Comparative Phytochemical Profiling and In Vitro Antibiofilm Activity of the Aerial and Underground Parts of Primula veris subsp. columnae (Ten.) Lȕdi

Abstract

The comparative UHPLC-MS/MS analysis of the underground and aerial parts of Primula veris subsp. columnae (Ten.) Lȕdi revealed a diverse range of secondary metabolites, including flavonoids and phenolic compounds and their glycosides, bisbibenzyl compounds and triterpene saponins. Phytochemical study of the aerial parts led to the isolation of 13 surface flavonoids and 6 flavonoid glycosides, of which 8,2′-dimethoxy-5-hydroxyflavone and 8,3′-dimethoxy-5-hydroxyflavone are new natural compounds. From the underground parts, a new triterpene saponin, primulasaponin VI, was isolated together with the known primulasaponins I, III and V; priverosaponin B 22-acetate; primulaverin; primverin; gaultherin; and 3-methoxy-4-primeverosylacetophenone. The structures of the new compounds were elucidated by NMR and HR-ESI-MS. The acetone extract obtained from the aerial parts and methanol extracts obtained from both aerial and underground parts were evaluated for their antibiofilm activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The methanol extract obtained from underground parts possessed the strongest overall antibiofilm activity, particularly against E. coli and S. aureus. While the acetone extract inhibited the biofilm formation by E. coli and S. aureus, it promoted biofilm formation by P. aeruginosa. All tested extracts exhibited low cytotoxicity toward human keratinocytes and melanoma cells.

1. Introduction

Primula veris L. (cowslip) is a well-known medicinal plant, and its medicinal properties are supported by the European Medicines Agency Committee on Herbal Medicinal Products. The root preparations stimulated airway secretions, reducing the viscosity of phlegm and facilitating its expectoration [1], while the flower preparations can be used as expectorants to alleviate coughs associated with colds [1,2] and for skin-related disorders [3]. P. veris extracts have also been reported to possess antioxidant, anti-inflammatory, diuretic, sedative and constrictive properties [4,5].

Biofilms represent structured microbial consortia capable of adhering to various surfaces, including biological tissues. They are able to synthesize an extracellular polymeric substance (EPS), which further enhances their resistance. This mode of organization allows microorganisms to function as a coordinated system with altered physiology compared with planktonic cells, including changes in gene expression and metabolic activity [6,7]. From a clinical perspective, biofilms play a crucial role in the development of chronic and recurrent infections. Typical representatives of biofilm-forming pathogens include Pseudomonas aeruginosa, commonly associated with chronic pulmonary infections in patients with cystic fibrosis; Staphylococcus aureus and Staphylococcus epidermidis, which are leading causes of infections associated with medical implants and catheters; and Escherichia coli, which is implicated in urinary tract infections [8,9]. Biofilm-associated infections are particularly difficult to treat because biofilms can evade the host immune response and persist within the organism. This is attributed both to the physical protection provided by the EPS matrix and to the presence of persisted cells with low metabolic activity [6]. A key characteristic of biofilms is their increased tolerance and resistance to antimicrobial agents. Furthermore, biofilms are recognized as a major factor contributing to infection chronicity, highlighting the need to develop novel prevention and treatment strategies aimed at disrupting biofilm structure and/or inhibiting the formation of these microbial consortia [6,9]. Data regarding the antibacterial properties of P. veris are limited and focused mainly on the aerial parts of the plant [10,11,12]. Recently, P. veris root extracts were reported to inhibit biofilm formation in several Gram-positive and Gram-negative strains [13].

Beyond its pharmacological significance, P. veris has been a subject of numerous phytochemical investigations. The aerial parts of P. veris are rich source of flavonoids and their glycosides, mainly derivatives of kaempferol, quercetin and isorhamnetin, together with polymethoxylated flavonoids [14,15,16,17,18,19]. The latter are accumulated on the external plant surface and serve as chemotaxonomic markers for the genus Primula [18]. The roots are characterized by the presence of various polyphenolic compounds such as primulaverin, primverin, acetophenones and their glycosides, bisbibenzyl compounds [5,13,18,20,21,22,23]. Another type of secondary metabolites, oleanane type triterpene saponins, occur in both roots and aerial parts [13,15,16,18,22,24]. Studies on P. veris constituents have mainly focused on particular plant organs, specific classes of secondary metabolites, or populations from limited geographical areas. During the last decade, the chemical profile of P. veris has mainly been investigated using HPLC-HRMS-based metabolomic approaches [14,15,16,17,22,25]. Although these studies have significantly expanded the knowledge of its metabolite profile, the identification of constituents has been based primarily on tentative annotation using mass spectrometric data. In contrast, the investigations involving compound isolation and structural elucidation by NMR spectroscopy remain scarce. As a result, the identities of many reported constituents cannot be considered unequivocally established.

Four subspecies of Primula veris L.—veris, columnae, macrocalyx and officinalis—are widely present across Europe in the mountainous landscapes, particularly in semi-natural grasslands and semi-open forests. The literature survey showed only one report on P. veris subsp. columnae, describing the isolation and structural elucidation of several bisbibenzyl compounds and acetophenone derivatives from the roots of the plant [20].

The limited information available on the chemical composition of P. veris subsp. columnae (Ten.) Lȕdi motivated us to perform a more complete characterization of the aerial and the underground parts of the plant by combining a classical phytochemical study with UHPLC-MS/MS-based metabolite profiling. In addition, the antibiofilm activity of extracts from different plant organs was evaluated to broaden the current understanding of the biological potential of cowslip.

2. Results and Discussion

2.1. Isolation and Identification of Individual Compounds from P. veris subsp. columnae

2.1.1. Surface Flavones from the Aerial Parts

Fresh aerial parts of P. veris subsp. columnae were washed with acetone in order to extract surface flavonoids. The purification of the acetone extract led to isolation of 13 individual compounds (Figure 1). Comparison of their ¹H NMR data (Figures S1–S11, Supplementary Materials) with the literature data allowed their identification as the known flavone (1) [26], 2′-methoxyflavone (2) [26], 3′-methoxyflavone (3) [26], 3′-hydroxy-4′-methoxyflavone (4) [26], 3′,4′-dimethoxyflavone (5) [26], 2′,5′-dimethoxyflavone (6) [26], 4′,5′-dimethoxy-3′-hydroxyflavone (7) [26], 3′,4′,5′-trimethoxyflavone (8) [26], 3′-methoxy-4′,5′-methylenedioxyflavone (9) [26], 5,6,2′,6′-tetramethoxyflavone (10) [26], and 8-methoxy-5-hydroxyflavone (11) [27].

Figure 1.

Structures of surface flavonoids 1–13.

Compound 12 exhibited a molecular ion peak [M + H]⁺ at m/z 299.0917 in the positive HR-ESI-MS, which corresponded to the molecular formula C₁₇H₁₄O₅ (Figure S20). The UV absorption maximum at 368 nm suggested a highly conjugated molecule. The ¹H NMR spectrum (Table 1) showed seven aromatic proton signals and two methoxy group singlets (Figure S12). According to the COSY spectrum, aromatic proton signals were grouped into two spin systems: H-6/H-7 and H-3′/H-4′/H-5′/H-6′ (Figure 2 and Figure S15). The singlet of H-3 at δ_H 7.18 exhibited HMBC correlation with the carbonyl carbon C-4 (δ_C 184.1), as well as with C-2 and C-1′ carbons (δ_C 161.8 and 133.5, respectively) (Figure 2). The same carbon signals, C-2 and C-1′, also showed HMBC correlations with H-6′ (δ_H 8.03) (Figure S19). The NOESY spectrum revealed the positions of the methoxy groups through H-7/8-OMe and H-3′/2′-OMe correlations (Figure 2, Figures S16 and S17). The multiplicity patterns given in Table 1, together with the COSY correlations and the remaining HMBC, revealed the structure of 8,2′-dimethoxy-5-hydroxyflavone for compound 12. A literature survey showed that 8,2′-dimethoxy-5-hydroxyflavone has previously been obtained by incomplete methylation of 5,8,2′-trihydroxyflavone [27]. However, no isolation of this compound from natural sources has been reported. Furthermore, its structural assignment relied solely on UV and MS data [27]. In the present work, the structure of 8,2′-dimethoxy-5-hydroxyflavone (12) was unambiguously determined by extensive NMR experiments and HRMS.

Table 1.

The ¹H (500 MHz) and ¹³C (125 MHz) NMR data of compounds 12 in CDCl₃ and 13 in (CD₃)₂CO (δ in ppm, multiplicity, J in Hz).

