Phytochemical Profiles and Antimicrobial Activity of Selected Populus spp. Bud Extracts

Buds of poplar trees (Populus species) are often covered with sticky, usually polyphenol-rich, exudates. Moreover, accessible data showed that some Populus bud extracts may be excellent antibacterial agents, especially against Gram-positive bacteria. Due to the fragmentary nature of the data found, we conducted a systematic screening study. The antimicrobial activity of two extract types (semi-polar—ethanolic and polar—ethanolic-water (50/50; V/V)) from 27 bud samples of different poplar taxons were compared. Antimicrobial assays were performed against Gram-positive (five strains) and Gram-negative (six strains) bacteria as well as fungi (three strains) and covered the determination of minimal inhibitory, bactericidal, and fungicidal concentrations. The composition of extracts was later investigated by ultra-high-performance liquid chromatography coupled with ultraviolet detection (UHPLC-DAD) and with electrospray-quadrupole-time-of-flight tandem mass spectrometry (UHPLC-ESI-qTOF-MS). As a result, most of the extracts exhibited good (MIC ≤ 62.5 µg/mL) or moderate (62.5 < MIC ≤ 500 µg/mL) activity against Gram-positives and Helicobacter pylori, as well as fungi. The most active were ethanolic extracts from P. trichocarpa, P. trichocarpa clone ‘Robusta’, and P. tacamahaca × P. trichocarpa. The strongest activity was observed for P. tacamahaca × P. trichocarpa. Antibacterial activity was supposedly connected with the abundant presence of flavonoids (pinobanksin, pinobanksin 3-acetate, chrysin, pinocembrin, galangin, isosakuranetin dihydrochalcone, pinocembrin dihydrochalcone, and 2′,6′-dihydroxy-4′-methoxydihydrochalcone), hydroxycinnamic acids monoesters (p-methoxycinnamic acid cinnamyl ester, caffeic acid phenethylate and different isomers of prenyl esters), and some minor components (balsacones).


Introduction
Poplars are high trees that belong to the genus Populus L. of Salicaceae Mirb.family due to the traditional organism systematics of Carolus Linnaeus [1,2], while the (Salix + Populus) clade is also classified into a higher clade of ((Goupiaceae + Violaceae) (Passifloraceae (Lacistemataceae + Salicaceae))) by Angiosperm Phylogeny Group [3].Different authors distinguished 6-7 sections of the Populus genus, but this classification is evolving [2].The deeper systematics of the genus Populus is complex and still discussed due to many factors.
It is claimed that Populus specimens are difficult to identify accurately.Moreover, most poplars are known for their ease of spreading and crossbreeding [1,2,4].A typical example is American P. balsamifera L., which quickly spread in Europe and produced intersectional hybrids with Europe-native P. nigra L. (black poplar) [4].As a result, pure specimens of black poplars are relatively rare today [1,4].Another issue is differences in statements of species classifications.According to World Flora Online [5], traditionally distinguished species like P. maximowiczii Henry, P. suaveolens Fisch.ex Loudon, and P. koreana Rehder are nowadays recognized as synonyms of P. suaveolens Fisch.A similar situation occurs for P. balsamifera and P. trichocarpa [2].In summary, different descriptions of Populus species exist in the literature.For this reason, the system of Populus species classification adopted for this work should be defined.In the present manuscript, a traditional division of species described by Bugała in his monograph elaboration and further modified by Korbik [1,2] was used.This decision was made due to the widespread use of traditional names in the literature, even in 2023.
Apart from systematics, the Salicaceae family is known for famous medicinal plants, especially willows (Salix genus).Willow bark contains biologically active compounds, especially salicylate-like phenolic glycosides [6].This group of metabolites includes glycosides and glycoside-esters (e.g., salicin and salicortin), aromatic acids (cinnamic and hydroxycinnamic acids), and others.It is well known that salicylates exhibit anti-inflammatory activity [6].Raw herbal materials from Salix species, especially bark, rich in salicylate-like phenolic glycosides, are still used in cold and mild rheumatic diseases.Moreover, the bark of willow species has its monograph (Salicis cortex) in many official pharmacopeias [7,8].
