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Article

Phytochemical Characterization and Anti-Biofilm Activity of Primula veris L. Roots

by
Antoaneta Trendafilova
1,*,
Desislava Raykova
1,
Viktoria Ivanova
1,
Miroslav Novakovic
2,
Paraskev Nedialkov
3,
Tsvetelina Paunova-Krasteva
4,
Ralitsa Veleva
5 and
Tanya Topouzova-Hristova
5
1
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
2
Institute of Chemistry, Technology and Metallurgy, National Institute of the Republic of Serbia, University of Belgrade, 11000 Belgrade, Serbia
3
Pharmacognosy Department, Faculty of Pharmacy, Medical University of Sofia, 1000 Sofia, Bulgaria
4
Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
5
Department of Cellular and Developmental Biology, Faculty of Biology, Sofia University St Kliment Ohridski, 1164 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Molecules 2025, 30(8), 1702; https://doi.org/10.3390/molecules30081702
Submission received: 26 February 2025 / Revised: 1 April 2025 / Accepted: 8 April 2025 / Published: 10 April 2025
(This article belongs to the Special Issue Discovery, Isolation, and Mechanisms of Bioactive Natural Products)
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Versions Notes

Abstract

:
In this study, three new undescribed triterpene saponins named primulasaponin III–V (19–21) were isolated from the roots of Primula veris L. of Bulgarian origin together with the known primulasaponin I. Their structures were elucidated via 1D and 2D NMR spectroscopy and HR-ESI-MS. In addition, 17 known phenolic compounds (six flavones, three acetophenones, four bisbibenzyls, and four phenolic glycosides) were identified in the chloroform and methanol extracts. Among them, flavone, 2′-methoxyflavone, 3′-methoxyflavone, 3′-hydroxy-4′,5′-dimethoxyflavone, 2′,5′-dimethoxyflavone, 3′-methoxy-4′,5′-methylendioxyflavone, paeonol, 2-primeverosyl-5-methoxy-acetophenone, and paeonolide were detected for the first time in the roots of P. veris. The minimum inhibitory and minimum bactericidal concentrations of the chloroform and methanol extracts of P. veris roots and the saponin-enriched fraction were determined, with MIC values ranging between 0.5 and 1 mg/mL. Additionally, the tested samples were evaluated for their ability to inhibit biofilm formation in the presence of sub-MICs. All tested samples showed better biofilm inhibition of Gram-negative strains compared to Gram-positive strains. The strongest effect was observed for the chloroform extract against the biofilm formation of Pseudomonas aeruginosa, while the saponin-enriched fraction showed the highest percentage of biofilm inhibition of Escherichia coli, Staphylococcus aureus, and Staphylococcus mutans. At the same time, chloroform extract showed lower cytotoxicity against human keranocyte cell line HaCaT, as compared with methanol extract and the saponin-enriched fraction.

Graphical Abstract

1. Introduction

Genus Primula is the largest one in the Primulaceae family and is distributed in the temperate and cold regions of the Northern Hemisphere [1]. Primula veris L. (syn. P. officinalis (L.) Hill), commonly known as cowslip, is an herbaceous perennial plant native to almost all of Europe, with its area of inhabitance extending from the northern parts of the Mediterranean to Scandinavia, as well as large parts of Asia [2,3]. In folk medicine, the leaf and root extracts (infusion, decoction, or tincture) of P. veris and other Primula species are used to treat various respiratory diseases, such as coughs, bronchitis and colds, skin problems, wound healing, ulcers; for toothaches; and against snake poison [1,4,5,6,7]. Primulae radix and Primulae flos are listed in the European pharmacopeia, and their extracts are incorporated in various expectorant and diuretic drugs [8]. Additionally, primrose is a valuable horticultural plant often used as an ornamental species [9].
Primula species are known to produce various polyphenolic compounds such as flavonoids; mono-, di-, and tri-glycosides; phenolic acids; phenolic glycosides (primulaverin and primeverin); and epicuticular flavonoids [1,5,10]. The latter are accumulated on the external surface of the plant and are mainly aglycones substituted with hydroxy, methoxy, methylenedioxy, and acetyl groups. These compounds exhibit anti-asthmatic, anti-inflammatory, antiviral, antioxidant, and antigenotoxic activities [11,12,13,14]. Epicuticular flavonoids are considered to be chemotaxonomic markers for the genus [15,16,17,18,19]. Aside from polyphenols, cowslip is also known for its species-specific saponins. So far, primula acids (primulasaponin) I and II, priverosaponin B, priverosaponin B-22-acetate, and primacrosaponin have been isolated from P. veris [9,10,20,21,22,23]. The secretolytic and expectorant activity of P. veris is attributed to the presence of saponins [24]. Other compounds found in the Primula genus are bisbibenzyls, which were initially considered as chemical markers of liverworts but were identified for the first time in higher plants, specifically in P. veris subsp. macrocalyx from Mt. Altay in Russia [25]. Recently, riccardin C and other bisbibenzyls were found in Russian, Armenian, and Iranian P. veris subsp. macrocalyx [26] and Serbian P. veris subsp. columnae [27]. Bisbibenzyl derivatives exhibited diverse biological activities such as antitumor and anti-leukemia [28] and antibacterial and antifungal activities [29].
The increasing prevalence of biofilm-associated infections, which often lead to chronic conditions, has drawn significant attention from the scientific community in the search for suitable inhibitors of bacterial biofilms. According to data from the U.S. National Institutes of Health, approximately 65% of all bacterial infections and 80% of chronic infections are associated with biofilm formation in patients [30]. The main reason for this is that antimicrobial agents can be up to 1000 times less effective against biofilms compared to free-living bacterial cells, making biofilm eradication particularly challenging [31,32]. Furthermore, biofilm tolerance is attributed to both the composition of the exopolysaccharide matrix and the phenotypic heterogeneity within the biofilm, which consists of metabolically active and inactive cells [33,34]. Despite the diverse bioactivities of Primula species and their metabolites, there is a lack of information regarding their antibiofilm potential. To the best of our knowledge, there is only one report on the antibiofilm activity of P. vulgaris root extract against Candida albicans [35]. Literature data on the antibacterial properties of Primula species are also scarce and focused mainly on extracts obtained from the aerial parts of P. veris [36,37,38], P. officinalis [39], P. vulgaris [35,40,41,42], and some non-European Primula species [43,44,45]. There are only two reports on the antibacterial activity of P. vulgaris and P. vulgaris subsp. rubra roots [35,42].
In Bulgarian flora, the genus Primula is represented by eight species, one of which is Primula veris L. [46,47]. A literature survey revealed only three reports on Bulgarian populations of P. veris: a study on the reproductive potential (embryological features and pollen and seed viability) and genetic diversity of P. veris [48], the establishment of ex situ collection of P. veris [49], and the presence of exudate flavonoids in the leaves of P. veris and their inhibitory activity on L. perrene seed germination [50].
The insufficient data for the chemical content of the Bulgarian Primula populations prompted us to conduct in-depth phytochemical investigations, starting with the roots of Primula veris L. The second goal of this work was to expand the knowledge of the biological activity of cowslip by studying its antibiofilm properties.

