Comparative Analyses of Phytochemical Variation Within and Between Congeneric Species of Willow Herb, Epilobium hirsutum and E. parviflorum: Contribution of Environmental Factors.

The plants in the Epilobium genus are considered to have several important medicinal properties due to their unique chemical composition. Although metabolic profiles of medicinal plants are mainly controlled by genetic factors, their production is also to some degree influenced by environmental factors, thus, variations in the levels of phytochemicals may represent long-term ecological and evolutionary interactions. In order to depict the magnitude of natural variation in level of chemical compounds among conspecific populations of Epilobium hirsutum (n = 31) and E. parviflorum (n = 16), metabolite profiling of aerial parts of plants was performed with gas chromatography/mass spectrometry analysis. Putative identification and structure annotation revealed the presence of 74 compounds including 46 compounds considered secondary metabolites categorized into flavonoids (n = 8), phenolic acids (n = 26), steroids (n = 3), and terpenes (n = 5) across all populations. Although there was a considerable natural variation among conspecific populations, principal component analysis revealed a clear separation of populations of each species based on the second main principal component which was highly correlated with eight secondary metabolites. The level of secondary metabolites was significantly correlated between species (r = 0.91), suggesting shared metabolic pathways underlying the production of chemical compounds. In addition, redundancy and variance partitioning analyses by including bioclimatic variables and altitude revealed a significant contribution of elevation in explaining the total variation of secondary metabolites in E. hirsutum. Two-thirds of all secondary metabolites were significantly correlated with altitude in E. hirsutum. The large-scale geographic analyses of populations revealed additionally detected flavonoids and terpenes (E. hirsutum and E. parviflorum) and steroids (E. hirsutum) for the first time. This study provides significant information on additional chemical compounds found across the distribution range of the two ecologically important species of willow herb and emphasizes the importance of geographic-wide sampling as a valuable strategy to depict intraspecific and interspecific variability in chemical traits.

Large Scale Geographical Mapping of Essential Oil Volatiles in Heracleum (Apiaceae): Identification of Novel Compounds and Unraveling Cryptic Variation.

Conspecific populations of plants in their native range are expected to show considerable variation due to long-term ecological and evolutionary factors. We investigated the levels of secondary metabolites in Heracleum including H. persicum a valuable medicinal plant to depict the magnitude of cryptic variation and the potential significance of novel chemical traits. The essential oil volatiles from fruits of 34 populations from different species of Heracleum in Iranian distribution range and a native of H. sphondylium and an invasive population of H. persicum from Norway were analyzed with GC/MS. Out of 48 compounds identified, a contrasting pattern in the level of two major compounds, octyl acetate and hexyl butyrate was found among all studied species. Interestingly, a significant geographic pattern was observed; the hexyl butyrate/octyl acetate ratio was high (range 1.8 - 3.2) in the northwestern Iranian populations of H. persicum compared to that in northern and central populations (range 0.3 - 0.9). Four populations from Zagros mountains also exhibited a unique composition. Anethole was found in two populations of H. persicum from central Zagros, which has not been previously reported for essential oil of fruits of Heracleum so far. The results suggest high efficiency of large scale sampling from distribution range of species in identifying novel compounds. The unique pattern of geographic structuring also provides novel information to unravel cryptic variation in Heracleum.

Molecular adaptations to phosphorus deprivation and comparison with nitrogen deprivation responses in the diatom Phaeodactylum tricornutum.

Phosphorus, an essential element for all living organisms, is a limiting nutrient in many regions of the ocean due to its fast recycling. Changes in phosphate (Pi) availability in aquatic systems affect diatom growth and productivity. We investigated the early adaptive mechanisms in the marine diatom Phaeodactylum tricornutum to P deprivation using a combination of transcriptomics, metabolomics, physiological and biochemical experiments. Our analysis revealed strong induction of gene expression for proteins involved in phosphate acquisition and scavenging, and down-regulation of processes such as photosynthesis, nitrogen assimilation and nucleic acid and ribosome biosynthesis. P deprivation resulted in alterations of carbon allocation through the induction of the pentose phosphate pathway and cytosolic gluconeogenesis, along with repression of the Calvin cycle. Reorganization of cellular lipids was indicated by coordinated induced expression of phospholipases, sulfolipid biosynthesis enzymes and a putative betaine lipid biosynthesis enzyme. A comparative analysis of nitrogen- and phosphorus-deprived P. tricornutum revealed both common and distinct regulation patterns in response to phosphate and nitrate stress. Regulation of central carbon metabolism and amino acid metabolism was similar, whereas unique responses were found in nitrogen assimilation and phosphorus scavenging in nitrogen-deprived and phosphorus-deprived cells, respectively.