In the case of the poplars (Populus genus), their organs (bark [9], leaves [10], buds [11,12]) also contain anti-inflammatory salicylate-like phenolic glycosides.For this reason, poplar's organs are also used in folk medicine to treat gout [13].However, unlike willows, poplars are not widely included in pharmacopeias.Leaves of poplars (Populi folium) are included in the national part of Polish Pharmacopeia [14].In case of buds, some species such as P. nigra L., P. balsamifera L., P. canadensis Marsh., P. laurifolia Ledeb., and P. suaveolens Fisch.are plant sources of Populi gemmae in the Russian Pharmacopeia [15].However, the Populi gemmae monograph is absent in European Pharmacopoeia 11 and USP-NF 2023.
Apart from salicylate-like phenolic glycosides, buds of many Populus species are covered by sticky, resinous exudates [1,16], an additional source of biologically active components.The amounts, seasons, and periods of resin production depend on species and environmental factors.Buds of some species (e.g., P. tremula L.) are only resinous for a short period before cracking in spring, while others are sticky almost all year [1].The composition of Populus resins is very complex but specific for species.For these reasons, a comparative analysis of bud exudates composition may be helpful for chemotaxonomic purposes [16].At this point, the main components of Populus resinous exudates were defined as phenols, volatile and non-volatile terpenes and terpenoids, and other substances [16,17].
Biologically, resins form a protective layer on buds, making them less sensitive to wetness, cold, and attacks of pathogenic microorganisms and parasites and less attractive for herbivores.On the one hand, poplar resins often contain relatively high concentrations of polar free phenolic acids; however, more apolar components such as flavonoid aglycones and phenolic acids esters are also detected [16,17].Surprisingly, one of the rarest substances in resins are glycosides, e.g., salicylate-like glycosides.Literature data have reported their presence in extracts from whole buds [11,12,18] but did not focus unambiguously on resins.Thus, one can guess that salicylates may be components of buds' interior green tissues, not bud exudates.
Resins of the Populus genus are plant precursors of a bee product known as propolis (or bee glue).It is well-known for multiple medicinal activities such as wound treatment, anti-inflammatory, antioxidant, and antimicrobial.These properties were also reported for poplar bud' extracts [11,[19][20][21].Researchers reported differences between propolis and poplar buds; however, it is impossible to claim if propolis or poplar buds have more potent medicinal properties.Moreover, Apis mellifera L. bees do not use resins from all available Populus species to produce propolis.It was observed that their preferences for local species may be so strong that foreign poplar trees are ignored.Sometimes, non-poplar species (e.g., birch) are preferred over foreign Populus specimens [22].The factors impacting bees' decisions remain unknown.It is suspected that components of some Populus species resins may be toxic or repellent to bees.That is why some poplars may contain highly active components not observed in propolis research.Moreover, literature data on Populus buds are limited compared to propolis or poplar leaves research.So far, phytochemical analyses for species other than P. nigra, P. balsamifera, and P. tremula have focused mainly on GC-MS profiling [23][24][25][26][27][28][29][30][31][32], while LC-MS and LC-DAD investigations are more limited [16,18,28,29].Moreover, the chemical composition of buds is not yet defined for every Populus species.
Our manuscript compares phytochemistry with antimicrobial properties of ethanol and ethanol/water (50/50; V/V) extracts of poplar buds.Populus species were selected due to the high production of resins (the viscosity of buds before cracking was evaluated in preliminary research) and the expected activity.Section 3 (Materials and Methods) contains a complete list of investigated poplar species.Instrumental analysis was performed using LC-UV-ESI-QTOF-MS/MS due to the expected high amounts of polyphenols and only a few similar works.Solvents used in extraction were chosen due to desired components and previous optimization.Ethanol dissolves buds' resins and their less polar components, such as flavonoid aglycones and hydroxycinnamic acids esters.More polar constituents (e.g., salicylate-like glycosides) from buds' green tissues were extracted by ethanol with water (50:50, V/V).Antimicrobial activity screening against bacterial (Gram-positive and Gramnegative) and fungal strains was based on previous experience and the expected activity of propolis [33], as well as results for P. nigra and P. tremula buds [20].To our knowledge, LC-MS-UV-ESI-qTOF-MS/MS analysis and antimicrobial screening were performed for the first time for poplar bud extracts of most Populus species, excluding P. nigra, P. balsamifera, and P. tremula.Moreover, the activity of all extracts against Helicobacter pylori was also tested for the first time.