2. Results and Discussion

2.1. Phytochemical Characterization of Primula veris

The air-dried and ground roots of P. veris were successively extracted with chloroform and methanol to obtain the corresponding extracts. Further purification of the chloroform extract led to the isolation of 13 compounds belonging to three different classes, namely flavones (1–6), acetophenones (7–9), and bisbibenzyls (10–13) (Figure 1). A comparison of their 1H NMR data (Supplementary Part I) led to the identification of flavones (1) [51], 2′-methoxyflavone (2) [51], 3′-methoxyflavone (3) [51], 2′,5′-dimethoxyflavone (4) [51], 3′-hydroxy-4′,5′-dimethoxyflavone (5) [51], 3′-methoxy-4′,5′-methylendioxyflavone (6) [51], 4-hydroxyacetophenone (7) [27], 4-hydroxy-3-methoxyacetophenone (8) [27], 2-hydroxy-4-methoxyacetophenone (paeonol) (9) [52], riccardin C (10) [27], 8′-oxoriccardin C (11) [27], 8′-hydroxyisomarchantin C (12) [27], and 8′-hydroxydihydroptyhantol A (13) [27]. To the best of our knowledge, flavones 1–6 are detected for the first time in the roots of P. veris, although they have been previously reported as constituents of the leaves of P. veris and other Primula species [1,50,51]. Paeonol (9) is also described for the first time in the roots of P. veris. It has been recently detected as one of the major volatile components in P. vulgaris Huds. subsp. vulgaris [53]. Acetophenones 7 and 8 and bisbibenzyls 10–13 have been recently isolated from Serbian P. veris subsp. columnae [27].
The methanol extract was subjected to polyamide column chromatography to give three main fractions, A–C. The subsequent separation of fraction B led to the isolation of two pairs of closely related compounds. A comparison of their 1H NMR data (Supplementary Part I.) with those published in the literature allowed for the identification of the phenolic glycosides primulaverin (14) [10], primeverin (15) [10], 2-primeverosyl-5-methoxy-acetophenone (16) [54], and paeonolide (paeonol 2-O-β-xylopyranosyl (1→6)-β-glucopyranoside, 17) [54,55]. Compounds 14/15 and 16/17 were detected in ratios of 1:0.07 and 1:0.2, respectively. Primulaverin (14) and primeverin (15) are characteristic compounds of P. veris roots, and the content of primulaverin is usually higher than that of primeverin [8,9,10,56]. These compounds degrade over storage time through the activity of the enzyme primverase and produce a typical odor which may serve as an indicator of the age of the plant material [56]. It is worth mentioning that 2-primeverosyl-5-methoxy-acetophenone (16) and paeonolide (17) are reported here for the first time in P. veris. These compounds have been previously reported as constituents of P. maximowiczii of Chinese origin [54].
Column chromatography of fraction C and further purification resulted in the isolation of the known primulasaponin I (primulic acid I) (18) [20] and three undescribed previously compounds, named primulasaponins III–V (19–21). Their structures were determined using NMR techniques (1H, 13C, COSY, HSQC, HMBC, and ROESY) and mass spectrometry (HR-ESI-MS) (Supplementary Part II). The NMR data of compound 19 (Table 1) showed the presence of seven signals corresponding to the tertiary methyls [δH/C 0.87/16.8 (H/C-24), 0.90/16.8 (H/C-25), 0.92/25.0 (H/C-30), 0.93/33.9 (H/C-29), 1.06/28.3 (H/C-23), 1.18/18.4 (H/C-26), and 1.22/19.9 (H/C-27)], ten signals corresponding to the methylene groups [δH 0.99 (m) and 1.74 (m)/δC 40.3 (C-1), δH 1.75 (m) and 2.02 (m)/δC 27.1 (C-2), δH 1.44 (m) and 1.50 (m)/δC 18.7 (C-6), δH 1.20 (m) and 1.56 (m)/δC 35.2 (C-7), δH 1.45 (m) and 1.66 (m)/δC 19.9 (C-11), δH 1.28 (m) and 2.03 (m)/δC 33.6 (C-12), δH 1.20 (m) and 1.99 (m)/δC 37.0 (C-15), δH 1.19 (m) and 2.37 (dd, 12.1 and 14.5 Hz)/δC 39.8 (C-19), δH 1.15 (m) and 2.06 (m)/δC 37.5 (C-21), δH 1.42 (m) and 1.94 (m)/δC 27.2 (C-22)], three methine signals [δH 0.72 (dd, 1.8 and 11.5 Hz/δC 56.8 (C-5), δH 1.22 (m)/δC 51.4 (C-9) and δH 1.67 (m)/δC 47.6 (C-18)], and three oxygenated methine signals [δH 3.20 (dd, 4.0 and 11.3 Hz)/δC 92.1 (C-3), δH 3.78 (m)/δC 77.3 (C-16) and δH 4.60 (s)/δC 99.7 (C-28)]. In addition, the positions of seven quaternary carbons were determined from the observed HMBC correlations (Figure 2) as follows: H-5, H-23, and H-24 with C-4 (δC 40.7); H-5 and H-25 with C-10 (δC 37.8); H-26 with C-8 (δC 43.34); H-26 and H-27 with C-14 (δC 43.9); H-27 and H-28 with C-13 (δC 88.9); H-16 and H-28 with C-17 (δC 48.7); and H-29 and H-20 with C-20 (δC 32.4). Furthermore, four glycosyl units were determined as glucuronyl [δH 4.45 (d, 7.7 Hz)/δC 105.8 (C-1′)], galactosyl [δH 5.20 (d, 7.8 Hz)/δC 100.8 (C-1″)], rhamnosyl [δH 5.28 (d, 1.2 Hz)/δC 102.0 (C-1‴)], and glucosyl [δH 4.88/δC 102.5 (C-1⁗)]. The coupling constants (Table 