Betula pendula Roth is one of the most common Betulaceae species in the world. It is broadly distributed in Europe, Asia, and North Africa and especially in northern Siberian forest-steppes. Leaves of B. pendula produce a variety of biologically active compounds such as phenylpropanoids, terpenoids, steroids, amino acids, carbohydrates, and fatty compounds that fulfill energy, structural, protective, regulatory, signaling, and other functions.

The qualitative and quantitative compositions of these compounds in addition to their dynamics, especially during early development, are extremely poorly studied [1]. The generation and analysis of such data is a critical problem for the chemistry of natural compounds and ecological chemistry. B. pendula in western Siberia is the principal food source of Lymantria dispar L., caterpillars of which can defoliate large areas of birch forest plantings and other woody plants. Young leaves are typically tender and have high contents of amino acids and water. This makes them attractive food sources for folivores and can help to increase their population [2].

Triterpenoids represent one of the principal groups of nonpolar and weakly polar secondary metabolites of B. pendula leaves, are involved in plant metabolic processes, and are key biosynthetic compounds for biologically active compounds such as vitamins, enzymes, steroidal hormones, etc. Fatty acids from phospholipids are used to construct biological membranes. Flavonoids bind to non-phenolic compounds in cell walls, help to strengthen them and thereby prevent intrusion of pathogens and uncontrolled water loss, and reduce the risk of photo-oxidative cell damage due to their antioxidant properties. Triterpenoids and flavonoids are also natural antifeedants that reduce the attractiveness and food value of plant tissues to folivores.

The chemical composition of woody-plant leaf secondary metabolites is also known to vary considerably during the whole growing season [3]. Herein, the dynamics of the contents of several biologically active compounds in B. pendula leaves during the first 20 d after their emergence are studied. This leaf development interval encompasses the minimum and maximum observed times of L. dispar larva appearance after the winter diapause when the insects start to feed on leaves. The results for the composition dynamics of biologically active metabolites from B. pendula leaves, especially flavonoids and triterpenoids with antifeedant activity, can be used to develop methods for predicting and controlling the L. dispar population.

Raw material collected in summer 2012 in Karasuksky District, Novosibirsk Oblast (53.42° N and 77.45° E) was used to investigate the dynamics of the contents of secondary metabolites from B. pendula leaves. Leaves were collected first at the start of bud emergence, i.e., the optimum phase for L. dispar feeding. The next collection occurred from 5 to 20 d after the start of emergence. The collection was described before in more detail [4]. Polar phenolic compounds were determined in aqueous EtOH and aqueous Me2CO extracts; nonpolar and weakly polar compounds, in the MeOH extract.

The contents of principal groups of lipophilic compounds from the MeOH extract of B. pendula leaves were identified and determined using GC-MS by comparing experimental and library mass spectra in the NIST 02 MS database and retention times of several authentic samples. A typical chromatographic profile of the MeOH extract of B. pendula leaves showed two distinctly different zones. The first (10–36 min) contained hydrocarbons, fatty acids, alcohols, and sterols; the second (42–60 min), triterpenoids. The triterpenoids, β-sitosterol, phytol, and hydrocarbons were determined in the untreated MeOH extract; fatty acids, in the extract after methylation by diazomethane.

The principal native triterpenoids in B. pendula leaves are known to be dammarane-type compounds with a C-3 malonate group (type 1 or 2) and a THF group or open side chain on C-17, respectively. Mass spectra with a characteristic strong ion with m/z 143 (100%) corresponding to rupture of the C-17–C-18 bond [5] were observed for type 1 triterpenoids during the analysis of native extracts of B. pendula leaves by GC-MS. Type 2 triterpenoids gave an ion with m/z 341 that corresponded to rupture of the C-20–C-22 bond and subsequent loss of a water molecule [6]. The other leaf triterpenoids had lupane- and oleanane-type skeletons [1].