Position	12		13
	δH	δC	δH	δC
2	-	161.8	-	165.0
3	7.18 s	111.4	6.96 s	106.8
4	-	184.1	-	184.4
5	-	153.6	-	154.2
6	6.73 d (9.0)	109.5	6.72 d (8.5)	110.5
7	7.17 d (9.0)	118.9	7.41 d (8.5)	120.1
8	-	140.6	-	141.6
9	-	146.5	-	146.6 *
10	-	111.5	-	112.0
1′	-	133.5	-	133.5
2′	-	158.5	7.65 brt (2.0)	112.7
3′	7.05 d (8.0)	112.0	-	161.2
4′	7.50 dt (8.5; 2.0)	133.0	7.22 dd (8.5; 2.0)	118.7
5′	7.14 bt (7.5)	121.1	7.54 d (8.5)	131.3
6′	8.03 dd (8.5; 2.0)	129.7	7.71 bd (8.5)	119.7
8-OMe	3.94 s	57.6	4.00 s	57.7
2′(3′)-OMe	3.95 s	55.9	3.94 s	55.9

* Determined from HMBC (H-7/C-9).

Figure 2.

Key NOE, COSY and HMBC correlations of compounds 12 and 13.

Compound 13 exhibited a molecular ion peak [M + H]⁺ at m/z 299.0919 in the positive HR-ESI-MS, corresponding to the molecular formula C₁₇H₁₄O₅ (Figure S28). The UV absorption maximum at 368 nm suggested a highly conjugated molecule. The ¹H NMR spectrum (Table 1) showed seven aromatic proton signals and two methoxy group singlets (Figure S21). Aromatic proton signals were grouped into two spin systems, H-6/H-7 and H-4′/H-5′/H-6′, according to the COSY spectrum (Figure 2 and Figure S24). The singlet of H-3 at δ_H 6.96 exhibited HMBC correlation with the carbonyl carbon C-4 (δ_C 184.4), as well as with C-2 and C-1′ carbons (δ_C 165.0 and 133.5, respectively) (Figure 2). The same carbons signals, C-2 and C-1′, showed HMBC correlations with H-2′ (δ_H 7.65) and H-6′ (δ_H 7.71) (Figure S27). The NOESY spectrum revealed the positions of the methoxy groups through H-7/8-OMe and H-2′, H-4′/3′-OMe correlations (Figure 2 and Figure S25). The multiplicity patterns given in Table 1, together with the COSY correlations and the remaining HMBC correlations, revealed the structure of 8,3′-dimethoxy-5-hydroxyflavone. According to the SciFinder and Reaxys databases, 13 is a new compound.

Previously, surface flavones 1–10 were isolated from cultivated P. veris obtained by in vitro propagation [26], while 5-hydroxyflavones 11–13 are found here for the first time in P. veris. The literature analysis revealed that compound 11 had been previously isolated from Primula edelbergii [28]. Compound 12 was erroneously reported by Valant-Vetschera et al., 2009 [29] as being previously known from P. capitata; however, only 5,8,2′-OH-flavone has been isolated from this species [30]. It is worth noting that flavonoids bearing a hydroxy or methoxy substituent at C-8, such as 5,8,2’-trihydroxyflavone, 5-hydroxy-2′-methoxyflavone and 5,8-dihydroxyflavone, have previously been reported in several Primula [18,29] and Dionysia species (Primulaceae) [31]. Furthermore, this study represents the first report of the natural occurrence of 8,2′-dimethoxy-5-hydroxyflavone (12) and the new 8,3′-dimethoxy-5-hydroxyflavone (13).

2.1.2. Flavonoid Glycosides from the Aerial Parts

From the flavonoid fraction of the methanol extract obtained from P. veris subsp. columnae air-dried aerial parts, six individual compounds were isolated (Figure 3). Comparison of their ¹H NMR data (Figures S29–S33, Supplementary Materials) with those published in the literature allowed the identification of rutin (quercetin-3-O-rutinoside, 14) [32], quercetin-3-O-β-glucopyranosyl-(1→2)-β-glucopyranosyl-(1→6)-β-glucopyranoside (15) [14,17], quercetin-3-O-[α-rhamnopyranosyl-(1→2)]-[α-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside (16) [33], kaempferol-3-O-β-glucopyranosyl-(1→2)-β-glucopyranosyl-(1→6)-β-glucopyranoside (17) [14], kaempferol-3-O-[α-rhamnopyranosyl-(1→6)]-[α-rhamnopyranosyl (1→2)]-β-glucopyranoside (18) [33], and isorhamnetin-3-O-β-glucopyranosyl-(1→2)-β-glucopyranosyl-(1→6)-β-glucopyranoside (19) [14,17].

Figure 3.

Structures of flavonoid glycosides 14–19.

Data regarding the presence of flavonoid glycosides in genus Primula are scarce and limited to few species [18,19]. The described flavonoids are usually quercetin, kaempferol and isorhamnetin glycosylated at C-3 with glucose, rhamnose and galactose as gentiobiose, neohesperose, robinobiose, etc. The literature survey showed that compounds 14, 15, 17 and 19 have been previously found in P. veris flowers of Greek origin [14,16,17], while flavonoids 16 and 18 are now isolated from Primula species for the first time. It seems that flavonoid triglycosides are the dominant compounds for P. veris growing on the Balkan Peninsula.

2.1.3. Isolation and Identification of Individual Compounds from the Underground Parts

Column chromatography of the methanol extract performed on Sephadex with methanol as eluting solvent afforded three main fractions, A–C. Further purification of fraction A resulted in the isolation and identification of the known primulasaponin I (primulic acid I) (20) [34], primulasaponin III (21) [13], primulasaponin V (22) [13] and priverosaponin B 22-acetate (23) [24] (Figure 4) (Figures S34–S37, Supplementary Materials).

Figure 4.

Structures of compounds 20–28 isolated from underground parts.

Compound 24 was isolated as a white amorphous powder. Its molecular formula was determined to be C₅₆H₉₀O₂₆ based on the HR-ESI-MS ion at m/z 1177.5655 ([M − H]⁻, calcd. 1177.5648 for C₅₆H₈₉O₂₆) and was supported by NMR spectroscopic data (Figures S38–S45, Supplementary Materials). The NMR data of compound 24 (Table 2) showed the presence of seven signals corresponding to the tertiary methyl groups (positions 23–27, 29 and 30), nine signals corresponding to the methylene groups (positions 1, 2, 6, 7, 11, 12, 15, 19 and 21), three methine signals (positions 5, 9 and 18), four oxygenated methine signals (positions 3, 16, 22 and 28) and signals for acetoxy group [δ_H 2.01 (s)/δ_C 21.27 and δ_C 172.75]. In addition, the positions of seven quaternary carbons were determined from the observed HMBC correlations of H-5, H-23 and H-24 with C-4 (δ_C 40.75); H-5 and H-25 with C-10 (δ_C 37.75); H-9, H-26 and H-27 with C-8 (δ_C 43.52); H-26 and H-27 with C-14 (δ_C 44.47); H-27 and H-28 with C-13 (δ_C 89.11); H-16, H-22 and H-28 with C-17 (δ_C 49.57); and H-19 (δ_H 2.43), H-21, H-29 and H-30 with C-20 (δ_C 33.79) (Figure 5). The position of the acetyl group at C-22 was established from the chemical shift of H-22 (δ_H 5.27) and HMBC correlations between H-22 and the carbonyl signal of the acetyl group at δ_C 172.75. Furthermore, four glycosyl units were determined as glucuronyl [δ_H 4.44 (d, 7.8 Hz)/δ_C 105.82 (C-1′)], galactosyl [δ_H 5.20 (d, 7.7 Hz)/δ_C 100.75 (C-1″)], rhamnosyl [δ_H 5.28 (d, 1.6 Hz)/δ_C 102.00 (C-1‴)] and glucosyl [δ_H 4.89 (d, 7.5 Hz)/δ_C 102.53 (C-1⁗)]. The coupling constants (Table 2) indicated β-configurations for the glucuronic acid, galactose and glucose residues and α-configuration for the rhamnose unit. In addition, the HMBC correlations H-3/C-1′, H-1″/C-3′, H-1‴/C-2″ and H-1⁗/C-2′ determined the linkages of the glycosyl units. Comparison of these data with those reported for priverosaponin B 22-acetate (23) [24] revealed an identical glycosyl part and almost the same triterpene framework. The only difference was the presence of an additional hydroxy group at C-28 in comparison with 23, and its position was unambiguously determined by the HMBC correlations H-28 with C-13, C-16, and C-17, respectively. The relative stereochemistry at C-3, C-16, C-22 and C-28 was determined by ROESY experiment (Figure 5). Thus, the observed correlations H-3/H-5 and H-5/H-9 revealed their α-axial orientation (i.e., β-orientation of the oxygen function at C-3), while the correlations H-25/H-26 and H-26/H-15 (δ_H 1.95) indicated the β-orientation of these protons. In addition, the correlations H-15 (δ_H 1.95)/H-28 and H-16/H-28 confirmed the α-position of the hydroxyl groups at C-16 and C-28. Further, the ROESY correlation H-22/H-30 assigned the α-orientation of the acetoxy group at C-22. Thus, the compound 24 was identified as 3-O-β-(β-glucopyranosyl-(1→2)-[α-rhamnopyranosyl-(1→2)-β-galactopyranosyl]-(1→3)-β-glucuronopyranosyl)-22α-acetoxy-16α,28α-dihydroxy-13,28-epoxyoleanane. According to the SciFinder database, this compound has not previously been reported, and hence, 24 was named primulasaponin VI.