1) revealed β-configuration for glucuronic acid, galactose, and glucose and α-configuration for rhamnose. In addition, the HMBC correlation between H-3 and an anomeric carbon at δC 105.75 (C-1′) indicated that the glycosyl part is attached at C-3 (Figure 2), while HMBC correlations - H-1″/C-3′ (δC 81.2), H-1‴/C-2″ (δC 76.2), and H-1⁗/C-2′ (δC 79.3) determined the linkage of the glycosyl units. The comparison of these data with those for primulasaponin I (18) showed the same tetrasaccharide moiety at C-3 and almost the same triterpene framework. The only difference was the presence of an additional hydroxy group at C-28, which was confirmed by the HMBC correlations H-28/C-13, H-28/C-16, and H-28/C-17. Additionally, the HR-ESI-MS of 19 displayed a deprotonated molecular ion peak [M−H]− at m/z 1119.55784 (calcd. 1119.55928), which corresponds to a compound with a molecular formula of C54H88O24, and differed from that of 18 with 16 Da ([M−H]− at m/z 1103.56257, Figure SII-9). The relative stereochemistry at C-3, C-16, and C-28 was determined using ROESY experiment (Figure 2A). 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 (δH 1.99) indicated that they were β-orientated. In addition, the correlations H-15 (δH 1.99)/H-28 and H-16/H-28 confirmed the α-position of the hydroxyl groups at C-16 and C-28. Therefore, compound 19 was identified as 3-O-β-(β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl]-(1→3)-β-D-glucuronopyranosyl)-16α,28α-dihydroxy-13β,28-epoxy-oleanane, named primulasaponin III.
Compound 20 had a molecular formula of C55H90O24, determined based on its deprotonated molecular ion peak [M−H]– (m/z 1133.57382, calcd. 1133.57493). Extensive 1D and 2D NMR analysis showed that compound 20 differed from 19 only in the aglycone part. Thus, the signal for H-28 in 19 was shifted upfield at δH 4.17 (s) and that for C-28 was shifteddownfield at δC 105.80 (Table 1). Furthermore, an additional signal for the methoxy group appeared at δH/C 3.31 (s)/55.36. The location of the latter at C-28 was deduced from the HMBC correlation between the C-28 and the methyl of a methoxy group at δH 3.31 as well as between H-28 and the carbon of the methoxy group at δC 55.4 (Figure 2B). Furthermore, the observed correlations H-25/H-26 and H-26/H-15 (δH 1.99) in the ROESY experiment indicated their β-orientation, while the correlations H-15 (δH 1.99)/H-28 and H-16/H-28 confirmed the α-position of the C-16 hydroxy and C-28 methoxy groups. Therefore, compound 20 was identified as 3-O-β-(β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl]-(1→3)-β-D-glucuronopyranosyl)-16α-hydroxy-28-methoxy-13β,28-epoxy-oleanane, named primulasaponin IV.
The HR-ESI-MS of compound 21 displayed a deprotonated molecular ion peak [M−H]− at m/z 1117.54210 (calcd. 1117.54363), which corresponds to a compound with a molecular formula of C54H86O24. The NMR data (Table 1) of 21 were very similar to those of compound 19 and showed a difference only in the aglycone part, namely at C-28. Thus, an additional signal at δC 182.02 appeared instead of the signals for an oxymethine group at δH 4.60 (s)/δC 99.7 (C-28). The presence of a carbonyl group was also confirmed by the downfield shift of C-13 (δC 94.9). Thus, the structure of compound 21 was elucidated as 3-O-β-(β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl]-(1→3)-β-D-glucuronopyranosyl)-16α-hydroxy-oleanan-28,13β-olide, named primulasaponin V.
Finally, the monosaccharides of compounds 18–21 obtained by acidic hydrolysis of a portion of the saponin-containing fraction C were identified as D-glucose, D-galactose, D-glucuronic acid, and L-rhamnose via TLC comparison with authentic standards.
The new saponins 19–21 could be described as oxygenated derivatives of primulasaponin I (18), differing in the nature of the substituent at C-28. It seems that oleanane triterpenoids with the 16α-hydroxy and 13β, 28-epoxy ring are characteristic for Primula species [57], while C-28 carbonyl derivatives (28→13 lactones) like compound 21 have not been found in Primulaceae species so far. The studied population of P. veris showed a different chemical profile from those reported previously for P. veris, P. veris subsp. macrocalyx, P. veris subsp. veris, and commercially available root extracts of Primula sp. [9,10,20,21,22,23]. Primulasaponin I (18) was found in the roots of all studied plants excluding P. veris subsp. macrocalyx [22]. It would be speculative to believe that intraspecific differences lead to different compositions, especially since some of the studies so far lack precise data on the taxonomic identity of the species. Moreover, environmental and ecological factors could also contribute to the differences in the main chemical components of plants.

