1. Introduction
Propolis (bee glue) is a viscous, aromatic substance produced by different species of bees, the most commercially available being the one produced by honey bee (
Apis mellifera L.). Bee glue is a mixture of plant exudates, bees wax and secretions of bees’ glands. Propolis is used by bees to insulate hive, repair any damage to hive structure and for hygienic purposes. The biological activities of bee glue are widely and well documented in the literature, including its medicinal properties such as antimicrobial, anti-inflammatory, antioxidative and wound-healing [
1,
2]. All of the biologically active components of propolis originate from plant exudates and resins, which are called plant precursors. Therefore, the species of those plants play a pivotal role in the properties of the final product [
1,
2].
Although bees are capable of foraging from a variety of plant sources, they show preference and prioritize certain species of plants, depending on a region. Therefore, composition of propolis is not completely random and it could be divided into several chemical types, based on main plant precursors [
1,
2,
3,
4].
In temperate climatic zone, exudates from buds of black poplar (
Populus nigra L.) are predominantly collected [
3,
4]. However, it was shown that other
Populus species could also be propolis precursors [
3,
4,
5,
6,
7], among them
P. tremula L. (aspen). It is significant that composition of aspens’ exudates is different from black poplars resins. [
3,
4,
5,
6,
7]. The presence of phenolic acids glycerides in aspen buds and aspen propolis constitutes the main difference [
3,
4,
5,
6,
7]. These components are absent in
P. nigra L. buds exudates [
3,
4,
5,
6,
7] which contain their own specific markers such as flavonoids (e.g., chrysin). Other mixed types of propolis such as aspen–poplar [
3,
4,
5] and aspen–birch–poplar [
6] are also known.
Composition of propolis and its plant precursors are not fully known, which is especially apparent in the area of comparative investigation of bee glue and its plant sources. Available data for comparative analysis between bee glue and
Populus from temperate climate include research performed using different techniques such as TLC (thin layer chromatography), TLC-MS (thin layer chromatography coupled with mass spectrometry) [
5], GC-MS (gas chromatography coupled with mass spectrometry) after sililation [
3,
4,
6,
7], and UHPLC-MS/MS (ultra-high performance liquid chromatography coupled with tandem mass spectrometry) [
8,
9]. However, comparative analysis of volatile components remains under-researched [
2]. Moreover, not all aspects of the composition of
Populus genus essential oils (EOs) have been studied. According to Jerković and Mastelić [
10],
P. nigra L. leaf buds contain mainly oxygenated sesquiterpenoids. As far as we know, the composition of volatile compounds of aspen and other
Populus buds is still not properly researched. Available information on
P. tremula L. essential oils includes only emission of terpenoids from leaves [
11] and bark [
12].
It is also noteworthy that propolis composition is unstable during vegetation season with reference to the data of polyphenolic extract of tropical samples (Brazilian green [
13], Brazilian red [
14], and Sonoran [
15]) and research from south Portugal [
16]. Similar investigation has not been performed for aspen–poplar propolis.
Therefore, as the biological activities of propolis are related to the concentration of polyphenols and volatile components, observation of their profiles may be useful in future investigations, for example in the search of cheaper or more easily available propolis replacements. Poplar is a known source of various folk remedies, effectiveness of which has sometimes been documented. Over time, such remedies fell into disuse and mostly replaced by propolis. Nevertheless, poplar derivates still could be used as food additives. As propolis is known for instability of both its composition and biological activity, usually with no clear cause and effect correlation between the two, Populus extracts could be a favorable replacement. Such a solution could even be economically viable considering the low cost of propagation and fast growth of the trees.
Due to a greater abundance and a generally higher stability of non-volatile components in propolis and Populus spp. exudates 70% ethanol in water extracts (70EE), we present a hypothesis that 70EE are better for identifying propolis plant precursors compared to essential oils (EOs).
2. Results
2.1. Composition of Essential Oils of Propolis and Its Plant Precursor
During preliminary research, hydrodistillation–extraction (HDE) using a Deryng apparatus and SDE (simultaneous distillation–extraction to dichloromethane) was performed to obtain volatile propolis derivates. Unlike HDE, SDE did not show any problems with clogging by waxy residue.
