Phenolic concentrations and carbon/nitrogen ratio in annual shoots of bilberry (Vaccinium myrtillus) after simulated herbivory.

Herbivory can be reduced by the production of defense compounds (secondary metabolites), but generally defenses are costly, and growth is prioritized over defense. While defense compounds may deter herbivory, nutrients may promote it. In a field study in boreal forest in Norway, we investigated how simulated herbivory affected concentrations of phenolics (generally a defense) and the carbon/nitrogen (C/N) ratio in annual shoots of bilberry (Vaccinium myrtillus), a deciduous clonal dwarf shrub whose vegetative and generative parts provide forage for many boreal forest animals. We measured concentrations of total tannins, individual phenolics, nitrogen and carbon following several types and intensities of herbivory. We identified 22 phenolics: 15 flavonoids, 1 hydroquinone and 6 phenolic acids. After high levels of herbivory, the total tannin concentration and the concentration of these 22 phenolics together (called total phenolic concentration) were significantly lower in bilberry annual shoots than in the control (natural herbivory at low to intermediate levels). Low-intensive herbivory, including severe defoliation, gave no significantly different total tannin or total phenolic concentration compared with the control. Many individual phenolics followed this pattern, while phenolic acids (deterring insect herbivory) showed little response to the treatments: their concentrations were maintained after both low-intensive and severe herbivory. Contrary to our predictions, we found no significant difference in C/N ratio between treatments. Neither the Carbon:Nutrient Balance hypothesis nor the Optimal Defense hypotheses, theories predicting plant resource allocation to secondary compounds, can be used to predict changes in phenolic concentrations (including total tannin concentration) in bilberry annual shoots after herbivory: in this situation, carbon is primarily used for other functions (e.g., maintenance, growth, reproduction) than defense.

Anti-Candida Activity of Extracts Containing Ellagitannins, Triterpenes and Flavonoids of Terminalia brownii, a Medicinal Plant Growing in Semi-Arid and Savannah Woodland in Sudan.

Various parts of Terminalia brownii (Fresen) are used in Sudanese traditional medicine against fungal infections. The present study aimed to verify these uses by investigating the anti-Candida activity and phytochemistry of T. brownii extracts. Established agar diffusion and microplate dilution methods were used for the antifungal screenings. HPLC-DAD and UHPLC/QTOF-MS were used for the chemical fingerprinting of extracts and for determination of molecular masses. Large inhibition zones and MIC values of 312 µg/mL were obtained with acetone, ethyl acetate and methanol extracts of the leaves and acetone and methanol extracts of the roots. In addition, decoctions and macerations of the leaves and stem bark showed good activity. Sixty compounds were identified from a leaf ethyl acetate extract, showing good antifungal activity. Di-, tri- and tetra-gallotannins, chebulinic acid (eutannin) and ellagitannins, including an isomer of methyl-(S)-flavogallonate, terflavin B and corilagin, were detected in T. brownii leaves for the first time. In addition, genipin, luteolin-7-O-glucoside, apigenin, kaempferol-4'-sulfate, myricetin-3-rhamnoside and sericic acid were also characterized. Amongst the pure compounds present in T. brownii leaves, apigenin and ß-sitosterol gave the strongest growth inhibitory effects. From this study, it was evident that the leaf extracts of T. brownii have considerable anti-Candida activity with MIC values ranging from 312 to 2500 µg/mL.

Alteration in Light Spectra Causes Opposite Responses in Volatile Phenylpropanoids and Terpenoids Compared with Phenolic Acids in Sweet Basil (Ocimum basilicum) Leaves.