Results and Discussion
Poplar buds, their resins, and propolis are similar, but they are not the same type of plant material and thus should not be replaced by each other.The Populus buds' composition and activity data are relatively sparse, particularly regarding propolis.Therefore, the extensive comparative studies of the phytochemistry and biological activity of poplar buds conducted in this study constitute a valuable contribution to this field.

LC-UV-ESI-qTOF-MS/MS Profile of Extracts
Complete results of LC-UV-ESI-qTOF-MS/MS are presented in Table 1 (identification of components in Populus bud extracts by LC-UV-ESI-qTOF-MS/MS) and in the supplement (Table S1.Relative abundance of extracts components and buds' extraction yield).A selection of chromatograms is given in Figure 1 (LC-MS chromatograms of Populus bud EtOH extracts represent five different chemical groups).The identification of components was based on retention times of chromatographic peaks and UV spectra, and calculated formulas of deprotonated molecular ions as well as MS/MS fragmentation spectra.Due to the different confidence levels, the obtained information was divided into four groups-A, B, C, and D (see Section 3 and Table 1 for details).Confidence levels A and B mean reliable identification, while levels C and D are tentative.More than 300 unique components were observed in UV and MS chromatograms.Because most of them remained at trace level (MS or UV peak), Table 1 and Supplementary Table S1 were limited to 223 components.Among them, 163 substances were identified (confidence levels A and B) or tentatively identified (confidence levels C and D).The substitution positions of glycerol by hydroxycinnamic acids were proposed by comparison with previous research [33] and literature [17,19,23].So far, it has been proved that more symmetric hydroxycinnamic acid glycerides dominate over non-symmetric glycerides in GC-MS research [17,19,23].For example, 2-acetyl-1,3-di-p-coumaroyl glycerol had a higher concentration than 3-acetyl-1,2-di-p-coumaroyl glycerol in P. tremula buds [23] and further in propolis [17,19]).Differences in concentration and ionization were used to identify isomers of caffeic acid p-coumaric acids methylbutenyl ester [33].
Regarding the components' relative abundance, poplar bud extracts were mainly rich in flavonoids, hydroxycinnamic acid monoesters, and hydroxycinnamic glycerides.Most observed substances easily produced ions in negative mode; therefore, their relative abundance was calculated from mass chromatograms.In contrast, tectochrysin, pinostrobin, and ferulic acid benzyl ester did not produce ions, or the signals were weak under standard conditions.For those substances, the relative abundance was obtained by comparison of UV max chromatograms in 280 nm.
Chromatographic profiles of ethanolic (EtOH) and water/ethanol (50/50; V/V) (W/E) extracts exhibited qualitative and quantitative differences.EtOH extracts were more abundant in a number of components than W/E, but some substances were present only in W/E.Most observed compounds remained at an ion trace level, and only unidentified component 2 (RT 0.87 min, [M−H] − at 195.0515 m/z) exhibited low and average relative abundance.Moreover, the most common components of EtOH and W/E extracts exhibited higher relative abundance in EtOH.Rarely were W/E extracts more abundant in common substances.For example, pinobanksin was more abundant in W/E of P.LA, P.M×P.B, and P.N3, while in EtOH for the rest of the samples.In summary, ethanol turned out to be a more appropriate solvent than water/ethanol (50/50; V/V) for batch extraction and further chemometric analysis of poplar buds regarding the number of extracted compounds and their relative abundance.
The relative abundance of most components was obtained due to deprotonated pseudomolecular ion intensity in a single chromatographic peak (see details in Section 3).Only pinostrobin and tectochrysin relative amounts were based on UV peak intensity due to their weak ionization in negative mode.
Populus bud extracts were divided into phytochemical groups.The division was based on the presence of dominant components.Domination was determined based on deprotonated pseudomolecular ion intensity (see Supplementary Table S1).Because chromatographic analyses of EtOH extracts had stronger and more numerous peaks of components, they were selected to direct the division.As a result, extracts were aggregated into several subtypes: (1) flavonoid, (2) flavonoid + hydroxycinnamic acid monoesters, (3) hydroxycinnamic acid monoesters, (4) hydroxycinnamic acid glycerides, and (5) mixed ones.
