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Article

Inula salicina L.: Insights into Its Polyphenolic Constituents and Biological Activity

by
Viktoria Ivanova
1,
Paraskev Nedialkov
2,
Petya Dimitrova
3,
Tsvetelina Paunova-Krasteva
3 and
Antoaneta Trendafilova
1,*
1
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
2
Pharmacognosy Department, Faculty of Pharmacy, Medical University of Sofia, 1000 Sofia, Bulgaria
3
Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Pharmaceuticals 2024, 17(7), 844; https://doi.org/10.3390/ph17070844
Submission received: 31 May 2024 / Revised: 20 June 2024 / Accepted: 24 June 2024 / Published: 27 June 2024
(This article belongs to the Special Issue Plant-Derived Natural Compounds as Bioactive Molecules with Beneficial Effects on Human Health)
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Abstract

:
In this study, UHPLC-HRMS analysis of the defatted methanol extract obtained from Inula salicina L. led to the identification of 58 compounds—hydroxycinnamic and hydroxybenzoic acids and their glycosides, acylquinic and caffeoylhexaric acids, and flavonoids and their glycosides. In addition, a new natural compound, N-(8-methylnepetin)-3-hydroxypiperidin-2-one was isolated and its structure was elucidated by NMR spectroscopy. The presence of a flavoalkaloid in genus Inula is described now for the first time. Chlorogenic acid was the main compound followed by 3,5-, 1,5- and 4,5-dicaffeoylquinic acids. The methanol extract was studied for its antioxidant potential by DPPH, ABTS, and FRAP assays and sun protective properties. In addition, a study was conducted to assess the effectiveness of the tested extract in inhibiting biofilm formation by Gram-positive and Gram-negative strains. Results from crystal violet tests revealed a notable decrease in biofilm mass due to the extract. The anti-biofilm efficacy was confirmed through the observation of the biofilm viability by live/dead staining. The obtained results showed that this plant extract could be used in the development of cosmetic products with antibacterial and sun protection properties.

Graphical Abstract

1. Introduction

Plants are a great source of compounds with distinct pharmacological properties that are effectively used in both traditional and official medicine for treatment of various diseases [1]. Oxygen metabolism is principal for human life, but it is also responsible for the production of reactive oxygen and reactive nitrogen species, which significantly affect the regulation of biological processes and cell functions [2]. Reactive oxygen species in the body often lead to an increase in the oxidative stress and the development of many chronic diseases such as cardiovascular, cancer, diabetes, obesity, etc.
Long exposure to UV-B (320–280 nm) radiation is mainly responsible for inducing the skin problems and increases the risk of skin diseases (cancer and photoallergic reactions) [3]. Using skin protectors is one way to stop or reduce UV radiation. In recent years, various plant extracts have been described as an alternative to synthetic sunscreens due to the content of phenolic compounds that possess absorption in the UV region and good antioxidant properties [4,5,6,7,8].
Biofilm is a highly effective microbial community resistant to antimicrobials or disinfectants posing significant challenges related to biofilm contamination on biotic or abiotic surfaces. Moreover, the contamination, difficult removal, and high tolerance of biofilms to antibiotics significantly increase the occurrence of infections, patient morbidity, hospitalization, or mortality rates [9]. Future efforts to control biofilm infections should focus on investigating the therapeutic potential of various inhibitors, including extracts from medicinal plants, and testing their anti-biofilm effects. The abundance of Bulgarian medicinal plants represents a rich yet largely unexplored resource for potential biofilm inhibitors. While many plant extracts are acknowledged for their effectiveness in modulating bacterial quorum sensing [10], the extent of their application as potential biofilm inhibitors remains an area of ongoing exploration.
The genus Inula (Asteraceae) is represented by approximately 100 species spread out mainly in Europe and Asia. Species from this genus are used in the traditional medicine (including traditional Chinese medicine) for the treatment of asthma, bronchitis, digestive disorders, urinary tract infections, and also skin [11,12]. Species of genus Inula possess various pharmacological properties, including anti-inflammatory, anti-allergic, anti-oxidative, anti-tumor, antimicrobial, anti-diabetic, gastroprotective, hepatoprotective, neuroprotective, cardioprotective, anti-aging, etc. [11,12,13,14,15], due to the biologically active compounds they contain, such as sesquiterpene lactones, flavonoids, mono- and dicaffeoyl esters of quinic acid, etc. [11,12,15,16,17].
Inula salicina L. (Irish fleabane, willow-leaved yellowhead) is distributed across Eurasia from Portugal to Japan [18,19]. The plant is used in folk medicine to treat angina, hernias, skin rashes, warts, and its leaves are useful as a wound healing agent [20] as well as a herbal tea in Spain [21]. The healing properties of this plant have not been clearly proven yet. The literature survey revealed only a few reports on biological activity of I. salicina. Thus, Yıldırım et al. reported low to moderate inhibitory activity against acetylcholinesterase, butyrylcholinesterase and α-amylase enzymes of the methanol extract of I. salicina ant its n-hexane, chloroform, ethyl acetate and aqueous methanol fractions and a good anti-inflammatory activity of the ethyl acetate fraction [22]. In the same study, all extracts had moderate effect against Candida species, while the chloroform fraction exhibited a notable antimicrobial activity against Staphylococcus aureus and S. epidermis [22]. Recently, chloroform and methanol extracts from five Inula species, including I. salicina have been studied for their anti-biofilm properties and pigment synthesis in C. violaceum [23]. Sevindik et al. reported the anti-urease activity of the ethanol extract of I. salicina which was better than thiourea (IC50 0.0122 vs. 0.0167 µg/mL) [24]. Different extracts of I. salicina from Turkey were also shown to have good to high antioxidant and free radical scavenging activities attributed to the presence of a significant amount of total phenolic compounds (TPC) [22,24]. The literature data regarding the chemical constituents are scarce. So far, chlorogenic and caffeic acids [25], apigenin [25], nepetin [26], hyperoside [25], three thymol derivatives [27], and two sesquiterpene lactones (alantolactone and isoalantolactone) [27] have been reported. In our recent study, no sesquiterpene lactones were detected in the chloroform extract I. salicina even in traces [28]. Instead, fifteen triterpene alcohols and their 3-O-esters (acetates and palmitates) were identified [28].
Within this context, our objectives were to perform a comprehensive phytochemical characterization of the methanol extract of I. salicina aerial parts and to evaluate its in vitro antioxidant, sun protective, and anti-biofilm properties. Through this study, our goal is to gain more in-depth information about the chemical composition of this extract and shed light on its potential health benefits.