Basil (Ocimum basilicum, cv. Dolly) grew under three different light spectra (A, B, and C) created by light-emitting diode lamps. The proportions of UV-A, blue, and green-yellow wavelengths decreased linearly from A to C, and the proportions of red and far-red wavelengths increased from A to C. Photosynthetic photon flux density was 300 µmol m-2 s-1 in all spectra. The spectrum C plants had highest concentrations of phenolic acids (main compounds: rosmarinic acid and cichoric acid), lowest concentrations and emissions of phenylpropanoid eugenol and terpenoids (main compounds: linalool and 1,8-cineole), highest dry weight, and lowest water content. Conversely, spectra A and B caused higher terpenoid and eugenol concentrations and emissions and lower concentrations of phenolic acids. High density of peltate glandular trichomes explained high terpenoid and eugenol concentrations and emissions. Basil growth and secondary compounds affecting aroma and taste can be modified by altering light spectra; however, increasing terpenoids and phenylpropanoids decreases phenolic acids and growth and vice versa.

Hydrolyzable tannins (ellagitannins), flavonoids, pentacyclic triterpenes and their glycosides in antimycobacterial extracts of the ethnopharmacologically selected Sudanese medicinal plant Combretum hartmannianum Schweinf.

In Sudanese traditional medicine, decoctions, macerations, and tonics of the stem and root of Combretum hartmannianum are used for the treatment of persistent cough, a symptom that could be related to tuberculosis (TB). To verify these traditional uses, extracts from the stem wood, stem bark, and roots of C. hartmannianum were screened for their growth inhibitory effects against Mycobacterium smegmatis ATCC 14468. Methanol Soxhlet and ethyl acetate extracts of the root gave the strongest effects (MIC 312.5 and 625 µg/ml, respectively). HPLC-UV/DAD and UHPLC/QTOF-MS analysis of the ethyl acetate extract of the root led to the detection of 54 compounds, of which most were polyphenols and many characterized for the first time in C. hartmannianum. Among the major compounds were terflavin B and its two isomers, castalagin, corilagin, tellimagrandin I and its derivative, (S)-flavogallonic acid dilactone, punicalagin, and methyl-ellagic acid xylopyranoside. In addition, di-, tri- and tetra-galloyl glucose, combregenin, terminolic acid, cordifoliside D, luteolin, and quercetin-3-O-galactoside-7-O-rhamnoside-(2→1)-O-ß-D-arabinopyranoside were characterized. Luteolin gave better growth inhibition against M. smegmatis (MIC 250 µg/ml) than corilagin, ellagic acid, and gallic acid (MIC 500-1000 µg/ml). Our study justifies the use of C. hartmannianum in Sudanese folk medicine against prolonged cough that could be related to TB infection. This study demonstrates that C. hartmannianum should be explored further for new anti-TB drug scaffolds and antibiotic adjuvants.

Accumulation of phenolics and growth of dioecious Populus tremula (L.) seedlings over three growing seasons under elevated temperature and UVB radiation.

Accumulation of secondary metabolites may exhibit developmentally regulated variation in different plant organs. Moreover, prevailing environmental conditions may interact with development-related variations in plant traits. In this study, we examined developmentally regulated variation in phenolic accumulation in the twigs of dioecious Populus tremula (L.) and how the effects of elevated temperature and ultraviolet B (UVB) radiation on growth and phenolics accumulation varied as the plants get older. In an open-field experiment, six female and six male genotypes were exposed to single and combined elevated temperature and UVB radiation treatments for three consecutive growing seasons. The concentrations of low molecular weight phenolics and condensed tannins did not show age-dependent variation in the twigs. In temperature-treated plants, diameter growth rate decreased, and concentration of condensed tannins increased as plants aged; there were no cumulative effects of elevated UVB radiation on growth and phenolic accumulation. Females maintained a higher concentration of low molecular weight phenolics throughout the experimental period; however, growth and phenolic concentration did not vary over time in females and males. Our results suggest that phenolic accumulation in perennial plants may not necessarily always exhibit age-dependent variation and the effects of elevated temperature on growth and phenolic may diminish as plants get older.

Genetic modification of the flavonoid pathway alters growth and reveals flexible responses to enhanced UVB - Role of foliar condensed tannins.