2.2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Primula veris Extracts and the Saponin-Enriched Fraction

In our study, we used four bacterial strains responsible for infections in the respiratory tract, the urogenital tract, skin, and oral cavity, as well as wound infections and implant-associated infections [34,58]. To determine the MIC of the methanol (ME) and chloroform (CE) extracts from Primula veris roots, as well as the saponin-enriched fraction (SF), the oxidation–reduction indicator resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) was used. This is a blue dye that transforms into resorufin upon changes in the metabolic activity of bacterial cells. It is known that this indicator dye is suitable for antibacterial screening of plant extracts and phytochemicals [59,60], essential oils [61], biosurfactants, and others. Data for the determination of the MIC and MBC are summarized in Table 2. The microdilution test showed that for both Gram-negative strains, MIC varied from 1 to 0.5 mg/mL for all three tested extracts. The lowest antimicrobial activity was recorded against P. aeruginosa after treatment with the methanol extract and the saponin fraction. For E. coli, all three tested extracts showed a MIC of 0.5 mg/mL. Among the Gram-positive strains, the lowest MIC of 0.5 mg/mL was observed for the saponin-enriched fraction. In comparison with the literature data, some differences in MIC values were observed in extracts from other Primula species.
Studies on the methanol extract from P. auriculata leaves reported antimicrobial activity at concentrations of 1 mg/mL against S. aureus and lower values of 0.5 mg/mL against E. coli [43]. After a broader study on five Primula species, namely P. denticulata, P. elliptica, P. macrophylla, P. rosea, and P. stuarti, it was found that MIC values of methanol extracts with a range from 0.5 mg/mL to 0.125 mg/mL depending on the species inhibited S. aureus [62]. A study on plant extracts from the roots of P. vulgaris subspecies rubra reported identical MIC values of 1.25 mg/mL for all three tested strains—S. aureus, E. coli, and P. aeruginosa. A difference was observed only in P. spina-christi for root extracts, where MIC values for S. aureus were determined at 0.625 mg/mL, while for the two Gram-negative strains, they remained within the same range as P. vulgaris at 1.25 mg/mL [35].

2.3. Inhibitory Effects of Sub-MICs of Primula veris Extracts and the Saponin-Enriched Fraction on Biofilm Formation

The inhibitory effects of the sub-MICs of ME, CE, and SF on biofilm formation were compared. The crystal violet staining method, which is widely used for biofilm analysis, was applied. The concentration-dependent antibiofilm effects were examined over a 24 h treatment period during biofilm formation and compared against an untreated control sample.
The results, illustrating the percentage of biofilm inhibition by the applied samples, are shown in Figure 3. The data revealed a clear trend of higher inhibition rates in Gram-negative strains compared to Gram-positive strains, where the highest inhibition by the saponin-enriched fraction barely reached 22%. As can be seen from Figure 3, all three tested 1/2 sub-MICs showed a significant inhibition percentage of P. aeruginosa‘s biofilm, with the highest effect recorded for the chloroform extract (62.8%), followed by the methanol extract (50.6%) and the saponin-enriched fraction (48.1%). In contrast, the SF (49.6%) and ME (42.4%) demonstrated better antibiofilm effects on E. coli than that of CE (37.1%). The observed effect for the two Gram-positive strains confirmed a general trend, with the highest inhibition percentage found in the saponin-enriched fraction and the lowest in the methanol extract. Nonetheless, an inhibition of over 10% was recorded in four of the tested 1/2 MICs for both Gram-positive strains.
Data on the effects of sub-minimal inhibitory concentrations of plant extracts on biofilm formation have also been reported by other authors [63,64,65]. But in comparison with our findings, we identified only one study reporting the antibiofilm activity of P. vulgaris root extract against C. albicans, an opportunistic pathogen responsible for vaginal candidiasis, with an MIC of 156 µg/mL [35]. The observed differences in the activity of the different extracts are not surprising as the extracts contained different types of compounds, which in turn influences their ability to inhibit biofilm formation. Similar results have been already reported in the literature for Myrsine umbellata (Primulaceae) [66], Cyclamen hederiflolium (Primulaceae) [67], Etlingera elatior [68], Inula sp. [69], etc. It is known that some phytochemicals, besides acting as potential biofilm inhibitors, can also suppress quorum-sensing signaling pathways, bioluminescence, and pigment production [69,70]. In certain cases, biofilm formation may have a protective role [71]. Regarding the flavonoid group, there is evidence of the group’s antibiofilm potential against pathogens such as S. aureus, a causative agent of infections in implantable devices, where targeted inhibition of Bap-mediated biofilm formation leads to a reduction in catheter colonization [72]. The antibiofilm and anti-amyloid activity of various flavonoids has also been demonstrated in S. mutans and E. coli [73,74]. Saponin-rich extracts and pure saponins are also reported to possess good antibiofilm activity against P. aeruginosa, E. coli, and S. aureus [67,75,76,77,78]. There are several hypotheses regarding the inhibitory properties of plant phytochemicals on biofilms. First, they penetrate the biofilm matrix by forming micropores and channels, allowing interaction with bacterial cell wall proteins and leading to its disruption, increased permeability, and the accumulation of the treatment compound in the cytoplasm. In some cases, cell lysis may occur, resulting in the leakage of intracellular components and subsequent cell death. Simultaneously, the adhesion potential of the cell is also disrupted. Phytochemicals can damage DNA and RNA structures and inhibit various enzymes involved in replication, transcription, and translation processes. As a result, gene expression, intracellular metabolism, and cell proliferation are inhibited. Their impact on translation leads to impaired synthesis of EPS, QS molecules, virulence factors, and motility structures. Moreover, efflux pumps are affected, and proton gradients are disrupted. Along with the formation of micropores, this facilitates the penetration of various antibiofilm agents [79,80]. Considering the above, we assume that the higher antibiofilm effectiveness of the plant extracts tested against Gram-negative bacteria may be due to two main reasons. On the one hand, their impact on biofilm cells is influenced by the composition of the bacterial cell wall. In Gram-positive bacteria, the cell wall consists mainly of a thick peptidoglycan layer, which may hinder the action of plant molecules and their access to the cell. In contrast, in Gram-negative bacteria, we assume that the penetration of treatment compounds into the cell is facilitated by the thinner peptidoglycan layer. Additionally, it is possible that a mechanism is activated to specifically inhibit certain pathways involved in exopolysaccharide matrix production, leading to its suppression and, consequently, the inability to form a biofilm, a phenomenon also confirmed by other authors [58,81]. In summary, the strategy of biofilm suppression using plant extracts appears promising. It could reduce reliance on conventional antibiotics and consequently limit antibiotic resistance.

