HPLC / MS-TOF Analysis of Surface Resins from Three Poplar Clones Grown in Serbia

DOI: https://doi.org/10.15177/seefor.16-12 (1) University of Novi Sad, Institute of Lowland Forestry and Environment, Antona Čehova 13d, RS-21000 Novi Sad, Serbia; (2) University of Belgrade, Faculty of Chemistry, Centre for Instrumental Analysis, Studentski trg 12-16, RS-11158, Belgrade, Serbia; (3) University of Belgrade, Institute of Chemistry, Technology and Metallurgy, Njegoševa 12, RS-11000, Belgrade, Serbia *Correspondence: e-mail: btrudic@uns.ac.rs © 2016 by the Croatian Forest Research Institute. This is an Open Access paper distributed under the terms of the Cr ative Commons Attribution License (http://creativecommons.org/licenses/by/4.0). inTRODUCTiOn The genus Populus from Salicaceae family consists of about 30 species, growing in southern and central Europe, in central Asia, Siberia, and North America, characterized and differentiated by the presence of different flavonoids, phenolic derivatives, and terpenoids in particular in young leaves, buds, and bud exudates [1]. Several species of Populus have been traditionally used in medicine, especially for their antiinflammatory properties [2]. Nowadays, large-scale poplar production is based on clone culture production of controlled hybrids. The complex chemistry of poplar bud exudates includes about a hundred different phenolic and terpenoid compounds [3], several of which are involved in biological processes [1]. Poplar biomass is a potential resource for natural production of bioactive molecules which could be substituted for some synthetic compounds associated with human and animal medicine [1]. Buds from Populus species are the main source of resins in propolis in Europe and North America (“poplar type” propolis) [4]. Resins comprising flavonoids and related phenolic acids represent approximately half of the propolis constituents [5]. Still, the chemical composition of the bee glue is extremely dependent on the plants found around the hive, as well on the geographic and climatic characteristics of the site. Citation: TRUDIĆ B, ANĐELKOVIĆ B, ORLOVIĆ S, TEŠEVIĆ V, PILIPOVIĆ A, CVETKOVIĆ M, STANKOVIĆ J 2016 HPLC/ MS-TOF Analysis of Surface Resins from Three Poplar Clones Grown in Serbia. South-east Eur for 7 (2): 129-133. DOI: https://doi.org/10.15177/seefor.16-12 Received: 26 Jul 2016; Revised: 26 Aug 2016; Accepted: 29 Aug 2016; Published

Less commonly, species such as Betula, Ulmus, Pinus, Quercus, Salix and Acacia are also used in the production of propolis by bees [6,7].For the first time, the chemical composition of the cuticular waxes of poplar clones from Serbia was done and the results of GC/MS analysis showed that there are no significant differences in presence of alkanes: n-nonacosane, n-hexacosane, n-untriacosane and n-octacosane within leaf wax of these three clones [8].
Chemical content of the surface resins from three poplar clones was in the focus of this research.This type of chemical analysis of the surface resins from any fast-growing tree species from Serbia has previously not been undertaken.

The Sampling of Plant Material
Five terminal twigs cuttings with healthy leaves of different development stage from two year old seedlings of M1 (Panonnia) poplar clone (Populus euramericana L.), cl.PE 19/66 and cl.B229 (both belonging to Populus deltoides) were sampled at the same time from the nursery collection of the Institute of Lowland Forestry and Environment, University of Novi Sad.The collections were grown as separate plantations, consisting of seedlings of the same age and origin, and influenced by the same climatic and weather conditions since they have been grown in the open.The two Populus deltoides clones B-229 and PE 19/66, and the hybrid P. x canadensis clone M-1 have previously been reported to be suitable for high biomass plantations [9][10][11].Populus deltoides clones such as PE 19/66 were showed greater net photosynthesis than hybrid poplars (e.g.M-1), but hybrids were superior in water use efficiency [12].The collections are part of the experimental Kać Forest estate located north-east from the city of Novi Sad, near the Kać village (N45° 17' 41" E19° 53' 30").The genetic structure of the M1 and B229 clones has previously been determined through SSR and AFLP marker system and published, confirming the specific DNA profile of two different species originating from this nursery collection [13].As the objective of the study was to examine the chemical composition and not the variability between single plants, the results were obtained for one pooled sample of terminal twigs cuttings with leaves from five plants per cultivar.

