Characterization of Secondary Metabolites of Leaf Buds from Some Species and Hybrids of Populus by Gas Chromatography Coupled with Mass Detection and Two-Dimensional High-Performance Thin-Layer Chromatography Methods with Assessment of Their Antioxidant Activity

Poplars provide medicinal raw plant materials used in pharmacy. Leaf buds are one of the herbal medicinal products collected from poplars, having anti-inflammatory and antiseptic properties, but there are no quality standards for their production and there is a need to determine their botanical sources. Therefore, the chemical compositions of the leaf buds from four species and varieties of poplars, Populus balsamifera, P. × berolinensis, P. × canadensis ‘Marilandica’, and P. wilsonii were investigated and compared using gas chromatography coupled with mass detection (GC-MS) and two-dimensional high-performance thin-layer chromatography (2D-HPTLC) in order to search for taxa characterized by a high content of biologically active compounds and with a diverse chemical composition that determines their therapeutic effects. The presence of 163 compounds belonging to the groups of flavonoids, phenolic acids derivatives, glycerides, and sesquiterpenes was revealed. Moreover, the conditions for the separation and identification of biologically active compounds occurring in analyzed leaf buds using 2D-HPTLC were optimized and used for metabolomic profiling of the studied poplars, enabling their fast and simple botanical identification. The total phenolic (TPC) and flavonoid (TFC) contents of examined extracts were determined and their antioxidant capacities were estimated by spectrophotometric DPPH, ABTS, and FRAP assays. Based on the analysis of phytochemicals and antioxidant activity, P. × berolinensis buds were selected as the raw plant material for medicinal purposes with the highest content of active compounds and the strongest antioxidant activity.


Introduction
Poplars are very common trees around the world belonging to the Salicaeae family.They grow in natural habitats but many of them are cultivated as a landscaping element or for commercial purposes [1].The species of the Populus genus are characterized by fast growth and usually have low soil requirements, so they are an economically efficient source of renewable energy and biomass for the paper industry [2].Because of this, in some countries (Germany, England, Italy, USA) they are cultivated as a short-rotation crop [1,3].Poplars are valuable medicinal plants used in traditional and conventional systems for the treatment of diverse diseases [4].Poplar buds (Populi gemmae) are one of three medicinal raw plant materials from the Populus genus characterized by anti-inflammatory and antiseptic properties, and they could be obtained as a by-product from the production of biomass for bioenergy purposes.Poplar ointment (Unguentum Populeon) was used in folk medicine to relieve skin inflammation, burns, and wounds, as well as muscle and rheumatic pain [5].