Table 2.

¹H (600 MHz) and ¹³C (150 MHz) NMR data of compound 24 in CD₃OD (δ in ppm, multiplicity, J in Hz).

Position	δH	δC	Position	δH	δC
1	1.00 m	40.28	26	1.17 s	18.84
	1.74 m		27	1.27 s	19.94
2	1.74 m	27.03	28	4.68 s	98.21
	2.04 m		29	0.97 s	33.45
3	3.21 m	91.97	30	1.00 s	25.72
4	-	40.75	OAc	2.01 s	21.27
5	0.74 dd (2.0, 11.6)	56.74			172.75
6	1.44 m	18.84	1′	4.44 d (7.8)	105.82
	1.47 m		2′	3.91 dd (7.9, 8.8)	79.38
7	1.22 m	35.08	3′	4.05 t (8.8)	81.04
	1.56 m		4′	3.59 m	72.3
8		43.52	5′	3.79 m	75.86
9	1.25 m	51.24	6′	-	176.21
10	-	37.75	1″	5.20 d (7.7)	100.75
11	1.45 m	19.86	2″	3.79 m	75.84
	1.66 m		3″	3.72 m	76.16
12	1.40 m	33.66	4″	3.70 m	71.94
	2.03 m		5″	3.52 m	76.89
13	-	89.11	6″	3.64 dd (4.1, 11.8)	62.77
14	-	44.47		3.82 m
15	1.27 m	36.82	1‴	5.28 d (1.6)	102
	1.95 m		2‴	3.95 dd (1.6, 3.4)	72.65
16	4.25 br d (5.2)	70.49	3‴	3.71	72.16
17	-	49.57	4‴	3.41 t (9.5)	73.76
18	1.69 m	47.68	5‴	4.12	70.28
19	1.18 m	38.9	6‴	1.27 d (6.5)	17.88
	2.43 dd (12.4, 14.5)		1⁗	4.89 d (7.5)	102.53
20	-	33.79	2⁗	3.23 dd (7.5, 9.5)	76.2
21	1.56 m	41.93	3⁗	3.34 m	77.88
	2.23 t (12.0)		4⁗	3.06 t (9.5)	72.6
22	5.27 dd (5.9, 12.4)	73.79	5⁗	3.38 m	78.14
23	1.06 s	28.22	6⁗	3.53 dd (8.0, 12.2)	63.51
24	0.87 s	16.75		3.86 dd (2.3, 12.2)
25	0.90 s	16.78

Figure 5.

Important HMBC and ROESY correlations for compound 24.

The literature survey showed that 3-O-β-(β-glucopyranosyl-(1→2)-[α-rhamnopyranosyl-(1→2)-β-galactopyranosyl]-(1→3)-β-glucuronopyranosyl)-oleanane triterpenoids with a 13β,28-epoxy ring are characteristic for Primula species [35]. The studied population of P. veris subsp. columnae exhibited a different saponin profile from those reported previously for P. veris, P. veris subsp. macrocalyx, P. veris subsp. veris, and commercially available root extract of Primula sp. [15,16,22,24,25,34]. Primulasaponin I (primulic acid I, 20) and priverosaponin B 22-acetate (23) are common constituents of the roots of the P. veris from Germany, Austria and Turkey [15,22,24], whereas priverosaponin B 22-acetate was not detected in wild growing populations of P. veris subsp. veris from Greece [16] and P. veris from Bulgaria [13]. Instead, primulasaponin III (21) and primulasaponin V (22) were recently isolated from Bulgarian P. veris [13]. The observed differences in saponin content may be attributed to intraspecific variation and/or differences in environmental and ecological conditions.

Subsequent purification of fraction B by column and preparative thin layer chromatography led to the isolation of four individual compounds (Figure 4). Comparison of their NMR data (Figures S46–S49, Supplementary Materials) with those published in the literature allowed their identification as primulaverin (25) [22], primverin (26) [22], gaultherin (methyl salicylate 2-O-β-xylopyranosyl-(1→6)-β-glucopyranoside, 27) [36] and 3-methoxy-4-primeverosylacetophenone (28) [37]. Compounds 25 and 26 are marker compounds for P. veris roots, as well as also being considered as indicators of the age of plant material [22]. Gaultherin (27) was recently detected in the roots of P. veris subsp. veris [16], whereas compound 28 has now been isolated from P. veris for the first time.

2.2. UHPLC-MS/MS Analysis of the Methanol Extracts of P. veris subsp. columnae Aerial and Underground Parts

Comparative UHPLC-MS/MS analysis of the methanolic extracts from the aerial and underground parts of P. veris subsp. columnae performed in negative ionization mode (Table 3, Figures S50 and S51, Supplementary Materials) enabled the identification of 57 compounds. Among these, 12 compounds were unambiguously identified with authentic standards, the structures of 18 compounds were confirmed by NMR following their isolation, and 27 compounds were tentatively identified by their chromatographic characteristics (m/z values, molecular formula, and fragmentation pattern) and comparison with data available in open-access LC-MS libraries and the literature. The identified compounds belonged to three main classes of metabolites: flavonoids (31 compounds), benzoic acid derivatives (11 compounds) and triterpenoids (7 compounds). In addition, two acetophenone derivatives, two bisbibenzyl compounds, two organic acids, one cinnamic acid and one sugar were also detected in analyzed extracts.

Free aglycones and mono-, di- and tri-glycosides of quercetin, kaempferol and isorhamnetin were recognized based on their mass-spectral fragmentation pattern. Quercetin (peak 40), luteolin (peak 41), kaempferol (peak 45) and isorhamnetin (peak 48) were identified by their [M − H]⁻ at m/z 301, 285, 285 and 315, respectively, as well as by comparison with authentic standards. The MS/MS spectra of quercetin, kaempferol and isorhamnetin 3-O-glycosides contained a base peak [Y₀ − H]^•− at m/z 300, 284 and 314, respectively, which is characteristic for flavonol 3-O-glycosides and results from the elimination of hexose (162 Da) (peaks 26, 32 and 35), rutinose (308 Da) (peaks 25, 30 and 33), dihexose (324 Da) (peaks 18 and 29), trihexose (486 Da) (peaks 14, 20 and 23), dirhamnose-hexose (454 Da) (peaks 17, 22 and 24) and rhamnose-dihexose (470 Da) (peak 15) from the corresponding [M − H]⁻. The structures of compounds 14–19 (peaks 25, 14, 17, 20, 22 and 23) were additionally confirmed by ¹H NMR after their isolation from the methanol extract of the aerial parts, while those of isoquercitrin (peak 26) and astragalin (peak 32) were confirmed by comparison with authentic standards. The mono- and dihexosides of dihydroxyflavone (peaks 36–38) were tentatively identified by the base peak at m/z 253 [Agl-H]‾ obtained by elimination of 162 and 324 Da from the respective [M − H]⁻ at m/z 415 and 577 [38]. Peaks 42, 46 and 47 had [M − H]⁻ at m/z 253 and were tentatively assigned as dihydroxyflavones, while peak 51, which had [M − H]⁻ at m/z 237, was assigned as hydroxyflavone. Peak 8 was tentatively identified as gallocatechin by comparison of its mass-spectral fragmentation with that reported in the literature [38].

Table 3.

Compounds in P. veris subsp. columnae aerial and underground parts identified by UHPLC-MS/MS.

