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Gas chromatographic-mass spectrometric examination of chemical composition of two Eurasian birch (Betula L.) bud exudates and its taxonomical implication
Biochemical Systematics and Ecology 52 (2014) 41–48
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Gas chromatographic-mass spectrometric examination of chemical composition of two Eurasian birch (Betula L.) bud exudates and its taxonomical implication b _ Valery Isidorov a, *, Lech Szczepaniak a, Ada Wróblewska b, Ewa Piroznikow , Lidia Vetchinnikova c a b c
Institute of Chemistry, Białystok University, 15-399 Białystok, Poland Institute of Biology, Białystok University, 15-950 Białystok, Poland Forest Research Institute, Karelian Research Centre of RAS, 185910 Petrozavodsk, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2013 Accepted 15 December 2013 Available online 9 January 2014
GC–MS was used to investigate the chemical diversity of bud exudates obtained from two species of white birch in Poland, Betula pendula Roth and Betula pubescens Ehrh. The ether extracts of the bud exudates contained more than 210 organic compounds including: terpenoids, phenylpropenoids of sesquiterpene alcohols and flavonoids. Headspace solidphase microextraction (HS-SPME) made it possible to detect as many as 140 volatile organic compounds (VOCs), mainly sesquiterpene hydrocarbons and their oxygen derivatives. The chemical compositions of both the ether extracts and the VOCs varied qualitatively and quantitatively between the species. The extracts from B. pendula buds contained predominantly triterpenoids. However, B. pubescens exhibited high amounts of sesquiterpenoids, flavonoid aglycones and phenylpropenoids of caryophyllane series sesquiterpenols. The differences in the composition of compounds in the bud exudates enabled the two species of birch to be differentiated. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Gas chromatography-mass spectrometry Birch buds Chemical composition Taxonomical implication
1. Introduction Birch (Betula L.) is an arborous tree found in the forests of the boreal and temperate zones of the Northern Hemisphere. The number of accepted birch species varies from 30 to over 60 (Keinänen et al., 1999), and such a great divergence clearly demonstrates taxonomic problems with this genus. In the European and East Siberian forests, birch is represented mostly by the commercially important species of white birch (Betula alba L. sensu Regel, 1865), silver birch (Betula pendula Roth.) and downy birch (Betula pubescens Ehrh.) (Hynynen et al., 2010). Recent morphological studies of leaf size, shape, and parameters of venation of B. pendula and B. pubescens along a 1600-km latitudinal zonal transect in the Urals and Western Siberia demonstrated their variability and dependence on the geographic location of the population (Migalina et al., 2010). These variations illustrate the problems in distinguishing between these white birches (Howland et al., 1995). To solve these problems, several chemotaxonomic studies have been accomplished (Hänsel and Hörhammer, 1954; Koshy et al., 1972; Wollenweber, 1975; Paw1owska, 1983; Meurer et al., 1988; Keinänen and Julkunen-Tiitto, 1998; Keinänen et al., 1999). The objects of these investigations were generally extracts from leaves, pollen and buds phenolics. More convincing
* Corresponding author. Tel.: þ48 85 745 7805. E-mail address: [email protected] (V. Isidorov). 0305-1978/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2013.12.008
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evidence for taxonomic implication of phenolics was obtained from investigations of foliar flavonoid glycosides (Keinänen and Julkunen-Tiitto, 1998; Keinänen et al., 1999). Chemical studies on birches have also been conducted because of their pharmacological interest. White birches belong to medical plants: bark tar and essential oils are used to cure skin diseases, whereas decoctions and extracts from dried leaves and buds are applied for treatment of inflammation and as diuretics (Hänsel and Hörhammer, 1954; European Pharmacopeia, 2010). For this reason, birch essential oils obtained by hydrodistillation of buds were the subject of numerous investigations (Holub et al., 1959; Demirci et al., 2000a,b,c; Demirci and Can Bas¸er, 2003; Isidorov et al., 2004; Klika et al., 2004; Can Bas¸er and Demirci, 2007; Orav et al., 2011). In contrast, only few works dealt with the chemical composition of extracts from birch buds (Kononenko et al., 1975a,b; Popravko et al., 1979; Vedernikov et al., 2007; Vedernikov and Roshchin, 2009) while there is virtually no information on compounds contained in birch bud exudates which may form the first line of plant protection against microorganisms and herbivores (Lahtinen et al., 2004). The main purpose of this study was to determine the composition of compounds in volatiles and extracts of the buds of two species of birch, B. pendula and B. pubescens and evaluate whether they could be used to differentiate between the species. 2. Experimental 2.1. Chemicals Triterpenoids, dammaradien-3-one, dipterocarpol and betulenol as well as pyridine and bis(trimethylsilyl)tri , Poland). Diethyl fluoroacetamide (BSTFA) containing 1% trimethylchlorosilane were purchased from Sigma–Aldrich (Poznan ether (POCH SA, Gliwice, Poland) was used after distillation. SPME Holder 57330-U and fused silica fibers with divinylbenzene/carboxen/polydimethylsiloxane (50/30 mm) stationary phase were purchased from Supelco Inc. (Bellefonte, PA, USA). 2.2. Plant material Buds of B. pendula (samples B-1 and B-2) and B. pubescens (samples Bp-1–Bp-3) were collected at the beginning of July, 2012 in the Biebrza National Park in northeastern Poland (53 320 N, 22 430 E). Additionally, buds of B. pendula (sample B-3) and B. pubescens (sample Bp-4) were collected from trees growing on the experimental field station of the Forest Research Institute near Petrozavodsk (Russia). Voucher specimens were deposited with the herbarium of the Geobotanical Station of Warsaw University (Bialowieza) and herbarium of the Biological Department of Petrozavodsk University. 2.3. Sample preparation and GC–MS analysis Buds (1–2 g) from each of the birch species were extracted by rinsing for 60 s in 15 mL of diethyl ether. The exudate extract was filtered through a paper filter and the solvent was evaporated to dryness on a rotor evaporator. About 5 mg of the residue was diluted with 220 mL of dry pyridine and 80 mL of BSTFA was added. The mixture was heated for 30 min at 60 C to form trimethylsilyl (TMS) derivatives. Volatiles from birch buds were determined by HS-SPME/GC–MS. 10 to 15 buds were transferred into a head-space vial of 16 mL in volume and immersed into a temperature controlled water bath (40 C). The septum of a screw-cap was picked by a needle protecting the SPME fiber, and the fiber coating was exposed to the headspace gas phase for 60 min. To improve the sorption conditions, the buds were constantly stirred. The volatiles collected on the fiber were desorbed by introducing the SPME fiber for 15 min into the injection port of the GC–MS apparatus. The analytes were separated on the Agilent 6890 gas chromatograph equipped with the MSD 5973 mass selective detector fitted with an electronic pressure control and a split/splitless injector. The separation was performed on the HP-5ms (30 m 0.25 mm; 0.25 mm film thickness) fused silica column at a helium flow rate of 1 mL/min. The initial column temperature was 40 C (3 min), rising to 220 C at 3 C/min. The injector worked in a splitless mode; the injector temperature was 250 C. The EIMS spectra were obtained at ionization energy of 70 eV, the source temperature being 230 C and that of the quadrulope being 150 C. Detection was performed in a full scan mode from 29 to 300 a.m.u. After integration, the fraction of each component in the total ion current (TIC) was calculated. To determine the retention times of reference compounds and to calculate linear temperature programmed retention indices (IT) of the analytes, the SPME fiber was inserted for 2–3 s into a vial with a mixture of C6–C18 n-alkanes. The separation of the alkanes was performed under the above conditions. TMS derivatives of the extracted compounds were separated and analyzed with the same GC–MS apparatus and column. Injection of 1 mL of the sample was performed with the HP 7683 autosampler. The injector worked in a split (1:50) mode. The MSD was set to scan at 40–600 a.m.u. Chromatograms were registered in a linear temperature programmed regime from 50 C to 320 C at the rate of 3 C/min. The extractions were repeated three times, so the precision of the method (expressed by a relative standard deviation, R.S.D) could be studied. The peak areas of the components obtained by replicate analyses were used for calculation of their R.S.D. values, which amounted to an average of 8% and 17% in assaying TMS derivatives and volatile compounds, respectively. The fairly high values for HS-SPME relative standard deviations may be due to a less precise procedure of compound extraction.