An analysis of mass spectra of triterpenoids from the MeOH extract of B. pendula leaves detected a group of seven compounds with retention times 42.53, 55.14, 55.54, 56.28, 56.65, 57.81, and 58.31 min. Their mass spectra were similar with strong fragment ions with m/z 143 (100%) that corresponded to type 1 epoxydammaranes [5]. Furthermore, compounds of this group that gave strong mass spectra showed an ion with m/z 341 (~4%). Total mass spectra of this group identified papyriferic acid (1, R1 = OCOCH2COOH; R2 = OAc) and its deacyl derivative (1, R1 = OCOCH2COOH; R2 = OH) with retention times of 58.31 and 57.81 min, respectively, in agreement with the literature [1].

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Compounds of a second group with retention times 43.16, 51.89, 53.10, and 54.05 min had similar mass spectra with strong ions with m/z 341 (25–40%). This was characteristic of native dammarane triterpenoids with a C-3 malonate and a C-17 open side chain [6]. This group of compounds was classified as type 2 dammarane triterpenoids based on the literature [1, 6] and experimental data.

A third group of triterpenoids with retention times 42.09, 44.03, 44.86, 45.40, and 50.96 min gave mass spectra with strong ions with m/z 135, 189, 191, and 203 and a typical cascade for pentacyclic triterpenoids in GC-MS analysis with ions with m/z 257, 271, 285, 297, and 299 [7]. These were triterpenoids with oleanane and lupane skeletons according to literature data for the triterpenoid composition of B. pendula leaves.

Fatty acids (as methyl esters) were identified from strong peaks for molecular ions (10–15%). Mass spectra of methyl esters of palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), and linolenic (C18:3) acids agreed with library spectra and had molecular ions with m/z of 270, 298, 296, 294, and 292, respectively. All C14–C26 acids were identified in the fatty acids. The principal ones were palmitic acid (~40% of total acids). Unsaturated acids consisted mainly of oleic, linoleic, and linolenic acids, the total contents of which were 25–30%.

Compounds with retention times of 21.13 and 35.81 min and molecular ions with m/z 296 and 414 were identified as phytol and β-sitosterol according to the agreement of total experimental and library mass spectra.

Hydrocarbons C21–C29, the principal ones of which were C25 and C27 with retention times 24.33 and 25.90 min, respectively, were identified from characteristic ions with m/z 57, 71, and 85 and the retention times of a known hydrocarbon mixture.

Table 1 lists the contents of flavonoids and hydroxycinnamic acids that were determined by HPLC-UV. Hydroxycinnamic acid glycosides included 3-caffeoylquinic (chlorogenic) acid and its isomers and p-coumaric and ferulic acid hexosides and pentosides, according to our earlier identification of phenolic compounds by HPLC-MS. Furthermore, a B. pendula leaf marker compound, 3,4′-dihydroxypropiophenone-3-β-D-glucopyranoside, was detected in the extract [8]. Flavonoid glycosides were mainly glucuronides, rhamnosides, arabinosides, and hexosides of myricetin, quercetin, and kaempferol; flavonoid aglycons, mono- and dimethyl ethers of tetrahydroxylated flavone and trimethyl ethers of pentahydroxylated flavone, quercetin, kaempferol, and apigenin. The total content of glycosylated flavonoids in the leaves was ~10 times greater than that of free flavonoids [4]. However, the main constituents on the leaf surfaces were methyl ethers of hydroxylated flavones [9]. Furthermore, soluble proanthocyanidins, the dynamics of the contents of which were related to the leaf age for many species of woody plants, were determined using a spectrophotometric method [10, 11].

Table 1 Identified Compounds and Groups of Compounds from Betula pendula Leaves during Early Development
Full size table

Table 1 presents data for the dynamics of the contents of lipophilic and phenolic compounds that were identified in B. pendula leaves. An analysis of the results found that the triterpenoid content, in contrast with that of phenylpropanoids, decreased nonlinearly during early leaf development. The halving of their contents on the 20th day was preceded by a 40% increase of their contents on days 5–10 as compared with their contents at the moment of emergence. A more detailed examination showed that the course of the dynamics was due mainly to epoxydammaranes, including papyriferic acid and its deacylated derivative (Table 1). The triterpenoid contents and deaths of insects feeding on leaves of various ages were clearly directly related. The greater the triterpenoid contents in B. pendula leaves was, the greater the lethality to L. dispar was. It was shown earlier that papyriferic acid fulfills a protective function in B. pendula by possessing antifeedant properties with respect to vertebrates that damage tree runners [12]. Thus, we propose based on our early investigations on L. dispar [4, 9] that triterpenoids are involved in B. pendula protection from leaf-eating invertebrates. The contents of fatty acids and hydrocarbons increased during early leaf development by 1.5 times; of phytol, by 3 times; of β-sitosterol, by 1.5 times by the 10th d (decreased to the starting level on the 20th d).