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Currently, Gilead balm is available on the UK pharmacy market, containing a poplar bud preparation intended for both oral and topical use.Internally, it is used as an antibacterial and expectorant in chronic inflammation of the upper respiratory tract, externally for superficial skin injuries, and as a disinfectant in laryngitis [6].However, the problem is determining the botanical source/origin of the raw plant material for its production.Some of the literature data indicate that balsam of Gilead is a tincture obtained from the buds of Populus candicans Ait.[7], but according to other sources, it contains preparations of leaves and flower buds of P. nigra L. or other species, including P. candicans Ait. and P. balsamifera L. [6].
Populi gemmae, due to its rich chemical composition, containing flavonoids, phenolic acids and their derivatives, and essential oil, is a valuable raw plant material with traditional use as an antiseptic and anti-inflammatory remedy [4].The anti-inflammatory and antioxidant properties of the buds of some poplar species have been confirmed in a number of in vitro tests.The antioxidant activity could be involved in the anti-inflammatory mechanism of extracts obtained from various raw plant materials.
In vitro studies on murine macrophages of the RAW 264.7 cell line showed a significant anti-inflammatory effect of the ethanol extract from P. × canadensis buds (10 and 20 µg/mL) stimulated with LPS (200 ng/mL) in combination with IFN-γ (10 ng/mL) and in vivo studies (25 and 100 mg/kg) on LPS-induced endotoxemia (1 mg/kg for 3 h) and acute lung injury in mice [8].The examined extract was standardized for the content of flavonoids (126.23 mg QE/g) and total phenolic compounds (145.54 mg GAE/g).HPLC analysis showed that flavonoids were dominated by chrysin, pinocembrin, and galangin (2.83-4.17g/100 g of extract), whereas in the group of phenolic acids, often found in poplar buds, caffeic and p-coumaric acids were absent.Both studies demonstrated that P. × canadensis poplar bud extract has anti-inflammatory effects, inhibiting the secretion of specific inflammatory cytokines IL-6, IL-10, MCP-1, and TNF-α, and blocking the activation of nuclear factor (NF)-κB.Moreover, lung histopathology showed that pretreatment with poplar bud extract at a higher dose (100 mg/kg) inhibited LPS-induced changes in murine lung tissues [8].
Our previous in vitro studies on human gingival fibroblasts (HGF-1), pro-inflammatory stimulated by silver nanoparticles (3.5 µg/mL), showed the anti-inflammatory properties of extracts from P. × berolinensis (7.5 µg/mL) and P. nigra (15 µg/mL) buds by decreasing the IL-6 and IL-1β release in HGF-1 cells and the downregulation of mRNA for both cytokines [9].The inhibition of pro-inflammatory cytokines was also demonstrated for two flavanones present in these extracts, inocembrin and pinostrobin (20 and 40 µM, respectively), in a dose-dependent manner.Moreover, the expression of COX-2 protein by pinocembrin and P. × berolinensis bud extract was demonstrated, which was distinguished from other extracts by its high flavanones content.These effects were not observed for buds from P. lasiocarpa, which did not contain flavonoids [9].
In vitro studies using LPS (100 ng/mL)-activated human leukemic monocytes THP-1 showed anti-inflammatory potential for the hydroalcoholic extracts from the buds of P. nigra (1.56 µg/mL), P. deltoides (3.12 µg/mL), and P. trichocarpa (6.25 µg/mL) [10].The analyzed extracts differed in total phenolic content (P.nigra 134.02 mg GAE/g, P. trichocarpa 60.07 mg GAE/g, and P. deltoides 58.19 mg GAE/g), as well as in the presence of individual compounds identified by the HPLC-DAD-MS method, belonging to flavonoids and derivatives of phenolic acids, mainly caffeic and p-coumaric acids.Each of the tested poplar bud extracts inhibited the production of pro-inflammatory cytokines, such as tumor necrosis factor-a/TNF-a, interleukin-6/IL-6, and IL-1β, as well as the extracellular release of the high-mobility group box 1 (HMGB1), a "damage-associated molecular pattern" (DAMP) molecule, that is involved in the pathogenesis of various inflammatory diseases.However, in both antioxidant capacity assays used, ABTS and DPPH, the highest activity was demonstrated by the extract from P. nigra buds, characterized by a total phenolic content twice as high as the others and the presence of caffeic acid derivatives, e.g., caffeic acid phenylethyl ether (CAPE), pinocembrin, and pinobanksin butyric and acetic esters [10].
The free radical scavenging ability of dry aqueous extract from P. nigra buds was proven by oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assay on normal human dermal fibroblasts (NHDF) [11].HPLC-DAD analysis of the tested extract showed the presence of salicin, caffeic and p-coumaric acids, and pinobanksin as the dominant compounds and the total phenolic content was 180 mg caffeic acid equivalent/g.Studies using the NHDF cell line demonstrated the transcriptional effect of poplar bud extract which modulated the expression of the antioxidant enzyme catalase gene, chemokine (C-C motif) ligand 5 (CCL5) gene, Krupper-like factor 10 (KLF10), transcription factor E2F4, and EGF response factor 1 (ZFP36L1) genes involved in antioxidant defense, inflammatory response, and cell renewal [11].
Many other data have demonstrated the antioxidant effects of P. nigra, P. balsamifera, and P. × canadensis buds in various tests [8,[12][13][14][15] and the dependence of their intensity on chemical composition; the presence of flavonoids and phenolic acids was especially observed.A number of studies have shown significant variability in the chemical composition of buds from different poplar species and varieties [16][17][18][19][20]; there is, therefore, a need to define/indicate Populus taxa that may be a source of medicinal raw material, valuable in terms of anti-inflammatory/antioxidant activity, and to develop methods for their standardization.Although poplar buds have monographs in the German Commission E and the British Herbal Compendium, they do not contain quality standards for this herbal product that correlate with its anti-inflammatory/antioxidant activity.
There are a lot of research data on the chemical composition of poplar buds [7,17,19,21,22], but many species and varieties remain unexplored, so there is a need to determine their composition using modern analytical methods.GC-MS is most often used in the analysis of the chemical composition of poplar buds because it allows one separation to identify most of the compounds from various chemical groups-polyphenols, tri-and sesquiterpenes, aliphatic acids, alcohols, etc.-present in these plant matrices [16,17,19,22,23].
The aim of the research was to recognize the chemical composition of the buds of poplar trees often grown in Poland and determine their antioxidant capacity in order to select taxa with the richest chemical compositions and highest antioxidant activities as sources of raw material for medicinal purposes.
Therefore, our research was focused on the analysis of the chemical composition of leaf buds collected from four species and varieties of poplars cultivated in Poland, namely Populus balsamifera, P. × berolinensis, P. × canadensis 'Marilandica', and P. wilsonii, using gas chromatography coupled with mass detection (GC-MS).Moreover, the conditions for the analysis of biologically active compounds from the poplar buds were optimized using two-dimensional high-performance thin-layer chromatography (2D-HPTLC), which is relatively fast and cheap compared to GC-MS, enabling the identification of their botanical origin by comparing the obtained chromatographic metabolomic profiles.

GC-MS Analysis
The chemical compositions of the leaf buds of four species and varieties of poplars occurring in Poland were examined using the GC-MS method, namely Populus balsamifera, P. × berolinensis, P. × canadensis 'Marilandica', and P. wilsonii.
GC-MS studies enabled the identification of a total of one hundred and sixty-three compounds occurring in the analyzed bud extracts, primarily from the group of flavonoids, phenolic acids and their derivatives, as well as glycerides and terpenes.There were significant differences in the chemical compositions of the tested leaf buds of different species and varieties of the Populus genus (Tables 1 and S1).The leaf buds of P. balsamifera, P. × berolinensis, and P. × canadensis 'Marilandica' were characterized by a high content of flavonoid compounds (41.28, 34.44, and 79.51% TIC (total ion current) respectively) and phenylpropanoids (30.85, 16.82, and 13.43% TIC, respectively), in contrast to P. wilsonii buds (1.38 and 3.57% TIC, respectively), which were distinguished by the presence of phenylpropanoid glycerides as the dominant ones (74.94% TIC).Moreover, the buds of P. × berolinensis, unlike the other analyzed taxa, contained more sesquiterpenes (23.68%TIC) (Figure 1, Table 1).The leaf buds of P. balsamifera, P. × berolinensis, and P. × canadensis 'Marilandica' were characterized by a high content of flavonoid compounds (41.28, 34.44, and 79.51% TIC (total ion current) respectively) and phenylpropanoids (30.85, 16.82, and 13.43% TIC, respectively), in contrast to P. wilsonii buds (1.38 and 3.57% TIC, respectively), which were distinguished by the presence of phenylpropanoid glycerides as the dominant ones (74.94% TIC).Moreover, the buds of P. × berolinensis, unlike the other analyzed taxa, contained more sesquiterpenes (23.68%TIC) (Figure 1, Table 1).A total of forty flavonoids were identified, of which pinocembrin from the flavanone group, galangin from the flavonols, and pinobanksin and its acetate from the flavanonols were present in all tested extracts.Flavonoids were the dominant group of compounds in P. × canadensis 'Marilandica' (79.51% TIC).This variety was characterized by the most diverse set of flavonoids (29 compounds identified), with the predominance of galangin (18.59%TIC), chrysin (12.38%TIC), pinobanksin (12.55%TIC) and its acetate (11.78%TIC), and pinocembrin (7.86% TIC) (Table 1).
Thirty-one compounds were identified in the group of phenolic acids and their derivatives.All tested poplar extracts contained caffeic, p-coumaric, and benzoic acid (Table 1).P. balsamifera buds were characterized by a high content of p-coumaric acid (15.36%TIC), whereas the remaining analyzed poplar taxa were characterized by a high content of caffeic acid (from 2.49 to 4.92% TIC).Moreover, P. × berolinensis extracts contained a significant content of 3,4-dimethoxycinnamic acid (3.65% TIC) and isoferulic acid (1.95% TIC) (Table 1).
A relatively high concentration of triterpenoids (11.76%TIC) was found in the analyzed extracts of P. wilsonii, in which the highest concentration was dipterocarpol (4.32% TIC), whereas the concentrations of lanosterol (0.53% TIC), αand β-amyrin (0.22 and 0.10% TIC), and triterpenol (0.08% TIC) were low.Moreover, it is worth noting that the presence of palmitic and α-linolenic acid was found in all tested poplar buds.

