No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Dry-air drying at room temperature - a practical pre-treatment method of tree leaves for quantitative analyses of phenolics?
Abstract
Introduction:
In ecological experiments, storage of plant material is often needed between harvesting and laboratory analyses when the number of samples is too large for immediate, fresh analyses. Thus, accuracy and comparability of the results call for pre-treatment methods where the chemical composition remains unaltered and large number of samples can be treated efficiently.
Objective:
To study if a fast dry-air drying provides an efficient pre-treatment method for quantitative analyses of phenolics.
Methodology:
Dry-air drying of mature leaves was done in a drying room equipped with dehumifier (10% relative humidity, room temperature) and results were compared to freeze-drying or freeze-drying after pre-freezing in liquid nitrogen. The quantities of methanol-soluble phenolics of Betula pendula Roth, Betula pubescens Ehrh., Salix myrsinifolia Salisb., Picea abies L. Karsten and Pinus sylvestris L. were analysed with HPLC and condensed tannins were analysed using the acid-butanol test.
Results:
In deciduous tree leaves (Betula, Salix), the yield of most of the phenolic compounds was equal or higher in samples dried in dry-air room than the yield from freeze-dried samples. In Picea abies needles, however, dry-air drying caused severe reductions in picein, stilbenes, condensed tannin and (+)-catechin concentrations compared to freeze-drying. In Pinus sylvestris highest yields of neolignans but lowest yields of acetylated flavonoids were obtained from samples freeze-dried after pre-freezing.
Conclusion:
Results show that dry-air drying provides effective pre-treatment method for quantifying the soluble phenolics for deciduous tree leaves, but when analysing coniferous species, the different responses between structural classes of phenolics should be taken into account.
... The results of the research of both Betula and Pinus pollen showed that the use of liquid nitrogen did not increase the content of phenolic compounds in the extracts. A study [59] froze Betula pendula, Betula pubescens leaves and Pinus sylvestris needles with liquid nitrogen; however, freezing did not affect the content of phenolic compounds significantly either. The research [8] showed that superfine grinding wall disruption hardly changes the total soluble phenolic contents when 60 Co-irradiation increases. ...
... This may have implications for the phenolic compounds accumulated in pollen and the antioxidant activity of bioactive compounds. Research [59] shows that the contents of phenolic compounds accumulated in the leaves of Betula pendula and Betula pubescens are similar, while the concentrations of the same group of phenolic compounds in the needles of Pinus sylvestris are lower than in the leaves of Betula. ...
... Chlorogenic acid, a compound with a strong antioxidant effect, was present in all Betula pollen samples, while it was found less frequently in Pinus. Chlorogenic acid, as one of the predominant phenolic acids, along with its derivatives are also found in other plant organs of the genus Betula [59,64]. Although [2] did not detect chlorogenic acid in Betula pendula pollen, they declared p-coumaric acid and ferulic acid to be bound to sporopollenin, and they identified p-coumaric acid as the main compound in the free PC fraction. ...
In this study, phenolic compounds and their antioxidant activity in the pollen of anemophilous Betula and Pinus were determined. Spectrophotometric, high-performance thin-layer and liquid chromatography methods were applied. Free phenolic compounds (free PC) and phenolic compounds bound to the cell wall (bound PC) were analysed in the pollen extracts. Regardless of the pollen species, their content was 20% higher than that in bound PC extracts. Pinus pollen extracts contained 2.5 times less phenolic compounds compared to Betula. Free PC extraction from the deeper layers of Pinus pollen was minimal; the same content of phenolic compounds was obtained in both types of extracts. The bioactivity of pollen (p < 0.05) is related to the content of phenolic compounds and flavonoids in Betula free PC and in bound PC, and only in free PC extracts of Pinus. Rutin, chlorogenic and trans-ferulic acids were characterised by antioxidant activity. Phenolic acids accounted for 70–94%, while rutin constituted 2–3% of the total amount in the extracts. One of the dominant phenolic acids was trans-ferulic acid in all the Betula and Pinus samples. The specific compounds were vanillic and chlorogenic acids of Betula pollen extracts, while Pinus extracts contained gallic acid. The data obtained for the phenolic profiles and antioxidant activity of Betula and Pinus pollen can be useful for modelling food chains in ecosystems.
... En los frutos de J. communis, se ha observado un mayor contenido de monoterpenos, diterpenos y flavonoides (Ben Mrid et al., 2019;Falasca et al., 2014;Jegal et al., 2017). Mientras que en P. sylvestris se han identificado principalmente monoterpenos, sesquiterpenos y diterpenos (Allenspach et al., 2020), flavonoides de tipo flavan-3-oles, acetilados, glicósilados, neolignanos y taninos condensados (Tegelberg et al., 2018). Así mismo, la corteza presenta cantidades importantes de compuestos fenólicos (Pap et al., 2021). ...