Interaction of soyasaponins with plant ingredients in diets for Atlantic salmon, Salmo salar L.

The effects of combining soyasaponins with plant ingredients on intestinal function and fish health were investigated in an 80 d study with Atlantic salmon (270 g) distributed thirty each into twenty-four tanks with seawater. Soyasaponins were supplemented (2 g/kg) to diets with maize gluten (MG), pea protein concentrate (PPC) and sunflower (SFM), rapeseed (RSM) or horsebean meals. A diet with soyabean meal (SBM) and another with wheat gluten and soyasaponins served as reference diets. Marked soyasaponin effects were observed when combined with PPC. This combination induced inflammation in the distal intestine (DI) similar to SBM, reduced feed intake, apparent digestibility of lipid, most amino acids and ash, decreased bile salt levels in intestinal chyme and decreased leucine aminopeptidase (LAP) activity but increased trypsin activity in the DI. No enteritis was observed in other diet groups, but small consistent negative soyasaponin effects were seen on lipid and fatty acid digestibility, faecal DM and LAP activity of the DI. Soyasaponin combination with RSM reduced digestibility of all nutrients including minerals. The mineral effect was also seen for SFM, whereas with MG and SFM a positive soyasaponin effect on feed intake was observed. Caution should be exercised to avoid ingredient combinations giving high saponin levels, a condition that appears to be a key factor in diet-induced enteritis together with certain plant ingredients.

Volatile constituents and biological activities of Pycnostachys abyssinica and Pycnostachys eminii extracts.

CONTEXT: Pycnostachys abyssinica Fresen and Pycnostachys eminii Gürke (Lamiaceae) are used in traditional Ethiopian medicine against eye and skin infections, "Mitch disease", and dysentery. OBJECTIVE: Our study was aimed at characterizing essential oil (EO), phytochemical groups, and antimicrobial and anthelmintic activity of extracts to underscore the species' indigenous medicinal use. MATERIALS AND METHODS: Plant organs of Pycnostachys species were subjected to hydrodistillation, and essential oils (EO) analyzed by GC-MS. Phytochemical compounds, antimicrobial (diffusion assay) and anthelmintic activity (bioassay) of gradient solvent extracts of different polarity were studied. RESULTS: In the stem and root EO of P. abyssinica, 25 (99%) and 30 (99.79%) compounds were detected respectively, with estragole (70.4%) (stem) and exo-fenchyl acetate (30.6%) (root) as the most abundant compounds. In leaf, stem and root EO of P. eminii, 30 (90.66%), 27 (90.59%) and 27 (99.96%) compounds were detected, respectively, with high levels of ß-caryophyllene (from 18.08% to 28.85%) and germacrene D (from 15.1% to 22.06%). Alkaloids, saponins, phytosterols, flavonoids, polyphenols, diterpenoids and carotenoids were detected in Pycnostachys. Petroleum ether, chloroform and methanol extracts showed distinct antimicrobial effects with generally higher potential activity of lipophilic and semi-lipophilic fractions. Leaf and root methanol extracts of both species showed lethal activity against earthworms. DISCUSSION: Identified EO constituents and phytochemical groups underscore the observed antifungal, antibacterial and anthelmintic activity of Pycnostachys gradient solvent extracts. CONCLUSION: EO analysis, phytochemical screening, and antimicrobial and anthelmintic assays indicate the biological potential of Pycnostachys species from Ethiopia, and emphasize their pharmacological and indigenous applications.

Constituents, antileishmanial activity and toxicity profile of volatile oil from berries of Croton macrostachyus.