The next group gathered two extracts with the domination of hydroxycinnamic acid monoesters.Both of them originated from P. nigra (P.N.1 and P.N.2).The main hydroxycinnamic acid monoesters in this group were derivatives of caffeic acid (2-methyl-2-butenyl, 3-methyl-2-butenyl, 3-methyl-3-butenyl ester, benzyl, phenethyl) and metoxycinnamic acid cinnamyl esters.Both samples also contained average signals of flavonoids (mainly pinocembrin and pinostrobin chalcone).It is essential to point out that these samples did not contain pinobanksin 3-acetate (the main flavonoid of other investigated samples) and had only traces of non-esterified pinobanksin when another sample (also classified as P. nigra) contained these flavanols at high concentrations.In the literature, both flavonoids are frequently reported in P. nigra buds [17].P.N.1 and P.N.2 were introduced to Arboretum from an old tree stand (Dęblin, on-Vistula river, Poland) and were previously evaluated as genetically pure P. nigra.As already mentioned in the Introduction, P. nigra easily crosses with available poplar species [1,4].Therefore, we hypothesize pinobanksin and pinobanksin 3-acetate presence in the so-called P. nigra bud sample is a result of P. nigra hybridization, unnoticeable by morphological examination methods.Another possibility is that the P.N.3 sample originates from a specific phenotype (chemotype) of P. nigra.
The last group was a (five) mixed type, including five EtOH extracts (P.M×P.TRI.-P.maximowiczii × P. trichocarpa; P.RO-P.trichocarpa clone 'Rochester'; P.TA×P.TRI.1,P.TA×P.TRI.2-P.tacamahaca × P. trichocarpa, sample 1 and 2; P.TRI-P.trichocarpa).It is worth pointing out that all samples in this group were P. trichocarpa and its crossbreed specimens with P. maximowiczii and P. tacamahaca.The lack of parent species-P.MAX, P.TA.1, and P.TA.2 in the (five) mixed group-led to a hypothesis that the impact of P. trichocarpa on secondary metabolites production in its hybrids is more substantial than the impact of other parent species.P.M×P.TRI, P.RO, P.TRI, and P.TA×P.TRI.2 revealed average and strong signals of p-coumaric acids, 2 ′ ,6 ′ -dihydroxy-4 ′ -methoxy dihydrochalcone and p-coumaric acid cinnamyl ester.Moreover, EtOH extracts also exhibited the presence of substances tentatively identified as balsacones (dihydrochalcones with additional phenylpropyl units).These components were previously isolated from P. balsamifera [36] but were absent in P.BA.EtOH extract.Readers need to remember that P. trichocarpa is sometimes classified as a subspecies of P. balsamifera [2].The rest of the components in the mixed type were more varied.P.RO had strong signals of pinocembrin, pinocembrin dihydrochalcone, and pinobanksin 3-acetate, while the rest of the five samples had no more eye-catching components.
In summary, batch negative mode LC-MS analysis of poplar bud extracts revealed the dominance of polyphenols' peaks with patterns specific enough to distinguish six groups.This method can be considered a promising strategy for poplar bud fingerprinting.Moreover, all EtOH extracts had stronger signals of polyphenols (from one to three relative levels of difference between considerable components; see Supplementary Table S1) than W/E, which can be practical information for further studies.
It is worth adding that buds of P.BA, P.KOM, P.MAX, P.M × P.TRI, P.N.3, P × PE.2, P.RO, P × RA, P.RO, P.TA.1, P.TA × P.TRI.1,P.TA × P.TRI.2, and P.TRI were relatively big (up to 4 cm), contained a lot of resins (organoleptic tests), and provided high extraction yield (>39% per dry mass of buds for EtOH extracts; see Supplementary Table S1.Finally, their extracts revealed strong signals of polyphenols.Therefore, they may be utilized as a source of specific components or extracts rich in polyphenols.

Antimicrobial Properties of Extracts
The antimicrobial properties of extracts are presented in Table 2 (Comparison of antimicrobial effect of ethanol and water/ethanol extracts of Populus buds).Performed research included determination of MIC (minimal inhibitory concentration) and MBC or MFC (minimal bactericidal/fungicidal concentration) as well as MBC/MIC or MFC/MIC ratio.Both extract types (EtOH and W/E) revealed relatively higher activity against Grampositive bacteria and fungi than against most Gram-negative ones.Only Helicobacter pylori violated this rule; therefore, it was described separately below.Similar profiles of activity were already reported for poplar propolis [19,20,33] as well as buds of P. nigra [19,20], P. balsamifera [11,37], P. tremula [19,20], and P. tremuloides [35].