Los suplementos alimenticios y/o remedios herbolarios pueden coadyuvar a minimizar la aparición de patologías, sin embargo, son necesarios estudios funcionales y toxicológicos para determinar su eficacia y posible toxicidad. El objetivo de la presente investigación fue evaluar el potencial antioxidante y biofuncional de suplementos alimenticios, hechos a base de extractos botánicos. Para esto se determinó la capacidad antioxidante, capacidad reductora total, contenido de flavonoides totales y toxicidad in vitro. Además, se identificaron los compuestos orgánicos volátiles mediante cromatografía de gases acoplado a espectrometría de masas y se evaluó su biofuncionalidad mediante estudios bioinformáticos. Se observó el potencial antioxidante de los suplementos, debido al contenido de compuestos con capacidad reductora total, además de su aporte de terpenos, ácidos grasos (ácido hexadecanoico) y fitoesteroles. El producto con mayor actividad antioxidante no presentó toxicidad en el modelo de Artemia salina. El estudio in silico arrojó que los posibles objetivos de los compuestos en el organismo se encuentren relacionados con la inhibición de la patogénesis de algunas de las enfermedades crónico-degenerativas con mayor incidencia y prevalencia en México como la diabetes e hipertensión.
... The mouth of the jar was covered with muslin cloth held with rubber bands and kept in the laboratory at 27 °C and 70% relative humidity. The setup was allowed to stand for 14 days before adding original adults Callosobruchus maculatus from the grains [24] . ...
The modern method of controlling insect pests, using synthetic insecticides has caused serious problems to human health and the environment. Hence, the need for alternative, cheap, readily available and acceptable insect pest control methods, among which are the use of plant leaf extracts. The aim of this study was to determine the bioinsecticidal efficacy of Eucalyptus camaldulensis and Hyptis suaveolens against cowpea weevils (Callosobruchus maculatus). The bioassay of the powdered methanol extracts of leaves of E. camaldulensis and H. suaveolens was carried varying quantities (3 g, 6 g, 9 g and 12 g) of powdered extracts of the respective plant samples were mixed separately with 50 g of cowpea. Synthetic insecticide, Al2(PO4)3 was used as positive control while the negative control contained no treatment. Proximate compositions of the treated and untreated cowpeas were done using standard procedures while the extracts were characterized using Gas Chromatography-Mass Spectroscopic. Although mortality rates of the C. maculatus in presence of the extracts did not follow any dose-dependant trend, the respective dosages used for treatments inhibited the growth and activities of Callosobruchus maculatus. A high rate of seed damage was observed in the untreated cowpea seeds. Gas Chromatography-Mass Spectroscopy of the extracts identified the presence of seven potential insecticidal compounds (Bargamotene, Globulol, Humulene, Linolein, Prasterone, Terpinen-4-ol and Traumatic acid) in Hyptis suaveolen. In the Eucalyptus camaldulensis extract, 13 potential insecticidal compounds (Barosma camphor, Carvacrol, Carbazon, Irone, Isoborneol, 5-methyl, 1,4-Naphthoquinone, Oleic Acid, Phytol, Piperitone, Pyrazole-3-carboxylic acid, Terpinen-4-ol, Viridiflorol) were identified. The bioinsecticidal activities of these plant extracts could be attributed to the presence of phytochemicals and isolated insecticidal compounds.
... In the laboratory, leaves were washed to remove attached material and air dried at room temperature (~ 25 °C) (Tegelberg et al. 2018). Dried leaves were milled to a fine powder and sieved through a 1.0 mm mesh size screen. ...
Full-text access: https://rdcu.be/cQhTj
Phenolic compounds (PC) of aquatic macrophytes are involved in relevant ecological interactions and ecosystems functions. However, they have been scarcely evaluated in tropical and subtropical floodplains, where hydrological connectivity could be a related variable due to its effects on environmental conditions. Accordingly, we analysed the foliar phenolic content of 16 common aquatic macrophyte species that are native to the subtropical floodplain of the Middle Paraná River to evaluate 1) intraspecific variability in relation with the hydrological connectivity of floodplain habitats; 2) differences among the main life forms occurring in the floodplain; and 3) PC release and production of chromophoric dissolved organic matter (CDOM) from leaves belonging to species characterized by different PC content. No species showed significant differences in PC between individuals collected in connected and isolated habitats. However, emergent macrophytes showed a higher PC content than free-floating macrophytes, which could be related with their higher frequency in isolated habitats. In this regard, biotic interactions, nutrient limitation, and water stress are known to be stronger in isolated habitats, which could favour the establishment of phenolic-rich species due to the beneficial functions of PC under these conditions. Finally, PC release and CDOM production were larger in phenolic-rich species, with the highest values for Ludwigia peploides. The high PC content of L. peploides could have important implications for the functioning of the Middle Paraná floodplain, where it is one of the most conspicuous macrophyte species; and could be involved in the major ecological problems that it causes outside its native distribution range.