V. Isidorov et al. / Biochemical Systematics and Ecology 52 (2014) 41–48
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The hexane solution of C10–C40 n-alkanes was separated under the above conditions. Linear temperature programmed retention indices were calculated from the results for this mixture and for the solutions of TMS derivatives of components extracted from birch buds surface. 2.4. Component identification To identify the components, both mass spectral data and the calculated retention indices were used. Mass spectrometric identification was carried out with an automatic system of GC–MS data processing supplied by NIST and home-made mass spectra libraries. The retention indices of the registered components were compared with those presented in the authors’
A Abundance TIC: Bet_Pubes1.D
2800000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000
Time-->
30.00
40.00
50.00
60.00
70.00
80.00
90.00
B Abundance
TIC: 120218bca08.D
2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000
Time-->
30.00
35.00
40.00
45.00
50.00
55.00
60.00
65.00
70.00
75.00
80.00
85.00
Fig. 1. Chromatograms of the ether extracts of downy birch (A) and silver birch (B) buds.
90.00
95.00
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V. Isidorov et al. / Biochemical Systematics and Ecology 52 (2014) 41–48
computer database containing IT values for more than 5000 compounds including more than 800 indices for TMS derivatives. This database contains our previous results (Isidorov and Vinogorova, 2003; Isidorov et al., 2006; Isidorov and Szczepaniak, 2009; Szczepaniak and Isidorov, 2011). The identification was considered reliable if the results of computer search at the mass spectra library were confirmed by the experimental IT values (if their deviation from the database values did not exceed 5 u.i.). 2.5. DNA isolation and sequencing The leaf fragments of B. pendula (B-1 and B-2) and B. pubescens (Bp-1–Bp-3) were dried in silica gel and then the genomic DNA was extracted. The plant material also consisted of two accessions of B. pendula (AJ535640.1) and B. pubescens (AJ535645.1) from the GenBank (NCBI). Alcohol dehydrogenase (ADH) was used to study the nuclear DNA sequences in the Betula genus. The procedure of DNA isolation and sequencing as well as the sequence alignment and phylogenetic analysis are presented in the Supplementary Information. 3. Results and discussion 3.1. Extractive components of birch buds The ether extracts of bud exudate from both birch species demonstrated 204 compounds, with the content being not less than 0.01% of TIC (Table 1S in the Supplementary Information provides a complete list of these components). Hence it follows Table 1 Relative composition (%) of birch buds exudate. 0.02 Compounds
nor-Sesquiterpenoids Including: - Birkenal - Birkenol Sesquiterpenoids Including: - b-Betulenol - 6-Hydroxy-b-caryophyllene - 14-Hydroxy-b-caryophyllene - 6-hydroxy-b-caryophyllene acetate - 14-Hydroxy-b-caryophyllene acetate - 14-Hydroxy-4,5-epoxy-b-caryophyllene acetate Triterpenoids Including: - Dammaradien-3-one - Dipterocarpol - Betulinol Phenylpropenoids Including: - p-Coumaric acid - 6-Hydroxy-b-caryophyllene p- coumarate - 14-Hydroxy-b-caryophyllene p-coumarate - 14-Hydroxy-b-caryophyllene ferulate - 14-Hydroxy-b-isocaryophylle-ne ferulate Flavonoids Including: - 5-Hydroxy-40 ,7-dimethoxyfla-vanone - Sakuranetin - Homoeriodictyol - Pectolinaringein - Acacetin - Kaempherid - Kaempherol - Apigenin - Catechin - 5,7,40 -Trihydroxy-4-methoxyfla-vanone Aliphatic acids Aliphatic alcohols Alkanes NNc Total b
trace – below 0.01% of TIC. a n.d. – not detected. c NN – not identified compounds.
B. pendula
B. pubescens
B-1
B-2
B-3
Bp-1
Bp-2
Bp-3
Bp-4
n.d.a
n.d.
n.d.
0.27 0.02
0.34 0.03
0.34 0.02
0.97 0.09
n.d. n.d. 0.11 0.02
n.d. n.d. 0.63 0.06
n.d. n.d. 0.59 0.06
0.22 0.02 0.31 0.03 0.27 0.02 0.97 0.09 0.05 0.01 0.04 0.01 0.07 0.01 n.d. 32.13 1.93 34.28 2.05 37.75 4.70 12.67 1.58
n.d. n.d. n.d. n.d. n.d. n.d. 90.23 8.12
n.d. n.d. n.d. n.d. n.d. n.d. 89.45 6.26
n.d. n.d. n.d. n.d. n.d. n.d. 84.52 5.49
1.66 7.76 1.52 0.40 3.85 0.30 2.68
0.72 0.07 17.21 1.12 2.14 0.17 n.d.
1.74 0.21 31.11 1.87 1.60 0.15 n.d.
2.07 0.23 19.84 1.14 1.49 0.13 Trace
n.d. 0.11 0.02 n.d. 14.67 1.61
n.d. n.d. n.d. 10.91 1.10
n.d. 0.03 0.01 n.d 6.36 0.76
n.d. n.d. n.d. 5.39 0.67
n.d. n.d. n.d. n.d. n.d. 1.40 0.15
n.d. n.d. n.d. n.d. n.d. 0.93 0.07
Trace n.d. n.d. n.d. n.d. 2.71 0.24
0.58 0.05 5.41 0.41 3.07 0.25 0.15 0.02 0.23 0.03 40.59 2.84
0.54 0.05 2.83 0.24 2.76 0.25 0.20 0.03 0.22 0.02 37.88 3.60
0.33 0.03 2.93 0.23 1.82 0.17 0.16 0.02 0.18 0.02 39.09 2.74
0.93 0.09 2.17 0.19 1.49 0.14 0.11 0.03 0.21 0.02 45.12 2.93
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.03 1.37 0.76 0.75 1.30 2.19 96.74
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.11 0.82 1.56 0.51 1.41 1.30 96.78
n.d. Trace n.d. 0.13 0.09 n.d. 0.04 n.d. 0.05 0.67 6.69 0.61 1.67 2.06 99.91
4.45 0.36 11.36 0.74 3.00 0.24 3.72 0.32 2.16 0.20 3.62 0.32 0.89 0.12 0.31 0.04 0.32 0.04 n.d. 1.62 0.13 0.68 0.06 1.96 0.11 4.68 0.56 99.86
2.22 5.69 3.25 2.19 4.33 2.19 1.81 0.92 0.66 n.d. 1.33 0.68 1.11 6.49 95.62
1.66 7.90 3.38 2.80 7.20 3.58 1.46 0.56 0.38 0.98 1.85 0.37 1.54 9.32 98.98
0.55 0.06 21.70 1.48 2.91 0.23 2.04 0.16 0.77 0.08 4.07 0.30 1.60 0.13 0.20 0.03 1.45 0.11 Trace 1.49 0.12 0.27 0.03 1.73 0.16 7.89 0.87 95.68
0.01 0.11 0.91 0.10 0.12 0.28
0.07 0.12 0.06 0.13 0.14
0.02 0.02 0.01 0.02 0.05 0.50 0.07 0.14 0.23
0.13 0.62 1.14 0.04 0.29 0.04 0.27
1.80 8.79 2.15 0.36 4.96 0.41 1.11
0.16 0.57 0.18 0.04 0.40 0.04 0.10
0.18 0.38 0.26 0.20 0.30 0.22 0.14 0.10 0.73 0.11 0.08 0.10 0.91
1.02 7.40 1.63 0.97 7.56 1.19 2.13
0.12 0.75 0.12 0.59 0.15 0.20
0.12 0.51 0.27 0.22 0.55 0.32 0.16 0.06 0.05 0.11 0.13 0.04 0.12 1.12
1.57 6.18 1.22 0.94 4.96 0.48 2.34
0.17 0.57 0.16 0.10 0.45 0.06 0.20
V. Isidorov et al. / Biochemical Systematics and Ecology 52 (2014) 41–48
45