2D-HPTLC Analysis
The developed 2D-HPTLC separation conditions included the use of high-performance silica gel as an adsorbent and a mixture of chloroform:methanol:formic acid (95:2:3, v/v/v) as the mobile phase for the first direction (1D) and a mixture of n-hexane:acetate ethyl:formic acid (60:40:1.3,v/v/v) in the second (2D) (Figure 2E).

2D-HPTLC Analysis
The developed 2D-HPTLC separation conditions included the use of high-performance silica gel as an adsorbent and a mixture of chloroform:methanol:formic acid (95:2:3, v/v/v) as the mobile phase for the first direction (1D) and a mixture of n-hexane:acetate ethyl:formic acid (60:40:1.3,v/v/v) in the second (2D) (Figure 2E).HPTLC plates were derivatized with a 2% methanol solution of AlCl3 or 0.5% solution of anisaldehyde in a 5% methanol solution of sulfuric acid (95 °C, 3 min) and analyzed under UV light at λ-366 nm or under visible light (λ-500 nm).Table 2 shows the retention parameters for the reference substances used and the colors of their zones obtained after derivatization with different spraying reagents.The developed 2D-HPTLC method enables the identification of caffeic and p-coumaric acid in all tested extracts (Figures 3-7).Moreover, from the group of phenolic acid derivatives, caffeic acid phenethyl ester (CAPE) was detected in the P. berolinensis bud extract (Figure 5G).Among the flavonoids, in the buds of P. balsamifera, P. × berolinensis, and P. × canadensis 'Marilandica', the presence of galangin, pinocembrin, and pinostrobin was confirmed.Pinobanksin and chrysin were also identified in the latter HPTLC plates were derivatized with a 2% methanol solution of AlCl 3 or 0.5% solution of anisaldehyde in a 5% methanol solution of sulfuric acid (95 • C, 3 min) and analyzed under UV light at λ-366 nm or under visible light (λ-500 nm).Table 2 shows the retention parameters for the reference substances used and the colors of their zones obtained after derivatization with different spraying reagents.The developed 2D-HPTLC method enables the identification of caffeic and p-coumaric acid in all tested extracts (Figures 3-7).Moreover, from the group of phenolic acid derivatives, caffeic acid phenethyl ester (CAPE) was detected in the P. berolinensis bud extract (Figure 5G).Among the flavonoids, in the buds of P. balsamifera, P. × berolinensis, and P. × canadensis 'Marilandica', the presence of galangin, pinocembrin, and pinostrobin was confirmed.Pinobanksin and chrysin were also identified in the latter two taxa, whereas kaempferol was detected in P. balsamifera and P. × canadensis 'Marilandica'.
two taxa, whereas kaempferol was detected in P. balsamifera and P. × canadensis 'Marilandica'.The developed 2D-HPTLC method was used to assess the antioxidant activity of the compounds from the tested poplar buds by bioautography using ABTS and DPPH solutions (Figures 4E,J, 5E,J, 6E,J and 7E,J).It was found that the compounds showing antioxidant activity in the obtained bioautograms were primarily flavonoids and phenolic acids and their derivatives.The buds of P. balsamifera, P. × berolinensis, and P. × canadensis  The developed 2D-HPTLC method was used to assess the antioxidant activity of the compounds from the tested poplar buds by bioautography using ABTS and DPPH solutions (Figures 4E,J, 5E,J, 6E,J and 7E,J).It was found that the compounds showing antioxidant activity in the obtained bioautograms were primarily flavonoids and phenolic acids and their derivatives.The buds of P. balsamifera, P. × berolinensis, and P. × canadensis 'Marilandica' contained numerous antioxidant substances, in contrast to the buds of P. wilsonii.

Antioxidant Capacity
The antioxidant capacity of the analyzed poplar bud extracts was estimated using DPPH, ABTS, and FRAP spectrophotometric tests and was expressed in mM Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid-TEA) per 1 g of dry weight (d.w.) of the tested plant material (Table 3).The highest capacity in the DPPH and FRAP tests was determined for the methanol extract from P. × berolinensis-7.5728and 24.675 mM TEA/g, respectively.Only in the ABTS study, the P. balsamifera extract (12.1 mM TEA/g) showed a slightly greater ability to scavenge free radicals.In turn, the lowest values of antioxidant potential in the DPPH and ABTS tests were recorded for the P. wilsonii bud extract-2.4102and 3.652 mM TEA/g, respectively.