2.4. Cytotoxicity Against Human Keratinocyte Cell Line HaCaT

The cytotoxicity assessment of the methanol (ME) and chloroform (CE) extracts of P. veris roots and the saponin-enriched fraction (SF) on a human keratinocyte cell line was performed using the CV test (crystal violet staining) to avoid the influence of enzyme-based cytotoxicity assays due to a reaction with polyphenols [82]. Our results showed the expected high cytotoxicity of the saponin-enriched fraction (SF), which is caused by the interaction of saponins with cell membranes [83]. The methanol extract also showed relatively high activity, while the chloroform extract was not toxic in the tested concentration range up to 300 μg/mL, and cell survival was above 80% (Figure 4). Combined with the high antibiofilm activity, this makes the chloroform extract of P. veris roots a promising subject for future research.
The effect of saponins in ethnomedicine is well known, and their action at the organism level has been studied to some extent [84]. Little is known about their action in vitro at the cellular level. There is evidence that saponins interact with cell membranes, causing temporary or permanent micropores in tumor and actively dividing cells, thus causing cell cycle arrest, disturbances in ionic balance and/or apoptosis in actively dividing cells, and they are potential candidates for application in antitumor therapies. There is evidence of specific effects on specific stress-associated signaling pathways and the activation of apoptosis in myeloid leukemia and other tumor cells by triterpenoid saponins and saikosaponin [85]. When applied externally, their contact with highly differentiated and non-dividing keratinocytes will not have a negative effect, while the development of melanomas or other skin diseases associated with increased proliferation and inflammation, such as psoriasis, for example, as well as wound healing, would be well influenced [4].

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 a sodium lamp at 20 °C by using a 0.5 dm quartz cell. The [α] 20 D is given in deg·cm3·g−1·dm−1 and concentration (c) 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 at 600 MHz (1H) and 150 MHz (13C) using the residual solvent’s signals (δH 7.26 in 1H and δC 77.00 ppm in 13C for CDCl3 and δH 3.31 in 1H and δC 49.3 ppm in 13C for CD3OD) as a reference. The chemical shifts (δ) are expressed in ppm and coupling constants (J) in Hz. HRESIMS spectra were acquired in negative mode on the Q Exactive Plus (Thermo Fisher Scientific, Inc., Bremen, Germany) mass spectrometer equipped with a heated HESI-II source. IR spectra were recorded on a Shimadzu IR Spirit FT-IR spectrometer using QATR-S as a single-reflection ATR measurement attachment.
Polyamide (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), 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 to monitor the separation of the extracts and for preparative TLC. The spots were visualized by spraying with concentrated H2SO4or with the NP reagent (1% diphenylboronic acid 2-aminoethyl ester in ethyl acetate), followed by heating at 105 °C. All solvents used were of HPLC grade.

3.2. Plant Material

The roots of Primula veris L. were collected in April 2023 in the vicinity of Assenovgrad town, Bulgaria (GPS 42.014051; 24.855882), at an altitude of 276 m. A voucher specimen (SOM 179 373) was deposited in the herbarium of the Institute of Biodiversity and Ecosystem research, Bulgarian Academy of Sciences, Sofia, Bulgaria. The plant was identified by Assoc. Prof. Dr Vladimir Vladimirov from the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria.