2. Results and Discussion

2.1. Identification of Compounds in Inula salicina by UHPLC-MS/MS and NMR

The methanol extract obtained from Inula salicina’s aerial parts was investigated by UHPLC-MS/MS (Figure 1). Since ESI in a negative ionization mode is more sensitive for phenolic compounds, the results from this analysis are presented in Table 1. In total, 58 compounds were identified based on their retention time, m/z values, molecular formula, fragmentation pattern and comparison with the literature data [29,30,31,32,33,34,35,36,37,38,39], and open access LC-MS libraries. Of them, 19 compounds were determined by direct comparison with standards and/or by 1H NMR (Table 1, Tables S1 and S2, Figure S1) after their isolation and 39 compounds were tentatively identified. The described above compounds belong to following main metabolite classes: hydroxycinnamic and hydroxybenzoic acids and their glycosides (1, 2, 4, 6, 7, 9, 14, and 21); acylquinic acids (3, 5, 8, 10–13, 15, 16, 23, 25, 26, 30, 31, 33, 36–43, 45, 49, 50); caffeoylhexaric acids (27, 32, 34, 35, 44, 46, 53, 54); and flavonoids and their glycosides (17–20, 22, 24, 28, 29, 47, 48, 51, 52, 55–58).
Compound 1 had [M-H]− at m/z 191 and characteristic for quinic acid fragment ions in its MS/MS [31]. Compounds 2, 9 and 14 displayed [M-H]− at m/z 153, 179 and 163 as well as [M-H-CO2]− at m/z 109, 135 and 119 in MS/MS, and were determined as protocatechuic, caffeic and p-coumaric acids, respectively. Compound 7 showed [M-H]− at m/z 193 and characteristic MS/MS ions at m/z 178 and 134 as a result of consequent elimination of CH3• and CO2 (15 and 44 Da, respectively) and was identified as ferulic acid [39]. Compounds 4 and 6 showed [M-H]− at m/z 341 and MS/MS ion at m/z 179 indicative for caffeoyl moiety [caffeic acid-H]− as a result from the elimination of 162 Da (hexose) and were tentatively identified as O-caffeoyl hexose isomers [31]. Compound 21 had [M-H]− at m/z 461 and MS/MS ions at m/z 323 [M-salicylic acid-H]−, 179 [caffeic acid-H]−, 161 [323-C6H10O5-H]− and 137 [salicylic acid-H]− and was tentatively identified as caffeoyl-(salicyl)-hexoside [38].
Further, 9 mono-, 16 di-, and 1 triacylquinic acids were also recognized by the characteristic for each subclass fragment ions [30,32,33,34,35,36,37,38]. Caffeoyl (3, 8, 10 and 11), p-coumaroyl (5, 12, 13 and 15) and feruloyl (16) quinic acids were recognized by their deprotonated molecular ion at m/z 353, 337 and 367, respectively. Further, the characteristic for C-5 substituted quinic acids’ base peak at m/z 191 in their MS/MS and led to determination of 8, 13 and 16 as 5-O-caffeoyl, 5-O-p-coiumaroyl and 5-O-feruloylquinic acids, respectively. 3,4-, 3,5-, 1,5- and 4,5-Dicaffeoylquinic acids (23, 25, 26 and 30) were identified by comparison with authentic standards. The structure of 8, 25 and 26 was additionally confirmed by 1H NMR (Table S1). Compounds 43 and 49 were identified as 3,5- and 4,5-di-O-p-coumaroylquinic acids based on their [M-H]− at m/z 483, a base peak at m/z 337, a cinnamate-derived peak at m/z 163 (in 43) and a diagnostic “dehydrated” quinic acid ion at m/z 173 (in 49). Six compounds (31, 33, 36, 37, 40 and 41) displayed the same [M-H]− at m/z 499 and fragment ions at m/z 353 and/or 337 with different intensity in their MS/MS spectra, corresponding to the elimination of caffeoyl and p-coumaroyl units. Some additional peaks at m/z 335 [353-H2O]− and 319 [337-H2O]−, and “dehydrated” quinic acid ion at m/z 173 allowed their identification as p-coumaroyl-caffeoylquinic acid isomers [36]. In addition, four feruloyl-caffeoylquinic acids (38, 39, 42 and 45) were identified by their [M-H]− at m/z 529 and characteristic fragmentation ions at m/z 367 [M-C9H6O3-H]−, 353 [M-C10H8O3-H]−, 349 [367-H2O]−, and 179 [caffeic acid-H]−. 3,4,5-Tricaffeoylquinic acid (50) was deduced from its [M-H]− at m/z 677 and the fragment ions at m/z 515, 353 and 191 due to the loss of three caffeoyl units. Their position at C-3, C-4 and C-5 was deduced from the peaks at m/z 173, 135 and 179 [30].
Four tricaffeoylhexaric acids (27, 32, 34 and 35) and a tetracaffeoylhexaric acid (44) were also detected. Their identification was based on their [M-H]− at m/z 695 and 857, respectively, the base peak at m/z 209 [hexaric acid–H]− and diagnostic fragment ions resulting from the sequential elimination of three of four caffeoyl moieties [38]. Compound 46 showed [M-H]− at m/z 617 and fragment ions at m/z 293 [M-2caffeoyl-H]− and 191 [M-2caffeoyl-C5H10O2]− due to the loss of two caffeoyl residues and a subsequent loss of 102 Da (2-methylbutiric acid/isovaleric acid). Therefore, compound 46 was tentatively identified as 2-methylbutanoyl/isovaleryl dicaffeoylhexaric acids. In the same manner, compound 54 was determined as 2-methylbutanoyl/isovaleryl tricaffeoylhexaric acid. Similarly, compound 53 was identified as isobutanoyl-tricaffeoylhexaric acid as it had [M-H]− at m/z 765, fragmentation ions due to the loss of three caffeoyl units and a base peak at m/z 279 as a result of elimination of 88 Da (C4H8O2) [38].
With exception of chlorogenic (8) and caffeic acids (9) [25] all other compounds are described now for the first time in I. salicina. As far as we know, there are only a few HPLC-MS/MS analyses of Inula species so far [29,38,40,41,42,43,44,45]. Among the identified acids and their derivatives, acylquinic acids dominated, especially mono- and dicaffeoyl quinic acids. Caffeoylquinic acid derivatives are common constituents of species of Asteraceae family [46], they could not be used as chemotaxonmical markers, but definitely contributed to the various biological activity of the plant extracts [47,48]. Caffeoylhexaric acids have been reported in I. sarana [38] and I. viscosa [44] only. However, it seems that these compounds are characteristic for the species of the tribe Inulae as they have been detected in some related genera such as Pulicaria, Carpesium, Xerolekia, etc. [49,50,51].
Free aglycones, flavonoid mono- and di-glycosides, including seven flavone and eight flavonol derivatives were recognized by their mass-spectral fragmentation pattern [29,38]. Compounds 47, 48 and 58 were identified as the free aglycones quercetin, luteolin and apigenin by their [M-H]− at m/z 301, 285 and 269, respectively, as well as by comparison with authentic standards. In addition, five methoxylated aglycones patuletin (51), nepetin (52), chrysoeriol (55), jaceosidin (56) and quercetagetin trimethyl ether (57) were identified from the corresponding deprotonated molecular ions and the characteristic fragmentations [29,38]. The structures of flavonoids 47, 48, 51, 52 and 58 were also confirmed by 1H NMR (Table S2). MS/MS spectra of quercetin glycosides rutin (17) and isoquercitrin (18) contained a peak at m/z 301, characteristic for quercetin and derived by the elimination of 308 (rutinose) and 162 (hexose) Da from the corresponding [M-H]− at m/z 609 and 463. The structure of 17 and 18 was additionally confirmed by comparison with authentic standards. Compounds 19, 20, 22, 24, and 29 showed similar fragmentation patterns yielding prominent peaks at m/z 285 (19 and 29), 331 (20) and 315 (22 and 24) resulting from the elimination of a hexose unit (162 Da) from the precursor ions. The presence of glucopyranosyl moiety and the aglycone part in the structures of 20, 21, 23, and 30 was further confirmed by 1H NMR (Table S3) after their isolation from the methanol extract. Compound 24 was tentatively determined as isorhamnetin hexoside. It is worth noting that with the exception of nepetin (52) and apigenin (58) [25,26], all other identified flavonoids are reported in I. salicina for the first time. The detection of 6-methoxyflavones and 6-methoxyflavonols in I. salicina is not surprising, as these compounds have been previously found in many Inula species [11,16,52,53,54,55] and they can be considered as a chemotaxonomic characteristic at the genus level.
Finally, the low-intensive deprotonated molecular ion [M-H]− at m/z 442.1150 and the base peak at m/z 327 [M-C5H8O2N-H]− suggested a flavonoid-alkaloid structure of compound 28 (Figure 2A). The lack of information in the literature and open access LC-MS libraries prompted us to perform further isolation of this compound and elucidate its structure. The HRESIMS in the positive mode of 28 showed [M + H]+ at m/z 444.1284 (calculated for C22H22O9N) pointing out a molecular formula C22H21O9N.
The 1H NMR spectrum (Table 2) contained two doublets at δH 6.94 (J = 2.2 Hz) and 7.62 (J = 8.4 Hz), a signal at δH 7.45 dd (J = 2.2 and 8.4 Hz), a singlet at δH 6.59, a methoxyl group signal at δ 3.89. These spectral data were very similar to that of nepetin (6-methoxyluteolin, 52) suggesting the same aglycone. Surprisingly, instead of the characteristic H-8 signal, additional signals for a benzyl methylene group (δH 4.72 and 4.58, δC 47.4) appeared in the 1H and 13C NMR. The observed HMBC correlations (Figure 2B) of these signals with C-8 (δC 97.3) and C-9 (δC 152.5) suggested a C-bound aliphatic portion attached to C-8. Further, 1H NMR, HSQC and HMBC spectra contained additional signals for three methylene groups (δH 3.57/3.36 and δC 53.4, δH 2.09/1.95 and δC 23.0, δH 2.48/2.18 and δC 28.8), a hydroxymethine group (δH 4.07 and δC 69.3) and a carbonyl group (δC 172.4). The observed COSY interactions H-3″/H-4″, H4″/H5″ and H5″/H6″ and HMBC correlations H-3″/C-2″, H-3″/C-5″, H-4″/C-2″, H-4″/C-6″ (Figure 2B) confirmed the proposed connectivity of this part of the molecule.
Further, the presence of 3-hydroxypiperidin-2-one moiety was confirmed by the base peak at m/z 329 in the MS/MS spectrum due to the loss of C5H8O2N unit (114 Da). The observed long range HMBC correlations (Figure 2B) of H-7″ with C-3″and C-6″ showed that the nitrogen atom is attached to C-2″, C-6″ and C-7″. Therefore, the new compound 28 was identified as N-(8-methylnepetin)-3-hydroxypiperidin-2-one. To the best of our knowledge, there is only one report for natural compounds with N-methyl-3-hydroxypiperidin-2-one residue in their structure. These compounds were two flavonol glycosides isolated from Astragalus monspessulantus from Bulgaria [56]. This is the first report of the presence of flavoalkaloids in the genus Inula. Flavoalkaloids are a unique group of structurally diverse secondary metabolites, consisting of a nitrogen-containing moiety attached to a flavonoid backbone at C-6 or C-8 positions [57], and are considered to be compounds with potential against various diseases such as cancer, inflammation, viral infections, etc. [58].

2.2. Quantitative Determination of Total Phenolics, Total Flavonoids, Chlorogenic and Dicaffeoylquinic Acids

Spectrophotometric methods are widely used to assess the total phenolic (TPC) and total flavonoid (TFC) content of the plant extracts as these compounds contribute significantly to their biological activity. In this study, TPC and TFC were found to be 215.57 ± 2.52 mg GAE/g DE and 87.44 ± 0.52 mg CE/g DE (Table 3). The results obtained for TPC were significantly higher than those found for the methanol extract of I. salicina from Turkey (143.80 mg GAE/g DE) and its n-hexane, chloroform, and aqueous methanol fractions (40.62, 166.20 and 126.10 mg GAE/g DE) and were twice lower than the ethyl acetate fraction (574.80 mg GAE/g DE) [22]. The TFC of the same extracts varied between 10.22 and 201.40 mg QE/g DE and increased in the following order: n-hexane > chloroform > methanol > aqueous methanol > ethyl acetate [22]. There is only one more study on TPC (58.54 μg GAE/mL) and no other data on TFC of I. salicina [24]. In our previous study on six Inula species, the TPC of the methanol extracts varied from 28.81 to 119.92 mg GAE/g DE with the highest phenolic level observed in the flower extract of I. ensifolia [17]. The study of methanol extracts of I. britannica from 11 different Bulgarian habitats revealed high variations in TPC (85.35–141.01mg GAE/g DE) and TFC (19.66–36.80 mg CE/g DE) [45]. Therefore, it can be concluded that the extract of I. salicina is the richest in phenolics and flavonoids among the representatives of the genus Inula growing in Bulgaria studied so far.
Further, the content of chlorogenic acid and four dicaffeoylquinic acid (DCQA) isomers was determined by the HPLC method (Figure 3). Chlorogenic acid (5-CQA) was the major component detected in the highest amount (103.39 ± 1.30 mg/g DE), while that of dicaffeoyl esters was significantly lower decreasing in the order 3,5-DCQA > 4,5-DCQA > 1,5-DCQA > 3,4-DCQA (Table 3).
In the study of some Hungarian Inula species, it has been reported that I. salicina was the richest in chlorogenic acid [25]. In addition, the ray florets of I. salicina were found to contain more chlorogenic acid than the disk florets unlike the other studied Inula species. It was interesting to compare the results of this study with those found for other Inula species growing in Bulgaria [17,45]. Thus, the amount of chlorogenic acid in I. aschersoniana var. aschersoniana, I. bifrons, I. conyza, I. ensifolia, I. germanica, and I. oculus-christi was significantly lower (5.48–28.44 mg/g DE) and I. ensifolia was the species with the highest amount detected [17]. The content of chlorogenic acid in the extracts of different I. britannica populations was also variable (14.99–51.41 mg/g DE) [45]. In these studies, the content of 5-CQA in the respective plants was lower than the total amount of dicaffeoyl esters of quinic acid [17]. Despite the different quantities of the individual DCQAs, 1,5-DCQA was the principal component in all studied species with exception of I. conyza [17]. 3,5-DCQA was the major compound in I. conyza [17] as the currently studied I. salicina. The latter contained a significant amount of 4,5-DCQA, similarly to I. ensifolia and I. oculus-christi. Therefore, it can be concluded that the extract of I. salicina is the richest in chlorogenic acid among the representatives of the genus Inula growing in Bulgaria studied so far.