The presence of 279 components (29% were tentatively identified and 71% were fully identified) was exhibited by GC-MS analysis. Only the components that exceeded at least 10% in single sample of those shown in
Table 1 and
Table 2 were used in statistical analyses. Full results are attached as
Supplementary Materials (Tables S1–S3). General amounts of compounds according to chemical classes are shown in
Table 3 and
Table 4.
Identified components were assigned to several groups (see
Table 3) and samples were characterized by the amounts of several component types. The highest amount of investigated essential oils was observed in black poplar sample: PN-3 (4.33%). The remaining
P. nigra samples exhibited a range of 0.50% to 1.50% of EOs.
P. tremula EOs’ content varied from trace amount (<0.05%) to 0.36%. Propolis contained 0.07% to 2.8% of EOs.
Black poplars buds exhibited different EOs profile, differing in both quality and quantity. Some buds contained mainly oxygenated sesquiterpenoids (83% in PN3 and 86% in PN5; large amount of α-, β- and γ-eudesmols), while two other samples were mainly composed of sesquiterpene hydrocarbons (73% in PN1 and 66% in PN2; main components were ar- and γ-curcumens and δ-cadinen). PN4, PN6 and PN7 showed a different profile, containing a mix of sesquiterpenes and sesquiterpenoids (45% in PN4, 32% in PN6, and 40% in PN7) and derivates of benzoic acid (49% in PN4, 54% in PN6 and 48% in PN7, mainly prenyl benzoate).
Aspen buds also exhibited different profiles of EOs. Four aspens contained mostly benzoic acid derivates (69% in PT4, 55% in PT1 and 51% in PT5). In PT1, benzyl benzoate was the main component (45.45%), while composition of PT4 and PT5 was more complex. Another pattern of composition was observed in PT2 and PT3, which contained mainly sesquiterpene derivates (91% in PT2 and 92% in PT3). EOs of PT2 were composed of similar amount of sesquiterpene hydrocarbons (cis-β-cariophyllene and α-guaiene) and oxygenated sesquiterpenoids (cariophyllene oxide), while PT3 contained higher amount of oxygenated sesquiterpenoids than sesquiterpenes. In the case of the last sample, PT6, a different type of EOs profile was observed:a mixture of 2-phenylethanol, unidentified aliphatic component, benzyl alcohol and eugenol.
Propolis’ EOs were usually composed of benzoic acid derivates (mainly benzoic acid and benzyl benzoate) and a mixture of oxygenated sesquiterpenoids (typical components were α-, β-, and γ-eudesmols). Only two samples (PR-LS6 and PR-GR) contained low amount of benzoic acid derivates. Moreover, in PR-GR, concentration of sesquiterpenes hydrocarbons (mainly ar- and γ-curcumens) was higher than oxygenated sesquiterpenoids.
2.2. Identification of Compounds Present in 70EE and UPLC-PDA-MS Profile of Propolis and Populus Buds
Results of polyphenols analysis are shown in
Table 5 and
Table 6. Complete results of the UPLC-PDA-MS (ultra-performance liquid chromatography coupled with photodiode array and mass spectrometry) analysis with identification data are attached as
Supplementary Materials (Tables S4–S7). Seventy-seven components were identified in all samples, most of which were identified based on literature data (see
supplement Table S4), according to the characteristic UV (ultraviolet light) absorption spectrum and mass fragmentation in negative ionization mode. In the case of most flavonoids, free phenolic acids and their monoesters, exhaustive literature data allow certain extent of identification. Phenolic acids glycerides proved difficult to identify, especially prediction of glycerol substitution position to phenolic acids due to a scarcity of HPLC-MS ion fragmentation literature data and a lack of available standards.