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.

1,6-Dehydropinidine Is an Abundant Compound in Picea abies (Pinaceae) Sprouts and 1,6-Dehydropinidine Fraction Shows Antibacterial Activity against Streptococcus equi Subsp. equi.

Knowledge about the defensive chemistry of coniferous trees has increased in recent years regarding a number of alkaloid compounds; in addition to phenolics and terpenes. Here, we show that Norway spruce (Picea abies (L.) H. Karst.), an important boreal zone tree species; accumulates 1,6-dehydropinidine (2-methyl-6-(2-propenyl)-1,6-piperideine) in its needles and bark. We reanalyzed previously published GC-MS data to obtain a full picture of 1,6-dehydropinidine in P. abies. 1,6-dehydropinidine appeared to especially accumulate in developing spring shoots. We used solid-phase partitioning to collect the alkaloid fraction of the sprouts and thin-layer chromatography to purify 1,6-dehydropinidine. The antibacterial properties of the 1,6-dehydropinidine fraction were tested using a broth microdilution method; with Streptococcus equi subsp. equi as a model organism. Based on our results 1,6-dehydropinidine is common in alkaloid extractions from P. abies (0.4 ± 0.03 mg g-1 dw in mature needles) and it is especially abundant in young spruce shoots (2.7 ± 0.5 mg g-1 dw). Moreover; 1,6-dehydropinidine extracted from P. abies sprouts showed mild antibacterial potential against Streptococcus equi subsp. equi (MIC 55 µg mL-1). The antibacterial activity of a plant compound thought of as an intermediate rather than an end-product of biosynthesis calls for more detailed studies regarding the biological function of these coniferous alkaloids.

HPLC-DAD and UHPLC/QTOF-MS Analysis of Polyphenols in Extracts of the African Species Combretum padoides, C. zeyheri and C. psidioides Related to Their Antimycobacterial Activity.

Combretum padoides Engl. & Diels, C. psidioides Welv. and C. zeyheri Sond. are used forthe treatment of infections and tuberculosis related symptoms in African traditional medicine. In orderto verify these uses, extracts were screened for their growth inhibitory eects against M. smegmatisATCC 14468. Ultra-high pressure liquid chromatography coupled to quadrupole time-of-flightmass spectrometry (UHPLC/QTOF-MS) and GC-MS were used to investigate the polyphenoliccomposition in the active extracts. The lowest minimum inhibitory concentration (MIC), 625 g/mL,was shown by a methanol extract of the stem bark of C. psidioides. A butanol extract of C. psidioidesgave large inhibition zone diameters (IZD 21 mm) and inhibited 84% of the mycobacterial growthat 312 g/mL. Combretastatin B-2 and dihydrostilbene derivatives were present in the methanolextract of C. psidioides, whereas the butanol extract of this species contained punicalagin, corilagin,and sanguiin H-4. Methanol and butanol extracts of the stem bark of C. padoides gave large inhibitionzone diameters (IZD 26.5 mm) and MIC values of 1250 and 2500 g/mL, respectively. C. padoidescontained an ellagitannin with a mass identical to punicalagin ([M-H]- 1083.0587) and a corilaginlike derivative ([M-H]- 633.0750) as well as ellagic acid arabinoside and methyl ellagic acid xyloside.A butanol extract of the roots of C. zeyheri showed mild antimycobacterial activity and containeda gallotannin at m/z [M-H]- 647.0894 as the main compound along with punicalagin and threeunknown ellagitannins at m/z [M-H]- 763.0788, 765.0566, and 817.4212. Our results indicate thatthe studied species of Combretum contain phenolic and polyphenolic compounds with possiblepotential as leads for antimycobacterial drugs or as adjuvants for conventional anti-TB drugs.

Potential Anti-Tuberculosis Activity of the Extracts and Their Active Components of Anogeissus Leiocarpa (DC.) Guill. and Perr. with Special Emphasis on Polyphenols.