Peak	RT	Compound	MF	[M − H]−	Δ, ppm	MS/MS Fragmentation Pattern	AP	UP	Ident.
1	0.93	Sucrose	C12H22O11	341.1089	−0.14	341, 179, 119, 89, 71, 59	+	+	[39]
2	0.97	Citric acid	C6H8O7	191.0189	−4.37	191, 111	+	+	[39]
3	1.95	Vanillic acid O-hexoside	C14H18O9	329.0881	0.82	329, 167, 152, 123, 108	+	+	[38]
4	2.4	Protocatechuic acid	C7H6O4	153.0183	−3.16	153, 109	+	+	st
5	2.65	Syringic acid O-hexoside	C15H20O10	359.0988	1.07	359, 197, 182, 167, 153, 138, 123	−	+	[38]
6	4.09	Salicylic acid	C7H6O3	137.0232	−4.88	293, 191, 151, 125, 89	+	+	[39]
7	4.23	Gentisic acid	C7H6O4	153.0184	−2.51	153, 109, 108	+	+	[39]
8	4.51	Gallocatechin	C15H14O7	305.0668	0.52	305, 261, 219, 179, 167, 137, 125	−	+	[38]
9	6.05	Caffeic acid	C9H8O4	179.0343	−3.71	179, 135	+	−	st
10	6.23	3-Methoxy-4-primeverosyl acetophenone (28)	C21H30O14	505.1568 $	1.09	293, 165, 150,122	−	+	NMR
11	6.4	Paeonolide	C21H30O14	505.1568 $	1.33	293, 165, 150, 122	−	+	NMR
12	7.48	Vanillic acid primeveroside	C19H26O13	461.1307	1.26	461, 167, 152, 108	−	+	[38]
13	9.75	Gaultherin (27)	C20H28O14	491.1408 $	1.46	293, 191, 151, 125, 89	+	+	NMR
14	10.18	Quercetin-3-O-β-glucopyranosyl-(1→2)-β-glucopyranosyl-(1→6)-β-glucopyranoside (15)	C33H40O22	787.1941	0.39	787, 301, 300, 271, 255	+	−	NMR
15	10.51	Kaempferol deoxyhexosyl-dihexoside	C33H40O20	755.2047	0.84	755, 285, 284, 255, 227	+	−	[38]
16	10.8	Primulaverin (25)	C20H28O13	521.1511 $	−0.25	293, 181, 166, 149, 131, 125, 89, 71	+	+	NMR
17	10.84	Quercetin-3-O-[α-rhamnopyranosyl-(1→2)]-[α-rhamnopyranosyl-(1→6)]-β-glucopyranoside (16)	C33H40O20	755.2045	0.6	755, 301, 300, 271, 255	+	−	[16]
18	10.94	Quercetin-3-O-gentiobioside	C27H30O17	625.1414	0.63	625, 301, 300, 271, 255	+	−	st
19	11.57	Quercetin-3-O-neohesperoside	C27H30O16	609.1466	0.72	609, 300, 271, 255, 245	+	−	[39]
20	11.7	Kaempferol-3-O-β-glucopyranosyl-(1→2)-β-glucopyranosyl-(1→6)-β-glucopyranoside (17)	C33H40O21	771.1995	0.77	771, 285, 284, 255, 227	+	−	NMR
21	12.08	Primverin (26)	C20H28O13	521.1512 $	−0.02	293, 181, 166, 149,131, 125, 89, 71	+	+	NMR
22	12.1	Kaempferol-3-O-[α-rhamnopyranosyl-(1→2)]-[α-rhamnopyranosyl (1→6)]-β-D-glucopyranoside (18)	C33H40O19	739.2097	0.83	739, 285, 284, 255, 227	+	−	NMR
23	12.32	Isorhamnetin-3-O-β-glucopyranosyl-(1→2)-β-glucopyranosyl-(1→6)-β-glucopyranoside (19)	C34H42O22	801.2103	1.04	801, 315, 314, 300, 271, 255	+	−	NMR
24	12.44	Isorhamnetin-3-O-dideoxyhexosyl-hexoside	C34H42O20	769.2202	0.63	769, 315, 314, 300, 271, 255	+	−	[16]
25	12.6	Quercetin-3-O-rutinoside (14)	C27H30O16	609.1466	0.72	609, 301, 300, 271, 255	+	+	st, NMR
26	12.95	Quercetin-3-O-glucoside	C21H20O12	463.0884	0.39	463, 301, 300, 271, 255	+	+	st
27	13.03	4-Hydroxybenzoic acid	C7H6O3	137.0232	−4.88	167, 152, 108	+	+	st
28	13.28	Isorhamnetin-3-O-gentiobioside	C28H32O17	639.1569	0.63	639, 315, 300, 271, 255	+	+	[14]
29	14.09	5-Methoxysalicylic acid	C8H8O4	167.034	−2.6	167, 152, 108	+	+	[39]
30	14.4	Kaempferol-3-O-rutinoside	C27H30O15	593.1517	0.81	593, 285, 284, 255, 227	+	−	st
31	14.48	Hydroxyflavone-O-dihexoside	C27H30O14	577.1566	0.48	577, 253, 133	+	−	[38]
32	14.77	Kaempferol-3-O-glucoside	C21H20O11	447.0931	−0.32	447, 284, 255, 227	+	−	st
33	14.93	Isorhamnetin-3-O-rutinoside	C28H32O16	623.1614	−0.61	623, 315, 300, 271, 255	+	−	st
34	15.35	Azelaic acid	C9H16O4	187.0966	−2.32	116	+	+	[39]
35	15.39	Isorhamnetin-3-O-glucoside	C22H22O12	477.1043	0.96	477, 314, 285, 271	+	−	[38]
36	15.91	Hydroxyflavone-O-hexoside	C21H20O9	415.1037	0.59	415, 253, 133	+	−	[38]
37	16.17	Hydroxyflavone-O-dihexoside	C27H30O14	577.1566	0.48	577, 253, 133	+	−	[38]
38	18.03	Dihydroxyflavone	C15H10O4	253.0505	−0.68	253, 133	+	−	[38]
39	19.39	Hydroxyflavone-O-hexoside	C21H20O9	415.1034	0.81	415, 253, 133	+	−	[38]
40	19.62	Quercetin	C15H10O7	301.0355	0.49	301	+	−	st
41	19.67	Luteolin	C15H10O6	285.0408	1.09	285	+	−	st
42	20.76	Dihydroxyflavone	C15H10O4	253.0505	−0.68	253, 133	+	−	[38]
43	20.8	Dihydroxy-methoxyflavone	C16H12O5	283.0613	0.51	283, 268, 240, 211, 148	+	−	[38]
44	21.95	Triterpene triglycoside	C47H74O19	987.4815 $	0.89	941, 779, 455	+	−
45	23.31	Kaempferol	C15H10O6	285.0407	0.77	285	+	−	st
46	23.62	Dihydroxyflavone	C15H10O4	253.0505	−0.68	253, 133	+	−	[38]
47	23.79	Dihydroxyflavone	C15H10O4	253.0507	−0.11	253, 133	+	−	[38]
48	24.24	Isorhamnetin	C16H12O7	315.0513	0.77	315, 300	+	−	st
49	24.67	Primulasaponin VI (24)	C56H90O26	1177.566	1.09	1177, 997, 529, 483, 423, 205	+	+	NMR
50	26.52	Primulasaponin III (21)	C54H88O24	1119.5610	1.57	1119, 939, 513, 471, 425, 205	+	+	NMR
51	27.15	Hydroxyflavone	C15H10O3	237.0554	−1.26	237, 209, 117	+	−	[38]
52	27.56	Priverosaponin B 22-acetate (23)	C56H90O25	1161.571	1.04	1161, 981, 513, 473, 471, 205	+	+	NMR
53	27.58	Primulasaponin V (22)	C54H86O24	1117.5449	1.05	1117, 937, 471, 469, 425, 205	+	+	NMR
54	27.8	Priverosaponin B	C54H88O24	1119.5613	1.79	1119, 939, 473, 423, 205	+	+	[40]
55	27.99	8-Oxo-riccardin C	C28H22O5	437.1397	0.95	437, 253, 133	+	+	NMR
56	28.19	Primulasaponin I (20)	C54H88O23	1103.5645	0.08	1103, 923, 457, 455, 205	+	+	NMR
57	28.95	Riccardin C	C28H24O4	423.1609	1.6	423, 299, 196, 121	+	+	NMR

st—confirmed with standard; NMR—confirmed by NMR; ^$ [M + HCOO]⁻ ion. The numbers in bold are the numbers of compounds, isolated in this study and represented in Figure 3 and Figure 4. AP—aerial part; UP—underground part; “+” indicates the presence of chemical entities, while “−” indicates their absence.

Benzoic acid derivatives were the second most abundant group of secondary metabolites detected in studied samples. Protocatechuic acid (peak 4), salicylic acid (peak 6), gentisic acid (peak 7), 4-hydroxybenzoic acid (peak 27), 5-methoxysalicylic acid (peak 28), vanillic acid hexoside (peak 3) and syringic acid hexoside (peak 5) were identified by comparison with standards or literature data [38]. Primulaverin (25, peak 16), primverin (26, peak 21) and gaultherin (27, peak 13) displayed [M + HCOO]⁻ at m/z 521 and 491, peak at m/z 293 [C₁₁H₁₇O₉]⁻, corresponding to primeverosyl moiety and characteristic fragment ions consistent with those reported in the literature [16,38]. Their structures were also confirmed by ¹H NMR. Another compound containing primeverosyl moiety (peak 12) was tentatively identified as vanillic acid primeveroside based on its [M − H]⁻ at m/z 521 and the base peak at m/z 167 [M − C₁₁H₁₇O₉]⁻.