that bud exudates are very complex mixtures, and not all components of these mixtures can be identified at present. The reason is the lack of available analytical parameters, electron impact mass spectra and/or chromatographic retention indices. However, in certain cases the mass spectrometric data allow to attribute the analytes to a definite class of organic compounds (group identification). In the case of downy birch exudates a positive identification of 91 compounds was possible, and their share in the TIC was about 70%. Compounds with the TIC portion of approximately 25% were tentatively identified as C15H22O (218 a.m.u.) and C15H24O2 (236 a.m.u.) sesquiterpenoids. The latter group was represented by compounds containing one or two –OH groups. Silanization of the former (monohydroxy sesquiterpenoids containing carbonyl- or epoxy-group) gave rise to TMS C18H32O2Si derivatives (308 a.m.u.), whereas that of the latter resulted in C21H40O2Si2 derivatives (380 a.m.u). The fraction of positively identified components extracted from buds of the second species, B. pendula, made up about 30% of TIC. According to mass spectrometric data, approximately 65% of TIC came from TMS derivatives of tetra- and pentacyclic triterpenoids of lanosterine and lupane series. In our investigation, only four commercially available substances were positively identified, tetracyclic b-sitosterol, dammaradien-3-one and dipterocarpol, as well as pentacyclic betulinol (lup-20(29)ene-3a,28-diol). Thus, the compositions of the two white birch exudates differed fundamentally, and these well-marked differences are illustrated on chromatograms in Fig. 1. Table 1 presents group composition, and main or characteristic for individual birch species compounds from these groups. While the above triterpenoids are specific for B. pendula, B. pubescens buds are characterized by the presence of the norsesquiterpenoids birkenal and birkenol (Klika et al., 2004; Domrachev and Tkachev, 2006), as well as sesquiterpene phenylpropenoids. The esters of sesquiterpene alcohols and p-coumaric acid were first identified by Russian authors in extracts from B. pendula buds (Vedernikov et al., 2007). To the best of our knowledge, the esters of sesquiterpenols and ferulic acid, detected by us, have not been identified previously in any natural objects. Another distinguishing feature of the exudate from B. pubescens buds is a high content of flavonoids (up to 45% of TIC) which are represented mainly by flavanones and flavonols. We detected 24 compounds of this class in the investigated samples, with sakuranetin being present in the greatest amount (28 14% of TIC, n ¼ 4). On the other hand, detectable amounts of only three flavonoids, 5,7,4-trihydroxy-4-methoxyflavanone and catechine and of one isomer of trimethoxy quercetine could be identified in B. pendula buds. There are qualitative and quantitative differences between our data on the composition of buds extractive components from the two birch species and earlier findings (Kononenko et al., 1975a,b; Popravko et al., 1979). Firstly, Kononenko et al. Abundance
A
TIC: 120705_B_1.D
450000 400000 350000 300000 250000 200000 150000 100000 50000 5.00
Abundance
10.00
15.00
B
20.00
25.00
30.00
35.00
40.00
25.00
30.00
35.00
40.00
TIC: 120628_B7.D
450000 400000 350000 300000 250000 200000 150000 100000 50000 5.00
10.00
15.00
20.00
Fig. 2. HS-SPME chromatograms of volatiles from downy birch (A) and silver birch (B) buds.
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V. Isidorov et al. / Biochemical Systematics and Ecology 52 (2014) 41–48
(1975a,b) and Popravko et al. (1979) reported a high content of flavonoids in B. pendula buds (they identified 15 flavonoids). Secondly, Vedernikov et al. (2007) detected phenylpropenoids in B. pendula buds, whereas in this study phenylpropenoids were detected only in B. pubescens buds (Table 1). It should be noted that in the cited earlier papers it is unclear as to whether the identification of the species was confirmed: both teams of the Russian authors examined buds of wild birch trees and did not provide any evidence for these species identities. In our opinion the authors erroneously determined the taxonomic categories of the investigated birch trees due to the morphological variability of white birches. We cannot also exclude the fact that the authors might have dealt with natural hybrids of B. pubescens and B. pendula. Such hybrids often occur in regions of cohabitation of these white birch species (Vetchinnikova, 2005). 3.2. Volatile components of birch buds The volatile emission of B. pubescens and B. pendula buds contained 85 and 81 compounds, respectively (Table 2S in the Supplementary Information lists these compounds). In contrast to the ether extracts, the share of positively identified substances was higher in the case of B. pendula (92% of TIC) compared to B. pubescens (79% of TIC) which emitted the above difficult-to-identify C15H22O and C15H24O2 sesquiterpenoids into the gas phase. As can be seen in Figs. 2A and B, the profiles of the chromatograms of both species (their “fingerprints”) are substantially different. Table 2 presents the group composition of volatiles and the relative composition of main (within these groups) compounds. A specific feature of the B. pendula volatiles is a high content of C15H24 sesquiterpene hydrocarbons and a low content of their oxygenated derivatives. On the contrary, the volatiles of B. pubescens are presented mainly by sesquiterpenoids, presumably with a caryophyllane-type skeleton. However, the more distinguishing feature of these species is a high content of nor-sesquiterpenoids, birkenal and birkenol. 3.3. Chemotaxonomic distinction between white birches The results of DNA isolation and sequencing provided a strong evidence for a relationship between the birch trees identified as B. pendula (B-1 and B-2, 100% bootstrap support), and the trees identified as B. pubescens (Bp-1–Bp-3, 94% bootstrap support, Fig. 3A). Table 2 Relative composition (%) of volatile compounds emitted from birch buds. Compounds
Monoterpenoids Including: - Limonene - (E)-b-ocimene - Linalool Sesquiterpene hydrocarbons Including: - a-Cubebene - a-Copaene - b-Caryophyllene - a-Humulene - Aromadendrene - Alloaromadendrene - g-Cadinene - d-Cadinene nor-Sesquiterpenoids including: - Birkenal - Birkenol Sesquiterpenoids Including: - b-Betulenal - 6-Hydroxy-b-caryophyllene - 14-Hydroxy-b-caryophyllene - 6-Hydroxy-b-caryophyllene acetate - 14-Hydroxy-b-caryophyllene acetate - 14-Hydroxy-a-humulene acetate - Caryophyllene oxide - Humulene epoxide II Aliphatic alcohols Aliphatic carbonyls Aliphatic acids ester Aromatics Total a
n.d. – not detected.
B. pendula
B. pubescens
B-1
B-2
B-3
Bp-1
Bp-2
Bp-3
Bp-4
0.93 0.19
4.66 0.84
1.02 0.22
0.55 0.12
0.51 0.12
0.43 0.10
0.22 0.04
0.03 0.02 0.43 0.09 0.07 0.02 86.35 13.82
0.45 0.09 0.21 0.04 1.46 0.03 72.05 11.53
0.41 0.09 0.32 0.09 0.12 0.03 78.02 12.48
0.07 0.02 0.17 0.04 0.07 0.02 17.60 0.98
0.06 0.02 0.09 0.02 0.02 0.01 16.79 3.19
0.09 0.02 0.02 0.01 0.02 0.01 18.87 3.40
0.05 0.03 0.01 9.06
1.16 0.24 15.77 2.84 3.41 0.68 3.97 0.72 11.33 1.93 6.35 1.21 3.22 0.71 6.65 1.33 n.d.
2.08 0.44 18.26 3.29 4.60 1.01 1.26 0.29 15.66 2.82 3.03 0.70 1.34 0.34 2.27 0.50 n.d.