In Sudanese traditional medicine, decoctions of the stem bark of Anogeissus leiocarpa are used for the treatment of tuberculosis (TB). However, this plant has not been investigated before for its antimycobacterial effects. Our screening results show, for the first time, that many extracts of various parts of A. leiocarpa exhibit growth inhibitory activity against Mycobacterium smegmatis. Minimum inhibitory concentration (MIC) values ranged between 625 and 5000 µg/mL, with an ethyl acetate extract of the root showing the lowest MIC value. The good antimycobacterial effects of the root part could be due to its high concentration of ellagic acid derivatives, ellagitannins, and flavonoids. Thin layer chromatography (TLC) fractionation resulted in some fractions with better activity than the starting point crude methanol extract (MIC 2500 µg/mL). Those fractions with the lowest MIC values contained a high number of antioxidant compounds. Fractions 3 and 4 (MIC 1500 and 1000 µg/mL, respectively) contained high concentrations of di-methyl ellagic acid ([M-H] - 329.0318). Fraction 6 (MIC 2000 µg/mL) contained a lower concentration of di-methyl ellagic acid and was not as growth inhibitory as fractions 3 and 4. Moreover, in fraction 3, an acetylated ellagic acid derivative ([M-H] - 343.0477) and di-methyl-ellagic acid xyloside ([M-H]- 461.0739) were tentatively characterized. Di-methyl ellagic acid xyloside was also present in fraction 4 and could strongly contribute to the antimycobacterial effect of this fraction. Additionally, protocatechuic acid ([M-H]-at m/z 153.0196) was present in fraction 4. Our antimycobacterial results obtained from this research justify the use of A. leiocarpa in Sudanese folk medicine against cough related to TB. Roots, stem bark, and leaves of A. leiocarpa are sources for new potent anti-TB drug lead compounds.

Long-term effects of elevated CO2, nighttime warming and drought on plant secondary metabolites in a temperate heath ecosystem.

BACKGROUND AND AIMS: Plant secondary metabolites play critical roles in plant stress tolerance and adaptation, and are known to be influenced by the environment and climate changes, yet the impacts and interactions of multiple climate change components are poorly understood, particularly under natural conditions. METHODS: Accumulation of phenolics and emissions of volatile organic compounds (VOCs) were assessed on heather, Calluna vulgaris, an abundant evergreen dwarf shrub in European heathlands, after 6 years of exposure to elevated CO2, summer drought and nighttime warming. KEY RESULTS: Drought alone had the strongest effects on phenolic concentrations and compositions, with moderate effects of elevated CO2 and temperature. Elevated CO2 exerted the greatest impact on VOC emissions, mainly by increasing monoterpene emissions. The response magnitudes varied among plant tissue types and chemical constituents, and across time. With respect to interactive effects of the studied climate change components, the interaction between drought and elevated CO2 was most apparent. Drought mainly reduced phenolic accumulation and VOC emissions, while elevated CO2 mitigated such effects. CONCLUSIONS: In natural ecosystems, co-occurring climate factors can exert complex impacts on plant secondary metabolite profiles, which may in turn alter ecosystem processes.

Phytochemical Shift from Condensed Tannins to Flavonoids in Transgenic Betula pendula Decreases Consumption and Growth but Improves Growth Efficiency of Epirrita autumnata Larvae.

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.

Responses in growth and phenolics accumulation to lateral bud removal in male and female saplings of Populus tremula (L.) under simulated climate change.