... Phenolic compounds are a good example of a secondary metabolite group susceptible to heating [57]. Even air drying at room temperature has an effect on some structural classes of phenolics in Scots pine and Norway spruce (Picea abies (L.) H. Karst) needles [58]. Our study showed that air-drying had the lowest effect on TT in Scots pine needles compared with microwaving and oven-drying at temperatures of 40 • C and 65 • C -Modification I. Oven-drying maintained the detectability of terpenoids, which is in line with the study conducted on Taraxacum officinale F.H. Wigg. ...
In ecological research, quantitative methods are often used to measure the total content of metabolites groups (i.e., phenols, carbohydrates). Until recently, there has been no simple and effective method to determine the total terpenoid content with satisfactory repeatability and sensitivity. The procedure proposed by Ghorai et al. (2012) requires the use of fresh plant material. That may be problematic when experimental units are located far from a laboratory. Our goals were to optimize the procedure, and to find the threshold of misestimation using the procedure adjusted to work with dried material. Needles of Pinus sylvestris were used to test the effect of changes in drying, grinding, storage, and extraction on determined total terpenoid content. All applied changes in material storage and grinding decreased the quantity of the terpenoids in needles. Only air-dried and ball-milled material produced similar results to those obtained with fresh material – can be recommended if the fresh material unattainable. Air-dried material may be stored for up to three months, but it resulted in greater variation and then greater sample size is needed. Lower sample mass and solvent volume have no impact on accuracy. Shorter extraction time, oven-drying or microwaving leads to unreliability of measurements.
... The leaves were thoroughly washed in distilled water to remove dust particles before being air-dried at laboratory room temperature (∼27°C) and in the absence of sunlight for 14 days. Dried leaves were then pulverized to powder form using a domestic blender and preserved in airtight containers until further use (Tegelberg, 2018). Pictorial representation of collection of plant leaves and preparation of extracts is presented in Fig. 1. ...
Biogenic iron oxide nanoparticles synthesized from a locally sourced, readily available plant — Azadirachta indica, were used synergistically with sodium persulphate (PS) to degrade total petroleum hydrocarbons (TPHs) in contaminated soil. A chemometric approach to optimizing the conditions for the degradation of TPH was developed using central composite design (CCD). Characterization of iron oxide nanoparticles was carried out with X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray (EDX) spectroscopy, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), and selected electron area diffraction (SAED). Analysis of TPH was carried out with gas chromatography flame ionization detector (GC-FID). The optimum condition for the complete degradation of TPH was achieved at pH 6.0 and oxidant dosage of 0.74 M within 14 h and 5 days. Iron nanoparticles AZA FeNP (1:1) and AZA FeNP (2:1) were synthesized by varying the ratio of extract/precursor. The nanoparticles displayed heterogeneous, amorphous morphology with increased agglomeration in AZA FeNP (1:1). AZA FeNP (2:1) XRD spectra exhibited characteristic peaks at 27.0°, 35.4° and 44.5°, which are attributed to iron nanoparticles. TEM and HR-TEM images confirmed spherical nanoclusters’ presence with an average size of 9.3 and 10.0 nm for AZA FeNP (1:1) and AZA FeNP (2:1), respectively. The EDX spectra displayed intense peaks of oxygen, carbon, and iron at 0.4, 0.6 and 6.4 KeV confirming the presence of FeNP. Under optimized conditions, PS alone degraded 68% TPH while 0.07 and 0.15 g/L FeNP achieved ∼93 and 95% degradation, respectively. Additionally, 0.07 and 0.15 g/L FeNP-activated PS achieved ∼99 and 100% TPH degradation, respectively. The outcome of these findings suggests that FeNP synthesized using A. indica successfully catalyzed PS for complete degradation of TPH in crude oil-contaminated soil.
... Samples for leaf and stem phenolics and condensed tannins, as well as samples for trichome density measurements, were taken after 51 days of UVB treatments. After measuring the fresh weight (FW), samples for chemical analysis and the remaining aboveground biomass were dry-air dried at room temperature (Tegelberg et al., 2018), while the samples for trichome density measurements were stored in +4°C and processed within 24 days. After measuring the FW and LA using a LICOR LI-3000C portable area meter (LICOR), leaves sampled for trichome analysis were prepared into microscope samples (Thitz et al., 2017). ...
