexoside, showing a precursor ion at m/z 547.1107. The fragmentation spectra analysis revealed several key ions at m/z 515, derived from methyl ester cleavage; at m/z 489, resulting from the fragmentation of the malonyl methyl ester to form the deprotonated kaempferol acetyl hexoside; at m/z 471, which arises from subsequent water loss; at m/z 447, indicating the loss of the malonyl methyl ester; and the ion at m/z 285 that matches with the kaempferol aglycon. The presence of the fragment ion at m/z 101, corresponding to the dehydrated methyl malonate ester, further confirms our hypothesis (Figure 2). To our knowledge, the malonyl methyl ester glycoside of kaempferol (48) is reported for the first time in the literature. O-glycosides of flavonols, such as quercetin and kaempferol, and particularly 3-O-glycosides, are abundant in alkanet plants [12,13,40]. In addition, O-acylation of sugar moieties is a common modification in flavonoids and anthocyanins, involving ester-linked aromatic (e.g., coumaroyl, caffeoyl) or aliphatic acyl groups [82]. Malonylation, one of the most common aliphatic forms of acylation, is critical for stabilizing labile structures, modulating lipophilicity, and protecting glycosyl groups from enzymatic degradation [83]. Across various species in the Boraginaceae family, the 6′′-O-malonylation is the most prevalent modification observed [84,85]. In Pulmonaria officinalis, kaempferol and quercetin 3-O-(6′′-O-malonyl)-glycosides were found [50], while, in Nemophila menziesii’s blue flowers, the presence of petunidin derivatives and apigenin with malonyl groups at the 6-O position of glucose residues has been reported [86].

Compound 52 was tentatively identified as kaempferol-O-caffeoyl-hexoside, showing a precursor ion at m/z 609.1264. In the MS/MS spectra, fragment ions were observed at m/z 447, corresponding to the kaempferol hexoside after the loss of a caffeoyl moiety; at m/z 323, corresponding to the dehydrated caffeoyl hexoside; and at m/z 285, which matches the kaempferol aglycon (Figure S3). The caffeoylation of flavonoid glycosides predominantly targets the sugar moiety, with most studies focusing on anthocyanins rather than other flavonoids [84,85]. Reported caffeoyl flavonol glycosides—primarily kaempferol and quercetin derivatives—are sporadically documented in the Asteraceae, Rosaceae, and Ranunculaceae families, with acylation sites varying across the sugar moiety [85,87,88,89]. Notably, the flavonoid kaempferol 3-O-(2′′-O-caffeoyl-6′′-O-acetyl)-β-D-glucopyranoside, isolated from Tournefortia sibirica, represents the first such derivative in the Boraginaceae family [90]. Based on these findings, the esterification site of the caffeoyl moiety of compound 52 remains to be determined.

Quercetin Derivatives

Compound 53 was identified as quercetin, exhibiting a parent ion at m/z 301.0346 and characteristic fragment ions at m/z 179, 151, and 107 [66]. Similarly, one O-hexoside (54) was isolated and identified as quercetin-3-O-β-glucoside, and two O-acetyl-hexosides (55, 56) of quercetin were also detected in AC extract. All of them exhibited characteristic fragment ions at m/z 300 or m/z 301, corresponding to quercetin aglycon [66]. Compound 57 was identified as the quercetin-O-malonyl-hexoside, with no previous reports in the Alkanna genus. It exhibited a fragmentation pattern similar to that of compound 47, which involved the decarboxylation of malonic acid, followed by the loss of the remaining acetylated hexose from the quercetin aglycone, resulting in ions at m/z 505 and 301 (Figure S3). Compound 58 was identified as rutin, with a precursor ion at m/z 609.1438 and a characteristic fragment ion at m/z 300 formed by the loss of the rutinose, already reported in A. orientalis, A. tinctoria, and A. kotschyana [12].