The triterpene saponins (peaks 49, 50, 52, 53, and 56) displayed [M − H]⁻ at m/z 1177, 1119, 1161, 1117, and 1103, respectively, and were identified by comparison of their mass-spectral data with those of reference compounds (20–24), isolated from the roots in this study and elucidated by NMR. MS/MS spectra of these compounds contained [M − 180]⁻ peaks derived by ^0,2A/^0,2X cross-ring cleavage of a neutral hexose linked by a 1→2 glycosidic bond to glucuronic acid. In the MS/MS spectra of all five saponins, fragment ions [M − 648]⁻ at m/z 529, 471, 513, 469, and 455 were also observed. The formation of these ions can be explained by the removal of the neutral sugar moiety of the molecules (C₂₄H₃₈O₂₀) with the simultaneous elimination of 2H and the formation, most likely, of a C2–C3 double bond in the negatively charged aglycone part of the molecule. Further, the presence of a fragment ion [sapogenin − H − CH₄O₂]⁻ at m/z 425 in the spectrum of compound 21 (peak 50) corresponded to the consequent loss of an epoxide moiety and H₂O [40]. The fragment ion [sapogenin − H − CO₂]⁻ at m/z 425 in the spectrum of compound 23 (peak 52) resulted from the CO₂ (44 Da) from the sapogenin moiety. The fragment ion at m/z 473 [sapogenin − H − 42]⁻ in the spectrum of compound 23 (peak 52) suggested the presence of an acetyl group in the sapogenin moiety [40]. The fragment ion at m/z 483 [sapogenin − H − CH₄O₂]⁻ and 423 [483 − C₂H₄O₂]⁻ in the spectrum of compound 24 (peak 49) corresponded to the consequent loss of an epoxide moiety H₂O and CH₃COOH. In the MS/MS spectra of all triterpene saponins, a fragmentation ion [C₈H₁₄O₆ − H]⁻ appeared at m/z 205, as a result of a complex cross-ring rearrangement of the sugar part of the molecule. Peak 54 displayed [M − H]⁻ at m/z 1119 and prominent fragment ions at m/z 939 [M − H − 180]⁻ and m/z 473 [M − H − 466]⁻ suggested the same tetrasaccharide moiety and sapogenin with a molecular formula C₃₉H₅₀O₃ and was tentatively annotated as priverosaponin B [40].

The acetophenone primeverosides (peaks 10 and 11) and bisbibenzyl compounds (peaks 55 and 57) were identified by comparison of their mass-spectral data with the respective standards with confirmed structure by ¹HNMR, which have been isolated from the roots of P. veris in our laboratories [13,20].

As shown in Table 3, flavonoids were detected only in the aerial parts of P. veris subsp. columnae, whereas acetophenone derivatives were exclusively found in the underground parts. Triterpene saponins, primulaverin, primverin, gaultherin and some other benzoic acid derivatives were identified in both the aerial and underground parts of P. veris subsp. columnae. In general, the chemical profile of the studied plant is consistent with that reported for P. veris subsp. veris from Greece [14,16] and P. vulgaris from Turkey [38]. However, acetophenone primeverosides (peaks 10 and 11), bisbibenzyl compounds (peaks 54 and 56) and triterpene saponins (peaks 49, 52 and 53) are annotated now for the first time in P. veris. Paeonolide (peak 11) was recently isolated from the roots of P. veris from Bulgarian origin [13], while 8-oxo-riccardin C and riccardin C (peaks 54 and 56) were isolated in our previous study of the underground parts of the plant collected from the same location [20].

2.3. Inhibitory Effect of P. veris subsp. columnae Extracts on Biofilm Formation

The present study evaluates the potential anti-biofilm activity of various plant extracts, including a methanolic extract obtained from underground parts and methanol and acetone extracts obtained from aerial parts, against three clinically significant bacterial species: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Preliminary results indicate that at a concentration of 500 µg/mL, the methanolic extract from underground parts exhibited the highest inhibitory activity against E. coli (74.7%) and S. aureus (64.3%), whereas lower activity was observed against P. aeruginosa (44.6%) (Figure 6A). In comparison, the extracts obtained from aerial parts showed moderate to weak activity, with the highest inhibition observed against S. aureus (39.62%) (Figure 6A). Similarly, high percentages of the inhibition of biofilm formation by S. aureus, P. aeruginosa, E. coli, Bacillus subtilis, and Enterococcus faecalis have also been reported for methanolic extracts of P. veris, Populus nigra, Populus alba, onion, olive leaves, and others [13,41,42,43]. Moreover, examination of the chemical profile of the extracts tested in our study revealed the presence of flavonoids. According to the literature, these compounds are known to inhibit biofilm formation, through mechanisms that include interference with cell adhesion, inhibition of quorum sensing systems, and blockage of the synthesis of components of the extracellular matrix, among others [44,45,46,47,48]. In support of our findings, numerous literature reports demonstrate that the secondary metabolites such as phenolic glycosides and triterpene saponins, as well as flavonoids such as quercetin and kaempferol, exhibit effective antibiofilm activities [44,45,49,50]. The tested acetone extract exhibited selective antibiofilm activity, with strong inhibitory effects against E. coli and S. aureus, but an opposite effect on P. aeruginosa biofilm (Figure 6B). Such behavior is consistent with the literature data highlighting the high adaptability of P. aeruginosa and its ability to modulate biofilm-related responses to phytochemicals from Tradescantia pallida often without inhibitory effects on biofilm formation [51]. Our findings place the observed anti-biofilm activity in the upper range reported for crude plant extracts, which typically show 20–70% inhibition depending on composition and experimental conditions, supporting the relevance of our results, particularly for the extract from underground parts. The lower percentage of inhibition, or the absence of inhibition, observed for all three extracts against P. aeruginosa is often associated with the biology of this strain, recognized as one of the most treatment-resistant biofilm-forming bacteria. This is mainly due to the dense structure of its polymeric matrix, an efficient quorum sensing system, and the activity of efflux pumps [52]. It is known that this microorganism possesses a hierarchical quorum sensing network (Las, Rhl, PQS, and IQS) that integrates population density and environmental stress signals. Evidence suggests that quorum sensing systems respond not only to cell density but also to external stressors, such as subinhibitory concentrations of antibiotics, which can shift regulatory output toward increased biofilm production. Because quorum sensing positively regulates EPS production and biofilm maturation, the stimulation of biofilm formation observed under experimental conditions may reflect activation or stress-induced modulation of quorum sensing, thereby promoting biofilm development [53].

Figure 6.

Anti-biofilm activity of P. veris subsp. columnae extracts on three bacterial strains: (A) methanol extracts from underground parts (MeOH Underground) and from aerial parts (MeOH Aerial); (B) acetone extract from aerial parts (Acetone Aerial). Statistically significant differences are indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001, n = 6).

The data on the biofilm inhibition in the Gram-positive bacterium S. aureus corroborated well with those reported in the literature. It is well known that plant extracts tend to exhibit greater efficacy against Gram-positive biofilms, likely due to the simpler structure of the cell wall and the absence of an outer membrane, which facilitates the penetration of phytochemicals [54,55]. Additionally, the higher inhibition observed with the acetone extract is not unusual. This is likely attributable to the presence of nonpolar flavonoids (aglycones), as confirmed by chemical analysis. These compounds are known to exhibit stronger anti-biofilm activity compared with their glycosylated counterparts. Their increased lipophilicity allows more effective interaction with bacterial membranes and biofilm matrices, leading to disruption of biofilm architecture, inhibition of quorum sensing, and suppression of virulence factors [56,57]. Moreover, an additional important aspect of the present study is the extraction solvent used. In our case, greater biofilm inhibition was observed with methanolic extracts than acetone extract. This is likely due to the synergistic action of phenolic acid glycosides and triterpene saponins identified in the chemical analysis. Phenolic glycosides primarily interfere with bacterial metabolism, adhesion, and oxidative balance, while triterpene saponins exert direct membrane-disrupting effects [58,59,60]. Their combined presence in plant extracts may enhance antibacterial and anti-biofilm activity, making them particularly suitable for application in anti-biofilm strategies. The findings are also consistent with our previous study [13].

Scanning electron microscopy (SEM) analysis showed that treatment with the plant extract led to significant morphological changes in the biofilms of Staphylococcus aureus, including reduced biofilm biomass, disrupted cell clustering, and visible surface damage to bacterial cells (Figure 7B). The biofilm appeared fragmented, with a weakly developed cohesive architecture, increased spacing between individual cells, and a lack of a multilayer structure. In contrast, the control micrographs showed a high density of biofilm projections characterized by typical mushroom-like vertical conformations (Figure 7A). Regarding morphological changes at the level of individual cells, isolated or widespread bipolar invaginations and changes in cell shape were observed (Figure 7B, white arrows). We assume that these morphological aberrations are consistent with those described in the literature and generally indicate damage to both the cell wall and the underlying membrane, which is indicative of the onset of cell lysis [61,62,63].

Figure 7.

Morphological analysis of biofilms treated with P. veris subsp. columnae extracts using scanning electron microscopy: (A) Control group S. aureus biofilms. (B) S. aureus biofilms treated with methanol extract from underground parts. (C) Control group E. coli biofilms. (D) E. coli biofilms treated with methanol extract from underground parts. Bars = 2–10 μm. White arrows indicate morphological invaginations.