2.3. Antioxidant Potential of Inula salicina Extract

DPPH radical scavenging, ABTS radical-ion and FRAP assays, based on different mode of actions are widely used for a preliminary study of the antioxidant potential of various compounds and plant extracts [59]. Thus, the antioxidant capacity of the studied extract measured by the DPPH and ABTS methods was 0.741 ± 0.006 and 0.711 ± 0.007 mM TE/g DE, respectively, while the ferric ion reducing antioxidant power in FRAP assay was found to be 5.77 ± 0.08 µM Fe2+/g DE). In a recent study on the antioxidant activity of Inula salicina from Turkey it has been found that the ethyl acetate fraction possessed the best DPPH and ABTS activity among the other fractions and the methanol extract and the measured IC50 in both assays was similar to that of known standards Trolox and ascorbic acid and better than the synthetic antioxidant BHA [22]. This is the first report on FRAP activity of I. salicina. There are reports in the scientific literature regarding antioxidant activity (DPPH, ABTS, FRAP, etc.) of other representatives of genus Inula [13,14,17,45,54,60,61]. Unfortunately, slight differences in the assays, measuring units and other factors make it difficult to compare the obtained data. However, it can be concluded that the Inula extracts which are richer in polyphenolics are better antioxidants.

2.4. In Vitro Sun Protection Factor (SPF) of Inula salicina Extract

The methanol extracts of I. salicina, chlorogenic and caffeic acids and rutin in four different concentrations were studied for their sun protection factor (SPF) (Figure 4) using the method described by Mansur [62]. As can be seen, all tested samples possessed good SPF at concentrations > 250 µg/mL (SPF 28–32), corresponding to 94–98% UV-B protection [4]. Further, I. salicina extract in concentration of 62.5 µg/mL had a SPF 10, corresponding to 90% UV-B protection; it was similar to that of rutin (SPF 9, 89% UV-B) and lower than that of chlorogenic and caffeic acids (SPF 18 and 28, 95 and 96%, respectively).
It can be assumed that phenolic compounds, particularly chlorogenic acid are responsible for the sun protective properties of I. salicina methanol extract [3,5,8]. It is worth mentioning that this is the first report on SPF of Inula species.

2.5. Biofilm Inhibition of Inula salicina Extract

To estimate the ability of I. salicina methanol extract to inhibit biofilm formation a crystal violet assay was conducted. The Gram-positive and Gram-negative strains utilized in this study are classified by the World Health Organization as a critical priority due to their antimicrobial resistance. It is well known that these strains cause infections such as otitis, sinusitis, infectious wounds, cystic fibrosis, chronic obstructive pulmonary diseases, osteomyelitis, endocarditis, chronic prostatitis, and others [9]. Their role in these diseases underscores the significance of understanding their pathogenesis and developing targeted treatment strategies. The analysis after 24 h interval of biofilm incubation demonstrated the inhibition effects of I. salicina extract on bacterial biofilms. The inhibition capacity of the plant extract was expressed as percentage of biofilm inhibition calculated relative to the control sample, which comprised M63 medium along with the corresponding bacterial strain. The highest anti-biofilm activity of a I. salicina methanol extract was detected in the P. aeruginosa strain (64.9% ± 0.06), followed by inhibition at S. aureus 43.4% ± 0.02. The lowest value was reported in E. coli (35.0% ± 0.04). These results are consistent with data from our previous study conducted with another Gram-negative strain, C. violaceum [23]. This research also underscores the synergistic effect of various extracts of Inula species against virulence factors of C. violaceum like biofilm formation, violacein production, and swarming motility. Furthermore, the study stands out as one of the few conducted with an Inula species, demonstrating the inhibitory efficacy of the tested extracts on biofilm formation and modeling their 3D structure [23]. The obtained results highlight the plant extracts such as Inula viscosa, Betula pendula, Galium odoratum, Urtica dioca, etc. are capable of reducing the biofilm formation of Escherichia coli, Candida albicans, and Candida glabrata [63,64,65]. Moreover, our previous study demonstrated the anti-biofilm activities of CaO/chitosan nanocomposites doped with different extracts from the leaves of A. indica and M. azedarach [66]. The obtained results showed a significant reduction in biofilms by the nanocomposites labeled with plant extracts in the tested model strains E. coli and S. aureus.

2.6. Live/Dead Biofilm Assays

Plant metabolites manifest different mechanisms of action, some of which can lead to anti-bacterial or anti-biofilm effects and subsequently to bacterial cell death [9]. Hence, it is extremely important to see how an extract affects bacterial viability both at the single-cell and biofilm consortium levels. To determine the bacterial cells viability within the biofilms cultivated in the presence of I. salicina methanol extract, we applied a live/dead fluorescent viability kit (Figure 5). The I. salicina extract is rich in chlorogenic acid and dicaffeoylquinic acids as described above. The scientific data of these metabolic ingredients showed that they have proven antibacterial effect on both Gram-negative and Gram-positive strains [67]. The remarkable anti-biofilm effects of I. salicina extract (crystal violet test), especially in P. aeruginosa, were also confirmed during the live/dead biofilm staining. In control groups, the formed biofilms consist of live, intact (green) cells. The fluorescence labeling provided in E. coli and S. aureus, showed that the biofilm is monolayered with the formation of local groups, while in P. aeruginosa, the biofilm tends to be multilayered, clustered, and dense, composed mainly of dead cells. Across the three bacterial strains, only single cells remain intact (green) without disruptions in bacterial cell membrane. However, during the assessment of both cell viability and biofilm formation upon the treatment, they are noticeably affected. The biofilm structure is loosened and reduced. The images of E. coli and S. aureus revealed distinct clustering, aggregation and a reduced biofilm layer, which were consistent with the results from the CV assay. In E. coli, the biofilm mainly consisted of non-viable cells, although some green cells were visible in certain areas. Treatment of S. aureus biofilms with I. salicina extract resulted in substantial cell aggregation, with varying sizes of clusters containing both dead and live cells, mostly in the upper layers.
This may be due to the inability of the phytochemicals to penetrate the dense structure of the biofilm matrix and reach all the bacterial cells. In another study, it was observed that treatment with 2% chlorogenic acid, also present in our extract, resulted in inhibition of the bacterial viability, which indicated cell membrane damage [68]. In contrast, in P. aeruginosa biofilm layers disintegrated without forming clusters. A significant part of the observed cells in P. aeruginosa were non-viable. However, there was a small portion of intact cells. This may be due to the presence of chlorogenic acid in the extract.

2.7. SEM on Pseudomonas aeruginosa Cell Morphology

Figure 6 shows the results of the SEM analysis of changes in the biofilm and the morphology of individual bacterial cells after treatment with the methanol extract. The electron microscopic analysis of the biofilms incubated with the extract clearly indicates a significant reduction in biofilm biomass and a lack of multilayer cell distribution compared to the control sample (Figure 6). Moreover, distinct deformations in the surface relief of individual cells are clearly defined (Figure 6B). A notable feature is the destruction of the surface relief with the presence of radial indentations along the length of the cells, indicated by a white triangle. In comparison to the control sample, where no changes in the relief are observed, the treated sample shows widespread invaginations at both poles of the bacterial cells, marked by a white arrow (Figure 6B). An interesting finding is the presence of a cell with a disrupted cell wall and likely release of cytoplasmic content, marked by a white star. Particularly, during the treatment in P. aeruginosa, chlorogenic acid can trigger the detachment of lipopolysaccharides from the outer membrane, resulting in high membrane permeability, depolarization, leakage of nutrients and metabolites and ultimately cell death [69]. These scientific data correlate with the SEM results obtained by us when treating the cells with the methanol extract (Figure 6B).
In conclusion, the tested methanol extract can affect both the process of biofilm formation, the cell viability and morphology in tested Gram-positive and Gram-negative strains. The effects varied among the bacterial strains, with an overall reduction in biofilm thickness, multilayer distribution and viability observed. In both Gram-negative strains, E. coli and P. aeruginosa, the cells were notably morphologically affected and most of them were non-viable. The trend was similar in S. aureus except for single intact cells. These differences between the Gram-positive and Gram-negative strains, may be related to the structure of the cell wall, as well as the mechanisms of action of the different bioactive constitutes of the extract and their concentrations [70]. This is once more supported by our viability and SEM assays through which a predominant accumulation of non-viable red and deformed bacterial cells was demonstrated, providing further evidence of the anti-biofilm and also antimicrobial effect of the applied plant extracts. This hypothesis has been confirmed by other researchers where the two active metabolic components—chlorogenic acid and dicaffeoylquinic acid—not only possess antibacterial activity due to their ability to disrupt membrane permeability and potential, but also a strong anti-biofilm effect [71]. Their anti-biofilm activity is probably due to different mechanisms of action. For instance, chlorogenic acid can disturb different processes such as quorum sensing, biofilm formation, bacterial flagella formation, etc. [69,72]. Moreover, it was found that 3,5-dicaffeoylquinic acid exhibits a strong anti-biofilm effect in P. aeruginosa [73].

3. Materials and Methods

3.1. Plant Material

The aerial parts of Inula salicina L. were picked up in full flowering stage near Dospat lake, Rhodopes Mts, Bulgaria in 2020, air-dried, grounded and kept in a dark place prior analysis. The species was identified by Assoc. Prof. PhD Ina Aneva (Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences) according to the following reference specimen (SOM 176701) deposited in the Herbarium of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences.

3.2. Preparation of the Methanol Extract

The plant material (54 g) was initially extracted with chloroform (3 × 500 mL) to remove non-polar compounds and chlorophylls followed by an extraction with methanol (3 × 500 mL). All extractions were performed at room temperature for 24 h each. Further filtration and evaporation of the solvent under reduced pressure yielded the methanol extract (4.06 g).