The profiles of 70% ethanol in water extract have shown certain variation between the samples. Propolis, aspens and black poplars exhibited qualitative and quantitative differences within their groups. In the case of P. nigra, four samples (PN1, PN2, PN3 and PN5) contained mainly flavonoid aglycones (chrysin, pinocembrin chalcone, galangin, pinocembrin-3-O-acetate and pinostrobin chalcone) and lower amount of free phenolic acid (p-coumaric acid) and their monoesters (benzyl and cinnamyl esters of p-coumaric acid). Different composition was observed for three P. nigra buds (PN4, PN6 and PN7), which contained a higher amount of free phenolic acids and their monoesters, but contained less flavonoid aglycones than PN4, PN6 and PN7.
Unlike black poplars, 70EE of aspens contained mainly free phenolic acids, phenolic acid glycerides (especially 2-acetyl-1,3-di-p-coumaroylglycerol and some phenolic acid monoesters (for example, p-coumaric acid benzyl ester). It is worth noting, however, that profiles of aspens were more uniform than those of black poplars.
Propolis samples had a composition resembling a mix of substances characteristic for aspens (phenolic acid glycerides) or poplar (flavonoids) (18 samples) or exhibited a profile similar to P. nigra (three samples: Polish (PR-LS6), German (PR-GR) and Canadian propolis (PR-CAN)).
2.3. Statistical Analysis of 70% Ethanol in Water Extracts and Essential Oils
Principal component analysis of 70EE has shown that a nine-principal-component model explained 99.3% of total variance, while 99.2% of total variance of EOs PCA analysis required a thirteen-principal-component model. Models limited to only two most important principal components explained 75.4% of 70EE total variance and 58.5% of EOs total variance. When poplars and aspens were excluded from analysis, the explanations of total variance were higher for two-component models (91.5% for 70EE and 79.2% for EOs).
Graphical results of PCA analysis (two-principal-component models: factor scores and their loading) are presented in
Figure 1 (PCA of 70EE) and
Figure 2 (PCA of EOs).
In PCA of 70EE, the first principal component was composed mainly of p-coumaric acid, 2-acetyl-1,3-di-p-coumaroylglycerol, p-coumaric acid benzyl ester, pinocembrin chalcone and pinostrobin chalcone. 2-acetyl-1,3-di-p-coumaroylglycerol, galangin, chrysin, pinobanksin-3-O-acetate and 2-acetyl-1-caffeoyl-3-di-p-coumaroylglycerol exhibited the highest effect on the second principal component of 70EE, with the strength of their impact decreasing in the order they were mentioned. For the EOs analyze, benzyl benzoate, benzoic acid, ar- and γ-curcumens and cis-β-caryophyllene were the main components of the first principal component. The second principal component of EOS was composed of α- and β-eudesmols, benzyl benzoate, γ-eudesmol and benzoic acid.
As shown in
Figure 1, 70EE samples may be divided in several groups (
P. nigra buds and black poplar propolis, mixed aspen–black poplar propolis and
P. tremula buds). It is significant that propolis samples were separated according to the plant origin (black poplar propolis or mixed aspen–poplar bee glue). Conversely, for EOs, propolis samples could not be divided according to plant origins and were separated according to their main chemical components. Moreover, some black poplars and aspens samples were close to each other. In PCA of EOs, black poplars samples reflect similar distribution to each other such as in PCA of 70EE (PN1 and PN2 created one sub-cluster; PN3 and PN5 created the second sub-cluster; and PN4, PN6 and PN7 created the third sub-cluster). In the case of propolis and aspen samples, their own clusters were not preserved in PCA of EOs (see
Figure 1 and
Figure 2 and compare the distribution of black poplars, aspens and propolis samples).
Analysis of Spearman’s coefficient rank exhibited more similarities between 70EEs compositions than EOs. More positive correlations between propolis and Populus buds were observed for 70EEs (42) than EOs (35). Numbers of positive correlations between same propolis samples (propolis vs. propolis) were 300 for 70EEs and 246 for EOs.
Moreover, propolis EOs were more often observed to exhibit positive correlation with Populus buds that were not their plant precursor than in 70EE analysis. The most outstanding result was observed for PR-LS2 bee glue. The 70EE of this sample was correlated with black poplar (PN1), but the EOs with aspens (PT1 and PT4).