2.14 0.43 17.5 2.80 3.90 0.76 2.18 0.46 13.45 0.84 5.08 0.98 2.14 0.46 4.43 0.93 n.d.
0.12 0.03 0.55 0.12 9.90 1.78 3.54 0.78 n.d.a n.d. n.d. n.d. 18.27 3.00
0.07 0.02 0.45 0.10 11.73 1.99 3.20 0.71 n.d. n.d. n.d. n.d. 14.06 2.39
0.04 0.01 0.47 0.10 13.00 2.60 3.81 0.72 n.d. n.d. n.d. n.d. 14.09 2.61
0.05 0.01 0.26 0.05 6.18 1.01 1.76 0.32 n.d. n.d. n.d. n.d. 11.00 1.98
n.d. n.d. 5.15 1.03
n.d. n.d. 2.34 0.49
n.d. n.d. 3.85 0.77
17.28 2.76 13.23 2.12 13.41 2.41 10.21 1.79 0.99 0.21 0.83 0.19 0.68 0.13 0.79 0.15 58.86 9.42 64.49 10.64 63.07 10.72 87.28 13.62
n.d. n.d. n.d. n.d. n.d. n.d. 2.19 0.99 0.95 4.57 0.68 2.15 91.24
0.93 0.18 10.81 1.73 3.39 0.64 2.32 0.46 24.24 3.88 1.21 0.23 5.25 0.89 1.88 0.37 0.72 0.15 0.85 0.15 0.08 0.02 0.15 0.04 97.08
n.d. n.d. n.d. n.d. n.d. n.d. 1.93 1.93 1.49 2.06 0.10 3.36 99.44
0.38 0.39 0.34 0.39 0.01 0.64
n.d. n.d. n.d. n.d. n.d. n.d. 0.92 0.72 0.37 13.7 0.72 3.24 97.17
0.19 0.17 0.08 2.19 0.14 0.61
0.42 0.21 0.20 0.87 0.15 0.42
1.95 0.39 14.47 2.46 4.95 0.85 3.41 0.75 17.82 2.99 1.20 0.28 5.89 1.09 2.08 0.40 0.69 1.15 0.26 0.06 0.08 0.02 1.46 0.32 98.34
2.02 0.44 13.04 2.42 5.51 1.05 2.91 0.61 20.23 3.05 1.06 0.25 5.50 1.04 2.56 0.50 0.88 0.18 0.27 0.06 0.17 0.04 0.26 0.06 98.04
0.01 0.01 0.01 1.81
6.42 1.16 13.96 2.30 4.63 0.88 4.05 0.88 21.80 3.64 2.84 0.65 5.56 1.05 2.86 0.50 0.42 0.09 0.19 0.05 0.09 0.02 0.55 0.12 97.81
V. Isidorov et al. / Biochemical Systematics and Ecology 52 (2014) 41–48
A
B
47
C
Fig. 3. Dendrograms for the chemical similarity of the birch species by chemotypes (A), composition of bud exudates extracts (B) and volatiles (C). AJ535640.1 and AJ535645.1 are the accessions of B. pendula and B. pubescens, respectively, from GenBank (NCBI).
To establish a relationship between the chemical composition of bud extracts/volatiles, on the one hand, and the taxonomic classification, on the other hand, a cluster analysis was applied to the matrix linking the chemical composition to the species identity. The dendrograms obtained from the cluster analysis (Fig. 3B and C) are clearly divided into two groups similar to the phylogenetic relationships of the trees (Fig. 3A). One group includes buds of B. pendula, whereas the other – buds of B. pubescens. Besides, in spite of the substantial differences in growing conditions, individuals from different populations (Polish and Russian) of the birch species tended to the appropriate genetically confirmed taxons: sample B-3 to silver birches (B-1 and B-2), and sample Be-4 to downy birches (Be-1–Be-3). It would be interesting to observe the lack of differences between the dendrograms obtained from ether extracts and HS-SPME analyses. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bse.2013.12.008 References Can Bas¸er, K.H., Demirci, B., 2007. Studies of Betula essential oils. ARKIVOC 7, 335–348. Demirci, F., Demirci, B., Can Bas¸er, K.H., Güven, K., 2000a. The composition and antifungal bioassay of the essential oils of different Betula species growing in Turkey. Chem. Nat. Comp. 36, 159–165. Demirci, B., Can Bas¸er, K.H., Özek, T., Demirci, F., 2000b. Betulenols from Betula species. Planta Med. 66, 490–493. Demirci, B., Can Bas¸er, K.H., Demirci, F., Hamann, M.T., 2000c. The composition and antifungal bioassay of the essential oils of different Betula species growing in Turkey. J. Nat. Prod. 63, 902–904. Demirci, B., Can Bas¸er, K.H., 2003. Essential oils from the buds of Betula ssp. growing in Turkey. Flavour Fragr. J. 18, 87–90. Domrachev, D.V., Tkachev, A.V., 2006. Absolute configuration of birkenal, a nor-sesquiterpene aldehyde from essential oil of Betula pubescens buds. Chem. Nat. Comp. 42, 304–306. European Pharmacopeia, 2010. 7th 260 Edition, Council of Europe, Strasbourg. Hänsel, R., Hörhammer, L., 1954. Verglechende Untersungen über die Flavonglykoside der Betulaceen. Arch. Pharm. 287, 117–126. Holub, M., Herout, V., Norak, M., Sorm, F., 1959. On terpenes. CIV. The constitution of betulenols from oil from the buds of white birch (Betula alba L.). Coll. Czech. Chem. Commun. 24, 3730–3738. Howland, D.E., Oliver, R.P., Davy, A.J., 1995. Morphological and molecular variation in natural population of Betula. New. Phytol. 130, 117–124.