The responses in growth and defense after tissue damage are highly variable in plants depending on species, damaged-tissue type and the intensity of damage. The prevailing abiotic conditions can also influence these responses. In this study, our aim was to examine how the removal of lateral vegetative buds affects the growth and accumulation of phenolics in saplings of the dioecious Populus tremula grown under simulated climate change. For three growing seasons, the saplings were grown under ambient conditions (control), elevated temperature (+2°C) and elevated UV radiation (30%) (UVB and UVA as its control), or a combination of these. In the fourth growing season, all saplings were grown under ambient conditions. The bud removal was performed twice - in summer and autumn - in the third year. The responses of growth and the accumulation of phenolics to the bud removal were measured at the end of the fourth growing season. Removal of 5% of the lateral buds resulted in higher leaf, stem and total plant biomass in both sexes of P. tremula saplings, compared to intact plants. The effects were greater in the temperature-treated plants, especially in the temperature-treated females. The concentrations of flavonoids and condensed tannins were higher in the bud-removed individuals. The concentration of condensed tannins was also higher in the males than in the females, opposite to the concentration of phenolic acids. There was no significant interaction between bud removal and UVB treatment on either growth or phenolics. Our results suggest that plants can allocate resources to both growth and defense simultaneously in response to tissue loss, and that global warming can modify the responses to some extent.

Fourier-transform infrared (FT-IR) spectroscopy analysis discriminates asymptomatic and symptomatic Norway spruce trees.

Conifer trees, including Norway spruce, are threatened by fungi of the Heterobasidion annosum species complex, which severely affect timber quality and cause economic losses to forest owners. The timely detection of infected trees is complicated, as the pathogen resides within the heartwood and sapwood of infected trees. The presence of the disease and the extent of the wood decay often becomes evident only after tree felling. Fourier-transform infrared (FT-IR) spectroscopy is a potential method for non-destructive sample analysis that may be useful for identifying infected trees in this pathosystem. We performed FT-IR analysis of 18 phloem, 18 xylem, and 18 needle samples from asymptomatic and symptomatic Norway spruce trees. FT-IR spectra from 1066 - 912 cm-1 could be used to distinguish phloem, xylem, and needle tissue extracts. FT-IR spectra collected from xylem and needle extracts could also be used to discriminate between asymptomatic and symptomatic trees using spectral bands from 1657 - 994 cm-1 and 1104 - 994 cm-1, respectively. A partial least squares regression model predicted the concentration of condensed tannins, a defense-related compound, in phloem of asymptomatic and symptomatic trees. This work is the first to show that FT-IR spectroscopy can be used for the identification of Norway spruce trees naturally infected with Heterobasidion spp.

Dual RNA-seq analysis provides new insights into interactions between Norway spruce and necrotrophic pathogen Heterobasidion annosum s.l.

BACKGROUND: Root and butt rot of conifer trees caused by fungi belonging to the Heterobasidion annosum species complex is one of the most economically important fungal diseases in commercial conifer plantations throughout the Northern hemisphere. We investigated the interactions between Heterobasidion fungi and their host by conducting dual RNA-seq and chemical analysis on Norway spruce trees naturally infected by Heterobasidion spp. We analyzed host and pathogen transcriptome and phenolic and terpenoid contents of the spruce trees. RESULTS: Presented results emphasize the role of the phenylpropanoid and flavonoid pathways in the chemical defense of Norway spruce trees. Accumulation of lignans was observed in trees displaying symptoms of wood decay. A number of candidate genes with a predicted role in the higher level regulation of spruce defense responses were identified. Our data indicate a possible role of abscisic acid (ABA) signaling in the spruce defense against Heterobasidion infection. Fungal transcripts corresponding to genes encoding carbohydrate- and lignin-degrading enzymes, secondary metabolism genes and effector-like genes were expressed during the host colonization. CONCLUSIONS: Our results provide additional insight into defense strategies employed by Norway spruce trees against Heterobasidion infection. The potential applications of the identified candidate genes as markers for higher resistance against root and butt rot deserve further evaluation.

Polyphenols from the sagebrush Artemisia tridentata ssp. tridentata affect the redox state of cultured hepatocytes by direct and indirect mechanisms.