3.3. Fractionation of the Methanol Extract and Isolation of Individual Compounds

The fractionation was worked out following the procedure described in [54]. Briefly, a portion of the extract (1.1 g) was re-dissolved in MeOH (10 mL), centrifuged (at 5800 rpm) and a clear methanolic solution was concentrated up to 5 mL. Further CC on Sephadex LH-20 with methanol as an eluent gave two main fractions I (0.62 g) and II (0.39 g). TLC (Silica gel 60 F254, EtOAc/HCOOH/CH3COOH/H2O, 100:11:11:26, spraying with NP reagent (1% diphenylboronic acid 2-aminoethyl ester in ethyl acetate) and UV visualization at 366 nm) of fraction II showed the presence of flavonoids (yellow to orange fluorescence) and caffeoylquinic acids (blue fluorescence) and was further separated by MPLC (LiChroprep RP-18) with H2O/CH3OH mixtures in different proportions. Next, selected fractions were purified by MPLC (LiChroprep RP-18, CH3OH/H2O, 50:50) and/or prep. TLC (Silica gel 60 F254, CHCl3/CH3OH, 10:1 or (RP-18, MeOH/H2O, 1:1 and 7:3) led to the isolation of 15 compounds: chlorogenic acid (8) (6.5 mg), rutin (17) (2.3 mg), isoquercitrin (18) (1.8 mg), luteolin-7-O-glucoside (19) (1.2 mg), patulitrin (20) (1.1 mg), nepitrin (22) (2.2 mg), 3,5-dicaffeoylquinic acid (25) (2.1 mg), 1,5-dicaffeoylquinic acid (26) (1.8 mg), N-(8-methylnepetin)-3β-hydroxypiperidin-2-one (28) (0.8 mg), kaempferol-3-O-glucoside (29) (1.2 mg), quercetin (47) (1.2 mg), luteolin (48) (5.6 mg), patuletin (51) (0.8 mg), nepetin (6-OMe-luteolin) (52) (1.2 mg), and apigenin (58) (1.1 mg).
N-(8-methylnepetin)-3-hydroxypiperidin-2-one (28):
Yellowish semisolid substance, UV (MeOH): λmax (log ε) 227.5 (2.72), 273.5 (1.80), 349.5 (1.73) nm; HRESIMS: m/z 444.12841 [M+H]+ (calcd. for C22H22O9N, 444.12891, Δppm = −1.12); 1H and 13C NMR: see Table 2.

3.4. NMR Analysis

The 1D and 2D NMR (1H, COSY, HSQC and HMBC) spectra were recorded on a Bruker Avance II+ 600 NMR spectrometer with operating frequency 600 (1H) and 150 (13C) using the residual solvent signal (δH/C 3.31 and 49.3 for CD3OD) as a reference. The known compounds were identified by comparison of their 1H NMR spectral data (Tables S1–S3) with literature data [34,74,75,76,77,78,79]. 1H and 13C NMR of compound 28 are presented in Table 2 and Figures S2–S7.

3.5. UHPLC-HRMS Analysis

The chromatographic and mass spectrometric conditions of the UHPLC-HRMS analysis were as given in the literature [80] with small modifications. The gradient of the chromatographic separations is given in Table 4. The system was kept at the initial condition for 5 min before each injection. The maximal injection time in full MS mode was set to 80 ms, while stepped normalized collision energy was 10, 15, and 20 (+p mode) as well as 10, 20, and 30 (−p mode).

3.6. HPLC-DAD Quantification of Caffeoylquinic Acids

The HPLC analysis was performed on Schimadzu Nexera-I LC-2040C 3D Plus liquid chromatograph equipped with a photodiode array detector (Schimadzu, Tokyo, Japan) on analytical column Force C18 (150 × 4.6 mm, 3 µm) at a temperature of 30 °C. The elution was performed in a gradient mode using a mixture of 0.1% of formic acid in water (A) and methanol (B) as follows: 0 min, 20% B; 5 min, 20% B; 37 min, 60% B; 38 min, 80% B; 42 min, 80% B; 43 min, 20% B; 47 min, 20% B. The injection volume was 2 µL, the flow rate was 0.6 mL/min and runs were monitored at 320 nm. Before analysis, samples were filtered through 0.22 µm syringe filter. Chlorogenic acid with Rt 12.6 min (0.019–0.305 mg/mL, R2 0.9999), 3,4-dicaffeoylquinic acid Rt 25.42 min (0.005–0.083 mg/mL, R2 0.9999), 3,5-dicaffeoylquinic acid Rt 25.70 min (0.019–0.308 mg/mL, R2 0.9999), 1,5-dicaffeoylquinic acid Rt 26.35 min (0.022–0.355 mg/mL, R2 0.9999) and Rt 29.13 min (0.008–0.135 mg/mL, R2 0.9999) were used as standards for preparation of the calibration curves (Figure 3). The experiment was performed in triplicate and the results are expressed as mg/g DE.

3.7. Evaluation of Total Phenolic (TPC) and Total Flavonoid (TFC) Contents

TPC was determined by Folin–Ciocalteu method [81] and the results were expressed as milligrams gallic acid equivalents per gram of dry extract (mg GAE/g DE). TFC was determined by Zhishen et al. colorimetric assay [82] and the results were expressed as milligrams of catechin equivalents per gram of dry extract (md CE/g DE).

3.8. Assessment of Antioxidant Potential

DPPH● (1,1-diphenyl-2-picrylhydrazyl) and ABTS●+ scavenging activities were determined using the procedure described by Thaipong et al. [83] and the results were expressed as mM Trolox equivalents per a gram of dry extract. FRAP (ferric ion reducing antioxidant power was performed according to procedure described in [84] and the results were expressed as µM Fe2+/g DE.

3.9. SPF and UV-B Photoprotective Study

The assay was performed according to the procedure described by Bojilov et al. [80] using different concentrations (1000, 250, 125, 62.5 and 15.6 µg/mL) of the sample and standards (caffeic acid, chlorogenic acid, and rutin). The sunscreen percentage absorption based on SPF (UV-B) was calculated as described in [4]: UV-B% = 100 − (100/SPF).

3.10. Biofilm Inhibition and Assessment of Biofilm Viability

3.10.1. Bacterial Strains and Growth Conditions

In this study, Gram-negative Escherichia coli 25922 and Gram-positive Staphylococcus aureus 29213 bacterial strains were utilized. The strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), while Pseudomonas aeruginosa PAO1 was sourced from the International Reference Panel [85]. All bacterial strains were stored in 8% DMSO at −80 °C and maintained at 4 °C on Nutrient Agar for E. coli (HiMedia, Bedford, PA, USA) and Tryptic Soy Agar (TSA, Sigma, Burlington, MA, USA) for S. aureus and Pseudomonas aeruginosa slants. For screening procedures, the strains were cultivated in Nutrient Broth (HiMedia, USA) and Tryptic Soy Broth (TSA, Sigma, Burlington, MA, USA) at 37 °C for 18 h.

3.10.2. Biofilm Inhibition

Overnight bacterial cultures were used as an initial inoculum to evaluate the anti-biofilm efficacy of I. salicina extract. The conditions and the analysis were the same as described in [23] using M63 media containing KH2PO4 (0.02 M), K2HPO4 (0.02 M), (NH4)2SO4 (0.02 M), MgSO4 (0.1 mM) and glucose (0.04 M). The bacterial inoculum diluted in M63 with 2% DMSO served as a control sample. The cultivation was performed for 24 h at 37 °C. Aqueous crystal violet (0.1%) was used for staining of the adherent bacteria and the optical density was measured at 570 nm. To confirm the quantitative data of biofilm inhibitions the experiment was performed in six replicates, and average values were calculated as percentages.

3.10.3. Assessment of Biofilm Viability Using Live/Dead Staining

The biofilms were cultivated on sterilized borosilicate cover glasses for evaluation of the plant extract effects on bacterial viability. Further, the biofilm was stained with Live/Dead BacLight Bacterial Viability Kits (Invitrogen, Carlsbad, CA, USA) according to the producer’s instructions, mounted on microscopic slides using Fluoromount Mounting Medium (Sigma, USA) and observed on a Nikon Ti-U confocal laser scanning microscope in epifluorescence mode with 60× oil Plan Apo objective, at excitation wavelengths of 488 nm and 543 nm. NIS-Elements software and the Icy bio-imaging program were used for processing the acquired images.

3.10.4. Scanning Electron Microscopy (SEM)

For SEM analysis, the biofilms were grown on sterile plastic pieces. The slides were then placed in 35 mm diameter Petri dishes, inoculated with bacterial suspension and addition of the extract, and allowed to develop biofilms over 24 h. After the incubation period, samples were washed with PBS and fixed for 4 h at 4 °C in 4% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.2). Further, the samples were washed with cacodylate buffer and post-fixed with 1% OsO4 for 1 h, dehydrated via a graded ethanol series, coated with gold and observed on Lyra/Tescan scanning electron microscope (TESCAN GROUP a.s., Brno, Czech Republic), with at least 10 randomly selected digital images taken per sample, and two samples analyzed per variant.

4. Conclusions

In this study we attempted to obtain more comprehensive information on the chemical composition and biological properties of Inula salicina. Qualitative phytochemical analysis showed the presence of various phenolic compounds-hydroxycinnamic and hydroxybenzoic acids and their glycosides, acylquinic acids, caffeoylhexaric acids and flavonoids and their glycosides, among which chlorogenic and dicaffeoylquinic acids were the predominant compounds. In addition, a new natural compound, N-(8-methylnepetin)-3β-hydroxypiperidin-2-one was discovered. The presence of flavoalkaloids in the studied species is described for the first time in genus Inula and could be of chemotaxonomic significance.
The methanol extract of I. salicina exhibited good radical scavenging activity, high SPF and notable anti-biofilm effects, especially in P. aeruginosa attributed to the presence of phenolic compounds and especially of chlorogenic acid. Our findings indicated the potential application of Inula salicina extract as biofilm inhibitor, offering promising prospects for treating various surfaces contaminated with biofilms, such as implants or for directly treating superficial wounds. The extract may also be a good candidate for the development of cosmetic products. However, additional experiments on skin models and evaluation of the cytotoxicity of I. salicina extract are needed to assess its safety potential.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ph17070844/s1, Table S1: 1H NMR data of caffeoylquinic acids; Table S2: 1H NMR data of flavonoid aglycones; Table S3: 1H NMR data of flavonoid glycosides; Figure S1: Structures of the isolated compounds; Figure S2: Full scan mass spectrum of compound 28 in positive ionization mode; Figure S3: 1H NMR spectrum of 28; Figure S4: COSY spectrum of 28; Figure S5: HSQC spectrum of 28; Figure S6: HMBC spectrum of 28; Figure S7: UV spectrum of 28.

Author Contributions

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

Funding

This research was funded by the National Science Fund, Ministry of Education and Science, Bulgaria; grant number KP-06-H41/8, 30 November 2020.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within this article.