4. Materials and Methods
4.1. Research Materials, Reagents and Standards
Twenty samples of propolis, seven of black poplars (PN1–7) and six of aspen (PT1–6) buds were analyzed in this work. Samples of bee glues were a mixture of material from different hives in the same apiary. These samples originated from Lower Silesia (12 samples: PR-LS1–6, PR-NW1–2, PR-ŚL1–2, PR-MR, and PR-NSW), West Pomerania (Szczecin, 4 samples: PR-SZ1–4), Podkarpacie (2 samples: PR-S1–2), Germany (commercial sample: PR-GR) and Canada (PR-CN).
P. nigra buds were collected in Szczodre (2013 and 2014), Wrocław (2015) (Lower Silesia, Poland) and Kórnik Arboretum of Institute of Dendrology of the Polish Academy of Sciences (2015) and bought from commercial sources. P. tremula buds were collected in Szczodre (2013, 2015 and 2016) and Arboretum Wojsławice, branch of Wrocław University Botanical Garden (2017). All of the samples were dried for three weeks prior to analysis.
Propolis was frozen in liquid nitrogen and mechanically ground in mortar. Populus buds were mechanically ground in an automatic mixer. Research materials were stored at −20 °C.
Ethanol and dichloromethane were purchased from POCH (Gliwice, Poland), Acetonitryl LC-MS from VWR Prolabo Chemicals (Leicestershire, UK) and 98% formic acid from Fluka (Buchs, Switzerland). Standards of n-alkanes (C8–C23) were obtained from Fluka (Buchs, Switzerland), those of polyphenols were purchased from Extrasynthese (Genay, France) which included acacetin, apigenin, caffeic acid, chrysin, ferulic acid, genkwanin, isoferulic acid, kempferol, and quercetin dihydrate. Pinobanksin was obtained from Sigma-Aldrich (Saint Louis, MO, USA).
4.2. Isolation and Analysis of Essential Oils (EOs)
Essential oils were obtained by hydro distillation–simultaneous extraction (SDE) according to Kujumgiev et al. [
18]. Distillation time was set at 2 h (from original 4 h). After distillation, dichloromethane was evaporated and the amount of EOs was evaluated as percentage (%) of whole sample mass. The procedure was repeated twice.
Next, essential oils were analyzed by GC-MS in Varian chromatograph with mass spectrometer (GC-MS CP 3800 + Saturn 2000, Varian, Palo Alto, CA, USA) equipped with Zebron ZB-1MS, GC Capillary column (Phenomenex, Torrance, CA, USA) according to methods of Szumny et al. [
42].
Qualitative and quantitative GC-MS analyses were performed on Varian CP 3800 + chromatographer (Varian, Palo Alto, CA, USA) coupled with mass spectrometer Saturn 2000 (Varian, Palo Alto, CA, USA). The system was equipped with capillary column Zebron ZB-1MS (10 m × 0.53 mm × 2.65 μm) (Phenomenex, Torrance, CA, USA). Collected data were analyzed by Varian MS Workstation (version 6.5.) (Varian, Palo Alto, CA, USA) equipped with NIST05 Mass Spectral Library with Search Program (National Institute of Standard and Technology, Gaithersburg, MD, USA) and MestreNova 9.0 (trial version, Mestrelab, Research, Santiago de Compostela, Spain).
The normalization of chromatograms of GC-MS was performed as manual correction of the peaks to the baseline. Peaks of multiple compounds were separated into singular peaks according to the main ion in GC-MS. For GC-MS standard mix of n-alkanes (C8–C23) was used.
For the statistical analysis of EOs, peaks of GC-MS chromatograms were integrated and their areas were calculated as a percentage (%) of combined area of all peaks.
Some overlapped peaks were described as single component (if one strongly dominated in peak) or multicomponent mix. Overlapped peaks were divided into single peaks according to major ions concentration.
Single components were identified by comparison of experimental mass spectra and retention indexes with standards of essential oils and literature. Identification data are available as
Supplementary Materials.