Basin big sagebrush (Artemisia tridentata Nutt. ssp. tridentata (Asteraceae)), is a widespread North American shrub which produces a variety of polyphenolic compounds. Although sagebrush has been used as a traditional remedy by natives and settlers to the region, the polyphenols in Artemisia tridentata ssp. tridentata have not been highly investigated for their bioactive properties. To determine whether these polyphenols affect the intracellular redox state, we measured their ability to neutralize radicals in vitro and in a human liver carcinoma cell line (HepG2), and their effects on intracellular glutathione levels. Extracts from Artemisia tridentata ssp. tridentata decreased the oxidation of 2'7'-dichlorofluorescin in vitro and in cultured cells. Cells treated with polyphenolic extracts showed increased levels of glutathione in a time and dose-dependent manner. Approximately 48 polyphenolic compounds were distinguishable in extracts, by HPLC/UV absorbance detection. Mass spectroscopy was used to identify thirteen compounds as aesculin, aesculetin, apigenin, apigenin-7-O-glucoside, axillarin, casticin, chlorogenic acid, isoscopoletin, kaempferol, luteolin, methyl-axillarin, quercetin, and scopoletin. These results indicate that polyphenols produced in Artemisia tridentata ssp. tridentata affect the redox state of living cells by multiple mechanisms.

Alkaloid-Rich Crude Extracts, Fractions and Piperamide Alkaloids of Piper guineense Possess Promising Antibacterial Effects.

Piper guineense is a food and medicinal plant commonly used to treat infectious diseases in West-African traditional medicine. In a bid to identify new antibacterial compounds due to bacterial resistance to antibiotics, twelve extracts of P. guineense fruits and leaves, obtained by sequential extraction, as well as the piperine and piperlongumine commercial compounds were evaluated for antibacterial activity against human pathogenic bacteria. HPLC-DAD and UHPLC/Q-TOF MS analysis were conducted to characterize and identify the compounds present in the extracts with promising antibacterial activity. The extracts, with the exception of the hot water decoctions and macerations, contained piperamide alkaloids as their main constituents. Piperine, dihydropiperine, piperylin, dihydropiperylin or piperlonguminine, dihydropiperlonguminine, wisanine, dihydrowisanine and derivatives of piperine and piperidine were identified in a hexane extract of the leaf. In addition, some new piperamide alkaloids were identified, such as a piperine and a piperidine alkaloid derivative and two unknown piperamide alkaloids. To the best of our knowledge, there are no piperamides reported in the literature with similar UVλ absorption maxima and masses. A piperamide alkaloid-rich hexane leaf extract recorded the lowest MIC of 19 µg/mL against Sarcina sp. and gave promising growth inhibitory effects against S. aureus and E. aerogenes as well, inhibiting the growth of both bacteria with a MIC of 78 µg/mL. Moreover, this is the first report of the antibacterial activity of P. guineense extracts against Sarcina sp. and E. aerogenes. Marked growth inhibition was also obtained for chloroform extracts of the leaves and fruits against P. aeruginosa with a MIC value of 78 µg/mL. Piperine and piperlongumine were active against E. aerogenes, S. aureus, E. coli, S. enterica, P. mirabilis and B. cereus with MIC values ranging from 39⁻1250 µg/mL. Notably, the water extracts, which were almost devoid of piperamide alkaloids, were not active against the bacterial strains. Our results demonstrate that P. guineense contains antibacterial alkaloids that could be relevant for the discovery of new natural antibiotics.

Climate Change Effects on Secondary Compounds of Forest Trees in the Northern Hemisphere.