Total Flavonoid (TFC) and Total Phenolic (TPC) Content
The total flavonoid content (TFC) was determined using a 1% solution of aluminum chloride (AlCl3) as a color reagent and expressed in rutin equivalents (RE).A very wide The developed 2D-HPTLC method was used to assess the antioxidant activity of the compounds from the tested poplar buds by bioautography using ABTS and DPPH solutions (Figure 4E,J, Figure 5E,J, Figure 6E,J and Figure 7E,J).It was found that the compounds showing antioxidant activity in the obtained bioautograms were primarily flavonoids and phenolic acids and their derivatives.The buds of P. balsamifera, P. × berolinensis, and P. × canadensis 'Marilandica' contained numerous antioxidant substances, in contrast to the buds of P. wilsonii.

Antioxidant Capacity
The antioxidant capacity of the analyzed poplar bud extracts was estimated using DPPH, ABTS, and FRAP spectrophotometric tests and was expressed in mM Trolox (6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid-TEA) per 1 g of dry weight (d.w.) of the tested plant material (Table 3).The highest capacity in the DPPH and FRAP tests was determined for the methanol extract from P. × berolinensis-7.5728and 24.675 mM TEA/g, respectively.Only in the ABTS study, the P. balsamifera extract (12.1 mM TEA/g) showed a slightly greater ability to scavenge free radicals.In turn, the lowest values of antioxidant potential in the DPPH and ABTS tests were recorded for the P. wilsonii bud extract-2.4102and 3.652 mM TEA/g, respectively.

Total Flavonoid (TFC) and Total Phenolic (TPC) Content
The total flavonoid content (TFC) was determined using a 1% solution of aluminum chloride (AlCl 3 ) as a color reagent and expressed in rutin equivalents (RE).A very wide range of TFC was found in the tested buds of four poplar taxa, ranging from 7.92 mg/g in Populus wilsonii to 65.01 mg/g in P. × berolinensis (Table 3).In the buds of the remaining studied poplars, P. balsamifera and P. × canadensis 'Marilandica', relatively high contents of flavonoids were revealed-44.76and 55.17 mg/g, respectively.No statistically significant differences were found between the TFC of P. × berolinensis and P. × canadensis 'Marilandica' and between the TFC of P. balsamifera and P. × canadensis 'Marilandica' (Table 3).
The total phenolic content (TPC) was determined using the Folin-Ciocalteu reagent and expressed in gallic acid equivalents (GAE).The highest concentration of phenols was found in the buds of P. × berolinensis (183.18mg/g) and P. balsamifera (166.17mg/g) (statistically insignificant difference p < 0.05).The lowest TPC was detected in the buds of P. wilsonii (64.96 mg/g) (statistically significant differences compared to TFC in the other tested poplar buds, p < 0.05) (Table 3).