Low activity against Gram-negative species may be a general rule for Populus bud extracts.It may result from components non-specifically eliminated via efflux pumps in many Gram-negative species [38], as well as differences in the structure of Gram-positive and Gram-negative cell barriers [39].The tested Gram-positive bacteria strains included Staphylococcus aureus, S. epidermidis, Micrococcus luteus, Bacillus subtilis, B. cereus, and Enterococcus faecalis.Comparison of MIC with MBC in pairs of EtOH and W/E extracts exhibited that the MBC/MIC ratio was usually equal to or lower than four for most strains and samples.These results showed that EtOH and W/E exhibit rather bactericidal than bacteriostatic effects.The bactericidal effect may result from a multifactorial mechanism of action.Antibacterial agents of poplar buds' resins, propolis, and flavonoids are known for disrupting cytoplasmic membrane function, inhibiting nucleic acid synthesis, and inhibiting the energy metabolism of bacterial strains [40].Similar effects on bacterial cell membranes were also caused by caffeic acid monoesters, especially CAPE (caffeic acid phenethyl ester), which is the most investigated [41].Potentially, ingredients that disrupt cell barrier function and stability may facilitate the penetration of active ingredients across the cell barrier.Active components may cause damage to bacterial cells in different ways, e.g., by increasing oxidative stress [42].Finally, a bacterial cell cannot be repaired and undergoes lysis.
There were noted differences between the activity of EtOH and W/E extracts in most cases.Moreover, EtOH extracts usually showed a stronger antibacterial activity than W/E.Among all extracts, the weakest activity was exhibited by extracts belonging to the hydroxycinnamic acid glycerides group (P.LAS, P.WIL, and P. × WCA; MICs from 250 to ≥1000 µg/mL against all Gram-positive strains).The remaining extracts exhibited varied MICs (from 31.3 to ≥1000 µg/mL) against different strains.In the case of propolis, previous research showed that the presence of hydroxycinnamic acid glycerides may be connected with weaker antimicrobial activity for 70% aqueous ethanol extracts [20,43].On the other hand, Isidorov et al. [19] showed that ethyl acetate extracts of P. tremula buds, rich in hydroxycinnamic acid glycerides, had better MIC against Gram-positive bacteria (Staphylococcus schleiferi, S. aureus, B. cereus, and B. thuringiensis).In another research, better activity of P. tremula methanolic extracts was exhibited against S. aureus and B. cereus [21].It seems important to point out that both reports revealed only a twofold difference between the MIC of P. tremula and P. nigra.In the case of disc-diffusion studies of P. tremula and P. nigra buds' antibacterial activity, it was shown that the more potent activity of P. tremula extracts was not a rule, and sometimes P. nigra was a better antibacterial agent [20].In summary, the impact of hydroxycinnamic acids glycerides on whole extracts' activity against Gram-positive bacteria is somewhat complex and probably depends on the presence and concentration of other components and their interactions.