Accumulation of certain phenolics is a well‐known response of plants to enhanced UVB radiation (280–315 nm), but few experiments have compared the relative importance of different phenolic groups for UVB resilience. To study how an altered phenolic profile affects the responses and resilience of silver birch (Betula pendula) to enhanced UVB, we used RNA interference (RNAi) targeting dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), or anthocyanidin reductase (ANR) to change the accumulation of phenolics. The unmodified control line and RNAi‐modified plants were grown for 51 days under ambient or +32% enhanced UVB dose in a greenhouse. RNAi greatly affected phenolic profile and plant growth. There were no interactive effects of RNAi and UVB on growth or photosynthesis, which indicates that the RNAi and unmodified control plants were equally resilient. UVB enhancement led to an accumulation of foliar flavonoids and condensed tannins, and an increase in the density of stem glands and glandular trichomes on upper leaf surfaces in both the control and RNAi‐modified plants. Our results do not indicate a photoprotective role for condensed tannins. However, decreased growth of high‐flavonoid low‐tannin DFRi and ANRi plants implies that the balance of flavonoids and condensed tannins might be important for normal plant growth.
... Immediately after each 48-h experiment, 2-6 undamaged mature leaves from the net-enclosed part of the plant were sampled for chemical and morphological analyses. After recording the FW, the sampled leaves were dry-air dried (relative humidity <20%) according to Tegelberg et al. (2018) and stored at −20°C. Out of the remaining leaves in each plant, partly eaten leaves were separated and the number of empty petioles (n pet ) was recorded. ...
Despite active research, antiherbivore activity of specific plant phenolics remains largely unresolved. We constructed silver birch (Betula pendula) lines with modified phenolic metabolism to study the effects of foliar flavonoids and condensed tannins on consumption and growth of larvae of a generalist herbivore, the autumnal moth (Epirrita autumnata). We conducted a feeding experiment using birch lines in which expression of dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS) or anthocyanidin reductase (ANR) had been decreased by RNA interference. Modification-specific effects on plant phenolics, nutrients and phenotype, and on larval consumption and growth were analyzed using uni- and multivariate methods. Inhibiting DFR expression increased the concentration of flavonoids at the expense of condensed tannins, and silencing DFR and ANR decreased leaf and plant size. E. autumnata larvae consumed on average 82% less of DFRi plants than of unmodified controls, suggesting that flavonoids or glandular trichomes deter larval feeding. However, larval growth efficiency was highest on low-tannin DFRi plants, indicating that condensed tannins (or their monomers) are physiologically more harmful than non-tannin flavonoids for E. autumnata larvae. Our results show that genetic manipulation of the flavonoid pathway in plants can effectively be used to produce altered phenolic profiles required for elucidating the roles of low-molecular weight phenolics and condensed tannins in plant–herbivore relationships, and suggest that phenolic secondary metabolites participate in regulation of plant growth.
... The leaves were thoroughly washed in distilled water and air-dried at room temperature fortwenty one (21) days (Figures 1a,b). The dried leaves were pulverized and preserved in air tight containers until further use [15,16]. Powdered leaves of C. indica (25 g) was extracted with 125 mL solvent to obtain three extracts: ethanolic, aqueous and ethanol/water (1:1) ( Figure 1). ...
Within the framework of determining capping and stabilizing bioactive components present in Canna indica towards nanoparticles synthesis, phytochemical screening, total phenolic content, infrared spectroscopy and chromatographic characterisation were carried out on the locally sourced plant. Extracts were prepared from C. indica leaves using ethanol, de-ionised water (DW) and ethanol/DW in ratio 1:1. Qualitative screening showed the presence of saponins, alkaloids, terpenoids, phenols and coumarins. Highest total phenolic content (TPC) was observed in the aqueous fraction and least in ethanol fraction. Characterisation was carried out using Fourier Transform - Infrared spectroscopy (FT-IR) and Gas Chromatography - Mass Spectrometry (GC-MS). Absorption bands observed from FT-IR analysis showed presence of aromatic O-H stretch (3300 cm ⁻¹ ) and aromatic C=C stretch (1451 and 1640 cm ⁻¹ ) respectively. GC-MS analysis of ethanolic extract indicated the presence of dl-.alpha.-tocopherol – a phenolic compound.