Acknowledgments

The authors are thankful to the National Science Fund at the Ministry of Education and Science, Bulgaria for the financial support of the project KP-06-H41/8. The authors acknowledge Ina Aneva from the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences for providing and identification of plant material and the Centre of Competence “Sustainable utilization of bio-resources and waste of medicinal and aromatic plants for innovative bioactive products” (BG05M2OP001-1.002-0012) for HPLC and NMR equipment used.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Szymanska, R.; Pospíšil, P.; Kruk, J. Plant-Derived Antioxidants in Disease Prevention 2018. Oxid. Med. Cell. Longev. 2018, 2018, 2068370. [Google Scholar] [CrossRef] [PubMed]
  2. Vara, D.; Pula, G. Reactive Oxygen Species: Physiological Roles in the Regulation of Vascular Cells. Curr. Mol. Med. 2014, 14, 1103–1125. [Google Scholar] [CrossRef] [PubMed]
  3. Ebrahimzadeh, M.A.; Enayatifard, R.; Khalili, M.; Ghaffarloo, M.; Saeedi, M.; Charati, J.Y. Correlation between Sun Protection Factor and Antioxidant Activity, Phenol and Flavonoid Contents of Some Medicinal Plants. Iran. J. Pharm. Res. 2014, 13, 1041–1048. [Google Scholar] [PubMed]
  4. Wilson, B.D.; Moon, S.; Armstrong, F. Comprehensive Review of Ultraviolet Radiation and the Current Status on Sunscreens. J. Clin. Aesthet. Dermatol. 2012, 5, 18. [Google Scholar]
  5. Li, L.; Chong, L.; Huang, T.; Ma, Y.; Li, Y.; Ding, H. Natural Products and Extracts from Plants as Natural UV Filters for Sunscreens: A Review. Anim. Model. Exp. Med. 2023, 6, 183–195. [Google Scholar] [CrossRef] [PubMed]
  6. Michalak, M. Plant Extracts as Skin Care and Therapeutic Agents. Int. J. Mol. Sci. 2023, 24, 15444. [Google Scholar] [CrossRef]
  7. He, H.; Li, A.; Li, S.; Tang, J.; Li, L.; Xiong, L. Natural Components in Sunscreens: Topical Formulations with Sun Protection Factor (SPF). Biomed. Pharmacother. 2021, 134, 111161. [Google Scholar] [CrossRef] [PubMed]
  8. Radice, M.; Manfredini, S.; Ziosi, P.; Dissette, V.; Buso, P.; Fallacara, A.; Vertuani, S. Herbal Extracts, Lichens and Biomolecules as Natural Photo-Protection Alternatives to Synthetic UV Filters. A Systematic Review. Fitoterapia 2016, 114, 144–162. [Google Scholar] [CrossRef]
  9. Damyanova, T.; Dimitrova, P.D.; Borisova, D.; Topouzova-Hristova, T.; Haladjova, E.; Paunova-Krasteva, T. An Overview of Biofilm-Associated Infections and the Role of Phytochemicals and Nanomaterials in Their Control and Prevention. Pharmaceutics 2024, 16, 162. [Google Scholar] [CrossRef]
  10. Dimitrova, P.D.; Damyanova, T.; Paunova-Krasteva, T. Chromobacterium Violaceum: A Model for Evaluating the Anti-Quorum Sensing Activities of Plant Substances. Sci. Pharm. 2023, 91, 33. [Google Scholar] [CrossRef]
  11. Seca, A.M.L.; Grigore, A.; Pinto, D.C.G.A.; Silva, A.M.S. The Genus Inula and Their Metabolites: From Ethnopharmacological to Medicinal Uses. J. Ethnopharmacol. 2014, 154, 286–310. [Google Scholar] [CrossRef] [PubMed]
  12. Zhao, Y.M.; Zhang, M.L.; Shi, Q.W.; Kiyota, H. Chemical Constituents of Plants from the Genus Inula. Chem. Biodivers. 2006, 3, 371–384. [Google Scholar] [CrossRef] [PubMed]
  13. Talebi, M.; Khoramjouy, M.; Feizi, A.; Ali, Z.; Khan, I.A.; Ayatollahi, N.A.; Ayatollahi, S.A.; Faizi, M. Novel Multi-Target Therapeutic Potential of the Genus Inula: Advances and Opportunities for Neuroprotection. Pharmacol. Res.-Mod. Chinese Med. 2023, 7, 100263. [Google Scholar] [CrossRef]
  14. Tavares, W.R.; Seca, A.M.L. Inula L. Secondary Metabolites against Oxidative Stress-Related Human Diseases. Antioxidants 2019, 8, 122. [Google Scholar] [CrossRef] [PubMed]
  15. Wang, G.W.; Qin, J.J.; Cheng, X.R.; Shen, Y.H.; Shan, L.; Jin, H.Z.; Zhang, W.D. Inula Sesquiterpenoids: Structural Diversity, Cytotoxicity and Anti-Tumor Activity. Expert Opin. Investig. Drugs 2014, 23, 317–345. [Google Scholar] [CrossRef] [PubMed]
  16. Seca, A.M.L.; Pinto, D.C.G.A.; Silva, A.M.S. Metabolomic Profile of the Genus Inula. Chem. Biodivers. 2015, 12, 859–906. [Google Scholar] [CrossRef] [PubMed]
  17. Trendafilova, A.; Ivanova, V.; Rangelov, M.; Todorova, M.; Ozek, G.; Yur, S.; Ozek, T.; Aneva, I.; Veleva, R.; Moskova-Doumanova, V.; et al. Caffeoylquinic Acids, Cytotoxic, Antioxidant, Acetylcholinesterase and Tyrosinase Enzyme Inhibitory Activities of Six Inula Species from Bulgaria. Chem. Biodivers. 2020, 17, e2000051. [Google Scholar] [CrossRef] [PubMed]
  18. Inula Salicina in Flora of China @ Efloras.Org. Available online: http://www.efloras.org/florataxon.aspx?flora_id=2&taxon_id=200024067 (accessed on 20 May 2024).
  19. Inula Salicina|Euro+Med-Plantbase. Available online: https://europlusmed.org/cdm_dataportal/taxon/e4ecad7f-1304-4bf9-9736-3a87a4da505b (accessed on 20 May 2024).
  20. AGFonds—Irish Fleabane (Inula salicina L.). Available online: https://www.agfonds.lv/herbs/herbs-i-j/irish-fleabane-inula-salicina-l/ (accessed on 20 May 2024).
  21. Tardío, J.; Pardo-De-Santayana, M.; Morales, R. Ethnobotanical Review of Wild Edible Plants in Spain. Bot. J. Linn. Soc. 2006, 152, 27–71. [Google Scholar] [CrossRef]
  22. Yıldırım, A.; Şen, A.; Hacıoğlu, M.; Seher, A.; Tan, B.; Şenkardeş, İ.; Bitiş, L. In Vitro Investigation of Antimicrobial, Enzyme Inhibitory and Free Radical Scavenging Activities of Inula salicina L. Int. J. Agric. Environ. Food Sci. 2022, 6, 389–395. [Google Scholar] [CrossRef]
  23. Dimitrova, P.D.; Ivanova, V.; Trendafilova, A.; Paunova-Krasteva, T. Anti-Biofilm and Anti-Quorum-Sensing Activity of Inula Extracts: A Strategy for Modulating Chromobacterium Violaceum Virulence Factors. Pharmaceuticals 2024, 17, 573. [Google Scholar] [CrossRef]
  24. Sevindik, E.; Aydin, S.; Paksoy, M.Y.; Sokmen, B.B. Anti-Urease, Total Phenolic Content and Antioxidant Activities of Some Inula L. (Asteraceae) Taxa in Turkey. Genetika 2020, 52, 825–834. [Google Scholar] [CrossRef]
  25. Péter, A.; Dósa, G. Detection of Phenoloids in Some Hungarian Inula and Centaurea Species. Acta Bot. Hung. 2002, 44, 129–135. [Google Scholar] [CrossRef]
  26. Wollenweber, E.; Dörr, M.; Fritz, H.; Valant-Vetschera, K.M. Exudate Flavonoids in Several Asteroideae and Cichorioideae (Asteraceae). Zeitschrift fur Naturforsch. Sect. C-J. Biosci. 1997, 52, 137–143. [Google Scholar] [CrossRef]
  27. Bohlmann, F.; Mahanta, P.K.; Jakupovic, J.; Rastogi, R.C.; Natu, A.A. New Sesquiterpene Lactones from Inula Species. Phytochemistry 1978, 17, 1165–1172. [Google Scholar] [CrossRef]
  28. Trendafilova, A.; Ivanova, V.; Todorova, M.; Staleva, P.; Aneva, I. Terpenoids in Four Inula Species from Bulgaria. J. Serbian Chem. Soc. 2021, 86, 1229–1240. [Google Scholar] [CrossRef]
  29. Gevrenova, R.; Zengin, G.; Sinan, K.I.; Zheleva-Dimitrova, D.; Balabanova, V.; Kolmayer, M.; Voynikov, Y.; Joubert, O. An In-Depth Study of Metabolite Profile and Biological Potential of Tanacetum balsamita L. (Costmary). Plants 2023, 12, 22. [Google Scholar] [CrossRef] [PubMed]
  30. Clifford, M.N.; Wu, W.; Kirkpatrick, J.; Kuhnert, N. Profiling the Chlorogenic Acids and Other Caffeic Acid Derivatives of Herbal Chrysanthemum by LC-MSn. J. Agric. Food Chem. 2007, 55, 929–936. [Google Scholar] [CrossRef]
  31. Shahzad, M.N.; Ahmad, S.; Tousif, M.I.; Ahmad, I.; Rao, H.; Ahmad, B.; Basit, A. Profiling of Phytochemicals from Aerial Parts of Terminalia Neotaliala Using LC-ESI-MS2 and Determination of Antioxidant and Enzyme Inhibition Activities. PLoS ONE 2022, 17, e0266094. [Google Scholar] [CrossRef]
  32. Clifford, M.N.; Johnston, K.L.; Knight, S.; Kuhnert, N. Hierarchical Scheme for LC-MSn Identification of Chlorogenic Acids. J. Agric. Food Chem. 2003, 51, 2900–2911. [Google Scholar] [CrossRef]
  33. Clifford, M.N.; Zheng, W.; Kuhnert, N. Profiling the Chlorogenic Acids of Aster by HPLC–MSn. Phytochem. Anal. 2006, 17, 384–393. [Google Scholar] [CrossRef]
  34. Tolonen, A.; Joustamo, T.; Mattlla, S.; Kämäräinen, T.; Jalonen, J. Identification of Isomeric Dicaffeoylquinic Acids from Eleutheracoccus Senticosus Using HPLC-ESI/TOF/MS and H-NMR Methods. Phytochem. Anal. 2002, 13, 316–328. [Google Scholar] [CrossRef] [PubMed]