GC-MS Analysis
The results of phytochemical tests conducted in this study confirmed previous literature data on significant differences in the chemical composition of buds of various Populus species and their hybrids (Figure 1, Table 1 and Table S1).So far, detailed phytochemical studies have included poplar buds originating mainly from species and varieties found in North America and Asia [18,21,22,24].Our research focused on identifying the chemical composition of buds from poplars grown in Europe.They confirm the need to select poplar buds in order to obtain medicinal raw plant materials with predictable and repeatable therapeutic effects, including anti-inflammatory ones.
Based on the data obtained from performed chromatographic analyses, the chemical composition of P. wilsonii buds was recognized for the first time [25], revealing the presence of phenylpropanoid glycerides as the dominant compounds (74.94% TIC), as well as several compounds from the triterpene group (11.76%TIC), phenolic acids (3.57% TIC), aliphatic acids (3.28% TIC), and flavonoids (1.38% TIC).Moreover, knowledge about the chemical composition of P. × berolinensis and P. × canadensis 'Marilandica' buds was significantly expanded.So far, only the presence of some flavonoid aglycones has been described in these raw plant materials based on TLC analyses [9,20,26].Furthermore, γand β-eudesmols, α-cadinol, α-calacorene, acorenol, α-muurolene, and ar-curcumene were identified as components of the essential oil from leaf buds of an undescribed variety of P. × canadensis [16].As a result of the research carried out, a number of compounds from the group of sesquiterpenes (23.68%TIC), cinnamic acids and their derivatives (16.82%TIC), triterpenes (6.66% TIC), and chalcones were identified in the buds of P. × berolinensis.In the buds of P. × canadensis 'Marilandica' phenylpropenoids (13.43% TIC), triterpenoids (1.71% TIC), aliphatic acids (1.29% TIC), and some new flavonoids and chalcones were detected, not previously described in this taxon.In turn, in the examined buds of P. balsamifera, the presence of several benzoic acid esters (3.04% TIC) and several new derivatives of pcoumaric acid (10.98%TIC) and caffeic acid, in addition to sesquicineol from sesquiterpene group, were found for the first time.The presence of flavonoids, chalcones, other phenylpropanoids, and sesquiterpenes previously described in this taxon was also confirmed (Table 1) [17,[27][28][29].
In most of the analyzed buds, the dominant group was flavonoids (34.44-79.51%TIC), including chalcones, with the exception of P. wilsonii buds, which were characterized by a low content of these compounds (1.38% TIC).Galangin, pinocembrin, chrysin, pinobanksin, and its acetate were identified in the flavonoid set of P. wilsonii buds.In addition to the 4-hydroxycinnamyl group of the A ring in the chemical structure of these compounds because compounds without this residue are inactive [29,38,39].
The confirmed high content of chalcones (14.5% TIC) in the flavonoid set from P. balsamifera buds (41.28%TIC) distinguishes this species from others and is of chemotaxonomic importance.
The GC-MS analyses showed the presence of caffeic, p-coumaric, and benzoic acids in all tested poplar buds.The buds of P. balsamifera were characterized by a high content of p-coumaric acid, whereas the remaining analyzed poplar taxa were characterized by a high content of caffeic acid and a significant content of 3,4-dimethoxycinnamic acid and isoferulic acid in P. × berolinensis (Table 1).
In vitro studies of the antioxidant properties of polyphenolic compounds identified in Populus nigra buds using the ORAC test showed higher activity of caffeic and p-coumaric acids compared to flavanone (pinocembrin) and flavanol aglycones (pinobanksin and its methyl derivative) and other tested phenolic acids (ferulic, isoferulic, and cinnamic).The observed differences in the activity of these compounds are related to the number and position of free hydroxyl groups in the aromatic ring.It was found that substituting one of the hydroxyl groups in the phenol ring reduces antioxidant activity, whereas the absence or blocking of all hydroxyl groups deprives this activity [11].
Phenolic acids, known for their antioxidant activity, are next to flavonoids, another group of secondary metabolites that may contribute to the anti-inflammatory effect of poplar bud extracts.
CAPE is the most frequently studied compound for the assessment of biological activity among those identified in the Populus genus.So far, this compound has been reported to have antioxidant, anti-inflammatory, immunomodulatory, antiviral, antiproliferative, anti-cancer, chemopreventive, and wound-healing properties [42].A number of studies have shown that the anti-inflammatory effect of CAPE is due to the reduction in ROS levels and the inhibition of the suppression of myeloperoxidase activity by NF-κB and the production of pro-inflammatory cytokines [42][43][44].Both in vitro and in vivo studies have shown that CAPE specifically targets genes involved in cell death, angiogenesis, cell cycle regulation, and metastasis.CAPE can reduce the proliferation of human cancer cells through the NF-κB-signaling pathway [43].
A similar composition in the buds of P. balsamifera, P. × berolinensis, and P. × canadensis 'Marilandica' of various groups of compounds with anti-inflammatory activity, demonstrated in the literature, makes these raw plant materials an interesting research model for comparing and assessing the anti-inflammatory effects of poplars in the context of disclosed quantitative differences in their chemical composition (Figure 1).The planned in vitro studies using cell lines will include the assessment of the impact of individual extracts on the activity of cyclooxygenase 1 and 2, and the gene expression of proteins involved in the inflammatory process, IL-1β, IL-6, IL-8, IL-10, TNF-α, etc.
One of the lesser-known groups of compounds found in leaf buds of the Populus genus is sesquiterpenes.So far, their occurrence has been demonstrated only in three poplar taxa, P. nigra, P. balsamifera, and P. × canadensis [16,23,28].Against the background of these rare literature data, the obtained results of GC-MS analyses expand the knowledge of the presence of sesquiterpenes in the buds of P. × berolinensis and P. wilsonii.The analyzed P. × berolinensis buds were characterized by a high concentration of sesquiterpenes (23.68%TIC) with the dominance of hinesol (9.0%TIC) and β-eudesmol (5.99% TIC).The remaining buds tested were characterized by a much lower content of these compounds.In this group of compounds, γ-curcumene dominated in the buds of P. balsamifera, whereas α-cadinol dominated in the plant material of P. × canadensis 'Marilandica'.On the other hand, in the examined P. wilsonii, sesquiterpenes occurred only in trace amounts (Table 1).
The leaf buds of P. × berolinensis are, compared to the other examined, an interesting plant material for assessing the impact of a high content of sesquiterpenes, in addition to a significant share of flavonoids and phenylpropanoids, on the biological activity of poplars.
The examined buds of P. wilsonii were distinguished by the presence of phenylpropenoid glycerides, among which 2-acetyl-1-p-coumaroyl-3-caffeoylglycerol (lasiocarpin B), 2-acetyl-1-caffeoyl-3-feruloylglycerol and 1, 3-di-p-coumaroyl-2-acetylglycerol (lasiocarpin A) were identified as the dominant ones (Table 1).Two of them, lasiocarpin A and B, were isolated from the buds of another representative of the Leucoides section of the Populus genus, namely P. lasiocarpa [45].This indicates that glycerol esters are compounds characteristic of the species of this section of poplar.Moreover, the presence of these compounds has so far been found in the buds of P. tremula from the Leuce section, P. szechuanica from Tacamahaca, and P. euphratica from Turanga [19,24,46,47].
The studied poplar buds constitute a valuable material for the isolation of a number of interesting chemical compounds from the group of phenylpropenoids and their glycerides, which are not commercially available.
The diversity of the chemical composition of Populus buds makes them an interesting plant material for testing the impact of the presence or absence of a given group of compounds on the biological properties and potential synergistic effects.Taking into account the traditional use of poplar buds and the demonstrated presence of compounds with antiinflammatory and antibacterial effects proven in the literature, the buds of P. balsamifera and P. × berolinensis should be selected from those examined as material for further in vivo studies to assess their effectiveness in the treatment of bacterial dermatitis.The detailed identification of the chemical composition of analyzed poplar buds is important for the interpretation of future planned research on their biological activity.