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Hynynen, J., Niemist, P., Viher-Aarnio, A., Brunner, A., Hein, S., Velling, P., 2010. Silviculture of birch (Betula pendula Roth and Betula pubescens Ehrh.) in northern Europe. Forestry 83, 103–119. Isidorov, V.A., Vinogorova, V.T., 2003. GC-MS analysis of compounds extracted from buds of Populus balsamifera and Populus nigra. Z. Naturforsch 58c, 355–360. Isidorov, V.A., Krajewska, U., Vinogorova, V.T., Vetchinnikova, L.V., Fuksman, I.L., Bal, K., 2004. Gas chromatographic analysis of essential oil from buds of different birch species with preliminary partition of components. Biochem. Syst. Ecol. 32, 1–13. Isidorov, V., Rafa1owski, K., Vinogorova, V., Reduta, L., Fuksman, I., 2006. GC-MS analysis of acidic components in aqueous extracts of coniferous trees’ needles. Chem. Anal. 51, 79–87. Isidorov, V.A., Szczepaniak, L., 2009. Gas chromatographic retention indices of biologically and environmentally important organic compounds on capillary columns with low-polar stationary phases. J. Chromatogr. A 1216, 8998–9007. Keinänen, M., Julkunen-Tiitto, R., 1998. High-performance liquid chromatographic determination of flavonoids in Betula pendula and Betula pubescens leaves. J. Chromatogr. A 793, 370–377. Keinänen, M., Julkunen-Tiitto, R., Rousi, M., Tahvanainen, J., 1999. Taxonomic implications of phenolic variation in leaves of birch (Betula L.) species. Biochem. Syst. Ecol. 27, 243–254. Klika, K.D., Demirci, B., Salminen, J.-P., Ovcharenko, V.V., Vuorela, S., Can Bas¸er, K.H., Pihlaja, K., 2004. New, sesquiterpenoid-type bicyclic compounds from the buds of Betula pubescens – ring-contracted products of b-caryophyllene? Eur. J. Org. Chem., 2627–2635 http://dx.doi.org/10.1002/ejoc.200300808. Kononenko, G.P., Popravko, S.A., Wulfson, N.S., 1975a. Flavonoid aglycones of the white birch (Betula verrucosa) buds. Bioorg. Khim. 1, 506–511 (in Russian). Kononenko, G.P., Popravko, S.A., Wulfson, N.S., 1975b. Secondary metabolites of birch. V. Mass spectrometric method of quantitative analysis of flavonoid aglycones in silver birch buds (Betula verrucosa). Bioorg. Khim 5, 1836–1842 (in Russian). Koshy, T.K., Grant, W.F., Brittain, W.H., 1972. Numerical chemotaxonomy of the Betula caerulea complex.Symp. Biol. Hung. 12, 201. Lahtinen, M., Salminen, J.-P., Kapari, L., Lempa, K., Ossipov, V., Sinkkonen, J., Valkama, E., Haukioja, E., Pihlaja, K., 2004. Defensive effect of surface flavonoids aglycones of Betula pubescens leaves against first instar Epirrita autumnata. larvae. J. Chem. Ecol. 30, 2257–2268. Meurer, B., Wiermann, R., Strack, D., 1988. Phenylopropanoid patterns in Fagales pollen and their phylogenetic relevance. Phytochemistry 27, 823–828. Migalina, S., Ivanova, L., Makhnev, A., 2010. Changes of leaf morphology in Betula pendula Roth and B. pubescens Ehrh. along a zonal-climatic transect in the Urals and Western Siberia. Russ. J. Ecol. 41, 293–301. Orav, A., Arak, E., Boikova, T., Raal, A., 2011. Essential oil in Betula spp. naturally growing in Estonia. Biochem. Syst. Ecol. 39, 744–748. Paw1owska, L., 1983. Biochemical and systematic study of the genus Betula L. Acta Soc. Bot. Pol. 52, 301–314. Popravko, S.A., Kononenko, G.P., Tikhomirova, V.I., Wulfson, N.S., 1979. Secondary metabolites of birch. IV. Identification of the group of flavonoid aglycones in birch buds (Betula verrucosa). Bioorg. Chem. 5, 1662–1667 (in Russian). Szczepaniak, L., Isidorov, V.A., 2011. Gas chromatographic retention indices of trimethylsilyl derivatives of terpene alcohols. J. Chromatogr. A 1218, 7061– 7064. Vedernikov, D.N., Galashkina, N.G., Roshchin, V.I., 2007. The esters of Betula pendula (Betulaceae) buds. Rastit. Resur 43, 84–92 (in Russian). Vedernikov, D.N., Roshchin, V.I., 2009. Extractive components of the buds of white birch Betula pendula Roth. (Betulaceae). The composition of aliphatic acids, hydrocarbons and esters. Khim. Rast. Syr 3, 69–73 (in Russian). Vetchinnikova, L.V., 2005. Karelian Birch and Other Rare Species of Betula L. Genus. Nauka Publ. House, Moscow, p. 302. Wollenweber, E., 1975. Flavonoidmuster im der Betulaceen. Biochem. Syst. Ecol. 3, 47–52.
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Gas chromatographic-mass spectrometric examination of chemical composition of two Eurasian birch (Betula L.) bud exudates and its taxonomical implication b _ Valery Isidorov a, *, Lech Szczepaniak a, Ada Wróblewska b, Ewa Piroznikow , Lidia Vetchinnikova c a b c
Institute of Chemistry, Białystok University, 15-399 Białystok, Poland Institute of Biology, Białystok University, 15-950 Białystok, Poland Forest Research Institute, Karelian Research Centre of RAS, 185910 Petrozavodsk, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2013 Accepted 15 December 2013 Available online 9 January 2014
GC–MS was used to investigate the chemical diversity of bud exudates obtained from two species of white birch in Poland, Betula pendula Roth and Betula pubescens Ehrh. The ether extracts of the bud exudates contained more than 210 organic compounds including: terpenoids, phenylpropenoids of sesquiterpene alcohols and flavonoids. Headspace solidphase microextraction (HS-SPME) made it possible to detect as many as 140 volatile organic compounds (VOCs), mainly sesquiterpene hydrocarbons and their oxygen derivatives. The chemical compositions of both the ether extracts and the VOCs varied qualitatively and quantitatively between the species. The extracts from B. pendula buds contained predominantly triterpenoids. However, B. pubescens exhibited high amounts of sesquiterpenoids, flavonoid aglycones and phenylpropenoids of caryophyllane series sesquiterpenols. The differences in the composition of compounds in the bud exudates enabled the two species of birch to be differentiated. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Gas chromatography-mass spectrometry Birch buds Chemical composition Taxonomical implication
1. Introduction Birch (Betula L.) is an arborous tree found in the forests of the boreal and temperate zones of the Northern Hemisphere. The number of accepted birch species varies from 30 to over 60 (Keinänen et al., 1999), and such a great divergence clearly demonstrates taxonomic problems with this genus. In the European and East Siberian forests, birch is represented mostly by the commercially important species of white birch (Betula alba L. sensu Regel, 1865), silver birch (Betula pendula Roth.) and downy birch (Betula pubescens Ehrh.) (Hynynen et al., 2010). Recent morphological studies of leaf size, shape, and parameters of venation of B. pendula and B. pubescens along a 1600-km latitudinal zonal transect in the Urals and Western Siberia demonstrated their variability and dependence on the geographic location of the population (Migalina et al., 2010). These variations illustrate the problems in distinguishing between these white birches (Howland et al., 1995). To solve these problems, several chemotaxonomic studies have been accomplished (Hänsel and Hörhammer, 1954; Koshy et al., 1972; Wollenweber, 1975; Paw1owska, 1983; Meurer et al., 1988; Keinänen and Julkunen-Tiitto, 1998; Keinänen et al., 1999). The objects of these investigations were generally extracts from leaves, pollen and buds phenolics. More convincing
* Corresponding author. Tel.: þ48 85 745 7805. E-mail address: [email protected] (V. Isidorov). 0305-1978/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2013.12.008