Among all poplar bud extracts, the most potent antibacterial agent against Grampositive bacteria was EtOH extract from P.TA×P.TRI.2 (MIC = 7.8 µg/mL vs. S. epidermidis and M. luteus, 15.6 µg/mL vs. E. faecalis and 31.3 µg/mL S. aureus, B. cereus and B. subtillis).Among others, only the EtOH extract of P.TA×P.TRI.2 exhibited stronger activity against B. cereus (MIC = 15.6 µg/mL).Other strong antibacterial agents included EtOH extracts of P.M × P.TRI, P.TA × P.TRI.1,P.TRI, and P.RO, as well as the W/E extract of P.TA × P.TRI.2, possessing significant activity against all tested Gram-positive strains (MIC ≤ 62.5 µg/mL).The potent extracts contained p-coumaric acid (P.TA × P.TRI.2,P.TRI, P.RO), pinocembrin (P.RO), isosakuranetin dihydrochalcone (P.TA × P.TRI.2),pinocembrin dihydrochalcone (P.TA × P.TRI.2), 2 ′ ,6 ′ -dihydroxy-4 ′ -methoxydihydrochalcone (P.TA × P.TRI.1,P.TA × P.TRI.2,P.TRI, P.RO), p-coumaric acid cinnamyl ester (P.TA × P.TRI.2,P.TRI, P.RO), and different components, tentatively identified as balsacones (P.TA × P.TRI.1,P.TA × P.TRI.2,P.TRI, P.RO).So far, it has been reported that balsacones exhibit activity against S. aureus strains [44].Therefore, the presence of balsacones was the main difference between the mixed group and the rest of the phytochemical groups; these components may play an important role in the antibacterial effect.The structures of balsacones and other compounds hypothetically responsible for high anti-Gram-positive bacteria activity are presented in Figure 2. In summary, Populus buds classified in a mixed group exhibited the most potent activity against Gram-positive bacterial strains (see Table S1).Moreover, all of them were clones of P. trichocarpa and its crossbreed species.mixed group and the rest of the phytochemical groups; these components may play an important role in the antibacterial effect.The structures of balsacones and other compounds hypothetically responsible for high anti-Gram-positive bacteria activity are presented in Figure 2. In summary, Populus buds classified in a mixed group exhibited the most potent activity against Gram-positive bacterial strains (see Table S1).Moreover, all of them were clones of P. trichocarpa and its crossbreed species.Research on propolis showed that significant amounts of p-coumaric acid are connected with lower antimicrobial activity [20,45].p-Coumaric acid alone showed antimicrobial activity but was an inferior antimicrobial agent to propolis flavonoids [46].Moreover, the high amounts of p-coumaric acid were correlated with a low abundance of flavonoids.As a result, it was suggested that the weaker activity of propolis with a high concentration of p-coumaric acid was caused by the lower amounts of flavonoids [20].In the literature, antibacterial effects against Gram-positive bacteria of propolis extracts were Research on propolis showed that significant amounts of p-coumaric acid are connected with lower antimicrobial activity [20,45].p-Coumaric acid alone showed antimicrobial activity but was an inferior antimicrobial agent to propolis flavonoids [46].Moreover, the high amounts of p-coumaric acid were correlated with a low abundance of flavonoids.As a result, it was suggested that the weaker activity of propolis with a high concentration of p-coumaric acid was caused by the lower amounts of flavonoids [20].In the literature, antibacterial effects against Gram-positive bacteria of propolis extracts were usually connected to the presence of some caffeic acid esters, such as CAPE [45], and flavonoids (pinobanksin 5-methyl ether, pinobanksin, chrysin, galangin, and pinobanksin 3-acetate) [43].Most of these components were present in P.TA × P.TRI.2 and the other most potent antibacterial agents (P.M × P.TRI, P.TA × P.TRI.1,P.TRI, P.RO).However, their signals were weaker than in samples with lower activity (flavonoid, hydroxycinnamic monoesters, and flavonoid + hydroxycinnamic monoester types).For this reason, it may be suspected that these components do not play a decisive role in the antibacterial effect of Populus bud extracts.In our opinion, the final activity against Gram-positive strains results from different interactions between specific components.MICs against Candida strains were generally higher than against Gram-positives.Moreover, most samples had a fungicidal rather than a fungistatic effect.In the literature, P. nigra bud extracts exhibited MICs = from 62.5 µg/mL (ethyl acetate extract) [19] to 1000 µg/mL (methanol extract) against C. albicans, while P. tremula buds were inactive (ethyl acetate extract) [19] or exhibited mild activity (methanol extract) (MIC = 500-1000 µg/mL) [19].Research on propolis from these two species (P.tremula and P. nigra) demonstrated similar results [43].A comparison of the experimental data with the literature allows us to suspect that apolar extracts of Populus buds should exhibit better activity against fungi polar extracts.However, data on the antifungal activity of propolis and Populus buds are not as widely available as for antibacterial activity.For these reasons and promising activity, further research is required (especially regarding the mechanism of action).