Other Flavonoid Derivatives

Compounds 59, 61–63 also correspond to flavonoids showing different degrees of hydroxylation and methylation, and their presence has been reported in several Alkanna species [12,40]. Compound 60 was characterized as isorhamnetin. In the MS/MS spectra, the fragment ion at m/z 300 ([M-H-CH₃]⁻) corresponded to the loss of a methyl group, while the abundance of the ion at m/z 271 ([M-H-CH₃-CO]⁻), resulting from demethylation combined with the loss of CO at position 3 of the C-ring, distinguished it from its isomeric flavonol rhamnetin [68]. Compound 64 was identified as a myricetin-O-hexoside, showing a parent ion at m/z 479.0863. In the MS/MS spectrum, the main fragment ion at m/z 317 ([M-H-162]⁻) corresponded to the aglycon of myricetin, following the loss of a hexose unit [69]. Furthermore, the O-hexosides of dimethoxy-trihydroxy (65) and trihydroxy-trimethoxy (66) flavones were tentatively identified by the observed fragment ions at m/z 329 ([M-H-162]⁻) and at m/z 359 ([M-H-162]⁻), respectively. Compound 66 has also been reported in A. tubulosa [40].

4.2. Molecular Network

The chemical profiling of AC was complemented by molecular networking analysis using the GNPS2 platform, integrated with CANOPUS-derived chemical class predictions, in order to map the diversity of secondary metabolites. While manual interpretation of MS and MS/MS fragmentation patterns remained central for compound identification, the GNPS-generated molecular network provided valuable insights into the chemical diversity and supported the dereplication step of metabolites sharing structural similarities.

Confirming the findings of the dereplication process, the two predominant clusters corresponded to hydroxycinnamic acid derivatives and flavonoids (Figure 1). Notably, two distinct subgroups were identified within the flavonoid cluster, corresponding to quercetin and kaempferol derivatives. In contrast, the hydroxycinnamic acid cluster did not exhibit any distinct subgroup, despite most features aligning with caffeic acid derivatives. This may be attributed to the diverse fragmentation patterns among caffeoyl oligomers, unlike flavonoids, which display significant similarity, particularly in the aglycone part. Nevertheless, compounds 31 (sebestenoid E) and 32 (lithospermic acid B), which share the same 2-aryl-benzofuran core structure, formed a small, distinct cluster.

4.3. PA Analysis

Among the detected PAs, three were tentatively identified as 1,2-saturated trachelanthamidine/isoretronecanol-type (PA 1, 3, 5) [78]. PA3, with a precursor ion at m/z 240.1594, was identified as 9-sarracinoyl- trachelanthamidine/isoretronecanol. The presence of the product ion at m/z 142 in MS/MS spectra corresponded to the necine base trachelanthamidine formed by the cleavage of the weak allylic ester bond at C9 and the subsequent loss of the sarracinoyl moiety (C₅H₇O₂) (Figure 3). PA5, with a [M + H] ⁺ ion at m/z 304.1758, was identified as a trachelanthamidine/isoretronecanol-hexoside. The presence of the fragment ion at m/z 142 in MS/MS spectra resulted from the loss of a hexose unit (m/z −162) (Figure 3). To our knowledge, the sarracinoyl ester PA3 and the hexoside of trachelanthamidine/isoretronecanol PA5 are reported for the first time in the literature.

PA2 was tentatively identified as the 1,2-saturated necine base, platynecine. It showed a precursor ion at m/z 158.1166 [M + H]⁺ and the characteristic fragment ions at m/z 122 and 140, both representing platynecine’s core structure [78]. Platynecine has already been reported in several Alkanna spp. (A.tinctoria, A. hellenica, A. graeca, A. sfikasiana) [17].

Retronecine-type and their respective N-oxides represented the majority of detected PAs. They were further classified into two subtypes (monoesters and open-chain diesters), based on the degree of esterification at positions 7 and 9. All PAs of this type shared a common fragment ion at m/z 138, except for the N-oxide of dihydroxy triangularine (PA9), which showed a fragment at m/z 136, typical of diester N-oxides [91]. Further characteristic fragment ions related to RETNO monoesters (leptanthine-N-oxide, PA7) and to RETNO open chain diesters (dihydroxy triangularine-N-oxide, PA9) were observed at m/z 172 and at m/z 254, respectively. RET monoesters showed a characteristic fragment ion at m/z 156, while both RET monoesters and diesters shared a common fragment ion at m/z 120. Fragmentation data were in good agreement with previously reported patterns for retronecine-type PAs and PANOs [17,91].