3.3. Extraction and Isolation of Individual Compounds

The air-dried and finely powdered roots of P. veris (52 g) were sequentially extracted with CHCl3 and MeOH (3 × 500 mL) at room temperature for 24 h each. The extracts were filtered and concentrated under vacuum using a rotary evaporator to obtain the corresponding CHCl3 (0.328 g) and MeOH (3.98 g) extracts.
A portion of the CHCl3 extract (0.200 g) was subjected to CC on the silica gel using the n-hexane/ethyl acetate mixture with increasing polarity (from 10:1 to 0:1). Fourteen fractions (F1–F14) were collected via TLC monitoring (Silica gel, n-hexane/ethyl acetate, 3:1 and RP-18 MeOH/H2O 8:2). Prep. TLC (Silica gel, n-hexane/diethyl ether, 2:1, twice development) of fr. F3 (12 mg) afforded compound 9 (1.2 mg). Prep. TLC (Silica gel, n-hexane/diethyl ether, 2:1) of fr. F5 (8.0 mg) and F7 (5.0 mg) yielded compounds 1 (5.3 mg) and 3 (3.0 mg). Prep. TLC (RP-18, H2O/MeOH, 2:8) of fr. F9 (6 mg) gave compound 2 (1.3 mg) and a mixture of compounds 7 and 8 (0.9 mg and a ratio of 0.3:1, as deduced from 1H NMR). Prep. TLC (RP-18, H2O/MeOH, 2:8) of fr. F11 (13 mg) afforded 4.6 mg of compound 10, 1.4 mg of compound 12, and a mixture of compounds 4 and 6 (0.7 mg and a ratio of 1:0.5, as deduced from 1H NMR). Prep. TLC (RP-18, H2O/MeOH, 2:8) of fr. F13 (10 mg) led to the isolation of compounds 11 (3.8 mg), 5 (1.1 mg), and 13 (0.9 mg).
A portion of the methanolic extract (3.0 g) was separated by CC on Polyamide using H2O/MeOH mixtures (from 0:1 to 1:0) to give 3 fractions A-C. MPLC on LiChroprep RP-8 of a portion of fr. B (60 mg) with H2O/MeOH (0:1 to 0:1) afforded a mixture of compounds 14 and 15 (23.6 mg in a ratio of 1:0.07, as deduced from 1H NMR) and afforded a mixture of compounds 16 and 17 (12.6 mg in a ratio of 1:0.2, as deduced from 1H NMR). Fr. C (360 mg) was subjected to MPLC on LiChroprep RP-8 (H2O/MeOH, 1:1 to 0:1) to give 7 subfractions C1–C7. MPLC on LiChroprep RP-8 (H2O/MeOH, 4:6 to 0:1) of fr. C2 afforded 8.12 mg of 19. Prep. TLC (RP-18, H2O/MeOH, 4:6) of fr. C3 afforded 5.2 mg of 21. MPLC on LiChroprep RP-8 (H2O/MeOH, 4:6 to 0:1) of fr. C5 yielded 25.2 mg of 18. MPLC on LiChroprep RP-8 (H2O/MeOH, 4:6 to 0:1) of fr. C6 gave 5.2 mg of 18 and 7.6 mg of 20.
Primulasaponin III (19): Amorphous white powder, [α] 20 D −0.09 (c 0.6, MeOH); FT-IR (ATR): νmax 3352, 2947, 1604, and 1037 cm−1; 1H (600 MHz, CD3OD) and 13C (150 MHz, CD3OD): Table 1; HR-ESI-MS m/z: 1119.55784 [M−H]− (calcd. for C54H87O24 1119.55928).
Primulasaponin IV (20): Amorphous white powder, [α] 20 D −0.01 (c 0.154, MeOH); FT-IR (ATR): νmax 3330, 2921, 1609, and 1043 cm−1; 1H (600 MHz, CD3OD) and 13C (150 MHz, CD3OD): Table 1; HR-ESI-MS m/z: 1133.57382 [M−H]− (calcd. for C55H89O24 1133.57493).
Primulasaponin V (21): Amorphous white powder, [α] 20 D −0.07 (c 0.375, MeOH); FT-IR (ATR): νmax 3350, 2924, 1744, 1604, and 1040 cm−1; 1H (600 MHz, CD3OD) and 13C (150 MHz, CD3OD): Table 1; HR-ESI-MS m/z: 1117.54210 [M−H]– (calcd. for C54H85O24 1117.54363).

3.4. Hydrolysis of Saponin-Enriched Fraction

A portion of fraction C (SF) (10 mg) that was dissolved in 1 mL of MeOH was mixed with 5% HCl (5 mL) and 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 the 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. Bacterial Strains, Growth Medium, and Cultural Conditions

This study included two Gram-negative and two Gram-positive bacterial strains. Pseudomonas aeruginosa 15692 (ATCC) is a strain from the International Reference Panel [86] isolated from wounds, Escherichia coli 25922 (ATCC), Staphylococcus aureus 29213 (ATCC), and Streptococcus mutans 35668 (ATCC). For long-term storage, the strains were frozen at −80 °C with the addition of the cryoprotectant 8% DMSO. Before experimental work, each strain was inoculated in a growth medium according to its specific requirements: P. aeruginosa and S. aureus in tryptic soy broth (TSA, Sigma, Burlington, MA, USA), E. coli in nutrient broth (HiMedia, Bedford, PA, USA), and S. mutans in brain heart infusion (HiMedia, Bedford, PA, USA).

3.6. Evaluation of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)

For the evaluation of MIC and MBC, standard culture media made of Mueller–Hinton broth (MHB) and Mueller–Hinton agar (MHA) (HiMedia, Bedford, PA, USA) were used. MIC was tested using the widely applied microdilution method, which relies on the oxidation–reduction indicator resazurin to assess changes in the metabolic activity of bacterial cells [59]. For the experiment, the test substances were diluted over a wide concentration range, from 2 mg/mL to 0.0156 mg/mL, with the addition of DMSO at a final concentration of 2% to avoid toxicity to bacterial cells. The initial concentration of bacterial strains was applied after densitometric measurement following the McFarland standard to a final cell concentration of 1 × 106 CFU/mL. Aliquots of 100 µL of the tested extracts (chloroform, methanol, and saponin fraction) at concentrations of 2 mg/mL were added to the first column of the plate, followed by serial two-fold dilutions in the MHB medium to the final test concentration. To each well, 10 µL of a 0.01% sterile aqueous resazurin solution (Sigma, New York, NY, USA) and 10 µL of bacterial suspension were added. The plates were incubated for 24 h at 37 °C under static conditions. As a positive control, 10 µL of ciprofloxacin (Sigma, New York, NY, USA) was added, while a sterile nutrient medium served as the negative control. After the incubation period, the lowest concentration of the test substance where a visual color change from blue to pink was observed was recorded as the minimum inhibitory concentration (MIC). To determine the MBC, samples were taken from the wells of the plate using a sterile swab and plated onto an MHA medium. The lowest concentration of the substances where no bacterial growth was observed was considered the MBC.

3.7. Biofilm Formation Experiments

To determine the antibiofilm effect of the applied plant extracts, the bacterial strains were cultivated in minimal salt medium M63 with the following composition: KH2PO4 (0.02 M), K2HPO4 (0.02 M), (NH4)2SO4 (0.02 M), MgSO4 (0.1 mM), and glucose (0.04 M), with a pH of 7.5. As a source of bacterial inoculum, the strains were incubated in the media described above for 18 h at 37 °C. The cultures were then diluted at a ratio of 1:100 in M63, and the plant extracts were applied in the presence of sub-MICs (1/2 MIC). The prepared suspensions were pipetted into 96-well U-bottomed polystyrene microtiter plates (Corning, Corning, NY, USA) with a final volume of 150 μL per well in six replicates per sample. The control sample contained only bacterial inoculum diluted in M63 with the addition of 2% DMSO (untreated probe). The prepared plates were incubated for 24 h at 37 °C under static conditions. After incubation, the non-adherent planktonic cells were removed by washing the wells with phosphate-buffered saline (PBS, pH 7.2), and the attached cells were stained with a 0.1% aqueous solution of crystal violet for 15 min. The final step involved extracting the dye with 70% ethanol for Gram-negative bacteria and a 95% ethanol–acetone (4:1) dilution for Gram-positive bacteria. Absorbance was measured at a wavelength of 595 nm using an ELISA plate reader (LTEK INNO, Gyeonggi-do, Republic of Korea). The quantitative data were analyzed as the means ± standard deviation (SD) using OriginPro 6.1 software.