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evidence for taxonomic implication of phenolics was obtained from investigations of foliar flavonoid glycosides (Keinänen and Julkunen-Tiitto, 1998; Keinänen et al., 1999). Chemical studies on birches have also been conducted because of their pharmacological interest. White birches belong to medical plants: bark tar and essential oils are used to cure skin diseases, whereas decoctions and extracts from dried leaves and buds are applied for treatment of inflammation and as diuretics (Hänsel and Hörhammer, 1954; European Pharmacopeia, 2010). For this reason, birch essential oils obtained by hydrodistillation of buds were the subject of numerous investigations (Holub et al., 1959; Demirci et al., 2000a,b,c; Demirci and Can Bas¸er, 2003; Isidorov et al., 2004; Klika et al., 2004; Can Bas¸er and Demirci, 2007; Orav et al., 2011). In contrast, only few works dealt with the chemical composition of extracts from birch buds (Kononenko et al., 1975a,b; Popravko et al., 1979; Vedernikov et al., 2007; Vedernikov and Roshchin, 2009) while there is virtually no information on compounds contained in birch bud exudates which may form the first line of plant protection against microorganisms and herbivores (Lahtinen et al., 2004). The main purpose of this study was to determine the composition of compounds in volatiles and extracts of the buds of two species of birch, B. pendula and B. pubescens and evaluate whether they could be used to differentiate between the species. 2. Experimental 2.1. Chemicals Triterpenoids, dammaradien-3-one, dipterocarpol and betulenol as well as pyridine and bis(trimethylsilyl)tri , Poland). Diethyl fluoroacetamide (BSTFA) containing 1% trimethylchlorosilane were purchased from Sigma–Aldrich (Poznan ether (POCH SA, Gliwice, Poland) was used after distillation. SPME Holder 57330-U and fused silica fibers with divinylbenzene/carboxen/polydimethylsiloxane (50/30 mm) stationary phase were purchased from Supelco Inc. (Bellefonte, PA, USA). 2.2. Plant material Buds of B. pendula (samples B-1 and B-2) and B. pubescens (samples Bp-1–Bp-3) were collected at the beginning of July, 2012 in the Biebrza National Park in northeastern Poland (53 320 N, 22 430 E). Additionally, buds of B. pendula (sample B-3) and B. pubescens (sample Bp-4) were collected from trees growing on the experimental field station of the Forest Research Institute near Petrozavodsk (Russia). Voucher specimens were deposited with the herbarium of the Geobotanical Station of Warsaw University (Bialowieza) and herbarium of the Biological Department of Petrozavodsk University. 2.3. Sample preparation and GC–MS analysis Buds (1–2 g) from each of the birch species were extracted by rinsing for 60 s in 15 mL of diethyl ether. The exudate extract was filtered through a paper filter and the solvent was evaporated to dryness on a rotor evaporator. About 5 mg of the residue was diluted with 220 mL of dry pyridine and 80 mL of BSTFA was added. The mixture was heated for 30 min at 60 C to form trimethylsilyl (TMS) derivatives. Volatiles from birch buds were determined by HS-SPME/GC–MS. 10 to 15 buds were transferred into a head-space vial of 16 mL in volume and immersed into a temperature controlled water bath (40 C). The septum of a screw-cap was picked by a needle protecting the SPME fiber, and the fiber coating was exposed to the headspace gas phase for 60 min. To improve the sorption conditions, the buds were constantly stirred. The volatiles collected on the fiber were desorbed by introducing the SPME fiber for 15 min into the injection port of the GC–MS apparatus. The analytes were separated on the Agilent 6890 gas chromatograph equipped with the MSD 5973 mass selective detector fitted with an electronic pressure control and a split/splitless injector. The separation was performed on the HP-5ms (30 m 0.25 mm; 0.25 mm film thickness) fused silica column at a helium flow rate of 1 mL/min. The initial column temperature was 40 C (3 min), rising to 220 C at 3 C/min. The injector worked in a splitless mode; the injector temperature was 250 C. The EIMS spectra were obtained at ionization energy of 70 eV, the source temperature being 230 C and that of the quadrulope being 150 C. Detection was performed in a full scan mode from 29 to 300 a.m.u. After integration, the fraction of each component in the total ion current (TIC) was calculated. To determine the retention times of reference compounds and to calculate linear temperature programmed retention indices (IT) of the analytes, the SPME fiber was inserted for 2–3 s into a vial with a mixture of C6–C18 n-alkanes. The separation of the alkanes was performed under the above conditions. TMS derivatives of the extracted compounds were separated and analyzed with the same GC–MS apparatus and column. Injection of 1 mL of the sample was performed with the HP 7683 autosampler. The injector worked in a split (1:50) mode. The MSD was set to scan at 40–600 a.m.u. Chromatograms were registered in a linear temperature programmed regime from 50 C to 320 C at the rate of 3 C/min. The extractions were repeated three times, so the precision of the method (expressed by a relative standard deviation, R.S.D) could be studied. The peak areas of the components obtained by replicate analyses were used for calculation of their R.S.D. values, which amounted to an average of 8% and 17% in assaying TMS derivatives and volatile compounds, respectively. The fairly high values for HS-SPME relative standard deviations may be due to a less precise procedure of compound extraction.
V. Isidorov et al. / Biochemical Systematics and Ecology 52 (2014) 41–48
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The hexane solution of C10–C40 n-alkanes was separated under the above conditions. Linear temperature programmed retention indices were calculated from the results for this mixture and for the solutions of TMS derivatives of components extracted from birch buds surface. 2.4. Component identification To identify the components, both mass spectral data and the calculated retention indices were used. Mass spectrometric identification was carried out with an automatic system of GC–MS data processing supplied by NIST and home-made mass spectra libraries. The retention indices of the registered components were compared with those presented in the authors’
A Abundance TIC: Bet_Pubes1.D
2800000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000
Time-->
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40.00
50.00
60.00
70.00
80.00
90.00
B Abundance
TIC: 120218bca08.D
2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000
Time-->
30.00
35.00
40.00
45.00
50.00
55.00
60.00
65.00
70.00
75.00
80.00
85.00
Fig. 1. Chromatograms of the ether extracts of downy birch (A) and silver birch (B) buds.
90.00
95.00
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computer database containing IT values for more than 5000 compounds including more than 800 indices for TMS derivatives. This database contains our previous results (Isidorov and Vinogorova, 2003; Isidorov et al., 2006; Isidorov and Szczepaniak, 2009; Szczepaniak and Isidorov, 2011). The identification was considered reliable if the results of computer search at the mass spectra library were confirmed by the experimental IT values (if their deviation from the database values did not exceed 5 u.i.). 2.5. DNA isolation and sequencing The leaf fragments of B. pendula (B-1 and B-2) and B. pubescens (Bp-1–Bp-3) were dried in silica gel and then the genomic DNA was extracted. The plant material also consisted of two accessions of B. pendula (AJ535640.1) and B. pubescens (AJ535645.1) from the GenBank (NCBI). Alcohol dehydrogenase (ADH) was used to study the nuclear DNA sequences in the Betula genus. The procedure of DNA isolation and sequencing as well as the sequence alignment and phylogenetic analysis are presented in the Supplementary Information. 3. Results and discussion 3.1. Extractive components of birch buds The ether extracts of bud exudate from both birch species demonstrated 204 compounds, with the content being not less than 0.01% of TIC (Table 1S in the Supplementary Information provides a complete list of these components). Hence it follows Table 1 Relative composition (%) of birch buds exudate. 0.02 Compounds
nor-Sesquiterpenoids Including: - Birkenal - Birkenol Sesquiterpenoids Including: - b-Betulenol - 6-Hydroxy-b-caryophyllene - 14-Hydroxy-b-caryophyllene - 6-hydroxy-b-caryophyllene acetate - 14-Hydroxy-b-caryophyllene acetate - 14-Hydroxy-4,5-epoxy-b-caryophyllene acetate Triterpenoids Including: - Dammaradien-3-one - Dipterocarpol - Betulinol Phenylpropenoids Including: - p-Coumaric acid - 6-Hydroxy-b-caryophyllene p- coumarate - 14-Hydroxy-b-caryophyllene p-coumarate - 14-Hydroxy-b-caryophyllene ferulate - 14-Hydroxy-b-isocaryophylle-ne ferulate Flavonoids Including: - 5-Hydroxy-40 ,7-dimethoxyfla-vanone - Sakuranetin - Homoeriodictyol - Pectolinaringein - Acacetin - Kaempherid - Kaempherol - Apigenin - Catechin - 5,7,40 -Trihydroxy-4-methoxyfla-vanone Aliphatic acids Aliphatic alcohols Alkanes NNc Total b
trace – below 0.01% of TIC. a n.d. – not detected. c NN – not identified compounds.
B. pendula
B. pubescens
B-1
B-2
B-3
Bp-1
Bp-2
Bp-3
Bp-4
n.d.a
n.d.
n.d.
0.27 0.02
0.34 0.03
0.34 0.02
0.97 0.09
n.d. n.d. 0.11 0.02
n.d. n.d. 0.63 0.06
n.d. n.d. 0.59 0.06
0.22 0.02 0.31 0.03 0.27 0.02 0.97 0.09 0.05 0.01 0.04 0.01 0.07 0.01 n.d. 32.13 1.93 34.28 2.05 37.75 4.70 12.67 1.58
n.d. n.d. n.d. n.d. n.d. n.d. 90.23 8.12
n.d. n.d. n.d. n.d. n.d. n.d. 89.45 6.26
n.d. n.d. n.d. n.d. n.d. n.d. 84.52 5.49
1.66 7.76 1.52 0.40 3.85 0.30 2.68
0.72 0.07 17.21 1.12 2.14 0.17 n.d.
1.74 0.21 31.11 1.87 1.60 0.15 n.d.