Early blight, caused by Alternaria solani is one of the destructive diseases of potato. An in vitro experiment was conducted using the poisoned food technique to evaluate the efficacy of acetone extracts of six plant species on the mycelial growth of A. solani. Plant extracts of Artemisia vulgaris, Melia azedarach, Parthenium hysterophorus, Datura stramonium, and Justicia adhatoda at concentrations of 0.1%, 0.5% and 5% prepared from Soxhlet extraction were mixed well in the molten potato dextrose agar (PDA) media and poured into petri plates (90 mm Ø). PDA media treated with a fungicide mancozeb was positive control while the untreated media was negative control. The poisoned plates were inoculated with a 4 mm diameter agar plug of seven days old actively growing A. solani and incubated at 26 ± 2 °C, The radial mycelial growth was recorded after 3rd, 5th, 7th and 9th days of incubation and percentage inhibition growth was calculated by comparing it with negative control plates. Artemisia vulgaris significantly inhibited colonial growth at 0.1% and 0.5% concentrations by 36.07% and 12.48%, respectively, at 5th day of inoculation (dai) as compared to negative control. M. azedarach (0.72%), P. hysterophorus (10.17%) and D. stramonium (10.20%) were moderately inhibitory in effect at 1% concentration while J. adhatoda did not possess inhibitory effect on the mycelial growth of A. solani at 5th dai. All the tested extracts were found with some antifungal activity at 5% concentration. A. vulgaris can be exploited as a potent alternative to fungicides for controlling A. solani.
This chapter introduces the supercritical fluid extraction of oleoresins and plant phenolics. It starts with the brief introduction on the various types of oleoresins and phenolic compounds from plant sources, followed by the different convention extraction methods performed. The important parameters that should be taken into account in order to optimize the extraction processes include: (1) the solubility of targeted compounds in supercritical fluid; (2) pretreatment of the sample before extraction (particle size, moisture content, and enzymatic pretreatment); (3) the use of cosolvent; and (4) operating parameters (pressure, temperature, carbon dioxide flow rate, and time. The use of cosolvents for the enhanced extraction of plant phenolics to increase the polarity of the supercritical fluid is discussed and the assisting technologies, namely, enzyme-assisted, ultrasound-assisted, and high hydrostatic pressure-supercritical fluid extraction for optimized supercritical fluid extraction of plant phenolics are explained.
The different assays used for tannin estimation in plant tissues can be classified into three groups according to the type of reaction involved: precipitation of proteins or alkaloids, reaction with phenolic rings, and depolymerization. The value of these methods for estimation of total tannins or of a particular group of tannins (proanthocyanidins, gallotannins or ellagitannins) is discussed. The importance of careful preparation of the tannin extract, including preparation and storage of the sample, extraction and fractionation of the isolates, is emphasized.
We studied the ability of tree seedlings to respond to two environmental factors, elevated ultraviolet B (UVB) radiation and availability of nitrogen (N), at the beginning of their development. Seeds of two birch species, Betula pubescens Ehrh. (common white birch) and B. pendula Roth (silver birch), were germinated and the seedlings grown in an experimental field in eastern Finland. The experimental design consisted of a constant 50% increase in UVB radiation (including a slight increase in UVA), a UVA control (a slight increase in UVA) and a control. The seedlings were fertilized with three levels of N. The experiment lasted for 2 months; aboveground biomass was measured and the most mature leaf of each seedling was taken for the analyses of phenolics. Growth of the seedlings was not significantly affected by enhanced UVB, but was increased by increasing N. Elevated UVB induced significant changes in phenolic compounds. Quercetin glycosides were accumulated in the leaves of both species in response to UVB; this is considered to be a protective response. However, the direction of the responses of individual phenolics to different N regimens differed. In addition, concentration of soluble condensed tannins was lower at moderate N than that at lower levels of N in both species; on the contrary, in B. pubescens the concentration of insoluble condensed tannins was highest at moderate N. No significant interaction between UV and N was detected, and the responses of the two species were highly similar to UVB, while the responses to N regimens varied slightly more between species.
The aim of this study was to evaluate the long-term effects of elevated CO2 concentration (doubling of ambient CO2 concentration) and temperature (2–6°C elevation) on the concentration and content of secondary compounds in the needles of
Scots pine (Pinus sylvestris L.) saplings grown in closed-top environmental chambers. The chamber treatments included (1) ambient temperature and CO2, (2) ambient temperature and elevated CO2, (3) elevated temperature and ambient CO2, and (4) elevated temperature and elevated CO2. The needle sampling and analyses of monoterpenes, HPLC-phenolics and condensed tannins in current- and 1-year-old needles
were made in two consecutive years. The results showed that the effects of elevation of CO2 and temperature were greatest on the monoterpene concentration in the needles while the concentration of HPLC-phenolics remained
almost unaffected by the changed growing conditions. Most of the observed decrease in monoterpene concentration was caused
by the CO2 enrichment while the effect of elevated temperature alone was not as significant. The accumulation of condensed tannins tended
to increase due to the elevation of CO2 alone compensating the reduced carbon allocation to monoterpenes. Overall, the responses of the concentrations of secondary
compounds to the elevation of CO2 and temperature are variable and depend strongly on the properties and characteristics of each compound as well as on the
interrelation between the production of these compounds and the primary production of trees.