Preparation of Resin Extracts
Terminal twigs cuttings were immediately transported in sterile plastic bottles (5 mL volumes) on ice to laboratories of the Centre for Instrumental Analysis in Belgrade.Poplar clones' surface resins were washed out from the terminal shoots with 10 mL dichlormethane.Afterwards, the extracts were evaporated under a stream of N 2 for 23 minutes and dissolved in methanol at a concentration of 10 mg/mL, respectively.

HPLC/MS-TOF Analysis
High-resolution Liquid Chromatography/Photo-Diode Array/Electro Spray/Time of Flight mass spectra (HRLC/PDA/ ESI/TOF MS) were measured on a HPLC instrument (Agilent 1200 Series) equipped with an autosampler, using a Zorbax Eclipse Plus C18 analytical column (1.8 μm particle size, 4.6 × 150 mm i.d., Agilent Technologies), and a PDA detector (DAD) coupled with a 6210 TOF LC/MS system (Agilent Technologies).
Exact mass measurements of pseudomolecular ions of analytes performed with a time-of-flight (TOF) mass spectrometer in negative polarity mode enabled the determination of molecular formula of most of the constituents.All identified compounds exhibited quasi-molecular ion [M-H]¯ in the negative mode, confirming the molecular mass.Peak identification was mainly tentative, by comparison of their retention time, mass, and UV spectra with the literature data.For nine compounds we had standards for additional conformation of identification.

ReSULTS AnD DiSCUSSiOn
Thirty-eight different compounds in resin extract of M1 clone and 24 common compounds within resin extract of B229 and PE 19/66 clones (Table 1, Figure 1) were identified.Fourteen compounds were unique for M1 clone, giving possibility to use them as potential species specific chemotaxonomic markers.Those are the compounds belonging to the caffeic, coumaryl and pinobaksin groups of compounds.
Our HPLC/MS-TOF analysis confirmed that Serbian poplar clones mainly consists of three groups of phenolic compounds: phenolic acids (caffeic acid, p-coumaric acid, cynammic acid), phenolic esters, and flavonoid aglycons of flavanones (pinocembrin, pinobaksin), flavonols (galangin, quercaetin and kaempferol), flavones (chrysin and its derivates, apigenin).We have also identified salycilate like populin.The results obtained in the experiment were compared with literature data on P. nigra HPLC bud and propolis analysis in the following text.Black poplar buds are coated with a viscous substance, an exudate which contains different varieties of phenolic compounds: flavonoid aglycons and their chalcones and phenolic acids and their esters [2].Also, the chemical characterization of bud exudate has allowed the identification among the flavonoid aglycons of some flavanones such as pinocembrin and pinostrobin, some flavonols such as galangin, quercetin and kaempferol, some flavones such as chrysin and apigenin [14][15][16] and some esters of phenolic acids, similar to our data when it comes to the phenolic content.Such compounds have also been reported in propolis [17].Bud extract of P. nigra was mainly composed of phenolic acids (caffeic, p-coumaric, ferulic, TAbLe 1.A list of identified compounds within surface resins of three poplar clones.Abbreviation NI means Not Identified.Compounds marked with * were previously reported in Trudić et al. [20].Bolded compounds are compounds identified by internal standards.isoferulic, di-O-methyl caffeic and cinnamic acids) (5.2%), followed by salicylates (salicin) (1.8%) and flavonoid aglycons (pinobaksin 5-methyl ethar, pinobaksin and pinocembrin) (1.5%) [18].

COnCLUSiOnS
Direct assessment of antioxidant activity of resins is required through FRAP, DPPH, ORAC, ABTS, lipid peroxidation and other tests to screen its radical scavenging capacity and correlate it with quantity analysis of its bioactive compounds.In this case, 70% ethanol should be used as a common and nontoxic solvent for extraction.It is also possible that isomers can be present, although further NMR analysis must be performed.
The results presented in this study shows application in:  1.
− Chemical compounds identified so far can be applied in pharmaceutical research and thus involve more nursery production of poplar clones' biomass as a resource of those molecules; − To understand the chemical profile of propolis, since resins are the starting material for its production by bees; − Chemotaxonomy, since we confirmed that there is a quite different chemical profile of surface resins between two species of poplars from our collection.In our study, 14 compounds were specifically characteristic for M1 clone (P.euramericana L) surface resin extract, indicating their species-specific significance.However, further comparative study with other poplar species is needed to determine the presence/absence of those compounds within genus.− To monitor and predict poplar's biomass pharmacological potential and label them as a nursery with ecosystem service significance.

FiGURe 1 .
FiGURe 1. DAD chromatograms of identified resin compounds measured on 280 nm, 65 minutes.Peak numbers refer to those reported in Table1.
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