Activity against Helicobacter pylori
Most of the EtOH extracts exhibited good activity (MIC ≤ 62.5 µg/mL) against H. pylori; only the activities of EtOH extracts from P.LAS, P.WIL, and P. × WCA were moderate (MIC from 250 to >1000 µg/mL).This suggests that a higher concentration of apolar flavonoids and phenolic acid monoesters increases the anti-Helicobacter activity of extracts.For propolis, it was proven that multiple polyphenolic substances can be connected with notable activity against H. pylori [46].As listed previously, they are pinobanksin, pinobanksin 5-methyl ether, pinobanksin 3-acetate, chrysin, pinocembrin, and galangin, as well as p-methoxycinnamic acid cinnamyl ester [46].Except for pinobanksin 5-methyl ether, these components were abundant in active extracts of Populus buds, and their presence correlated with anti-Helicobacter activity.Antibacterial agents of poplar buds may attack the cell barrier, disrupt metabolism, inhibit energy production, and cause oxidative stress in bacterial cells.The anti-Helicobacter effect of flavonoids was documented [47].Krzy żek et al. [48] proved that myricetin slows the process of transformation into coccoid forms, reduces biofilm formation of H. pylori, and exhibits additive effects with clarithromycin and metronidazole.Other anti-Helicobacter properties were recorded by González et al. [49].In their research, flavonoids such as chrysin inhibited the function of HsrA (one of the transcriptional regulators essential for cell viability) [49].Moreover, it was proven that flavonoid-rich propolis extracts [46] and single flavonoids isolated from propolis [49] inhibit the urease of H. pylori.Urease increases the low pH of gastric juice, which allows the survival of H. pylori.This effect may be potentially used in anti-Helicobacter therapies.From the clinical point of view, it is also important that Korean propolis exhibits an anti-inflammatory effect on gastric mucous membranes (infected gastric mucosal injury mice model) [50].In summary, all earlier propolis research suggests that poplar bud extracts may be used in anti-Helicobacter therapy in the future.Herewith, we report the observations in this field systematically.

Preparation of Populus Bud Extracts
The extraction process was based on our previous research on propolis and poplar buds [16,20].Ground plant material was extracted with ethanol (96%, V/V) or with 50/50 ethanol in water (V/V) at the ratio of 1:10 (1.0 g of buds per 10.0 mL of solution).The extraction yield was provided in Supplementary Table S1.Extraction was performed in an ultrasonic bath (Sonorex, Bandelin, Berlin, Germany).Extraction conditions were set to 20 • C (initial temperature) for 15 min and 756 W (90% of ultrasonic bath power).The process was repeated thrice (total extraction time was 45 min).The temperature during all the process did not exceed 45 • C. Obtained extracts were stored at room temperature for 12 h for stabilization purposes (precipitation of potentially co-extracted wax).Next, extracts were filtered through the Whatman No. 10 paper (Cytiva, Marlborough, MA, USA), and ethanol was evaporated under reduced pressure.Next, extracts were frozen and lyophilized in Alpha 2-4 LD Plus lyophilizer (Christ, Osterode am Harz, Germany).Extraction yield was evaluated as a gram of lyophilized extract per gram of dried buds (see Supplementary Table S1).

UHPLC-DAD-MS/MS Profiling of Populus Bud Extracts
UHPLC analyses were performed as previously described [33] with a Thermo Scientific UltiMate 3000 system (Thermo Scientific™ Dionex™, Sunnyvale, CA, USA), coupled with an autosampler and DAD detector recording spectral data in the 200-600 nm range and monitoring at 280, 320, and 360 nm.UHPLC-MS/MS was carried out using a Compact ESI-qTOF MS/MS detector (Bruker Daltonics, Bremen, Germany).MS detector was used in electrospray negative mode.Conditions of analysis were ion source temperature was set to 210 • C, nebulizer gas pressure to 2.0 bar, and dry gas (nitrogen) flow to 8.01 L/min.The capillary voltage was 4.5 kV.The collision energy was set to 8.0 eV.Internal calibration was obtained run by run with a 10 mM sodium formate solution.For ESI-MS/MS experiments, collision energy was set at 35.0 eV, and nitrogen was used as collision gas.The scan range was set between 30 and 1300 m/z.
Identification of components was based on several parameters, such as retention times of chromatographic peaks and UV spectra, calculated formulas of deprotonated molecular ions, and MS/MS fragmentation spectra of deprotonated molecular ions.These values were compared with previous research (the same LC-ESI-UV-qTOF-MS/MS methods were used) [33], standards, and literature.The standards were used directly in current investigations (see list in Section 3.1) or in our previous research on propolis, a poplar resin mixed with beeswax [33].For this reason, propolis may be partially used as a plant reference standard for poplar bud extracts.Literature about propolis LC-MS research is significantly more abundant than about poplar buds.For this reason, it is a valuable resource for comparisons.