This chapter serves as an introduction to the most commonly found phenolics. A phenolic is a chemical compound characterized by at least one aromatic ring bearing one or more hydroxyl groups. Among the commonly found phenolics, the predominant structures in higher plants are the flavonol glycosides, hydroxycinnamate conjugates and condensed tannins in the leaves, and the anthocyanins in petals and fruits. Many phenolics occur as derivatives formed by condensation or addition reactions. Most of the thousands of phenolics known to date are of plant origin. Phenolics are of great importance as cellular support materials. They form an integral part of cell wall structures, mainly in polymeric materials such as lignins and suberins for mechanical support and barriers against microbial invasion. Lignins, after cellulose, are the second most abundant organic structures on earth. With their formation, plants became adapted to terrestrial life by building rigid organs such as woody stems and conducting cell elements for water transport. This chapter begins with a discussion on shikimate/argogenate pathway, and phenylalanine/ hydroxycinnamate pathway. The chapter also explains the chemistry of hydroxycinnamate conjugates, hydroxycoumarins, hydroxybenzoates, flavonids, lignins, lignans, neolignans, tannins, and quinines in detail.
Simultaneously with warming climate, other climatic and environmental factors are also changing. Here, we investigated for the first time the effects of elevated temperature, increased ultraviolet-B (UVB) radiation, fertilization and all combinations of these on the growth, secondary chemistry and needle structure of 1-year-old Norway spruce (Picea abies (L.) Karst.) seedlings in an outdoor experiment. After one growing season, elevated temperature increased root : shoot ratio and concentrations of needle piperidine alkaloids, while concentrations of needle catechins and acetophenones and bark flavonoids decreased compared with ambient temperature seedlings. UVB-radiation increased concentrations of bark condensed tannins, while fertilization increased total biomass and concentrations of needle catechins. In addition to the main effects, concentrations of some individual phenolic compounds showed UV × temperature or UV × temperature × fertilization interactions, and fertilization modified temperature response on root : shoot ratio. All the treatments described here affected the defence chemistry profiles of the seedlings, which may imply some changes in plant-herbivore interactions in connection with changing climate. The interactions between treatments indicate a need for further experiments involving several simultaneously affecting environmental changes.
Members of the Salicaceae often produce phenolic glycosides and condensed tannins. There is much debate on the best method for the preservation of leaf material prior to chemical analysis. Published results indicate freeze-drying, a method commonly used for tannin analysis, may be inappropriate for phenolic glycosides, unless done in a manner to prevent thawing during the drying process. Another commonly employed method, air-drying, is appropriate for phenolic glycosides but inappropriate for condensed tannins. I present evidence using willow leaves that demonstrates that: (1) leaves freeze-dried in external flasks without temperature control contain lower concentrations of phenolic glycosides (salicortin and 2'-cinnamoylsalicortin), (2) air-dried leaves have reduced concentrations of condensed tannins, while (3) vacuum-dried fresh leaves have high concentrations of both phenolic glycosides and condensed tannins. Freeze-drying caused salicortin and 2'-cinnamoyl salicortin concentrations to drop by 20 mg/g and 4 mg/g, respectively. Salicin, a product of salicortin and 2'-cinnamoyl salicortin degradation, is absent in vacuum-dried leaves, present in air-dried leaves and very high in freeze-dried leaves. Thus, the presence of salicin in this system is an artifact of the preservation technique. Condensed tannin concentrations dropped nearly 20 mg/g when leaves were air-dried. Thus, vacuum-drying fresh leaves allows researchers to quantify phenolic glycosides and condensed tannins from the same leaf material.
Systematische Untersuchungen über die Glykosidführung der einheimischen Salicaceen mit Hilfe moderner chromatographischer Untersuchungsverfahren haben zur Isolierung einer Reihe bisher unbekannter Verbindungen geführt, die z. T. als acylierte Derivate des Salicins, z. T. als phenolische Glykoside mit Bindung des Zuckers über eine primäre alkoholische Hydroxylgruppe charakterisiert werden konnten.