2D-HPTLC Analysis
As mentioned in the introduction, a basic and very important element of the standardization of a medicinal raw plant material (herbal medicine) is its correct botanical and phytochemical identification.For this purpose, in addition to morphological and anatomical analysis, chromatographic methods, including TLC, are used.Taking into account the complex chemical composition of poplar buds revealed and described above, comprising numerous compounds from the phenolic group (alcohols, phenolic acids, and flavonoids) and various substances such as terpenes, sesquiterpenes, aliphatic acids, and glycerol derivatives (Table 1), the technique 2D-TLC (two-dimensional thin-layer chromatography) was used.
The developed method enabled the separation of approximately 30-35 compounds, depending on the examined poplar variety, whereas in the 1D-TLC systems used, only 16 compounds were separated (unpublished data).Optimization experiments (Figure 2) were performed based on 2D-TLC separation conditions previously used in the quantitative analysis of flavonoids and phenolic acids in propolis samples [48] and the results of our previous work on TLC separation of flavonoid aglycones in extracts from poplar buds [20].Balsam poplar bud extract was used for this study, which, according to the results of GC-MS analyses, was characterized by a very rich and diverse chemical composition.
In the next stage, the suitability of various detection reagents indicated in the literature as characteristic for phenolic acids and flavonoids was tested [49][50][51].Chromatograms developed using the spraying reagents were analyzed at daylight and UV λ-366 nm.The most effective in the analysis of poplar bud extracts were 2% methanol solution of AlCl 3 and 0.5% solution of anisaldehyde in 5% methanol solution of sulfuric acid (Table 2).It should be noted that the use of a reagent-containing anisaldehyde requires activation of the HPTLC plate (washing with chloroform and then drying at 100 • C for 30 min) in order to remove factory impurities of the adsorbent.Otherwise, as a result of developing the chromatogram in a given mobile phase, they accumulate in its upper right corner and, as a result of the derivatization process, turn purple, covering the spots of compounds migrating in this part, including pinostrobin (Figures 2E and 3F).
Greater variation in the color of spots of individual compounds within groups was observed when using a detection reagent containing anisaldehyde than when using aluminum chloride.Additionally, the obtained chromatograms were observed in visible and ultraviolet light at λ-366 nm, which extended the possibilities of identifying a given compound due to its different color in visible light and fluorescence at λ-366 nm (Figure 3E,F, Table 2).In visible light, spots of phenolic acids, including cinnamic acid derivatives, appear as greyish-pink, among which p-coumaric acid stands out with an intense pink color.This is a very valuable property when identifying this compound in TLC chromatograms because it does not give selective color reactions with other reagents used.Most spots of flavonoid compounds are yellow in color, whereas hydroxyl derivatives of flavanone (pinocembrin, naringenin, eriodictyol) and flavanonol (pinobanksin) are orange in color.However, the methoxy derivative of pinocembrin-pinostrobin and its chalcone-were violet in color (Figure 3F).In contrast, under ultraviolet light, after derivatization with anisaldehyde, flavanones exhibit brownish-red fluorescent zones, clearly distinguishing them from the greenish-yellow or light blue fluorescent zones of flavones and flavonols (Figure 3E, Table 2).
On the other hand, the use of a 2% AlCl 3 and the detection of compounds under UV light with a wavelength of λ-366 nm results in an intense blue fluorescent zone of caffeic acid and differentiates the fluorescence of galangin and chrysin-light blue and greenish-yellow, respectively (Figure 3B,C, Table 2).When using a spraying reagent with anisaldehyde, the fluorescence of this phenolic acid is less intense and similar to other compounds.
The optimized 2D-HPTLC method enabled the separation of methanol extracts from the analyzed poplar buds, which in most cases are multi-component mixtures (Figures 4-7).Among the standards used, the separation of chrysin and galangin as well as p-coumaric acid from caffeic acid phenethyl ester was achieved, unlike the 1D-HPTLC method (Figure 3C,F).Moreover, it was noted that the spot identified as pinocembrin, as a result of the previously performed 1D-HPTLC separation of the extract from P. balsamifera buds, is a mixture of two compounds (Figure 4).
The developed 2D-HPTLC method is a fast, simple, and relatively cheap method for assessing the botanical identity of poplar buds, which involves identifying characteristic compounds present in a given Populus species or variety.At the same time, in the tested poplar buds, it enables bioautographic visualization of compounds showing antioxidant activity using ABTS and DPPH solutions ( Figures 4E,J, 5E,J, 6E,J and 7E,J).
The buds of P. balsamifera, P. × berolinensis, and P. × canadensis 'Marilandica' contained a number of compounds with the ability to scavenge free radicals, which belonged primarily to the group of simple phenols and polyphenols.In turn, ABTS and DPPH bioautograms of buds of P. wilsonii, a species rich in glycerol derivatives, showed only the presence of a few compounds with antioxidant properties.

Antioxidant Capacity
The antioxidant activity of the tested buds of Populus ranged from 2.41 to 7.57 mM TEA/g d.w. in the DPPH assay, 3.65 to 12.10 mM TEA/g d.w. in ABTS, and 13.39 to 24.68 mM TEA/g d.w. in the FRAP test.Statistically significant differences in the antioxidant capacity were found between all tested bud extracts in the DPPH and ABTS tests, whereas in the FRAP test, only the extract from P. × berolinensis differed from the others (Table 3).However, what is interesting and requires explanation in further research is the fact that in the FRAP test, no statistically significant differences were found between extracts from the buds of P. wilsonii, P. balsamifera, and P. × canadensis 'Marilandica' (Table 3).