3.8. Cytotoxicity Assessment

The cytotoxicity assessment of methanol (ME) and chloroform (CE) extracts from P. veris roots and the saponin-enriched fraction (SF) on a human keratinocyte cell line was performed via the CV assay (crystal violet staining) as previously described [87]. All experiments were performed in triplicate. The optical density 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 a percentage of cell viability compared to the control of untreated cells. The data were analyzed with OriginPro 9.0 and presented as a mean value ± SE. Statistical significance is according to one-way ANOVA at the 0.05 level (* p < 0.05).

4. Conclusions

In summary, the current study showed the presence of various phenolic compounds (flavones, acetophenones, phenolic glycosides, and bisbibenzyls) and saponins closely related to primulasaponin I in the roots of Primula veris of Bulgarian origin. Among them, three triterpene saponins were found to be new compounds undescribed so far. This investigation contributes to the phytochemical characterization of cowslip in Europe, but detailed phytochemical analyses of more populations of P. veris are needed to understand the intraspecific and interspecific variability of this well-known medicinal plant. Additionally, the antibiofilm properties of the chloroform and methanol extracts and the saponin-enriched fraction obtained from the roots of P. veris were studied for the first time. The obtained results revealed better biofilm inhibition of the tested samples of Gram-negative bacterial strains (P. aeruginosa and E. coli) than Gram-positive ones (S. aureus and S. mutans). Expanding knowledge on biofilm inhibition mechanisms, particularly the modulation of bacterial virulence traits by plant extracts, provides advantages for discovering other agents with similar effects and potential applicability in medical practice for treating biofilm-related infections.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules30081702/s1, Supplementary Part I. Figures SI-1–SI-13: 1H NMR spectra of the known compounds 1–17; Supplementary Part II. Figures SII-1–SII-36: Spectral data (1H and 13C NMR, DEPT, COSY, HSQC, HMBC, ROESY, HR-ESI-MS, and IR) of primulasaponin I (18) and new saponins 19–21.

Author Contributions

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

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

The authors are thankful to the bilateral project of the Bulgarian Academy of Sciences and the Serbian Academy of Sciences and Arts “Phytochemical and metabolomics investigation of biologically active compounds from Primula species and medicinal mushrooms” and to Vladimir Vladimirov for the identification of the plant material.

Conflicts of Interest

The authors declare no conflicts of interest.