Among RET derivatives, PA4 was tentatively identified as retronecine pentoside. It showed a precursor ion at m/z 288.1435 [M + H]⁺ and fragment ions at m/z 138, related to the loss of a pentose unit, and at m/z 156 and m/z 120, consistent with the retronecine core structure (Figure 3) [92]. PA4 is reported for the first time in the literature, while its N-oxide (retronecine-N-oxide pentoside) has been recently reported in Alkanna species [17]. In addition, compounds PA6 (leptanthine) and PA7 (leptanthine-N-oxide), previously found in species such as Onosma leptantha [93], represent new reports within Alkanna spp., unlike dihydroxy triangularine (PA8) and its N-oxide (PA9), as well as 7-senecioyl-9-(3-acetoxy-2-hydroxy-2-methylbutyryl) retronecine N-oxide (PA10), which have been previously reported in several alkanet species [16,17].

Glycosidic PAs are relatively rare in the literature. Thesinine glycosides were described from Borago officinalis and Tephroseris kirilowii, while retrorsine and its N-oxide glycosides, alongside senecionine and seneciphylline hexosides, were reported from Senecio vulgaris [94]. More recently, PA analysis of A. graeca, A. hellenica, A. tinctoria, A. sfikasiana, Eupatorium cannabinum, and S. vulgaris revealed the presence of a wide range of glycosidic PAs, mainly related to retronecine N-oxide hexosides [17].

4.4. Evaluation of TPC and TFC

As presented in Table 3, TPC was found to be 74.45 mg GAE/g extract, while TFC was 46.66 mg RE/g extract. These results are comparable to those obtained from the aerial parts of Alkanna species, although extraction techniques appear to significantly influence these quantitative data. Different extracts of A. trichophila displayed TPC values ranging from 34.9 to 53.4 mg GAE/g and TFC values between 30.8 and 69.6 mg RE/g, while extracts of A. tubulosa had TPC values between 22.5–93.3 mg GAE/g, with corresponding TFC values ranging from 7.14 to 20.1 mg RE/g [40,75]. The methanolic extracts of A. tinctoria, A. kotschyana, and A. orientalis showed TPC values ranging from 34.9 to 53.4 mg GAE/g and TFC values between 15.97 and 25.90 mg RE/g [12]. Comparing the results of the current study with those referred to in the literature, it is evident that AC is characterized by a high content of both phenolics and flavonoids.

4.5. Evaluation of Biological Activity

4.5.1. Antioxidant Activity

Under various physicochemical conditions or pathological states, the body produces free radicals and reactive oxygen and nitrogen species, which, when excessive, lead to oxidative stress. This imbalance is associated with diseases such as atherosclerosis, inflammation, certain cancers, and aging. Natural antioxidants can neutralize free radicals and prevent further cellular damage [95].

The methanolic extract of AC exhibited considerable antioxidant activity (Table 4). When compared to other species within the genus that have undergone similar evaluations, AC exhibited the highest radical scavenging activities. Indeed, AC values for DPPH (227.01 mg TE/g) and ABTS (450.91 mg TE/g) were significantly higher than those of A. tinctoria, previously reported with the highest radical scavenging activity among related species (DPPH 211.6 mg TE/g; ABTS 366.9 mg TE/g) [12]. Similarly, A. trichophilla and A. tubulosa demonstrated a range of antioxidant capacities across different extracts considerably lower than those found in AC [40,75]. Regarding FRAP, CUPRAC, and phosphomolybdenum assays, AC exhibited values of 365.59 mg TE/g extract for FRAP, 462.42 mg TE/g extract for CUPRAC, and 2.11 mmol TE/g extract for the phosphomolybdenum assay. Among the species examined in similar studies, comparable or lower values were detected [12,40,75]. Finally, the AC exhibited a metal chelating capacity of 7.91 mg EDTAE/g. In comparison to the potential of the methanolic extract of A. orientalis (19.11 mg EDTAE/g), it showed a significantly lower metal chelating capacity [12]. Similarly, extracts from A. tubulosa and A. trichophilla demonstrated substantial metal chelation abilities, with values 8.71 to 20.57 mg EDTAE/g extract, and 17.51 to 19.84 mg EDTAE/g extract, respectively [40,75].