Total Flavonoid (TFC) and Total Phenolic (TPC) Content
Quantitative analysis of flavonoids (TFC) in the tested poplar buds confirms the preliminary results of analyses using 2D-HPTLC and GC-MS methods.Extracts from P. balsamifera, P. × canadensis 'Marilandica', and P. × berolinensis contained 44.76, 55.17, and 65.01 mg/g RE, respectively, whereas Populus wilsonii contained only 7.92 mg/g RE.Unlike other taxa, only a few flavonoids were found in P. wilsonii by chromatographic methods (Table 1, Figure 7).
The determined content of flavonoid compounds in the buds of Populus balsamifera (44.76 mg/g) was very similar to the content in raw plant material from Lithuania (39.33-45.26mg/g) [13,14].
Significant differences were also found in the TPC of the analyzed poplar buds.The extract of P. wilsonii contained 64.96 mg/g GAE, whereas in the remaining taxa tested, TPC ranged from 141.34 to 183.113 mg/g GAE (Table 3).
The determined TPC is similar to the TPC in the buds of P. nigra from Romania (106.57mg/g GAE [12]), and P. balsamifera and P. nigra buds (196.81 mg/g and 95.02 mg/g) from Lithuania (expressed in p-coumaric acid equivalents) [14].In the buds of Populus × berolinensis, P. × canadensis 'Marilandica', and P. wilsonii, both TFC and TPC were determined for the first time.
The conducted research has shown that the leaf buds of P. × berolinensis are characterized by the most diverse chemical composition, the highest content of flavonoids and phenols, and the highest antioxidant capacity, which allows us to assume that they may constitute a potential herbal medicine with anti-inflammatory effects.However, further in vitro and in vivo studies using different inflammation models are needed to verify this hypothesis.
Demineralized water was prepared using a Merck Millipore Water Purification System, Millipore (Molsheim, France).

Methanol Extracts
Dried buds of poplars (1 g) were manually crushed and then extracted three times with methanol (30 mL, 60 • C) on a magnetic stirrer (45 min).The obtained extracts were combined and evaporated to dryness and then dissolved in methanol (15 mL).

Ether Extracts
Dried buds of poplars (2 g) were manually crushed and then extracted with diethyl ether (15 mL) by shaking (60 s).The obtained extract was filtered with a paper filter and the solvent was evaporated to dryness.

GC-MS Analysis 4.4.1. Preparation of TMS Derivatives and Their GC-MS Analysis
For the preparation of TMS derivatives, 5−10 mg of the ether bud extract was put into a vial of 2 mL in volume and dissolved in 240 µL of pyridine and then 60 µL of BSTFA was added.The reaction mixture was heated for 0.5 h at 60 • C to form trimethylsilyl (TMS) derivatives.
The solutions of TMS derivatives were analyzed by GC−MS on an HP 7890A gas chromatograph with a 5975C VLMSD mass selective detector (MSD) with a Triple-Axis Detector (Agilent Technologies, Santa Clara, CA, USA).The apparatus was fitted with an HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) with an electronic pressure control and a split/splitless injector.The latter worked at 220 • C in the split (1:50) mode.The helium flow rate through a column was 1 mL/min in a constant flow mode.The sample volume was 1.0 µL.The initial column temperature was 50 • C, rising at 3 • C/min to 320 • C. To identify the components, both mass spectral data and the calculated retention indices were used.The MSD acquisition parameters were as follows: transfer line temperature was 280 • C, MS source temperature was 230 • C, and MS Quard temperature was 150 • C. The EI mass spectra were obtained at an ionization energy of 70 ev.The MSD was set to scan 41-600 a.m.u.After integration, the fraction of each component in the total ion current (TIC) was calculated.The described procedure was carried out in triplicate.

Retention Indices Determination and Component Identification
Linear temperature programmed retention indices (I T ) were calculated from the results of the separation of the hexane solution of the C 8 -C 40 n-alkanes calibration standard and prepared samples of derivatized extracts.At present, the n-alkanes calibration standards are commercially available.However, high-temperature capillary chromatography allows for the separation of high-boiling compounds with I T values higher than 4000.To calculate the retention indices for such components, one needs the corresponding reference compounds, n-alkane homologs with n > 40.For this purpose, we extracted (using n-hexane) Parafilm M ® foil containing small amounts of higher n-alkanes [52].The retention times of linear C 40 -C 45 homologs were registered as the single ion (m/z 57).
The components were identified using an automatic data-processing system included with the equipment used.Mass spectrometric identification was carried out with an automatic system of GC-MS data processing supplied by NIST mass spectra library and the recently published database of retention indices and mass spectra of TMS derivatives [52].The latter contains more than 1800 spectra and I T values of TMS derivatives prepared from authentic preparations of flavonoids and other phenolics, as well as terpenoids, aliphatic acids, alcohols, and carbohydrates.
The hexane solution of C 10 -C 40 n-alkanes was separated under the above conditions.The linear temperature-programmed retention indices of the registered components were calculated from the results of the separation of this solution and silanized bud extracts and were compared with the collections [52,53].The identification was considered reliable if the results of the computerized search of the mass spectra library were confirmed by the experimental I T values, i.e., if their deviation from the averaged published index value did not exceed ±10 u.i.In the case of a greater deviation of the I T values from those given in databases [52,53], or in the absence of the literature values for this independent parameter, the identification is considered tentative and the name of the compound is marked with a question mark.
As a result of the above experiments, developed and dried plates were analyzed in daylight (λ-500 nm) and UV light at λ-366 nm, before and after the derivatization by 2% methanolic solution of AlCl 3 or 0.5% solution of anisaldehyde in 5% sulfuric acid in methanol (95 • C, 3 min).Spraying reagents were spread on developed and dried plates using an automated spraying device-Derivatizer (Camag, Muttenz, Switzerland).Documentation of chromatograms was performed using the TLC Visualizer (Camag, Muttenz, Switzerland).Then, the plates were stored for 30 min in a dark place and analyzed in daylight.Compounds with scavenging properties of the DPPH free radical were visible as yellow spots on the purple background of the chromatograms [54].

ABTS Test
A 7 mM solution of ABTS was created by dissolving 38.41 mg of ABTS into 8 mL of demineralized water.A 2.45 mM solution of sodium persulfate (K 2 S 2 O 8 ) was made by dissolving 66.2 mg of K 2 S 2 O 8 into 10 mL of demineralized water.Then, 1 mL of 2.45 mM K 2 S 2 O 8 was added to 7 mM ABTS.The obtained mixture was filled up to 10 mL with water and left in the refrigerator (4 • C) for 16 h.After this time, developed and dried HPTLC plates were sprayed with the use of an automated spraying device, the Camag Derivatizer.Next, the plates were stored for 6 min in a dark place and analyzed in daylight.Bright spots of compounds with antiradical properties were observed on the blue background of the chromatograms [55].
The documentation of chromatograms and bioautograms was performed using the TLC Visualizer (Camag, Muttenz, Switzerland).