Plant secondary compounds (PSCs), also called secondary metabolites, have high chemical and structural diversity and appear as non-volatile or volatile compounds. These compounds may have evolved to have specific physiological and ecological functions in the adaptation of plants to their growth environment. PSCs are produced by several metabolic pathways and many PSCs are specific for a few plant genera or families. In forest ecosystems, full-grown trees constitute the majority of plant biomass and are thus capable of producing significant amounts of PSCs. We summarize older literature and review recent progress in understanding the effects of abiotic and biotic factors on PSC production of forest trees and PSC behavior in forest ecosystems. The roles of different PSCs under stress and their important role in protecting plants against abiotic and biotic factors are also discussed. There was strong evidence that major climate change factors, CO2 and warming, have contradictory effects on the main PSC groups. CO2 increases phenolic compounds in foliage, but limits terpenoids in foliage and emissions. Warming decreases phenolic compounds in foliage but increases terpenoids in foliage and emissions. Other abiotic stresses have more variable effects. PSCs may help trees to adapt to a changing climate and to pressure from current and invasive pests and pathogens. Indirect adaptation comes via the effects of PSCs on soil chemistry and nutrient cycling, the formation of cloud condensation nuclei from tree volatiles and by CO2 sequestration into PSCs in the wood of living and dead forest trees.

Fertilization Changes Chemical Defense in Needles of Mature Norway Spruce (Picea abies).

Nitrogen availability limits growth in most boreal forests. However, parts of the boreal zone receive significant levels of nitrogen deposition. At the same time, forests are fertilized to increase volume growth and carbon sequestration. No matter the source, increasing nitrogen in the boreal forest ecosystem will influence the resource situation for its primary producers, the plants, with possible implications for their defensive chemistry. In general, fertilization reduces phenolic compound concentrations in trees, but existing evidence mainly comes from studies on young plants. Given the role of the phenolic compounds in protection against herbivores and other forest pests, it is important to know if phenolics are reduced with fertilization also in mature trees. The evergreen Norway spruce is long-lived, and it is reasonable that defensive strategies could change from the juvenile to the reproductive and mature phases. In addition, as the needles are kept for several years, defense could also change with needle age. We sampled current and previous year needles from an N fertilization experiment in a Norway spruce forest landscape in south-central Norway to which N had been added annually for 13 years. We analyzed total nitrogen (N) and carbon (C), as well as low-molecular phenolics and condensed tannins. Needles from fertilized trees had higher N than those from controls plots, and fertilization decreased concentrations of many flavonoids, as well as condensed tannins in current year needles. In previous year needles, some stilbenes and condensed tannins were higher in fertilized trees. In control trees, the total phenolic concentration was almost five times as high in previous year needles compared with those from the current year, and there were great compositional differences. Previous year needles contained highest concentrations of acetophenone and stilbenes, while in the current year needles the flavonoids, and especially coumaroyl-astragalins dominated. Condensed tannins did not differ between current and previous year needles from control trees. In conclusion, the phenolic defense of current year needles of mature P.abies trees was strongly changed upon fertilization. This may imply that nitrogen deposition and forest fertilization leave forests less robust in a time when pests may take advantages of a changing climate.

Variability in the composition of phenolic compounds in winter-dormant Salix pyrolifolia in relation to plant part and age.

The phenolic phytochemicals of winter-dormant Salix pyrolifolia were determined from the vegetative buds, and the bark and wood of different-aged twigs by HPLC-DAD and UHPLC-QTOF-MS analyses. All the plant parts were composed of salicylate glucosides and the other Salix-specific, simple phenolic glucosides as well as of phenolic acids, flavonoids and the high molecular-weight condensed tannins. The flavonoid composition was most diverse in buds and they also contained a large amount of chlorogenic acid (5-caffeoylquinic acid IUPAC), while salicylate glucosides and simple phenolic glucosides predominated in bark. The wooden interior part of the twigs contained fewer components and the lowest concentrations of compounds. Salicortin was the main compound in winter-dormant S. pyrolifolia (over 10% of bark biomass), but the concentrations of picein, salireposide, isosalipurposide, catechin and condensed tannins were also high. The flavonoid composition was highly naringenin- and quercetin-biassed. The composition of phytochemicals was organ-specific and remained relatively similar between different-aged trees. However, there were compound-specific fluctuations in the concentrations of phytochemicals with the age of the trees and within plant parts. Generally, the one-year-old plants differed from the older trees in their high concentration of condensed tannins in all the plant parts studied and in the highest concentration of isosalipurposide in bark, while the total amounts of salicylate glucosides in plant parts, and of naringenin glucosides in buds, tended to be highest in 20 year-old-trees.