4.3. Preparation and UPLC-PDA-MS Analysis of 70% Ethanol in Water Extract (70EE)
Previously ground research material (propolis and buds) was extracted by 70% ethanol in water (proportion: 1.0 g/10 mL) in an ultrasonic bath. Extraction conditions were set at 400 °C for 45 min and 756 W (90% of ultrasound bath power). Next, extracts were stored at room temperature for 12 h and then filtrated through Wattman No. 10 filtrate paper.
Composition of obtained 70EE was analyzed by Waters Acquity UPLC system (Waters, Milford, CT, USA) equipped with PDA 200–500 nm, mass spectrometer Xevo-Q-TOF (Waters, Milford, CT, USA) and column BEH C18 130 Å, (1.7 μm, 2.1 mm × 100 mm) (Waters, Milford, CT, USA). We used modified method of Shi et al. [
42,
43]. The technique adopted in this research differed in terms of elution parameters and electrospray ionization parameters (only ESI-NEG mode was used—electrospray ionization in negative mode).
The elution system consisted of acetonitrile/0.1% solution formic acid in water. The gradient elution program began with 20% acetonitrile, and was increased to 30% in 10.70 min → 31% in 15.30 min → 31% in 15.90 min → 32% in 17.00 min → 34% in 18.00 min → 36% in 20.30 min → 40% in 21.50 min → 45% in 25.50 min → 50% in 29.70 min → 100% in 33.00 min → 100% in 36.00 min → 20% in 38.00 min.
Parameters of ESI-NEG were set at capillary voltage of 2.80 kV, sampling cone of 66 kV and extraction cone of 4.0 kV. Collision energy was set at 0, 20, 20–30, 30 and 30–50 kV.
Data were processed using Masslynx 2.0 (Waters, Milford, CT, USA) and MestreNova 9.0 (trial version, Mestrelab, Research, Santiago de Compostela, Spain). Single components were identified by comparison of experimental mass, UV absorption spectra and retention time to standards and literature data. Identification data are available as
Supplementary Materials.
The normalization of chromatograms of UPLC was performed as manual correction of the peaks to the baseline. Peaks of multiple compounds were separated into singular peaks according to the spectrum of the main component and main ion in UPLC. UPLC peaks were read at 200–500 nm spectrum for the reason of acquiring average values. For UPLC a mix of acacetin, apigenin, caffeic acid, chrysin, p-coumaric acid, ferulic acid, genkwanin, isoferulic acid, kempferol, quercetin dihydrate and pinobanksin was used. As standards of some of the major compounds present in the samples and contributing to the UV spectrum significantly, mostly phenol acid glycerides and monoesters, proved themselves to be unobtainable, it was decided that the analysis could only be reliably carried out as comparison of peak areas calculated as a percentage of total chromatogram peak area.
For statistical analysis of 70EE, peaks of UV chromatograms were integrated in the range of 200–500 nm. Area of integrated peaks was calculated as a percentage (%) of combined area of all peaks.
Some overlapped peaks were described as single component (if one strongly dominated in peak) or multicomponent mix. These data were used in the principal component analysis of 70EE.
4.4. Statistical Analysis of 70EE and EOS Composition
Statistical analysis included principal component analysis (PCA) (according to Statsoft, Inc., Kraków, Poland [
44]) and Spearman’s coefficient rank [
44]. UPLC-PDA (for 70EE analyze compounds) and GC-MS (for EOS analyze) peaks (at least ≥10% in one sample) were used as variables. Reductive analyses were carried out, namely those considering peaks constituting ≥1%, ≥2%, ≥3%, ≥5%, and ≥10% of total area. Meta-analysis of the results demonstrated that ≥10% model explained the data sufficiently, while simultaneously eliminating excessive data input and preventing excessively close groupings of the points, both of which posed a problem in the remaining models, albeit to varying degrees. The authors felt it best to present only the last model in the publication not to make the interpretation of the results excessively bothersome and improve clarity.
Calculations were performed on Statistica 12.5 software (Statsoft, Inc., Kraków, Poland). Calculations of Spearman’s coefficient rank were performed using two models. The first model encompassed Populus buds and propolis and the second model contained only some propolis samples.