Data for E. coli revealed a significant reduction in biofilm formation, likely due to suppression of surface colonization, as evidenced by the presence of isolated, predominantly dispersed bacterial cells. In contrast, the control samples showed a well-structured mature biofilm with densely adhered cells (Figure 7C). Additionally, significant morphological alterations were detected, including cell deformation manifested as radial invaginations (along the entire length of the cell) or bipolar invaginations (Figure 7D, white arrows). The observed findings in both strains correlated well with the quantitative inhibition data (74.7% for E. coli and 64% for S. aureus) and suggest that the extract exerts its anti-biofilm activity through a combination of extracellular matrix disruption, anti-adhesion effects, and morphological damage of the cells (Figure 7B,D). We hypothesize that phenolic acid glycosides and triterpene saponins exert different but complementary effects on bacterial morphology. Phenolic glycosides primarily induce sublethal structural and metabolic changes, leading to reduced adhesion and biofilm formation, while triterpene saponins cause direct membrane disruption, cytoplasmic leakage, and cell lysis [58,59,64].

2.4. Cytotoxicity of P. veris subsp. columnae Extracts on Human Melanoma and Keratinocytes

All three tested extracts showed low cytotoxicity toward human keratinocytes and melanoma cells (Figure 8). During 24 h treatment with an extract obtained from the aerial parts of the plant, signs of cellular stress were observed, including an increase in the protein content of the cytoplasm, especially pronounced in melanoma cells even at concentrations above 5 μg/mL. Non-cancerous cells were more sensitive to the extract obtained from underground parts, whereas melanoma cells exhibited high resistance even at concentrations up to 150 μg/mL of all extracts. In keratinocytes, significant cell loss was observed only at concentrations above 50 μg/mL. Flavonoid aglycones present in the acetone extract again had no significant effect on cell viability and showed low cytotoxicity only at high concentrations and only in keratinocytes.

Figure 8.

Cytotoxicity of P. veris subsp. columnae extracts on HaCaT (A,C) and A375 (B,D) cells after treatment for 24 (A,B) and 72 h (C,D) according to CV assay. Vehicle control is marked as 0 μg/mL. Viability of treated cells is compared with untreated control and expressed as mean% ± SE. Statistically significant differences are indicated with an asterisk (* p < 0.05). The color of the asterisks corresponds to the color of the samples in the graph.

Longer treatment did not result in a decrease in cell mass, indicating that the extracts probably had no effect on the cell cycle in either human cell type. Overall, these findings suggest that P. veris subsp. columnae extracts exhibit low cytotoxicity towards human cells at concentration ranging from 1 to 50 μg/mL. The strongest effect on cell survival was observed for saponin-rich extract obtained from the underground parts of the plant. However, even at higher concentrations (up to 200 μg/mL), its cytotoxic effect was reversible, and after prolonged treatment (72 h), recovery of the cell population to approximately 80% of the control was observed. These results indicate that the extracts exhibit promising preliminary antibiofilm activity together with low cytotoxicity in the tested cell models. Nevertheless, further mechanistic, microbiological and safety studies are required before their practical application can be considered. The biological activities of natural and synthetic saponins have been studied by other research groups, particularly with respect to their antitumor properties [65]. For saponins with antitumor activity, the reported effective concentrations generally range from 1 to 13 μg/mL. In our study, no significant differences were observed in the direct toxicity (up to 24 h of treatment) of the extract obtained from underground parts between cancerous and non-cancerous cells. This extract, which is rich in primulasaponins, caused a significant decrease in cell viability only at concentrations above 75 μg/mL for keratinocytes and above 150 μg/mL in melanoma cells. The relatively low cytotoxicity of the extracts toward cancer cells may be due to the antagonistic action of other constituents present in the extract in addition to the saponins. Extracts containing polar and non-polar flavonoids did not significantly affect cell viability even at concentrations up to 200 μg/mL, which is consistent with the results reported by other authors [66]. Although the highest concentration tested for cytotoxicity (200 μg/mL) was lower than the concentration that exhibited antibiofilm activity (500 μg/mL), the extracts still demonstrated low cytotoxicity, highlighting their potential as promising candidates for further investigation in biomedical application.

3. Materials and Methods

3.1. General Experimental Procedures

Specific optical rotation values were measured on a Jasco P-2000 polarimeter (Jasco, Tokyo, Japan) at the D line of sodium lamp at 20 °C by using a 0.5 dm quartz cell. The [α]$\begin{array}{c}20\\ D\end{array}$ values are given in deg·cm³·g⁻¹·dm⁻¹, and concentration (c) is given in g·cm⁻³. The 1D and 2D NMR (¹H and ¹³C NMR, DEPT, COSY, HSQC, HMBC, and ROESY) spectra were recorded on a Bruker Avance NEO 600 spectrometer (Biospin GmbH, Rheinstetten, Germany) with the operating frequencies 600 MHz (¹H) and 150 MHz (¹³C) and a Bruker Avance III 500 (Bruker, Darmstadt, Germany) spectrometer at 500 MHz for ¹H and 125 MHz for ¹³C, with CD₃OD, (CD₃)₂CO and CDCl₃. The chemical shifts (δ) are expressed in ppm, and coupling constants (J) are expressed in Hz. HR-ESI-MS spectra of the new compounds were recorded using a system consisting of a liquid chromatograph (1290 Infinity II LC system; Agilent Technologies, Waldbronn, Germany) with a binary pump, a thermostated column oven compartment, and an autosampler connected to a QToF mass detector (6546 LC/Q-TOF, Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent dual Jet Stream (AJS) electrospray ion source (ESI). A methanol solution of the compound was introduced into the mass spectrometer using a mobile phase composed of solvents A, water, and B, acetonitrile, both containing 0.1% formic acid, at a flow rate of 0.20 mL min⁻¹. Mass spectra of positive or negative ions were recorded in MS mode in the m/z range of 80–1700 under the following conditions: capillary voltage, 3500 V; fragmentor voltage, 100 V; nozzle voltage, 1000 V; skimmer, 65 V; octupole 1 RF Vpp, 750 V; sheath gas temperature (nitrogen), 300 °C; sheath gas flow (nitrogen), 11 L min⁻¹; drying gas (nitrogen), 11 L min⁻¹; and nebulizer, 35 psi. Ions m/z 121.0508 and 922.0097 in positive ion mode and 112.9855, 966.0007 and 1033.9881 in negative ion mode were used to lock the masses for precise mass measurements. A personal computer system with Agilent MassHunter software (revision 10.0) was used for data collection and processing. IR spectra were recorded on a Shimadzu IR Spirit FT-IR spectrometer (Shimadzu Corporation, Kyoto, Japan) using QATR-S as a single-reflection ATR measurement attachment. UV spectra were recorded on a GBC Cintra UV/Vis spectrometer with acetone as the solvent in the concentration range 2−5 × 10⁻⁵ M.

Sephadex LH-20 (BioChemika, Fluka Chemie GmbH, Buchs, Switzerland), Silica gel 60 (70-230 mesh ASTM) (Merck KGaA, Darmstadt, Germany), and LiChroprep^® RP-18 (40–63 µm) (Merck KGaA, Darmstadt, Germany) were used as adsorbents for column chromatography (CC). MPLC was performed on LiChroprep^® RP-8 (Merck, Darmstadt, Germany). Thin-layer chromatography (TLC) on Silica gel 60 F₂₅₄ (Merck, Darmstadt, Germany) and Silica gel RP-18 (Merck, Darmstadt, Germany) plates was used for monitoring the separation of the extracts and for preparative TLC. Preparative TLC was performed on Silica gel 60 F₂₅₄ glass plates (20 × 20 cm, Merck, Darmstadt, Germany). The spots were visualized by spraying with concentrated H₂SO₄ or with NP reagent (1% diphenylboronic acid 2-aminoethyl ester in ethyl acetate) followed by heating at 105 °C. All solvents used were of HPLC grade. Semipreparative HPLC separation was performed on an Agilent Instrument 1100 Series equipped with a DAD (Agilent Technologies Inc., Santa Clara, CA, USA). The column used was a Zorbax Eclipse XDB C18 (9.4 mm × 250 mm, 5 μm, Agilent Technologies, Waldbronn, Germany). Solvents for HPLC separation were of chromatographic grade. Solvents for CC were freshly distilled.

3.2. Plant Material

Primula veris subsp. columnae (Ten.) Lȕdi was collected in April of 2022 in the village of Gostilje (43.667421° N, 19.818758° E) on Mt. Zlatibor, Serbia. The plant material was determined by biologist Dr. Marjan Niketić, Natural History Museum, Belgrade, Serbia. A voucher specimen (BEOU17833) was deposited in the Herbarium of the Institute of Botany and Botanical Garden “Jevremovac”, University of Belgrade (BEOU), Belgrade, Serbia. The plant material was air-dried at room temperature in a dark place for 2 weeks and then stored at room temperature in a paper bag.

3.3. Preparation of Extracts

The fresh aerial parts (leaves, stems and flowers) of P. veris subsp. columnae (188 g) were washed without milling with 3 L of acetone for 3 min in an ultrasonic bath. After evaporation of acetone using rotavapor, 2.6 g of the crude acetone extract was obtained (the yield was 1.38%).