Während in den Blättern der untersuchten Salix–Arten nur z. T. Phenolglykoside (Salicin, Salicortin) nachgewiesen werden konnten, enthielten die Rinden Glykosidgemische sehr unterschiedlicher Zusammensetzung. Das bisher als Hauptglykosid der Salicaceen angesehene Salicin konnte zwar in allen untersuchten Rinden nachgewiesen werden, aber meist nur in geringen Konzentrationen und niemals als Hauptglykosid. Demgegenüber wurde in einigen Arten das Vorkommen größerer Mengen acylierter Salicinderivate beobachtet. Weitere Untersuchungen erstreckten sich auf die Glykosidakkumulation in den einzelnen Pflanzenorganen in Abhängigkeit vom Zeitpunkt der Ernte.
Summary
Extensive studies of indigenous Salicaceae resulted in the isolation of several new glycosides. Some of the latter proved to be acylated salicins while others are phenolic glycosides in which the sugar is linked with a primary alcoholic hydroxyl.
Leaves do not always contain phenolic glycosides (salicin, salicortin) but barks contain complex mixtures of glycosides differing in composition with species and season. Salicin was detected in the bark of all species investigated but it is never the main glycoside. Some species contain acylated salicins as main glycosides in their bark.
Elevations of carbon dioxide, temperature and ultraviolet-B radiation in the growth environment may have a high impact on the accumulation of carbon in plants, and the different factors may work in opposite directions or induce additive effects. To detect the changes in the growth and phytochemistry of silver birch (Betula pendula) seedlings, six genotypes were exposed to combinations of ambient or elevated levels of CO2 , temperature and UVB radiation in top-closed chambers for seven weeks. The genotypes were relatively similar in their responses, and no significant interactive effects of three-level climate factors on the measured parameters were observed. Elevated UVB had no effect on growth, nor did it alter plant responses to CO2 and/or temperature in combined treatments. Growth in all plant parts increased under elevated CO2 , and height and stem biomass increased under elevated temperature. Increased carbon distribution to biomass did not reduce its allocation to phytochemicals: Condensed tannins, most flavonols and phenolic acids accumulated under elevated CO2 and elevated UVB, but this effect disappeared under elevated temperature. Leaf nitrogen content decreased under elevated CO2 . We conclude that, as a result of high genetic variability in phytochemicals, B. pendula seedlings have potential to adapt to the tested environmental changes. The induction in protective flavonoids under UVB radiation together with the positive impact of elevated CO2 and temperature mitigates possible UVB stress effects, and thus atmospheric CO2 concentration and temperature are the climate change factors that will dictate the establishment and success of birch at higher altitudes in the future.
Sample preparation methods for high-performance liquid chromatographic analysis of birch leaf phenolics were compared. The methods tested were: (1) air-drying at ambient temperature, (2) oven-drying at 40 °C, (3) oven-drying at 80 °C, (4) freeze-drying, prefreezing with liquid N2, (5) freeze-drying, prefreezing at −18 °C, (6) freeze-drying without prefreezing, (7) storing frozen for 12 days without drying, and (8) immediate extraction of fresh samples. Although there were significant differences among methods in absolute concentrations of phenolics, none of the methods altered considerably the phenolic profile of birch leaves. For quantitative analysis, the samples should be analyzed immediately. Alternatively, the samples may be stored fresh frozen, at least for a short time. Among the drying methods, the highest concentrations of phenolics were obtained by freeze-drying of slowly frozen leaves (method 5). Freeze-drying without prefreezing resulted in significantly lower concentrations of flavonoid glycosides than method 5. Oven-drying did not prove to be a good alternative to conventional freeze-drying, nor did it show any advantage over simple air-drying. Keywords: Betula pendula; sample preparation; drying methods; flavonoid glycosides; phenolic compounds
A high-performance liquid chromatography (HPLC) procedure based on a water–methanol gradient with tetrahydrofuran for simultaneous analysis of flavonoids and other phenolics of differing polarities in birch leaves was developed. Mobile phases with and without tetrahydrofuran or orthophosphoric acid provided additional information for tentative identification of the compounds. Forty-five compounds were determined from Betula pendula and B. pubescens leaves. Five of the isolated flavonol glycosides have not been reported earlier from B. pendula. B. pendula leaves have a range of myricetin and quercetin glycosides with the same sugar moieties in roughly the same proportions. Flavonoid aglycones were deposited on leaf surfaces.