Terminalia laxiflora and Terminalia brownii contain a broad spectrum of antimycobacterial compounds including ellagitannins, ellagic acid derivatives, triterpenes, fatty acids and fatty alcohols.

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia laxiflora Engl. & Diels, (Sudanese Arabic name: Darout الدروت) and Terminalia brownii Fresen (Sudanese Arabic name: Alshaf ألشاف) (Combretaceae) are used in Sudanese traditional folk medicine and in other African countries for treatment of infectious diseases, TB and its symptoms, such as cough, bronchitis and chest pain. AIM OF STUDY: Because of the frequent use of T. laxiflora and T. brownii in African traditional medicine and due to the absence of studies regarding their antimycobacterial potential there was a need to screen extracts of T. laxiflora and T. brownii for their growth inhibitory potential and to study the chemical composition and compounds in growth inhibitory extracts. MATERIALS AND METHODS: The plant species were collected in Sudan (Blue Nile Forest, Ed Damazin Forestry areas) and selected according to their uses in traditional medicine for the treatment of bacterial infections, including TB. Eighty extracts and fractions of the stem bark, stem wood, roots, leaves and fruits of T. laxiflora and T. brownii and nine pure compounds present in the active extracts were screened against Mycobacterium smegmatis ATCC 14468 using agar diffusion and microplate dilution methods. Inhibition zones and MIC values were estimated and compared to rifampicin. HPLC-UV/DAD, GC/MS and UHPLC/Q-TOF MS were employed to identify the compounds in the growth inhibitory extracts. RESULTS: The roots of T. laxiflora and T. brownii gave the best antimycobacterial effects (IZ 22-27 mm) against Mycobacterium smegmatis. The lowest MIC of 625 µg/ml was observed for an acetone extract of the root of T. laxiflora followed by methanol and ethyl acetate extracts, both giving MIC values of 1250 µg/ml. Sephadex LH-20 column chromatography purification of T. brownii roots resulted in low MIC values of 62.5 µg/ml and 125 µg/ml for acetone and ethanol fractions, respectively, compared to 5000 µg/ml for the crude methanol extract. Methyl (S)-flavogallonate is suggested to be the main active compound in the Sephadex LH- 20 acetone fraction, while ellagic acid xyloside and methyl ellagic acid xyloside are suggested to give good antimycobacterial activity in the Sephadex LH-20 ethanol fraction. RP-18 TLC purifications of an ethyl acetate extract of T. laxiflora roots resulted in the enrichment of punicalagin in one of the fractions (Fr5). This fraction gave a five times smaller MIC (500 µg/ml) than the crude ethyl acetate extract (2500 µg/ml) and this improved activity is suggested to be mostly due to punicalagin. 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, triacontanol, sitostenone and ß-sitosterol were found in antimycobacterial hexane extracts of the stem bark of both studied species. Of these compounds, 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, triacontanol, sitostenone have not been previously identified in T. brownii and T. laxiflora. Moreover, both plant species contained friedelin, betulinic acid, ß-amyrine and two unknown oleanane-type triterpenoids. Of the listed compounds, friedelin, triacontanol and sitostenone gave a MIC of 250 µg/ml against M. smegmatis, whereas stigmasterol and ß-sitosterol gave MIC values of 500 µg/ml. CONCLUSIONS: Our results show that T. laxiflora and T. brownii contain antimycobacterial compounds of diverse polarities and support the traditional uses of various parts of T. laxiflora and T.brownii as decoctions for treatment of tuberculosis. Further investigations are warranted to explore additional (new) antimycobacterial compounds in the active extracts of T. laxiflora and T. brownii.