The dried aerial parts of P. veris subsp. columnae (65 g) were ground and extracted 3 times with 300 mL of CHCl₃ for 48 h at room temperature in an ultrasonic bath during the last hour of each extraction. After evaporation of the solvents using rotavapor, the crude extract weighed 2.31 g (the yield was 3.54%). The remaining plant material was extracted 3 times with 300 mL of CH₃OH for 48 h at room temperature in an ultrasonic bath during the last hour of each extraction. After evaporation of combined methanol extracts, 3.42 g was obtained (the yield was 5.26%). TLC comparison of the chloroform extract with the acetone extract showed the same chemical profile regarding flavonoids 1–13, and it was not further worked up for isolation of individual compounds.

The air-dried underground parts (roots and rhizomes) of P. veris subsp. columnae (200 g) were ground and extracted 3 times with 500 mL of CHCl₃ for 48 h at room temperature in an ultrasonic bath during the last hour of each extraction. After evaporation of the solvent, the crude extract weighed 2.2 g (the yield was 1.1%). The remaining plant material was extracted 3 times with 500 mL of CH₃OH for 48 h at room temperature in an ultrasonic bath during the last hour of each extraction. After evaporation of combined methanol extracts, 18.6 g was obtained (the yield was 9.40%). TLC of the chloroform extract showed the presence of the same bisbibenzyl and acetophenone compounds isolated from the plant from the same location and described in our previous study [20], and it was not further worked up for isolation of individual compounds.

3.4. Isolation of Individual Compounds

3.4.1. Isolation of Surface Flavonoids from the Acetone Extract of P. veris subsp. columnae

Crude acetone extract was dissolved in acetone and fractionated using reversed phase semi-preparative HPLC equipped with a DAD detector (254 nm, 280 nm) (Agilent Technologies Inc., Santa Clara, CA, USA) and Zorbax Eclipse XDB C-18 column (250 × 9.4 mm, 5 µm, Agilent Technologies, Waldbronn, Germany). The eluent was H₂O/CH₃CN, and the flow rate was 4 mL/min. The basic HPLC method 1 was as follows: 0–15 min, 35% CH₃CN; 16–25 min, 35–100% CH₃CN; and 25–30 min, 100% CH₃CN. After the first fractionation, 9 fractions were obtained: fr. 1 (t_R = 10.9 min), fr. 2 (t_R = 14.4 min), fr. 3 (t_R = 20.0 min), fr. 4 (t_R = 20.5 min), fr. 5 (21.2 min), fr. 6 (t_R = 21.8 min), fr. 7 (t_R = 22.2 min), fr. 8 (t_R = 23.9 min), and fr. 9 (24.1 min). Fr. 1 and 2 were pure compounds 4 (24 mg) and 10 (25 mg), respectively, while fr. 2, 3, 5, and 8 were additionally purified into pure compounds 7 (17 mg), 5 (6 mg), 8 (18 mg) and 11 (4 mg), respectively, using the same method 1. Fr. 6 was separated using isocratic HPLC method 2: 0–15 min, 50% CH₃CN, and 16–20 min, 100% CH₃CN, to give compounds 1 (17 mg) and 9 (12 mg). Separation of Fr. 7 using isocratic method 3, 0–24 min, 40% CH₃CN, and 25–30 min, 100% CH₃CN, afforded compounds 2 (27 mg) and a mixture of 3 and 6, which were further purified by method 2 to give 3 (18 mg) and 6 (5 mg). Fr. 9 was separated into two compounds, 12 (1.2 mg) and 13 (1.4 mg), using method 2.

8,2′-Dimethoxy-5-hydroxyflavone (12): light yellow amorphous substance. UV/Vis (MeOH, λ_max, nm) (log ε): 278 (4.15), 317sh (3.69), 368sh (3.34). IR (capillary film, ν_max, cm⁻¹): 3068, 2978, 2920, 2851, 1664, 1617, 1588, 1579, 1511, 1489, 1438, 1401, 1290, 1253, 1212, 1149, 1065, 1008, 868, 809, 766, 692. ¹H and ¹³C NMR, see Table 1. HR-ESI-MS m/z 299.0917 [M + H]⁺ (calcd for C₁₇H₁₅O₅, 299.0914).

8,3′-Dimethoxy-5-hydroxyflavone (13): light yellow amorphous substance. UV/Vis (MeOH, λ_max, nm) (log ε): 278 (4.15), 317sh (3.69), 368sh (3.34). IR (capillary film, ν_max, cm⁻¹): 3068, 2988, 2923, 2852, 1660, 1623, 1592, 1579, 1508, 1492, 1439, 1402, 1291, 1249, 1211, 1147, 1063, 1006, 868, 806, 778, 764, 694. ¹H and ¹³C NMR, see Table 1. HR-ESI-MS m/z 299.0919 [M + H]⁺ (calcd for C₁₇H₁₅O₅, 299.0914).

3.4.2. Isolation of Flavonoids from the Methanol Extract of P. veris subsp. columnae Aerial Parts

The methanol extract (1.030 g) of the aerial part was subjected to CC on a Sephadex LH 20, and 12 fractions (Fr. 1–12) were obtained. MPLC on LiChroprep^® RP-8 with H₂O/MeOH (6:4 to 0:1) of Fr. 5 (130 mg) yielded 7 subfractions (S1–S7). Further purification of S1 (6.5 mg) and S2 (8mg) by prep. TLC (RP-18, MeOH/H₂O, 1:1) gave compounds 15 (2.09 mg) and 17 (1.8 mg), respectively. Prep. TLC (Silica gel, CHCl₃/MeOH/H₂O, 61:32:7) of S4 (22 mg) yielded 1.5 mg of 18. MPLC on LiChroprep^® RP-8 of fr. 6 (175 mg) with H₂O/MeOH (7:3 to 0:1) afforded 7 subfractions (S6-1–S6-7). Prep. TLC (Silica gel, CHCl₃/MeOH/H₂O, 61:32:7) of subfr. S6-4 and S6-5 (77 mg) afforded 15 (9.7 mg) and 16 (2.5 mg). Subfr. S6-7 (16.9 mg) separated by prep. TLC (Silica gel, CHCl₃/MeOH/H₂O, 61:32:7) gave 1.5 mg of 14, 1.8 mg of 18, and 4.2 mg mixture of 17 and 19 (in ratio1:0.4 by NMR).

3.4.3. Isolation of Compounds from the Methanol Extract of P. veris subsp. columnae Underground Parts

A portion of the methanol extract from underground parts (1 g) was subjected to CC on Sephadex LH-20 using MeOH as eluent, and 3 main fractions were collected: A (0.56 g), B (0.40 g) and C (0.033 g). MPLC on LiChroprep^® RP-8 with H₂O/MeOH (from 4:1 to 0:1) of a portion of fr. A (0.28 g) gave 8 fractions, A1–A8. MPLC on LiChroprep^® RP-8 (H₂O/MeOH, 2:1) fr. A2 afforded compound 24 (5.7 mg). MPLC on LiChroprep^® RP-8 (H₂O/MeOH, 1:1 and 0:1) of fr. A3 yielded 24 (15 mg) and 21 (37 mg). Fr. A4 (9.8 mg) contained 21. MPLC on LiChroprep^® RP-8 (H₂O/MeOH, 1:1 to 0:1) of fr. A5 and further prep. TLC (RP-18 MeOH/H₂O 7:3 (×2)) afforded 7.6 mg of compound 22. Fr. A6 (70 mg) contained compound 23. MPLC on LiChroprep^® RP-8 (H₂O/MeOH, 1:1 to 0:1) of fr. A7 gave 10 mg of 23 and 40 mg of 20. A portion of fr. B (0.15 g) was subjected to MPLC on LiChroprep^® RP-8 with H₂O/MeOH (from 4:1 to 0:1) to give 6 main fractions, B1–B6. Further purification of fr. B3 (16.7 mg) by prep. TLC (Silica gel, CHCl₃/MeOH/H₂O, 60:15:4) afforded 28 (2.4 mg). Fr. B4 (4.9 mg) was a mixture of 25 and 27 in a ratio of 1:1.7 (deduced by ¹H NMR), fr. B5 (113.97 mg) contained pure 25 and fr. B6 (7.3 mg) was a mixture of 25 and 26 in a ratio of 1.4:1 (deduced by ¹H NMR).

Primulasaponin VI (24): Amorphous white powder, [α]$\begin{array}{c}20\\ D\end{array}$ −32.8 (c 0.45, MeOH); FT-IR (ATR): ν_max 3351.8, 2925.3, 1707.3, 1603.2, 1031.2 cm⁻¹; ¹H (600 MHz, CD₃OD) and ¹³C (150 MHz, CD₃OD): Table 2; HR-ESI-MS m/z: 1177.5655 ([M − H]⁻, calcd. 1177.5648 for C₅₆H₈₉O₂₆).