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Molecules 30 01702 g001
Figure 1. Structures of isolated compounds from P. veris roots.
Figure 1. Structures of isolated compounds from P. veris roots.
Molecules 30 01702 g001
Molecules 30 01702 g002
Figure 2. Important HMBC and ROESY correlations for compounds 19 (A) and 20 (B).
Figure 2. Important HMBC and ROESY correlations for compounds 19 (A) and 20 (B).
Molecules 30 01702 g002
Molecules 30 01702 g003
Figure 3. Inhibition of biofilm formation under the influence of methanol (ME) and chloroform (CE) extracts of P. veris roots and the saponin-enriched fraction (SF). The antibiofilm effects were examined after a 24 h treatment period and calculated as a percentage of the biofilm in the untreated control sample. (A) P. aeruginosa; (B) E. coli; (C) S. aureus; (D) S. mutans.
Figure 3. Inhibition of biofilm formation under the influence of methanol (ME) and chloroform (CE) extracts of P. veris roots and the saponin-enriched fraction (SF). The antibiofilm effects were examined after a 24 h treatment period and calculated as a percentage of the biofilm in the untreated control sample. (A) P. aeruginosa; (B) E. coli; (C) S. aureus; (D) S. mutans.
Molecules 30 01702 g003
Molecules 30 01702 g004
Figure 4. Inhibition of cellular viability under the influence of the methanol (ME) and chloroform (CE) extracts of P. veris roots and the saponin-enriched fraction (SF). Cytotoxicity was examined at after a 24 h treatment period and calculated as a percentage of the untreated control cells (* p < 0.05).
Figure 4. Inhibition of cellular viability under the influence of the methanol (ME) and chloroform (CE) extracts of P. veris roots and the saponin-enriched fraction (SF). Cytotoxicity was examined at after a 24 h treatment period and calculated as a percentage of the untreated control cells (* p < 0.05).
Molecules 30 01702 g004
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data of compounds 19–21 in CD3OD (δ in ppm, multiplicity, and J in Hz).
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data of compounds 19–21 in CD3OD (δ in ppm, multiplicity, and J in Hz).
Position192021
δCδHδCδHδCδH
140.30.99 m 140.30.98 m40.21.02 m
1.74 m1.75 m1.75 m
227.11.75 m27.11.75 m27.01.75 m
2.02 m1.95 m2.01 m
392.13.20 dd (4.0, 11.3)92.33.19 dd (4.0, 11.3)91.93.21 dd (4.0, 11.3)
440.7-40.7-40.7-
556.80.72 dd (1.8, 11.5)56.80.73 dd (1.8, 11.5)56.80.76 dd (1.8, 11.5)
618.71.44 m18.71.43 m18.71.43 m
1.50 m1.50 m1.55 m
735.21.20 m35.21.22 m34.91.24 m
1.56 m1.55 m1.59 m
843.3-43.4-43.0-
951.41.22 m51.41.23 m50.81.34 m
1037.8-37.8-37.8-
1119.91.45 m19.91.45 m19.41.55 m
1.66 m1.65 m1.65 m
1233.61.28 m33.71.28 m32.21.56 m
2.03 m2.00 m2.15 m
1388.9-89.0-94.9-
1443.9-44.0-43.0-
1537.01.20 m37.41.20 m37.41.20 m
1.99 m1.99 m1.99 m
1677.33.78 m77.23.82 m76.23.79 m
1748.7-48.9 49.1-
1847.61.67 m48.01.68 m52.61.92 m
1939.81.19 m39.81.17 m39.21.30 m
2.37 dd (12.1, 14.5)2.28 dd (12.1, 14.5)2.49 dd (12.1, 14.5)
2032.4-32.4-32.2-
2137.51.15 m37.11.15 m36.71.21 m
2.06 m2.01 m2.09 m
2227.21.42 m26.51.42 m28.71.63 m
1.94 m1.61 m1.83 m
2328.31.06 s28.21.06 s28.21.07 s
2416.80.87 s16.80.87 s16.70.88 s
2516.80.90 s16.80.91 s16.80.91 s
2618.81.18 s18.91.18 s18.31.08 s
2719.91.22 s19.81.21 s19.71.38 s
2899.74.60 brs106.94.17 brs182.0-
2933.90.93 s33.90.92 s33.50.96
3025.00.92 s24.90.88 s24.90.92
OCH3 55.43.31 s
1′105.84.45 d (7.7)105.84.48 d (7.7)105.84.46 d (7.7
2′79.33.92 dd (7.7, 8.8)79.13.93 dd (7.7, 8.8)79.33.92 dd (7.7, 8.8)
3′81.24.06 t (8.8)81.04.05 t (8.8)81.24.07 t (8.8)
4′72.23.59 m72.23.61 m72.33.59 m
5′75.93.79 m75.93.78 m75.93.79 m
6′176.6- -
1″100.85.20 d (7.8)100.95.20 d (7.8)100.85.20 d (7.8)
2″76.23.79 m76.13.79 m76.23.79 m
3″75.93.72 m76.03.74 m76.03.74 m
4″71.93.72 m71.83.72 m71.83.72 m
5″76.93.53 m76.93.53 m76.93.53 m
6″62.73.65 dd (4.1, 11.5)62.83.65 dd (4.1, 11.5)62.73.67 dd (4.1, 11.5)
3.81 m3.81 m3.81 m
1‴102.05.28 d (1.2)102.15.28 d (1.2)102.05.29 d (1.2)
2‴72.63.97 dd (1.2, 3.5)72.63.96 dd (1.2, 3.5)72.63.96 dd (1.2, 3.5)
3‴72.33.71 dd (3.5, 9.5)72.33.71 dd (3.5, 9.5)72.23.71 dd (3.5, 9.5)
4‴73.73.44 t (9.5)73.83.45 t (9.5)73.73.43 t (9.5)
5‴70.34.10 dq (9.5, 6.5)70.24.11 dq (9.5, 6.5)70.24.11 dq (9.5, 6.5)
6‴17.91.27 d (6.5)17.91.27 d (6.5)17.91.27 d (6.5)
1⁗102.54.88102.64.88102.64.88
2⁗76.23.22 dd (7.2, 9.5)76.13.22 dd (7.2, 9.5)76.13.22 dd (7.2, 9.5)
Glu-377.93.35 m77.93.35 m77.93.35 m
4⁗72.43.05 t (9.5)72.63.05 t (9.5)72.63.05 t (9.5)
5⁗78.13.40 m78.13.40 m78.13.40 m
6⁗63.53.54 dd (8.0, 12.5)63.53.54 dd (8.0, 12.5)63.53.55 dd (8.0, 12.5)
3.87 dd (2.5, 12.5)3.87 dd (2.5, 12.5)3.88 dd (2.5, 12.5)
1 Multiplicity: m—multiplet; d—doublet; dd—doublet of doublets; t—triplet; s—singlet; brs—broad singlets.
Table 2. Determination of the MIC and MBC (mg/mL) of P. veris extracts and the saponin-enriched fraction.
Table 2. Determination of the MIC and MBC (mg/mL) of P. veris extracts and the saponin-enriched fraction.
Sample 1P. aeruginosaE. coliS. aureusS. mutans
MICMBCMICMBCMICMBCMICMBC
CE1.02.00.50.51.02.01.02.0
ME0.51.00.51.01.02.01.02.0
SF0.51.00.51.00.52.00.52.0
1 Chloroform extract (CE), methanol extract (ME), and saponin-enriched fraction (SF).
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Trendafilova, A.; Raykova, D.; Ivanova, V.; Novakovic, M.; Nedialkov, P.; Paunova-Krasteva, T.; Veleva, R.; Topouzova-Hristova, T. Phytochemical Characterization and Anti-Biofilm Activity of Primula veris L. Roots. Molecules 2025, 30, 1702. https://doi.org/10.3390/molecules30081702

AMA Style

Trendafilova A, Raykova D, Ivanova V, Novakovic M, Nedialkov P, Paunova-Krasteva T, Veleva R, Topouzova-Hristova T. Phytochemical Characterization and Anti-Biofilm Activity of Primula veris L. Roots. Molecules. 2025; 30(8):1702. https://doi.org/10.3390/molecules30081702

Chicago/Turabian Style

Trendafilova, Antoaneta, Desislava Raykova, Viktoria Ivanova, Miroslav Novakovic, Paraskev Nedialkov, Tsvetelina Paunova-Krasteva, Ralitsa Veleva, and Tanya Topouzova-Hristova. 2025. "Phytochemical Characterization and Anti-Biofilm Activity of Primula veris L. Roots" Molecules 30, no. 8: 1702. https://doi.org/10.3390/molecules30081702

APA Style

Trendafilova, A., Raykova, D., Ivanova, V., Novakovic, M., Nedialkov, P., Paunova-Krasteva, T., Veleva, R., & Topouzova-Hristova, T. (2025). Phytochemical Characterization and Anti-Biofilm Activity of Primula veris L. Roots. Molecules, 30(8), 1702. https://doi.org/10.3390/molecules30081702

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