Although several climatic factors are expected to change simultaneously in the future, the effect of such combined changes on plants have seldom been tested under field conditions. We report on a field experiment with dark-leaved willow, Salix myrsinifolia, subjected to enhancements in ultraviolet-A (UVA), UVB radiation and temperature, setup in Joensuu, Eastern Finland. S. myrsinifolia is a dioecious species, known as an important food plant for many herbivores. Cuttings of eight clones, four of each sex, of dark-leaved willow were planted in the field in spring 2009. In both 2009 and 2010, the total biomass increased significantly with temperature, and in 2010 there was an additive effect of UVB radiation. Both height and diameter increased with temperature in 2009, while the effect on height growth ceased in 2010. Males had greater diameter growth than females in 2010. Most phenolic compounds in the leaves decreased under enhanced temperature in both growing seasons. In 2010, four of six salicylates increased in response to enhanced temperature. Some quercetin derivatives increased under enhanced UVB radiation. Females had higher concentrations of chlorogenic acids than males, and while enhanced temperature reduced chlorogenic acid in females only, luteolins were reduced only in males. In summary, the combined enhancements gave no effects in addition to those that appeared under the single-factor treatments, except for the additive effect of UVB on temperature-increased biomass. The few gender-related differences found in response to climate change do not allow any marked expectations of future climate-induced changes in sex ratios.
In this study, we compared the effects of several preservation methods on the secondary phenolics of the mature leaves of purple willow (Salix purpurea L., salicaceae) with results obtained with fresh leaf analyses. Conventional freeze-drying, in which the leaves were first frozen with liquid nitrogen and then placed in a freeze-dryer, produced substantial qualitative and quantitative changes in purple willow flavonoids and salicylates. Modified freeze-drying, in which leaves were put into a freeze-dryer without being prefrozen, gave concentrations that, for most secondary components, were comparable with those found in fresh leaves. Reducing the freeze-dryer chamber temperature hindered the decomposition of phenolics in prefrozen leaves and in leaves dried without prefreezing. Heat drying induced substantial changes in the composition of all phenolics, except for apigenin-7-glucoside. Vacuum drying at room temperature gave the highest concentrations for nearly all phenolics, while room-drying with desiccation gave results that were comparable with those obtained by fresh leaf analyses.
In this study, I investigated the effects of different methods of sample drying and storage, and the choice of extraction solvent and analysis method on the concentrations of 14 individual hydrolyzable tannins (HTs), and insoluble ellagitannins in birch (Betula pubescens) leaves. Freeze- and vacuum-drying of birch leaves were found to provide more reliable results than air- or oven-drying. Storage of leaves at -20 degrees C for 3 months before freeze-drying did not cause major changes in tannin content, although levels of 1,2,3,4,6-penta-O-galloylglucose and isostrictinin were altered. Storage of dried leaf material at -20 degrees C is preferred because 1 year storage of freeze-dried leaves at 4 degrees C and at room temperature decreased the concentration of the pedunculagin derivative, one of the main ellagitannins of birch. Furthermore, storage at room temperature increased the levels of isostrictinin and 2,3-(S)-HHDP-glucose, indicating possible HT catabolism. Of the extraction solvents tested, aqueous acetone was superior to pure acetone, or aqueous or pure methanol. The addition of 0.1% ascorbic acid into 70% acetone significantly increased the yield of ellagitannins. presumably by preventing their oxidation. By comparing the conventional rhodanine assay and the HPLC-ESI-MS assay for quantification of leaf galloylglucoses, the former tends to underestimate total concentrations of galloylglucoses in birch leaf extract. On the basis of the outcomes of all the method and solvent comparisons, their suitability for qualitative and quantitative analysis of plant HTs is discussed, emphasizing that each plant species, with its presumably unique HT composition, is likely to have a unique combination of ideal conditions for tissue preservation and extraction.
Analytical methods are reviewed for the determination of simple biophenols in forest trees such as Acer
(maple), Betula
(birch), Coniferus, Eucalyptus, Juniperus
(cedar), Picea
(spruce) and Quercus
(oak). Data are limited but nevertheless clearly establish the critical importance of sample preparation and pre-treatment in the analysis. For example, drying methods invariably reduce the recovery of biophenols and this is illustrated by data for birch leaves where flavonoid glycosides were determined as 12.3 ± 0.44 mg g−1 in fresh leaves but 9.7 ± 0.35 mg g−1 in air-dried samples (data expressed as dry weight). Diverse sample handling procedures have been employed for recovery of biophenols. The range of biophenols and diversity of sample types precludes general procedural recommendations. Caution is necessary in selecting appropriate procedures as the high reactivity of these compounds complicates their analysis. Moreover, our experience suggests that their reactivity is very dependent on the matrix. The actual measurement is less contentious and high performance separation methods particularly liquid chromatography dominate analyses whilst coupled techniques involving electrospray ionization are becoming routine particularly for qualitative applications. Quantitative data are still the exception and are summarized for representative species that dominate the forest canopy of various habitats. Reported concentrations for simple phenols range from trace level (<0.1 µg g−1) to in excess of 500 µg g−1 depending on a range of factors. Plant tissue is one of these variables but various biotic and abiotic processes such as stress are also important considerations.
- Harborne
Tannin analysis. Oxford, OH
- Hagerman AE