  35. Jaiswal, R.; Halabi, E.A.; Karar, M.G.E.; Kuhnert, N. Identification and Characterisation of the Phenolics of Ilex Glabra L. Gray (Aquifoliaceae) Leaves by Liquid Chromatography Tandem Mass Spectrometry. Phytochemistry 2014, 106, 141–155. [Google Scholar] [CrossRef] [PubMed]
  36. Clifford, M.N.; Marks, S.; Knight, S.; Kuhnert, N. Characterization by LC-MS(n) of Four New Classes of p-Coumaric Acid-Containing Diacyl Chlorogenic Acids in Green Coffee Beans. J. Agric. Food Chem. 2006, 54, 4095–4101. [Google Scholar] [CrossRef] [PubMed]
  37. Jaiswal, R.; Sovdat, T.; Vivan, F.; Kuhnert, N. Profiling and Characterization by LC-MSn of the Chlorogenic Acids and Hydroxycinnamoylshikimate Esters in Maté (Ilex paraguariensis). J. Agric. Food Chem. 2010, 58, 5471–5484. [Google Scholar] [CrossRef] [PubMed]
  38. Zengin, G.; Nilofar; Yildiztugay, E.; Bouyahya, A.; Cavusoglu, H.; Gevrenova, R.; Zheleva-Dimitrova, D. A Comparative Study on UHPLC-HRMS Profiles and Biological Activities of Inula Sarana Different Extracts and Its Beta-Cyclodextrin Complex: Effective Insights for Novel Applications. Antioxidants 2023, 12, 1842. [Google Scholar] [CrossRef] [PubMed]
  39. Sinosaki, N.B.M.; Tonin, A.P.P.; Ribeiro, M.A.S.; Poliseli, C.B.; Roberto, S.B.; da Silveira, R.; Visentainer, J.V.; Santos, O.O.; Meurer, E.C. Structural Study of Phenolic Acids by Triple Quadrupole Mass Spectrometry with Electrospray Ionization in Negative Mode and H/D Isotopic Exchange. J. Braz. Chem. Soc. 2020, 31, 402–408. [Google Scholar] [CrossRef]
  40. Brahmi-Chendouh, N.; Piccolella, S.; Crescente, G.; Pacifico, F.; Boulekbache, L.; Hamri-Zeghichi, S.; Akkal, S.; Madani, K.; Pacifico, S. A Nutraceutical Extract from Inula Viscosa Leaves: UHPLC-HR-MS/MS Based Polyphenol Profile, and Antioxidant and Cytotoxic Activities. J. Food Drug Anal. 2019, 27, 692–702. [Google Scholar] [CrossRef]
  41. Zhao, Q.; Li, Y.; Li, S.; He, X.; Gu, R. Comparative Bioactivity Evaluation and Metabolic Profiling of Different Parts of Duhaldea Nervosa Based on GC-MS and LC-MS. Front. Nutr. 2023, 10, 1301715. [Google Scholar] [CrossRef] [PubMed]
  42. Peng, J.; Xie, J.; Shi, S.; Luo, L.; Li, K.; Xiong, P.; Cai, W. Diagnostic Fragment-Ion-Based for Rapid Identification of Chlorogenic Acids Derivatives in Inula Cappa Using UHPLC-Q-Exactive Orbitrap Mass Spectrometry. J. Anal. Methods Chem. 2021, 2021, 6393246. [Google Scholar] [CrossRef] [PubMed]
  43. Stojakowska, A.; Malarz, J.; Kiss, A.K. Hydroxycinnamates from Elecampane (Inula helenium L.) Callus Culture. Acta Physiol. Plant. 2016, 38, 1–5. [Google Scholar] [CrossRef]
  44. Rechek, H.; Haouat, A.; Hamaidia, K.; Pinto, D.C.G.A.; Boudiar, T.; Válega, M.S.G.A.; Pereira, D.M.; Pereira, R.B.; Silva, A.M.S. Inula viscosa (L.) Aiton Ethanolic Extract Inhibits the Growth of Human AGS and A549 Cancer Cell Lines. Chem. Biodivers. 2023, 20, e202200890. [Google Scholar] [CrossRef] [PubMed]
  45. Ivanova, V.; Todorova, M.; Rangelov, M.; Aneva, I.; Trendafilova, A. Phenolic Content and Antioxidant Capacity of Inula Britannica from Different Habitats in Bulgaria. Bulg. Chem. Commun. 2020, 52, 168–173. [Google Scholar]
  46. Fraisse, D.; Felgines, C.; Texier, O.; Lamaison, J.-L. Caffeoyl Derivatives: Major Antioxidant Compounds of Some Wild Herbs of the Asteraceae Family. Food Nutr. Sci. 2011, 2, 181–192. [Google Scholar] [CrossRef]
  47. Wianowska, D.; Gil, M. Recent Advances in Extraction and Analysis Procedures of Natural Chlorogenic Acids. Phytochem. Rev. 2018, 18, 273–302. [Google Scholar] [CrossRef]
  48. Liu, W.; Li, J.; Zhang, X.; Zu, Y.; Yang, Y.; Liu, W.; Xu, Z.; Gao, H.; Sun, X.; Jiang, X.; et al. Current Advances in Naturally Occurring Caffeoylquinic Acids: Structure, Bioactivity, and Synthesis. J. Agric. Food Chem. 2020, 68, 10489–10516. [Google Scholar] [CrossRef]
  49. Kłeczek, N.; Michalak, B.; Malarz, J.; Kiss, A.K.; Stojakowska, A. Carpesium Divaricatum Sieb. & Zucc. Revisited: Newly Identified Constituents from Aerial Parts of the Plant and Their Possible Contribution to the Biological Activity of the Plant. Molecules 2019, 24, 1614. [Google Scholar] [CrossRef]
  50. Malarz, J.; Michalska, K.; Galanty, A.; Kiss, A.K.; Stojakowska, A. Constituents of Pulicaria Inuloides and Cytotoxic Activities of Two Methoxylated Flavonols. Molecules 2023, 28, 480. [Google Scholar] [CrossRef] [PubMed]
  51. Kłeczek, N.; Malarz, J.; Gierlikowska, B.; Kiss, A.K.; Stojakowska, A. Constituents of Xerolekia speciosissima (L.) Anderb. (Inuleae), and Anti-Inflammatory Activity of 7,10-Diisobutyryloxy-8,9-Epoxythymyl Isobutyrate. Molecules 2020, 25, 4913. [Google Scholar] [CrossRef] [PubMed]
  52. Ivanova, V.; Trendafilova, A.; Todorova, M.; Danova, K.; Dimitrov, D. Phytochemical Profile of Inula Britannica from Bulgaria. Nat. Prod. Commun. 2017, 12, 153–154. [Google Scholar] [CrossRef]
  53. Wollenweber, E.; Christ, M.; Dunstan, R.H.; Roitman, J.N.; Stevens, J.F. Exudate Flavonoids in Some Gnaphalieae and Inuleae (Asteraceae). Z. Naturforsch. C. 2005, 60, 671–678. [Google Scholar] [CrossRef]
  54. Trendafilova, A.; Todorova, M.; Ivanova, V.; Aneva, I. Phenolic Constituents and Antioxidant Capacity of Inula Oculus-Christi from Bulgaria. Bulg. Chem. Commun. 2017, 49, 176–180. [Google Scholar]
  55. Ivanova, V.; Todorova, M.; Nedialkov, P.; Trendafilova, A. A New Flavonol Acylglucoside from Inula Aschersoniana Janka Var. Aschersoniana. Comptes Rendus L’Academie Bulg. des Sci. 2021, 74, 514–520. [Google Scholar] [CrossRef]
  56. Krasteva, I.; Bratkov, V.; Bucar, F.; Kunert, O.; Kollroser, M.; Kondeva-Burdina, M.; Ionkova, I. Flavoalkaloids and Flavonoids from Astragalus Monspessulanus. J. Nat. Prod. 2015, 78, 2565–2571. [Google Scholar] [CrossRef]
  57. Ilkei, V.; Hazai, L.; Antus, S.; Bölcskei, H. Flavonoid Alkaloids: Isolation, Bioactivity, and Synthesis. Stud. Nat. Prod. Chem. 2018, 56, 247–285. [Google Scholar] [CrossRef]
  58. Khadem, S.; Marles, R.J. Chromone and Flavonoid Alkaloids: Occurrence and Bioactivity. Molecules 2011, 17, 191–206. [Google Scholar] [CrossRef] [PubMed]
  59. Munteanu, I.G.; Apetrei, C. Analytical Methods Used in Determining Antioxidant Activity: A Review. Int. J. Mol. Sci. 2021, 22, 3380–3410. [Google Scholar] [CrossRef] [PubMed]
  60. Özcan, F.Ş.; Özcan, N.; Dikmen Meral, H.; Çetin, Ö.; Çelik, M.; Trendafilova, A. Extraction of Sesquiterpene Lactones from Inula Helenium Roots by High-Pressure Homogenization and Effects on Antimicrobial, Antioxidant, and Antiglycation Activities. Food Bioprocess Technol. 2024, 1–12. [Google Scholar] [CrossRef]
  61. Ceylan, R.; Zengin, G.; Mahomoodally, M.F.; Sinan, K.I.; Ak, G.; Jugreet, S.; Cakır, O.; Ouelbani, R.; Paksoy, M.Y.; Yılmaz, M.A. Enzyme Inhibition and Antioxidant Functionality of Eleven Inula Species Based on Chemical Components and Chemometric Insights. Biochem. Syst. Ecol. 2021, 95, 104225. [Google Scholar] [CrossRef]
  62. Mansur, J.S.; Breder, M.N.; Mnasur, M.C.; Azulay, R.D. Determinacao Do Fator de Protecao Solar Por Espectrofotometria. An. Bras. Dermatol 1986, 61, 121–124. [Google Scholar]
  63. Kurz, H.; Karygianni, L.; Argyropoulou, A.; Hellwig, E.; Skaltsounis, A.L.; Wittmer, A.; Vach, K.; Al-Ahmad, A. Antimicrobial Effects of Inula Viscosa Extract on the In Situ Initial Oral Biofilm. Nutrients 2021, 13, 4029. [Google Scholar] [CrossRef]
  64. Asraoui, F.; El Mansouri, F.; Cacciola, F.; Brigui, J.; Louajri, A.; Simonetti, G. Biofilm Inhibition of Inula viscosa (L.) Aiton and Globularia alypum L. Extracts Against Candida Infectious Pathogens and In Vivo Action on Galleria Mellonella Model. Adv. Biol. 2023, 7, 2300081. [Google Scholar] [CrossRef]
  65. Wojnicz, D.; Kucharska, A.Z.; Sokół-Łętowska, A.; Kicia, M.; Tichaczek-Goska, D. Medicinal Plants Extracts Affect Virulence Factors Expression and Biofilm Formation by the Uropathogenic Escherichia Coli. Urol. Res. 2012, 40, 683–697. [Google Scholar] [CrossRef]
  66. Paunova-Krasteva, T.; Hemdan, B.A.; Dimitrova, P.D.; Damyanova, T.; El-Feky, A.M.; Elbatanony, M.M.; Stoitsova, S.; El-Liethy, M.A.; El-Taweel, G.E.; El Nahrawy, A.M. Hybrid Chitosan/CaO-Based Nanocomposites Doped with Plant Extracts from Azadirachta Indica and Melia Azedarach: Evaluation of Antibacterial and Antibiofilm Activities. Bionanoscience 2023, 13, 88–102. [Google Scholar] [CrossRef]