Due to information collected from the literature, four levels of identification confidence were obtained: A (comparison of UV and MS/MS spectra with standards; the highest level of confidence), B (comparison of MS/MS and/or UV spectrum with literature; good level of confidence), C (component was identified according to deprotonated molecular ion formula and prediction from MS/MS spectra detected in Populus genus in literature, but there are no sufficient MS and UV data; average/weak level of confidence), and D (component was identified according to deprotonated molecular ion and prediction from MS spectra, but there are no sufficient MS/MS and UV data and substances were not reported in Populus genus literature; the weakest level of confidence).In the case of high-resolution mass spectrometry and calculations of the formulas, those with errors higher than 5 ppm were disqualified.

Determination of Antimicrobial Activity
The propolis extracts dissolved in dimethylsulfoxide (DMSO) were screened for antibacterial and antifungal activities by microdilution broth method according to both the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (www.eucast.orgaccessed on 3 January 2023) using Mueller-Hinton broth or RPMI with MOPS for growth of fungi, as we described elsewhere [51].Minimal inhibitory concentrations (MICs) of the tested extracts were evaluated for the wide panel of the reference microorganisms, including Gram-negative bacteria (Salmonella Typhimurium ATCC 14028, Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 9027 and Helicobacter pylori), Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 10240, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 10876, and Enterococcus.faecalis ATCC 29212), and fungi (Candida glabrata ATCC 90030, Candida albicans ATCC 102231, Candida parapsilosis ATCC 22019).The sterile 96-well polystyrene microtitration plates (Nunc, Roskilde, Denmark) were prepared by dispensing 100 µL of appropriate dilution of the tested extracts in broth medium per well by serial twofold dilutions to obtain final concentrations of the tested extracts ranging from 1000 to 1.95 mg/L The inocula were prepared with fresh microbial cultures in sterile 0.85% NaCl to match the turbidity of 0.5 McFarland standard and were added to wells to obtain final density of 5 × 10 5 CFU/mL for bacteria and 5 × 10 4 CFU/mL for yeasts (CFU, colony forming units).After incubation (35 • C for 24 h), the MICs were assessed visually as the lowest concentration of the extracts that shows complete growth inhibition of the reference microbial strains.Appropriate DMSO control (at a final concentration of 10%), a strain growth control (inoculum without the tested extracts), and medium sterility control (the tested extracts without inoculum) were included on each microplate.The MIC for H. pylori ATCC 43504 was determined using a twofold microdilution method in MH broth with 7% of lysed horse blood at extract concentration ranging from 1000 to 1.95 mg/L with bacterial inocula of 3 McFarland standard.After incubation at 35 • C for 72 h under microaerophilic conditions (5% O 2 , 15% CO 2 , and 80% N 2 ), the growth of H. pylori was visualized with the addition of 10 µL of 0.04% resazurin to each well.The MIC endpoint was recorded after 4 h incubation as the lowest concentration of extract that completely inhibits growth [52].
Abbreviations: A(%)-relative abundance; RT-retention time; A -identification by comparison of UV and MS/MS spectra with standards (the highest level of confidence); B -identification by comparison of MS/MS and/or UV spectrum with literature (good level of confidence); C -component was identified according to deprotonated molecular ion formula and prediction from MS/MS spectra detected in Populus genus in literature, but there are no sufficient MS and/or UV data (average/weak level of confidence); D -component was identified according to deprotonated molecular ion and prediction from MS spectra, but there are no sufficient MS/MS, and UV data and components have not been reported in poplars in literature (the weakest level of confidence); [M + H]!-components does not produce ions in ESI-negative mode; therefore, positive fragmentation was presented; ND/--UV maximum was not determined due to low concentration, overlapping peaks or lack of UV absorption by components; *-UV maximum was evaluated approximately due to low concentration or overlapping peaks.

Table 2 .
Comparison of antimicrobial effect (MIC and MBC (µg/mL)) of ethanolic and ethanolic-water extracts of Populus buds.