2.07 0.23 19.84 1.14 1.49 0.13 Trace
n.d. 0.11 0.02 n.d. 14.67 1.61
n.d. n.d. n.d. 10.91 1.10
n.d. 0.03 0.01 n.d 6.36 0.76
n.d. n.d. n.d. 5.39 0.67
n.d. n.d. n.d. n.d. n.d. 1.40 0.15
n.d. n.d. n.d. n.d. n.d. 0.93 0.07
Trace n.d. n.d. n.d. n.d. 2.71 0.24
0.58 0.05 5.41 0.41 3.07 0.25 0.15 0.02 0.23 0.03 40.59 2.84
0.54 0.05 2.83 0.24 2.76 0.25 0.20 0.03 0.22 0.02 37.88 3.60
0.33 0.03 2.93 0.23 1.82 0.17 0.16 0.02 0.18 0.02 39.09 2.74
0.93 0.09 2.17 0.19 1.49 0.14 0.11 0.03 0.21 0.02 45.12 2.93
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.03 1.37 0.76 0.75 1.30 2.19 96.74
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.11 0.82 1.56 0.51 1.41 1.30 96.78
n.d. Trace n.d. 0.13 0.09 n.d. 0.04 n.d. 0.05 0.67 6.69 0.61 1.67 2.06 99.91
4.45 0.36 11.36 0.74 3.00 0.24 3.72 0.32 2.16 0.20 3.62 0.32 0.89 0.12 0.31 0.04 0.32 0.04 n.d. 1.62 0.13 0.68 0.06 1.96 0.11 4.68 0.56 99.86
2.22 5.69 3.25 2.19 4.33 2.19 1.81 0.92 0.66 n.d. 1.33 0.68 1.11 6.49 95.62
1.66 7.90 3.38 2.80 7.20 3.58 1.46 0.56 0.38 0.98 1.85 0.37 1.54 9.32 98.98
0.55 0.06 21.70 1.48 2.91 0.23 2.04 0.16 0.77 0.08 4.07 0.30 1.60 0.13 0.20 0.03 1.45 0.11 Trace 1.49 0.12 0.27 0.03 1.73 0.16 7.89 0.87 95.68
0.01 0.11 0.91 0.10 0.12 0.28
0.07 0.12 0.06 0.13 0.14
0.02 0.02 0.01 0.02 0.05 0.50 0.07 0.14 0.23
0.13 0.62 1.14 0.04 0.29 0.04 0.27
1.80 8.79 2.15 0.36 4.96 0.41 1.11
0.16 0.57 0.18 0.04 0.40 0.04 0.10
0.18 0.38 0.26 0.20 0.30 0.22 0.14 0.10 0.73 0.11 0.08 0.10 0.91
1.02 7.40 1.63 0.97 7.56 1.19 2.13
0.12 0.75 0.12 0.59 0.15 0.20
0.12 0.51 0.27 0.22 0.55 0.32 0.16 0.06 0.05 0.11 0.13 0.04 0.12 1.12
1.57 6.18 1.22 0.94 4.96 0.48 2.34
0.17 0.57 0.16 0.10 0.45 0.06 0.20
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that bud exudates are very complex mixtures, and not all components of these mixtures can be identified at present. The reason is the lack of available analytical parameters, electron impact mass spectra and/or chromatographic retention indices. However, in certain cases the mass spectrometric data allow to attribute the analytes to a definite class of organic compounds (group identification). In the case of downy birch exudates a positive identification of 91 compounds was possible, and their share in the TIC was about 70%. Compounds with the TIC portion of approximately 25% were tentatively identified as C15H22O (218 a.m.u.) and C15H24O2 (236 a.m.u.) sesquiterpenoids. The latter group was represented by compounds containing one or two –OH groups. Silanization of the former (monohydroxy sesquiterpenoids containing carbonyl- or epoxy-group) gave rise to TMS C18H32O2Si derivatives (308 a.m.u.), whereas that of the latter resulted in C21H40O2Si2 derivatives (380 a.m.u). The fraction of positively identified components extracted from buds of the second species, B. pendula, made up about 30% of TIC. According to mass spectrometric data, approximately 65% of TIC came from TMS derivatives of tetra- and pentacyclic triterpenoids of lanosterine and lupane series. In our investigation, only four commercially available substances were positively identified, tetracyclic b-sitosterol, dammaradien-3-one and dipterocarpol, as well as pentacyclic betulinol (lup-20(29)ene-3a,28-diol). Thus, the compositions of the two white birch exudates differed fundamentally, and these well-marked differences are illustrated on chromatograms in Fig. 1. Table 1 presents group composition, and main or characteristic for individual birch species compounds from these groups. While the above triterpenoids are specific for B. pendula, B. pubescens buds are characterized by the presence of the norsesquiterpenoids birkenal and birkenol (Klika et al., 2004; Domrachev and Tkachev, 2006), as well as sesquiterpene phenylpropenoids. The esters of sesquiterpene alcohols and p-coumaric acid were first identified by Russian authors in extracts from B. pendula buds (Vedernikov et al., 2007). To the best of our knowledge, the esters of sesquiterpenols and ferulic acid, detected by us, have not been identified previously in any natural objects. Another distinguishing feature of the exudate from B. pubescens buds is a high content of flavonoids (up to 45% of TIC) which are represented mainly by flavanones and flavonols. We detected 24 compounds of this class in the investigated samples, with sakuranetin being present in the greatest amount (28 14% of TIC, n ¼ 4). On the other hand, detectable amounts of only three flavonoids, 5,7,4-trihydroxy-4-methoxyflavanone and catechine and of one isomer of trimethoxy quercetine could be identified in B. pendula buds. There are qualitative and quantitative differences between our data on the composition of buds extractive components from the two birch species and earlier findings (Kononenko et al., 1975a,b; Popravko et al., 1979). Firstly, Kononenko et al. Abundance
A
TIC: 120705_B_1.D
450000 400000 350000 300000 250000 200000 150000 100000 50000 5.00
Abundance
10.00
15.00
B
20.00
25.00
30.00
35.00
40.00
25.00
30.00
35.00
40.00
TIC: 120628_B7.D
450000 400000 350000 300000 250000 200000 150000 100000 50000 5.00
10.00
15.00
20.00
Fig. 2. HS-SPME chromatograms of volatiles from downy birch (A) and silver birch (B) buds.
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(1975a,b) and Popravko et al. (1979) reported a high content of flavonoids in B. pendula buds (they identified 15 flavonoids). Secondly, Vedernikov et al. (2007) detected phenylpropenoids in B. pendula buds, whereas in this study phenylpropenoids were detected only in B. pubescens buds (Table 1). It should be noted that in the cited earlier papers it is unclear as to whether the identification of the species was confirmed: both teams of the Russian authors examined buds of wild birch trees and did not provide any evidence for these species identities. In our opinion the authors erroneously determined the taxonomic categories of the investigated birch trees due to the morphological variability of white birches. We cannot also exclude the fact that the authors might have dealt with natural hybrids of B. pubescens and B. pendula. Such hybrids often occur in regions of cohabitation of these white birch species (Vetchinnikova, 2005). 3.2. Volatile components of birch buds The volatile emission of B. pubescens and B. pendula buds contained 85 and 81 compounds, respectively (Table 2S in the Supplementary Information lists these compounds). In contrast to the ether extracts, the share of positively identified substances was higher in the case of B. pendula (92% of TIC) compared to B. pubescens (79% of TIC) which emitted the above difficult-to-identify C15H22O and C15H24O2 sesquiterpenoids into the gas phase. As can be seen in Figs. 2A and B, the profiles of the chromatograms of both species (their “fingerprints”) are substantially different. Table 2 presents the group composition of volatiles and the relative composition of main (within these groups) compounds. A specific feature of the B. pendula volatiles is a high content of C15H24 sesquiterpene hydrocarbons and a low content of their oxygenated derivatives. On the contrary, the volatiles of B. pubescens are presented mainly by sesquiterpenoids, presumably with a caryophyllane-type skeleton. However, the more distinguishing feature of these species is a high content of nor-sesquiterpenoids, birkenal and birkenol. 3.3. Chemotaxonomic distinction between white birches The results of DNA isolation and sequencing provided a strong evidence for a relationship between the birch trees identified as B. pendula (B-1 and B-2, 100% bootstrap support), and the trees identified as B. pubescens (Bp-1–Bp-3, 94% bootstrap support, Fig. 3A). Table 2 Relative composition (%) of volatile compounds emitted from birch buds. Compounds
Monoterpenoids Including: - Limonene - (E)-b-ocimene - Linalool Sesquiterpene hydrocarbons Including: - a-Cubebene - a-Copaene - b-Caryophyllene - a-Humulene - Aromadendrene - Alloaromadendrene - g-Cadinene - d-Cadinene nor-Sesquiterpenoids including: - Birkenal - Birkenol Sesquiterpenoids Including: - b-Betulenal - 6-Hydroxy-b-caryophyllene - 14-Hydroxy-b-caryophyllene - 6-Hydroxy-b-caryophyllene acetate - 14-Hydroxy-b-caryophyllene acetate - 14-Hydroxy-a-humulene acetate - Caryophyllene oxide - Humulene epoxide II Aliphatic alcohols Aliphatic carbonyls Aliphatic acids ester Aromatics Total a
n.d. – not detected.