The antioxidant activity can be primarily associated with the presence of PCs [96]. The high phenolic and flavonoid content observed in the AC extract likely explains the strong antioxidant activities.

4.5.2. Enzyme Inhibitory Activity

AChE and BChE play a crucial role in the pathophysiology of Alzheimer’s disease (AD). In AD, the accumulation of intraneuronal β-amyloid (Aβ) leads to the degeneration of basal forebrain cholinergic neurons, reducing acetylcholine (ACh) levels, which, in turn, results in memory deficits [97]. Inhibiting AChE and BChE—catabolic enzymes that degrade ACh—can help counteract this deficiency. The AC extract exhibited inhibitory activities of 1.43 mg GALAE/g for AChE and 0.38 mg GALAE/g for BChE (Table 5). These results are comparable to those reported for A. tinctoria and A. kotschyana [12]. In contrast, methanolic extracts of A. trichophila showed higher inhibition values both for AChE of (2.70 mg GALAE/g) and BChE (5.53 mg GALAE/g) [75]. Meanwhile, A. tubulosa, which was tested across different extracts, displayed inhibitory values ranging from 3.36 to 3.48 and 3.29 to 15.19 mg GALAE/g extract for AChE and BChE, respectively, with the highest activity observed in the ethanolic extract [40].

α-Amylase and α-glucosidase are pivotal enzymes involved in carbohydrate metabolism. The excessive activity of these enzymes leads to a disturbance in the absorption of glucose and its accumulation in the blood [98]. Plant extracts that inhibit these enzymes represent valuable sources of compounds with a promising therapeutic potential for preventing and treating diabetes. The AC extract exhibited inhibitory activities of 0.47 and 6.65 mmol ACAE/g extract for the α-amylase and α-glucosidase, respectively (Table 5). The inhibition of α-amylase by AC was comparable to values reported in the literature. Extracts of A. trichophila showed inhibition ranging from 0.33 to 0.69 mmol ACAE/g, with the highest activity observed in the methanolic extract [67]. The ethyl acetate extract of A. tubulosa showed an inhibition of 0.50 mmol ACAE/g, while methanolic extracts of A. orientalis, A. tinctoria, and A. kotschyana exhibited values between 0.45 and 0.61 mmol ACAE/g, with the highest value corresponding to A. orientalis [12,40,75]. However, AC exhibited considerably strong inhibition against α-glucosidase, which was similar only to that observed in A. orientalis (6.49 mmol ACAE/g extract), unlike related species that exhibited inhibition of 3.69–4.46 mmol ACAE/g extract [12].

The presence of PCs such as rosmarinic acid and quercetin glycosides may be associated with the enhanced inhibition of α-glucosidase, since they have been reported to interact with the enzyme [99,100]. On the other hand, 6-O-caffeoyl-hyperoside, along with other acylated flavonoids from the Spiraea genus, has been identified as an inhibitor of α-amylase activity [87]. The presence of multiple acylated flavonoids in the AC extract may be associated with the observed inhibitory effect on this enzyme.

5. Conclusions

In this study, a comprehensive phytochemical analysis of the aerial parts of AC was performed, identifying PCs and PAs as the predominant chemical classes. The rich phenolic profile is consistent with previous reports on other Alkanna species. Utilizing an untargeted MS approach allowed the detection of novel compounds, highlighting its value in studying unexplored species and enabling the discovery of previously unknown metabolites that merit further investigation through targeted analyses. Despite methodological differences among studies on Alkanna species, comparative analysis with the existing literature provides a valuable context for understanding the phytochemical diversity of AC. The evaluation of antioxidant and enzyme inhibitory activities demonstrated the interesting bioactivity of the AC extract, indicating its potential health benefits. Optimizing extraction methods could yield extracts with enhanced potency, provided that PA levels are controlled to comply with EU safety standards for human exposure.