  67. Chen, K.; Peng, C.; Chi, F.; Yu, C.; Yang, Q.; Li, Z. Antibacterial and Antibiofilm Activities of Chlorogenic Acid Against Yersinia Enterocolitica. Front. Microbiol. 2022, 13, 885092. [Google Scholar] [CrossRef] [PubMed]
  68. Sun, J.; Wang, D.; Sun, Z.; Liu, F.; Du, L.; Wang, D. The Combination of Ultrasound and Chlorogenic Acid to Inactivate Staphylococcus Aureus under Planktonic, Biofilm, and Food Systems. Ultrason. Sonochem. 2021, 80, 105801. [Google Scholar] [CrossRef]
  69. Wang, L.; Pan, X.; Jiang, L.; Chu, Y.; Gao, S.; Jiang, X.; Zhang, Y.; Chen, Y.; Luo, S.; Peng, C. The Biological Activity Mechanism of Chlorogenic Acid and Its Applications in Food Industry: A Review. Front. Nutr. 2022, 9, 943911. [Google Scholar] [CrossRef] [PubMed]
  70. Hemeg, H.A.; Moussa, I.M.; Ibrahim, S.; Dawoud, T.M.; Alhaji, J.H.; Mubarak, A.S.; Kabli, S.A.; Alsubki, R.A.; Tawfik, A.M.; Marouf, S.A. Antimicrobial Effect of Different Herbal Plant Extracts against Different Microbial Population. Saudi J. Biol. Sci. 2020, 27, 3221. [Google Scholar] [CrossRef]
  71. Lou, Z.; Wang, H.; Zhu, S.; Ma, C.; Wang, Z. Antibacterial Activity and Mechanism of Action of Chlorogenic Acid. J. Food Sci. 2011, 76, M398–M403. [Google Scholar] [CrossRef]
  72. Miao, M.; Xiang, L. Pharmacological Action and Potential Targets of Chlorogenic Acid. Adv. Pharmacol. 2020, 87, 71–88. [Google Scholar] [CrossRef]
  73. D’Abrosca, B.; Buommino, E.; D’Angelo, G.; Coretti, L.; Scognamiglio, M.; Severino, V.; Pacifico, S.; Donnarumma, G.; Fiorentino, A. Spectroscopic Identification and Anti-Biofilm Properties of Polar Metabolites from the Medicinal Plant Helichrysum Italicum against Pseudomonas Aeruginosa. Bioorg. Med. Chem. 2013, 21, 7038–7046. [Google Scholar] [CrossRef]
  74. Garayev, E.; Di Giorgio, C.; Herbette, G.; Mabrouki, F.; Chiffolleau, P.; Roux, D.; Sallanon, H.; Ollivier, E.; Elias, R.; Baghdikian, B. Bioassay-Guided Isolation and UHPLC-DAD-ESI-MS/MS Quantification of Potential Anti-Inflammatory Phenolic Compounds from Flowers of Inula Montana L. J. Ethnopharmacol. 2018, 226, 176–184. [Google Scholar] [CrossRef] [PubMed]
  75. Stefanakis, M.K.; Tsiftsoglou, O.S.; Mašković, P.Z.; Lazari, D.; Katerinopoulos, H.E. Chemical Constituents and Anticancer Activities of the Extracts from Phlomis × commixta Rech. f. (P. Cretica × P. Lanata). Int. J. Mol. Sci. 2024, 25, 816. [Google Scholar] [CrossRef] [PubMed]
  76. Ma, T.; Sun, Y.; Wang, L.; Wang, J.; Wu, B.; Yan, T.; Jia, Y. An Investigation of the Anti-Depressive Properties of Phenylpropanoids and Flavonoids in Hemerocallis Citrina Baroni. Molecules 2022, 27, 5809. [Google Scholar] [CrossRef] [PubMed]
  77. Hajiaghaee, R.; Monsef-Esfahani, H.R.; Khorramizadeh, M.R.; Saadat, F.; Shahverdi, A.R.; Attar, F. Inhibitory Effect of Aerial Parts of Scrophularia Striata on Matrix Metalloproteinases Expression. Phyther. Res. 2007, 21, 1127–1129. [Google Scholar] [CrossRef] [PubMed]
  78. Krzyzaniak, L.M.; Antonelli-Ushirobira, T.M.; Panizzon, G.; Sereia, A.L.; De Souza, J.R.P.; Zequi, J.A.C.; Novello, C.R.; Lopes, G.C.; De Medeiros, D.C.; Silva, D.B.; et al. Larvicidal Activity against Aedes Aegypti and Chemical Characterization of the Inflorescences of Tagetes Patula. Evid.-Based Complement. Altern. Med. 2017, 2017, 9602368. [Google Scholar] [CrossRef] [PubMed]
  79. Kim, S.B.; Hwang, S.H.; Suh, H.W.; Lim, S.S. Phytochemical Analysis of Agrimonia Pilosa Ledeb, Its Antioxidant Activity and Aldose Reductase Inhibitory Potential. Int. J. Mol. Sci. 2017, 18, 379. [Google Scholar] [CrossRef] [PubMed]
  80. Bojilov, D.; Manolov, S.; Ahmed, S.; Dagnon, S.; Ivanov, I.; Marc, G.; Oniga, S.; Oniga, O.; Nedialkov, P.; Mollova, S. HPLC Analysis and In Vitro and In Silico Evaluation of the Biological Activity of Polyphenolic Components Separated with Solvents of Various Polarities from Helichrysum Italicum. Molecules 2023, 28, 6198. [Google Scholar] [CrossRef] [PubMed]
  81. Yoo, K.M.; Lee, C.H.; Lee, H.; Moon, B.K.; Lee, C.Y. Relative Antioxidant and Cytoprotective Activities of Common Herbs. Food Chem. 2008, 106, 929–936. [Google Scholar] [CrossRef]
  82. Jia, Z.; Tang, M.; Wu, J. The Determination of Flavonoid Contents in Mulberry and Their Scavenging Effects on Superoxide Radicals. Food Chem. 1999, 64, 555–559. [Google Scholar] [CrossRef]
  83. Thaipong, K.; Boonprakob, U.; Crosby, K.; Cisneros-Zevallos, L.; Hawkins Byrne, D. Comparison of ABTS, DPPH, FRAP, and ORAC Assays for Estimating Antioxidant Activity from Guava Fruit Extracts. J. Food Compos. Anal. 2006, 19, 669–675. [Google Scholar] [CrossRef]
  84. Trendafilova, A.; Ivanova, V.; Trusheva, B.; Kamenova-Nacheva, M.; Tabakov, S.; Simova, S. Chemical Composition and Antioxidant Capacity of the Fruits of European Plum Cultivar “Čačanska Lepotica” Influenced by Different Rootstocks. Foods 2022, 11, 2844. [Google Scholar] [CrossRef] [PubMed]
  85. De Soyza, A.; Hall, A.J.; Mahenthiralingam, E.; Drevinek, P.; Kaca, W.; Drulis-Kawa, Z.; Stoitsova, S.R.; Toth, V.; Coenye, T.; Zlosnik, J.E.A.; et al. Developing an International Pseudomonas Aeruginosa Reference Panel. Microbiologyopen 2013, 2, 1010–1023. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Full-scan LC-MS chromatograms of Inula salicina: (A). TIC in positive mode; (B). Base peak in negative mode.
Figure 1. Full-scan LC-MS chromatograms of Inula salicina: (A). TIC in positive mode; (B). Base peak in negative mode.
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Figure 2. Structure (A) and key HMBC and 1H-1H COSY correlations (B) of N-(8-methylnepetin)-3-hydroxypiperidin-2-one (28).
Figure 2. Structure (A) and key HMBC and 1H-1H COSY correlations (B) of N-(8-methylnepetin)-3-hydroxypiperidin-2-one (28).
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Figure 3. HPLC chromatogram of I. salicina extract and standard mixture at 320 nm, 5-CQA–chlorogenic acid, DCQA–dicaffeoylquinic acid.
Figure 3. HPLC chromatogram of I. salicina extract and standard mixture at 320 nm, 5-CQA–chlorogenic acid, DCQA–dicaffeoylquinic acid.
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Figure 4. Sun protection factor (SPF) and UV-B absorption (line in orange) of I. salicina extract, chlorogenic and caffeic acids and rutin. Subscripts of different letters symbolize a statistically significant difference between the samples (p < 0.05).
Figure 4. Sun protection factor (SPF) and UV-B absorption (line in orange) of I. salicina extract, chlorogenic and caffeic acids and rutin. Subscripts of different letters symbolize a statistically significant difference between the samples (p < 0.05).
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Figure 5. Evaluation of the viability of Gram-positive and Gram-negative bacterial cells within the biofilms. The first column represents the fluorescence microscopy images of E. coli, S. aureus and P. aeruginosa biofilms in the control group. The second column demonstrates the anti-biofilm efficacy of the applied metabolic product of Inula salicina-IS2. Bars = 50 μm.
Figure 5. Evaluation of the viability of Gram-positive and Gram-negative bacterial cells within the biofilms. The first column represents the fluorescence microscopy images of E. coli, S. aureus and P. aeruginosa biofilms in the control group. The second column demonstrates the anti-biofilm efficacy of the applied metabolic product of Inula salicina-IS2. Bars = 50 μm.
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Figure 6. Evaluation of bacterial cell morphology within biofilms by scanning electron microscopy. (A) Control group; (B) P. aeruginosa biofilms incubated with methanol extract from I. salicina. Bars = 2 µm. White triangle point to radial indentations, white arrow point to invaginations, white star to disrupted cell wall.
Figure 6. Evaluation of bacterial cell morphology within biofilms by scanning electron microscopy. (A) Control group; (B) P. aeruginosa biofilms incubated with methanol extract from I. salicina. Bars = 2 µm. White triangle point to radial indentations, white arrow point to invaginations, white star to disrupted cell wall.