The flavonoid and hydroxycinnamic acid contents in leaves decreased linearly during the observation period by 2 and 1.5 times, respectively. Because more mature B. pendula leaves have a negative effect on insects [4], it could be assumed that these classes of phenolic compounds were apparently not involved in protecting the plant from insect folivores based on a phenological divergence during the development of the insects and plant. The contents of soluble proanthocyanidins increased gradually as the leaves aged. This is often encountered in woody plants.

Plant phenolic compounds are active metabolites. One of their most important functions is participation in redox reactions caused by endogenous and exogenous factors. They are capable of exhibiting antioxidant properties and neutralizing active oxygen species generated during unfavorable abiotic and biotic episodes. A modified Folin–Ciocalteau method [13] was used to estimate the contents of easily oxidized phenolic compounds, i.e., the potential oxidative capacity of B. pendula leaf phenolic compounds. The amount of phenols could be determined in parallel with the total phenol content under alkaline conditions that were optimal for the oxidation. The resulting difference defined the concentration of easily oxidized phenols. Table 1 presents the contents of phenolic compounds that could be easily oxidized to quinones, which have high biological toxicity, and total phenols. We suppose that the contents of easily oxidized phenolic compounds could be viewed as a toxicity parameter for L. dispar caterpillars. Mature leaves contain 1.5–2 times more easily oxidized phenolic compounds and are more toxic to insects than recently emerged young leaves. The total phenol contents were practically unchanged during the studied period of early leaf development.

Thus, the investigations showed that the contents of triterpenoids, phenylpropanoids, other phenolic compounds, fatty acids, phytol, hydrocarbons, and β-sitosterol undergo substantial changes during 20 d from the time of emergence of B. pendula leaves. The course of the concentration changes and its composition are determined not only by the class of secondary metabolites but also actual compounds. This emphasizes the importance of identifying compounds during phytochemical analysis of plant leaves. The dynamics of the contents of various biologically active compounds from B. pendula during early leaf development can be used for a more detailed study of ecological and biochemical aspects of the life cycles of B. pendula and L. dispar and their interaction and for the development of methods for predicting and controlling the L. dispar population.

EXPERIMENTAL

Extracts were analyzed by HPLC on an Agilent 1100 liquid chromatograph with a diode-matrix and Agilent 1200 mass-selective detectors. The analysis conditions for Agilent 1100 were steel column (4.6 × 150 mm) packed with Zorbax Eclipse XDB-C8 reversed-phase sorbent (5 μm), gradient elution using MeOH (A) and aqueous trifluoroacetic acid solution (0.1%) (B) from 0 to 100% A from 0 to 20 min and 100% A from 20 to 25 min, and eluent flow rate 0.8 mL/min. Detection was made simultaneously at four wavelengths (254, 280, 320, and 360 nm).

The HPLC-MS system comprised an Agilent 1200 liquid chromatograph and micrOTOF-Q hybrid quadrupole–timeof-flight mass spectrometer (Bruker). The column (2.1 × 50 mm) contained Zorbax XDB-C8 (3.5 μm). The eluent consisted of MeOH (A) and formic acid (2%) (B) with 50% A (5 min), from 50 to 100% A (5–25 min), and 100% A (25–35 min) at flow rate 0.2 mL/min.

Mass Detector Operating Parameters. Ionization method, electrospray at atmospheric pressure (API-ES). Negative and positive ions scanned for m/z = 100–1000. Drying gas (N2) flow rate, 8 L/min; temperature, 230°C, sprayer pressure, 1.6 bar.