Antioxidant Capacity Assays
Results of all the tests described below are expressed as mM Trolox equivalents per g of the dry weight of buds (mM TE/g d.w.).

DPPH Assay
The DPPH assay was performed using a method previously described by Tuberoso et al. [56] with some modifications.The freshly prepared 0.04 mM solution of DPPH in methanol was subjected to ultrasound (5 min) and then placed in the refrigerator (4 • C) for 60 min.The standard curve was linear between 0.02 and 0.1 mM Trolox.
Then, 350 µL of the plant extracts or Trolox dilution were mixed with 2.5 mL of 0.04 mM DPPH solution in brown glass bottles and stored in a dark place for 30 min.The blank sample consisted of 350 µL of methanol and 2.5 mL of a 0.04 mM DPPH solution.Spectrophotometric measurements were made at λ-517 nm.

FRAP Assay
The FRAP assay was carried out using a method previously described by Benzie and Strain [57] with some modifications.To perform the FRAP test, the following reagents were made: a 300 mM acetate buffer (pH 3.6), a 10 mM solution of TPTZ in 40 mM HCl, and 20 mM of FeCl 3 × 6H 2 O.The prepared solutions were mixed in a ratio of 10:1:1 (v/v/v) and then heated to 37 • C (3 min) in a water bath.The standard curve was linear between 0.02 and 0.48 mM Trolox.
For testing, 150 µL of the analyzed extracts or Trolox dilution were mixed with 3 mL of previously prepared FRAP reagent.Samples were incubated in the dark for 30 min.The blank sample consisted of 150 µL of water and 3 mL of FRAP reagent.Absorbance was measured at λ-593 nm.

ABTS Assay
The ABTS assay was performed using a method previously described by Thaipong et al. [58], with some modifications.For this test, 2 mL of 7 mM ABTS solution was mixed with 0.35 mL of 140 mM sodium persulfate and the prepared mixture was incubated for 15 h in a refrigerator (4 • C) with no access to light.After this time, the reagent was diluted with water in a ratio of 1:90 (v/v) while maintaining a constant absorbance, i.e., 0.7 ± 0.02.The standard curve was linear between 0.02 and 0.12 mM Trolox.
For testing, 200 µL of the analyzed extracts or appropriate Trolox dilution were taken and 2 mL of previously prepared ABTS reagent was added.The blank sample consisted of 200 µL of water and 2 mL of ABTS reagent.The absorbance was measured after 6 min at λ-734 nm.

Total Phenolic Content (TPC)
The TPC was estimated according to the Folin-Ciocalteu method [59] and was expressed as mg of gallic acid equivalent (GAE) per g of dry weight (d.w.) in relation to the calibration curve of GA (y = 1.2405x + 0.0250; r 2 = 0.9996) in a concentration range of 0.2-1.0mg/mL.

Total Flavonoid Content (TFC)
The TFC was determined using the method described by Barman et al. [60] and was expressed as mg of rutin equivalent (RE) per g of d.w. in relation to the calibration curve (y = 0.0034x + 0.0096; r 2 = 0.9997) in a concentration range of 6.4-102.4mg/mL.
Absorbance in all the above tests was measured by spectrophotometer UV-1800 (Shimadzu, Kyoto, Japan).

Statistical Analysis
The mean difference between the TFC, TPC, and the DPPH, ABTS, and FRAP antioxidant capacity was controlled using the one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests.All statistical analyses were performed using Statistica 12 (StatSoft, Kraków, Poland).

Conclusions
The research carried out showed significant differences in the chemical composition (GC-MS identification, TPC and TFC determination) and antioxidant capacity (spectrophotometric DPPH, ABTS, and FRAP assays) of the buds of the studied Populus taxa.The composition of P. wilsonii buds was analyzed for the first time and the experiments performed provided new information about the types of biologically active compounds found in buds of P. balsamifera, P. × berolinensis, and P. × canadensis 'Marilandica'.
The 2D-HPTLC method for metabolomic profiling of the studied poplars was developed and could be used in a pharmacopoeial monograph of Populi gemmae for their botanical identification.This method can also be used to assess the botanical origin of propolis.
The plant material from P. × berolinensis had the richest chemical composition and the highest content of flavonoids and phenols, which determines the highest antioxidant capacity.In our previous studies [9], the buds of this poplar hybrid showed the strongest anti-inflammatory effect, and the results obtained confirm the influence of their rich chemical composition on biological activity.The above data indicate that P. × berolinensis buds may be a potential herbal medicine with valuable anti-inflammatory effects.Further research will be conducted to explain the mechanisms of action of this plant material at the molecular level.

Figure 1 .
Figure 1.The content (% of TIC) of main group of compounds identified by GC-MS method in analyzed poplar buds.

Figure 1 .
Figure 1.The content (% of TIC) of main group of compounds identified by GC-MS method in analyzed poplar buds.

Table 2 .
hRf (Rf × 100) values of compounds identified in poplar buds extracts by 2D-HPTLC method and the colors of their zones in visible light and fluorescence at UV light, detected using different spraying reagents.

Table 2 .
hRf (R f × 100) values of compounds identified in poplar buds extracts by 2D-HPTLC method and the colors of their zones in visible light and fluorescence at UV light, detected using different spraying reagents.

Table 3 .
Total flavonoid (TFC) and phenolic (TPC) content, and antioxidant capacity of methanol extracts from poplar buds.

Table 3 .
Total flavonoid (TFC) and phenolic (TPC) content, and antioxidant capacity of methanol extracts from poplar buds.