B. pendula
B. pubescens
B-1
B-2
B-3
Bp-1
Bp-2
Bp-3
Bp-4
0.93 0.19
4.66 0.84
1.02 0.22
0.55 0.12
0.51 0.12
0.43 0.10
0.22 0.04
0.03 0.02 0.43 0.09 0.07 0.02 86.35 13.82
0.45 0.09 0.21 0.04 1.46 0.03 72.05 11.53
0.41 0.09 0.32 0.09 0.12 0.03 78.02 12.48
0.07 0.02 0.17 0.04 0.07 0.02 17.60 0.98
0.06 0.02 0.09 0.02 0.02 0.01 16.79 3.19
0.09 0.02 0.02 0.01 0.02 0.01 18.87 3.40
0.05 0.03 0.01 9.06
1.16 0.24 15.77 2.84 3.41 0.68 3.97 0.72 11.33 1.93 6.35 1.21 3.22 0.71 6.65 1.33 n.d.
2.08 0.44 18.26 3.29 4.60 1.01 1.26 0.29 15.66 2.82 3.03 0.70 1.34 0.34 2.27 0.50 n.d.
2.14 0.43 17.5 2.80 3.90 0.76 2.18 0.46 13.45 0.84 5.08 0.98 2.14 0.46 4.43 0.93 n.d.
0.12 0.03 0.55 0.12 9.90 1.78 3.54 0.78 n.d.a n.d. n.d. n.d. 18.27 3.00
0.07 0.02 0.45 0.10 11.73 1.99 3.20 0.71 n.d. n.d. n.d. n.d. 14.06 2.39
0.04 0.01 0.47 0.10 13.00 2.60 3.81 0.72 n.d. n.d. n.d. n.d. 14.09 2.61
0.05 0.01 0.26 0.05 6.18 1.01 1.76 0.32 n.d. n.d. n.d. n.d. 11.00 1.98
n.d. n.d. 5.15 1.03
n.d. n.d. 2.34 0.49
n.d. n.d. 3.85 0.77
17.28 2.76 13.23 2.12 13.41 2.41 10.21 1.79 0.99 0.21 0.83 0.19 0.68 0.13 0.79 0.15 58.86 9.42 64.49 10.64 63.07 10.72 87.28 13.62
n.d. n.d. n.d. n.d. n.d. n.d. 2.19 0.99 0.95 4.57 0.68 2.15 91.24
0.93 0.18 10.81 1.73 3.39 0.64 2.32 0.46 24.24 3.88 1.21 0.23 5.25 0.89 1.88 0.37 0.72 0.15 0.85 0.15 0.08 0.02 0.15 0.04 97.08
n.d. n.d. n.d. n.d. n.d. n.d. 1.93 1.93 1.49 2.06 0.10 3.36 99.44
0.38 0.39 0.34 0.39 0.01 0.64
n.d. n.d. n.d. n.d. n.d. n.d. 0.92 0.72 0.37 13.7 0.72 3.24 97.17
0.19 0.17 0.08 2.19 0.14 0.61
0.42 0.21 0.20 0.87 0.15 0.42
1.95 0.39 14.47 2.46 4.95 0.85 3.41 0.75 17.82 2.99 1.20 0.28 5.89 1.09 2.08 0.40 0.69 1.15 0.26 0.06 0.08 0.02 1.46 0.32 98.34
2.02 0.44 13.04 2.42 5.51 1.05 2.91 0.61 20.23 3.05 1.06 0.25 5.50 1.04 2.56 0.50 0.88 0.18 0.27 0.06 0.17 0.04 0.26 0.06 98.04
0.01 0.01 0.01 1.81
6.42 1.16 13.96 2.30 4.63 0.88 4.05 0.88 21.80 3.64 2.84 0.65 5.56 1.05 2.86 0.50 0.42 0.09 0.19 0.05 0.09 0.02 0.55 0.12 97.81
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A
B
47
C
Fig. 3. Dendrograms for the chemical similarity of the birch species by chemotypes (A), composition of bud exudates extracts (B) and volatiles (C). AJ535640.1 and AJ535645.1 are the accessions of B. pendula and B. pubescens, respectively, from GenBank (NCBI).
To establish a relationship between the chemical composition of bud extracts/volatiles, on the one hand, and the taxonomic classification, on the other hand, a cluster analysis was applied to the matrix linking the chemical composition to the species identity. The dendrograms obtained from the cluster analysis (Fig. 3B and C) are clearly divided into two groups similar to the phylogenetic relationships of the trees (Fig. 3A). One group includes buds of B. pendula, whereas the other – buds of B. pubescens. Besides, in spite of the substantial differences in growing conditions, individuals from different populations (Polish and Russian) of the birch species tended to the appropriate genetically confirmed taxons: sample B-3 to silver birches (B-1 and B-2), and sample Be-4 to downy birches (Be-1–Be-3). It would be interesting to observe the lack of differences between the dendrograms obtained from ether extracts and HS-SPME analyses. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bse.2013.12.008 References Can Bas¸er, K.H., Demirci, B., 2007. Studies of Betula essential oils. ARKIVOC 7, 335–348. Demirci, F., Demirci, B., Can Bas¸er, K.H., Güven, K., 2000a. The composition and antifungal bioassay of the essential oils of different Betula species growing in Turkey. Chem. Nat. Comp. 36, 159–165. Demirci, B., Can Bas¸er, K.H., Özek, T., Demirci, F., 2000b. Betulenols from Betula species. Planta Med. 66, 490–493. Demirci, B., Can Bas¸er, K.H., Demirci, F., Hamann, M.T., 2000c. The composition and antifungal bioassay of the essential oils of different Betula species growing in Turkey. J. Nat. Prod. 63, 902–904. Demirci, B., Can Bas¸er, K.H., 2003. Essential oils from the buds of Betula ssp. growing in Turkey. Flavour Fragr. J. 18, 87–90. Domrachev, D.V., Tkachev, A.V., 2006. Absolute configuration of birkenal, a nor-sesquiterpene aldehyde from essential oil of Betula pubescens buds. Chem. Nat. Comp. 42, 304–306. European Pharmacopeia, 2010. 7th 260 Edition, Council of Europe, Strasbourg. Hänsel, R., Hörhammer, L., 1954. Verglechende Untersungen über die Flavonglykoside der Betulaceen. Arch. Pharm. 287, 117–126. Holub, M., Herout, V., Norak, M., Sorm, F., 1959. On terpenes. CIV. The constitution of betulenols from oil from the buds of white birch (Betula alba L.). Coll. Czech. Chem. Commun. 24, 3730–3738. Howland, D.E., Oliver, R.P., Davy, A.J., 1995. Morphological and molecular variation in natural population of Betula. New. Phytol. 130, 117–124.
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