GC-MS analysis of extracts used a 6890N GC with a 5975N quadrupole mass-selective detector, 7683B autosampler (Agilent Technologies), and an HP-5ms quartz capillary column (copolymer of 5% diphenyl- and 95% dimethylsiloxane) (30 m × 0.25 mm) with stationary phase thickness 0.25 μm. The carrier gas was He at flow rate 1.0 mL/min. The sample was injected in splitless mode. The injector and detector temperatures were 280°C. The column temperature varied as follows: 2 min, 50°C, increase to 280°C at 10°C/min, and steady at that temperature for 40 min. Component contents were calculated using the internal standard method without correction coefficients. Compounds were identified using the NIST 02 MS database and known samples of betulin, β-sitosterol, and methyl stearate.

Total contents of phenols, easily oxidized phenols, and soluble proanthocyanidins were determined on an Agilent 8453 spectrophotometer.

Preparation of Aqueous EtOH Extract for Determination of Phenylpropanoid Content. Air-dried B. pendula leaves (accurate weight, ~40 mg) were extracted with EtOH (70%, 2 mL) at 50–60°C and stirred for 10 min. The extract was filtered through Diapak C16 reversed-phase sorbent and used for HPLC analysis.

Preparation of MeOH Extract for Determination of Contents of Lipophilic Compounds. Air-dried B. pendula leaves (accurate weight, ~100 mg) were extracted with MeOH (3 × 3 mL) at 20–25°C with stirring for 24 h. The extract was evaporated to dryness (yield 39–42%). Solutions (2 mg/mL) in MeOH were prepared for GC-MS analysis.

Methylation of MeOH Extract of B. pendula Leaves. MeOH extract (1 mL, 4 mg/mL) was treated carefully with freshly prepared diazomethane in Et2O (~2 mL) until N2 was not evolved when new portions of diazomethane solution were added. The mixture was stirred and shaken, left for 12–15 h at 20–25°C, and treated with MeOH (2 mL). The solution was used for GC-MS analysis of the fatty acids in the extract.

Preparation of Aqueous Me 2 CO Extract for Determination of Contents of Total Phenols, Easily Oxidized Phenols, and Soluble Proanthocyanidins [13]. Air-dried B. pendula leaves (accurate weight, ~10 mg) were extracted with aqueous Me2CO (70%, 3 × 0.8 mL) at 20–25°C, stirred, centrifuged for 10 min at 14,000 rpm, and left to evaporate at 20–25°C. The residue was lyophilized. The dry residue was treated with H2O (250 μL) and dissolved with stirring. The insoluble precipitate was separated by centrifugation for 20 min at 14,000 rpm. The resulting solution was used for the analysis.

Determination of Total Contents of Phenolic Compounds and Easily Oxidized Phenolic Compounds. The analysis used a modified Folin–Ciocalteau method [13] and buffer solutions A, carbonate buffer (50 mM) at pH 10; B, formic acid (0.6%); and C, a mixture of solutions A and B (9:5, v/v). The standard for calculating phenolic compound contents was gallic acid.

Extract (20 μL) was treated with solution C (280 μL). The resulting solution (50 μL) was treated with Folin–Ciocalteau reagent (50 μL, 1 N) and sodium carbonate solution (20%, 100 μL) and held at 25°C for 60 min, stirring and shaking every 10 min for 10 s. Absorption of the resulting solution was measured at 730 nm, from which the total contents of phenols (mg/g of a.d.m.) were calculated.

Extract (20 μL) was treated with solution A (180 μL) and held at 25°C for 90 min with stirring and shaking for 10 s every 10 min. The resulting alkaline solution was neutralized by adding solution B (100 μL), after which the aforementioned procedure for determining phenols was repeated. Absorption was measured at 730 nm, from which the contents of unoxidized phenols (mg/g of a.d.m.) were calculated.

The contents of easily oxidized phenols were calculated as the difference between the total contents of phenols and the contents of phenols that were unoxidized at alkaline pH values.

Determination of Contents of Soluble Proanthocyanidins. A spectrophotometric method based on oxidation of proanthocyanidins to anthocyanidins in BuOH–HCl that was adapted to B. pendula leaves was used to determined their contents [10]. A freshly prepared mixture of BuOH and HCl (95:5, 6.08:0.32 mL) was treated with extract (100 μL) and H2O (0.6 mL), thoroughly mixed, heated on a water bath at 95°C for 50 min, and cooled to 20–25°C. The contents of soluble proanthocyanidins was determined at 555 nm. The standard was cyanidin chloride.