3.4.4. Acid Hydrolysis of Compound 24

Compound 24 (5 mg) dissolved in 1 mL of MeOH was mixed with 5% HCl (5 mL) and was refluxed for 4 h. The reaction mixture was concentrated under reduced pressure to remove MeOH. After extraction with CH₂Cl₂, the aqueous solution was concentrated under vacuum to obtain sugar residue and compared with authentic standards (D-glucose, D-galactose, D-glucuronic acid and L-rhamnose) by TLC (Silica gel, CH₃CN/H₂O, 85:15).

3.5. UHPLC/HRMS Analysis

UHPLC–HRMS/MS analysis was carried out following the protocol reported in our previous study [67] using a Vanquish UHPLC system coupled to a Q Exactive Plus Orbitrap^® mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Separation was achieved on an Accucore C18 column (150 × 2.1 mm, 2.6 μm) using water containing 0.1% formic acid (A) and acetonitrile (B) as mobile phases under gradient elution. The flow rate was set at 0.3 mL/min, and the injection volume was 3 μL.

Mass spectrometric detection was carried out in negative ionization mode with a spray voltage of 2.90 kV and a capillary temperature of 320 °C. Full-scan MS spectra were acquired over an m/z range of 120–1200 at a resolution of 70,000, while MS/MS spectra were obtained using data-dependent acquisition (Top 5) with stepped collision energies of 20, 40, and 70. Nitrogen was used as both the nebulizing and the collision gas.

The extracts were initially dissolved in methanol to obtain stock solutions of 5000 mg/L and further diluted to 400 mg/L using a 50:50 (v/v) water/methanol mixture prior to analysis. Instrument operation and data acquisition were carried out with Xcalibur software (version 4.2 SP1), whereas data processing and compound identification were performed using FreeStyle software (version 1.5) from Thermo Fisher Scientific.

For compound identification, authenticated analytical standards were used. The following standards were obtained from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany): caffeic acid (≥98%, CAS: 501-16-6), kaempferol (≥95%, CAS: 520-18-3), luteolin (≥95%, CAS: 491-70-3), quercetin (≥95%, CAS: 117-39-5), isorhamnetin (≥95%, CAS: 480-19-3), quercetin 3-O-gentiobioside (≥95.0%, CAS: 7431-83-6), kaempferol 3-glucoside (≥95%, CAS: 480-10-4), quercetin 3-glucoside (≥95%, CAS: 482-35-9), kaempferol 3-rutinoside (≥95%, CAS: 17650-84-9), and isorhamnetin 3-rutinoside (≥95%, CAS: 604-80-8). Additional standards were purchased from Sigma-Aldrich (Darmstadt, Germany): protocatechuic acid (≥99%, CAS: 99-50-3) and 4-hydroxybenzoic acid (≥99%, CAS: 99-96-7).

3.6. Inhibition of Biofilm Formation

The efficacy of the plant extracts used for inhibiting biofilm formation was evaluated using the crystal violet staining method [68]. Briefly, 18 h bacterial cultures from Gram-negative and Gram-positive strains—Pseudomonas aeruginosa 15692 (ATCC), Escherichia coli 25922 (ATCC), and Staphylococcus aureus 29213 (ATCC)—were grown in Trypticase soy broth or Nutrient broth according to their metabolic characteristics at 37 °C. Biofilms were cultivated in M63 media with the following composition: 0.02 M KH₂PO₄, 0.04 M K₂HPO₄, 0.02 M (NH₄)₂SO₄, 0.1 mM MgSO₄, and 0.04 M glucose. For the assay, plant extracts at a final concentration of 500 µg/mL were mixed with an overnight bacterial culture (1 × 10⁵ CFU/mL) diluted 1:100 in M63 medium and transferred into 96-well microtiter plates in six replicates. A bacterial inoculum diluted in M63 medium containing 2% DMSO was used as the control. Plates were incubated at 37 °C for 24 h under static conditions. After incubation, wells were washed three times with phosphate-buffered saline (PBS) to remove non-adherent cells. Biofilms were stained with 0.1% crystal violet for 15 min and washed, and the dye was solubilized with 70% ethanol. Absorbance was measured at 570 nm, using an ELISA plate reader (LTEK INNO, Gyeonggi-do, Republic of Korea). To confirm the quantitative data on biofilm inhibition, statistical significance was indicated for all tested samples compared with the control using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test (* p < 0.05; ** p < 0.01; *** p < 0.001, sample number n = 6). Significant differences are indicated by asterisks. The data are reported as means ± standard deviation (SD) using OriginPro 9.0 software.

The biofilm inhibition assays were calculated based on the following formula:

Biofilm inhibition (%) = (control − test sample)/control × 100

3.7. Scanning Electron Microscopy (SEM)

An 18 h culture of E. coli 25922, S. aureus 29213 was prepared in the presence of plant extracts (500 μg/mL) and applied onto pre-treated sterile polystyrene fragments. After incubation at 37 °C for 24 h to allow biofilm formation, samples were washed and fixed with 4% glutaraldehyde, followed by post-fixation with 1% osmium tetroxide. The biofilms were then dehydrated through a graded ethanol series, mounted on SEM stubs, air-dried, and gold-coated. Finally, the samples (three independent) were examined using scanning electron microscopy (SEM) at 20 kV. To visualize the structure of the biofilm, six independent microscopic fields were analyzed for each sample.

3.8. Cytotoxicity Assessment of P. veris subsp. columnae Extracts

The cytotoxicity of P. veris subsp. columnae extracts on human keratinocyte cells (HaCaT cell line, ATTC) and human melanoma cells (A375 cell line, ATCC) was evaluated by CV assay (crystal violet staining) as previously described [69]. The dried extracts were dissolved in DMSO as stock solutions, and then the stock was diluted in cell culture medium (DMEM with 10% FBS) to a final concentration between 1 and 200 μg/mL. The maximum DMSO concentration in test solutions was used as the vehicle control. All experiments were performed in triplicate. The absorbance of the samples was measured at 570 nm using an Epoch Microplate Spectrophotometer (BioTek^® Instruments Inc., Winooski, VT, USA) with Gen5™ Data Analysis software, version 1.11.5. The results are presented as the percentage of cell viability compared with untreated control cells. Data were analyzed with OriginPro 9.0 and presented as mean ± SE. The statistical significance was determined by one-way ANOVA at the 0.05 level (* p < 0.05).

4. Conclusions

The present comparative UHPLC-MS/MS study of the aerial and underground parts of Primula veris subsp. columnae revealed the presence of surface flavonoids, flavonoids and their glycosides, phenolic compounds (phenolic acids, acetophenones and bisbibenzyls) and their glycosides, as well as triterpene saponins. The results confirmed that flavonoids are predominately accumulated in the aerial parts of the plants, while triterpene saponins and other phenolic compounds dominated in the underground parts. In addition, the structures of 28 compounds were confirmed by NMR after their isolation from the corresponding extracts. The identification of the two previously unreported natural products, 8,3′-dimethoxy-5-hydroxyflavone and primulasaponin VI, together with the first report of 8,2′-dimethoxy-5-hydroxyflavone and) from a natural source, represents a contribution to the phytochemistry of this well-known medicinal plant.

Furthermore, the obtained results clearly demonstrate that the antibiofilm efficacy of the investigated P. veris subsp. columnae extracts is strongly influenced by the extraction solvent, the plant part from which the extracts were obtained, and the tested microbial strain. The substantial differences observed between the acetone and methanol extracts highlight not only the pronounced activity of the bioactive compounds but also their selectivity toward different bacterial species. The highest activity of the methanol extract obtained from the underground parts was recorded against E. coli, whereas the acetone extract showed no inhibitory activity against P. aeruginosa. Overall, the results confirm that the investigated P. veris subsp. columnae extracts possess significant antibiofilm potential, and further elucidation of their mechanisms of action will be essential for the effective suppression of biofilm formation. Moreover, the extracts obtained from the aerial parts exhibited low in vitro cytotoxicity toward human cells at concentrations of up to 200 μg/mL, whereas the cytotoxicity observed for the extract from the underground parts was reversible. These findings suggest that the identified bioactive substances may be suitable candidates for the development of products with potential biomedical applications. Future studies could contribute to the development of novel natural antibiofilm agents for use in medicine and in the prevention and control of biofilm-associated bacterial infections.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants15152259/s1.

Author Contributions

Conceptualization, A.T. and M.N.; methodology, A.T., M.N., T.P.-K. and T.T.-H.; resources, M.N.; investigation and data curation, A.T., M.N., M.J., P.S., V.I., P.D., R.V., T.P.-K. and T.T.-H.; writing—original draft preparation, A.T., M.N., T.P.-K. and T.T.-H.; writing—review and editing, A.T., M.N., M.J., P.S.,V.I., P.D., R.V., T.P.-K. and T.T.-H.; visualization, A.T., M.N., R.V. and T.P.-K.; supervision, A.T. and M.N.; project administration, A.T., M.N. and T.P.-K.; funding acquisition, T.P.-K. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the National Science Fund at the Ministry of Education and Science, Bulgaria (Research Grant KP-06-H91/13; 4 December 2025).