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Table 1. Identification of compounds by UHPLC-MS/MS in Inula salicina methanol extract.
Table 1. Identification of compounds by UHPLC-MS/MS in Inula salicina methanol extract.
NoRt (min)Compound NameFormula[M-H]−, m/zΔ, ppmMS/MS FragmentsIdentification *
10.75Quinic acidC7H11O6191.05541.64191, 173, 163, 145, 129, 115, 101MS
23.27Protocatechuic acidC7H5O4153.0183−3.49153, 109MS
34.14Neochlorogenic acid (3-O-caffeoylquinic acid)C16H17O9353.0878−0.10353, 191, 179, 135MS, St
45.75O-Caffeoyl hexoseC15H17O9341.08790.16341, 179, 135MS
56.073-O-p-Coumaroylquinic acidC16H17O8337.0929−0.08337, 191, 163MS
66.85O-Caffeoyl hexose isomerC15H17O9341.0877−0.29341, 281, 251, 221, 179, 161, 135MS
76.97Ferulic acidC10H9O4193.0499−3.26193, 178, 149, 134MS
87.47Chlorogenic acid (5-O-caffeoylquinic acid)C16H17O9353.08810.76353, 191, 179, 135MS, St, NMR
97.89Caffeic acidC9H7O4179.0342−1.08179, 135MS, St
109.41Chlorogenic acid isomerC16H17O9353.0877−0.45353, 191, 179, 161MS
119.95Chlorogenic acid isomerC16H17O9353.0876−0.62353, 191, 179, 161, 135MS
1210.154-O-p-Coumaroylquinic acid C16H17O8337.09341.55337, 191, 173, 163MS
1310.655-O-p-Coumaroylquinic acid C16H17O8337.09321.01337, 191, 173, 163MS
1411.76p-Coumaric acidC9H7O3163.0388−4.58163, 135, 119MS
1512.44p-Coumaroylquinic acid isomerC16H17O8337.09331.28337, 191, 163MS
1612.555-O-Feruloylquinic acidC17H19O9367.1033−0.58367, 191, 173MS
1717.81Rutin (quercetin 3-O-rutinoside)C27H29O16609.14650.41609, 301MS, St, NMR
1818.39Isoquercitrin (quercetin 3-O-glucoside)C21H19O12463.08820.06463, 301, 300, MS, St, NMR
1918.55Luteolin 7-O-glucosideC21H19O11447.09330.09447, 285, 284MS, St, NMR
2018.87Patulitrin (patuletin 7-O-glucoside)C22H21O13493.09920.91493, 331, 330, 316MS, NMR
2119.35Caffeoyl-(salicyl)-hexosideC22H21O11461.1089−0.04461, 323, 221, 179, 161, 137MS
2219.63Nepitrin (nepetin 7-O-glucoside)C22H21O12477.1036−0.51477, 315MS, NMR
2319.663,4-Di-O-caffeoylquinic acidC25H23O12515.1191−0.81515, 353, 335, 191, 179, 173MS, St
2420.12Isorhamnetin hexosideC22H21O12477.1038−0.12477, 315, 299MS
2520.563,5-Di-O-caffeoylquinic acidC25H23O12515.1191−0.81515, 353, 191, 179MS, St, NMR
2620.671,5-Di-O-caffeoylquinic acidC25H23O12515.1191−0.81515, 353, 191, 179MS, St, NMR
2720.87Tricaffeoylhexaric acidC33H27O17695.12611.84695, 533, 371, 209, 191MS
2820.94N-(8-methylnepetin)-3-hydroxypiperidin-2-oneC22H20O9N442.11501.37442, 327, 312, 284, 256MS, NMR
2921.28Kaempferol 3-O-glucoside (astragallin)C21H19O11447.09370.97447, 285, 284, 255MS, NMR
3022.164,5-Di-O-caffeoylquinic acidC25H23O12515.1190−1.05515, 353, 191, 179, 173MS, St
3122.193-O-p-Coumaroyl-4-O-caffeoylquinic acidC25H23O11499.12500.77499, 353, 337, 335, 319, 173, 163MS
3222.20Tricaffeoylhexaric acid isomerC33H27O17695.12611.84695, 533, 371, 209, 191MS
3322.363-O-Caffeoyl-4-O-p-coumaroylquinic acidC25H23O11499.12490.53499, 353, 337, 335, 319, 173, 163MS
3422.39Tricaffeoylhexaric acid isomerC33H27O17695.12611.84695, 533, 371, 209, 191MS
3522.53Tricaffeoylhexaric acid isomerC33H27O17695.12611.84695, 533, 371, 209, 191MS
3622.573-O-p-Coumaroyl-5-O-caffeoylquinic acidC25H23O11499.12521.26499, 353, 337, 191, 163MS
3722.673-O-Caffeoyl-5-O-p-coumaroylquinic acidC25H23O11499.12511.08499, 353, 337, 191, 179, 163MS
3822.79Caffeoylferuloyl quinic acidC26H25O12529.13530.20529, 367, 161MS
3923.03Caffeoylferuloyl quinic acid isomerC26H25O12529.13571.01529, 367, 353, 191MS
4023.094-O-p-Coumaroyl-5-O-caffeoylquinic acidC25H23O11499.12500.89499, 337, 191, 173, 163MS
4123.204-O-Caffeoyl-5-O-p-coumaroylquinic acidC25H23O11499.12500.83499, 353, 337, 191, 179, 173MS
4223.34Caffeoylferuloyl quinic acid isomerC26H25O12529.13520.08529, 367, 179, 161MS
4323.523,5-di-O-p-Coumaroylquinic acidC25H23O10483.12980.19483, 337, 319, 191, 163MS
4423.66Tetracaffeoylhexaric acidC42H33O20857.15751.11857, 695, 533,371, 209, 191MS
4523.69Caffeoylferuloyl quinic acid isomerC26H25O12529.13530.20529, 367, 179, 161MS
4623.762-Methylbutanoyl/isovaleryl dicaffeoylhexaric acidC29H29O15617.1505−1.1617, 455, 293, 191, 179MS
4723.81QuercetinC15H9O7301.0353−0.22301, 179, 151MS, St
4823.85LuteolinC15H9O6285.0404−0.20285MS, St, NMR
4923.894,5-di-O-p-Coumaroylquinic acidC25H23O10483.12980.19483, 337, 191, 173, 163MS
5023.913,4,5-Tricaffeoylquinic acidC34H29O15677.15161.34677, 515, 353, 335, 191, 179, 173, 161MS
5123.95Patuletin (6-methoxyquercetin)C16H11O8331.0459−0.22331, 316, 287, 271MS, NMR
5224.18Nepetin (6-methoxyluteolin)C16H11O7315.05110.29315, 301, 300MS, NMR
5324.32Isobutanoyl tricaffeoylhexaric acidC37H33O18765.16791.59765, 603, 441, 279, 261, 191MS
5424.852-Methylbutanoyl/isovaleryl tricaffeoylhexaric acid isomerC38H35O18779.18300.80779, 617, 455, 293, 275, 191MS
5525.12ChrysoeriolC16H11O6299.05630.75299, 284MS
5625.31JaceosidinC17H13O7329.0667−0.08329, 314, 299MS
5725.49Quercetagetin trimethyl etherC18H15O8359.07730.25359, 344, 329, 301MS
5825.99ApigeninC15H9O5269.04570.63269MS, St, NMR
* MS—The compounds were tentatively identified; St—the identity of the compounds was confirmed by injecting authentic samples; NMR—the compounds were isolated and their identity was confirmed by NMR experiments.
Table 2. 1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of N-(8-methylnepetin)-3-hydroxypiperidin-2-one (28) recorded in CD3OD (δ in ppm, multiplicity, J in Hz).
Table 2. 1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of N-(8-methylnepetin)-3-hydroxypiperidin-2-one (28) recorded in CD3OD (δ in ppm, multiplicity, J in Hz).
№δHδC *№δHδC *
2 164.53′ 145.8
36.59 (s)101.94′ 150.3
4 182.55′6.94 (d, 8.4)115.6
5 168.56′7.45 (dd, 2.2, 8.4)119.0
6 132.5OMe3.89 (s)59.6
7 160.52” 172.4
8 97.33”4.07 (dd, 5.5, 9.3)69.3
9 152.54″2.18 (m)/2.48 (m)28.8
10 103.15″2.09 (m)/1.95 (m)23.0
1′ 122.16″3.57 (m)/3.36 (m)53.4
2′7.62 (d, 2.2)113.57″4.58 (d, 13.3)/4.72 (d, 13.3)47.4
* Deduced from HSQC and HMBC experiments.
Table 3. Content of total phenolics (TPC), total flavonoids (TFC) and individual compounds, and antioxidant potential (DPPH, ABTS and FRAP) of I. salicina.
Table 3. Content of total phenolics (TPC), total flavonoids (TFC) and individual compounds, and antioxidant potential (DPPH, ABTS and FRAP) of I. salicina.
TPC aTFC b5-CQA c3,4-DCQA c3,5-DCQA c1,5-DCQA c4,5-DCQA cDPPH dABTS dFRAP e
Mean215.5787.44103.395.38±30.6218.5823.330.7410.7115.77
SD2.520.521.300.260.790.690.500.0060.0070.08
a expressed as mg GAE/g DE; b expressed as mg CE/g DE; c expressed as mg/g DE; d expressed as mg TE/g DE; e expressed as mg/g DE µM Fe2+/g DE; Mean and SD from 3 measurements.
Table 4. The gradient of the chromatographic separation.
Table 4. The gradient of the chromatographic separation.
Time (min)Solvent A * (%)Solvent B * (%)
0→1955
1→2095→825→18
20→2482→6018→40
24→2760→3040→70
27→2930→570→95
29→31595
* A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile.
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Ivanova, V.; Nedialkov, P.; Dimitrova, P.; Paunova-Krasteva, T.; Trendafilova, A. Inula salicina L.: Insights into Its Polyphenolic Constituents and Biological Activity. Pharmaceuticals 2024, 17, 844. https://doi.org/10.3390/ph17070844

AMA Style

Ivanova V, Nedialkov P, Dimitrova P, Paunova-Krasteva T, Trendafilova A. Inula salicina L.: Insights into Its Polyphenolic Constituents and Biological Activity. Pharmaceuticals. 2024; 17(7):844. https://doi.org/10.3390/ph17070844

Chicago/Turabian Style

Ivanova, Viktoria, Paraskev Nedialkov, Petya Dimitrova, Tsvetelina Paunova-Krasteva, and Antoaneta Trendafilova. 2024. "Inula salicina L.: Insights into Its Polyphenolic Constituents and Biological Activity" Pharmaceuticals 17, no. 7: 844. https://doi.org/10.3390/ph17070844

APA Style

Ivanova, V., Nedialkov, P., Dimitrova, P., Paunova-Krasteva, T., & Trendafilova, A. (2024). Inula salicina L.: Insights into Its Polyphenolic Constituents and Biological Activity. Pharmaceuticals, 17(7), 844. https://doi.org/10.3390/ph17070844

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