The chemical composition of the volatile oil from berries of Croton macrostachyus Hochst. ex Del. was determined by GC and GC/MS. The oil was tested for its in vitro antileishmanial activity on two Leishmania strains, and its toxicity on the human monocytic leukemia (THP-1) cell line and erythrocytes from sheep blood. The main constituents of the oil were benzyl benzoate (51.8%), linalool (10.1%), gamma-muurolene (9.3%), (E,E)-alpha-farnesene (3.2%), delta-cadinene (2.8%) and alpha-curcumene (2.7%). The oil was effective against L. donovani and L. aethiopica promastigotes (MIC = 0.08 microL/mL and 0.16 microL/mL, respectively) and axenic amastigote stages (EC50 = 20.00 nL/mL and 6.66 nL/mL, respectively). The CC50 value for the oil was 10.00 nL/mL on THP-1 cells with selectivity index values of 0.5 for L. donovani and 1.5 for L. aethiopica. The median lethal concentration (LC50) of the oil was 2.45 microL/mL. Thus the observed high efficacy and moderate toxicity of the volatile oil from C. macrostachyus, makes the plant a promising source of new lead compounds in the search for safe and effective antileishmanial drugs.

Essential-oil composition, antileishmanial, and toxicity study of Artemisia abyssinica and Satureja punctata ssp. punctata from Ethiopia.

Essential oils of Artemisia abyssinica and Satureja punctata ssp. punctata from Ethiopia were analyzed by GC and GC/MS, and screened for leishmanicidal activity against promastigote and axenic amastigotes of Leishmania donovani and L. aethiopica, including toxicity studies on human monocytic leukemia cells (THP-1) and erythrocytes in vitro. GC/MS of A. abyssinica oil revealed 67 compounds (99.94%) with the major constituents yomogi alcohol (38.47%), artemisyl acetate (24.88%), and artemisia alcohol (6.70%), and oxygenated monoterpenes (84.00%) as the dominant group. The oil of S. punctata contained 67 compounds (99.49%) with the main constituents geranial (27.62%), neral (21.72%), alpha-bisabolol (13.62%), and (E)-nerolidol (4.82%), of which oxygenated mono- and sesquiterpenes (58.39 and 26.91%, resp.) showed highest abundance. Both oils showed effect on promastigotes (MIC 76.5 to 312.5 nl/ml) and amastigotes (EC(50) 4.06 to 131.00 nl/ml) of L. donovani and L. aethiopica, and varying toxicities on THP-1 cells (CC(50) 0.013 to 350 nl/ml with selectivity index between 0.001 and 28) and erythrocytes (with LC(50) 0.35 to 1.52 microl/ml). S. punctata oil exerted highest activity against both Leishmania sp. and toxicity. The revealed antileishmanial activities support further isolation and investigation of oil constituents for in vitro/in vivo evaluation.

Potent in vitro inhibition of CYP3A4 and P-glycoprotein by Rhodiola rosea.

Six clones of RHODIOLA ROSEA, obtained from plants originating from widely different areas in Norway, were investigated for their IN VITRO inhibitory potential on CYP3A4-mediated metabolism and P-gp efflux transport activity. Presumed active constituents in the ethanol extracts of the different clones were quantified. C-DNA baculovirus expressed CYP3A4 and Caco-2 cells were used for inhibitory assays, and as positive control inhibitors ketoconazole and verapamil were applied, respectively. A validated HPLC methodology was used to quantify the formation of 6-beta-OH-testosterone and scintillation counting was used to quantify the transport of (3)H-digoxin in Caco-2 cells. All clones showed potent inhibition of CYP3A4 and P-gp activities, with IC (50) values ranging from 1.7 to 3.1 microg/mL and from 16.7 to 51.7 microg/mL, respectively, being below that reported for other herbs and some known classic drug inhibitors, such as St. John's wort and fluoxetine. RHODIOLA ROSEA might thus be a candidate for clinically relevant drug interactions. The concentration of presumed biologically active constituents in the different clones varied considerably, but this variation was not related to the clones' inhibitory potential on CYP3A4 or P-gp activities. Other constituents might thus be responsible for the observed inhibitory properties. The place of origin seemed to be of minor importance for CYP3A4 or P-gp inhibition.

Volatile profiling of Arabidopsis thaliana - putative olfactory compounds in plant communication.

Arabidopsis thaliana from the Brassicaceae family has arisen as the model organism in plant biology research. The plant's genome has been characterized and worldwide studies are conducted at the genetic, protein and metabolic level to unravell the function of genes involved in growth, reproduction, biosynthesis, and plant communication. As part of the multidisciplinary project BIOEMIT at NTNU, metabolomic studies of Arabidopsis T-DNA knock-out mutants and ecotypes have been carried out. Volatile profiles of autolyzed, intact plants and single plant organs were obtained by solid-phase microextraction coupled with gas chromatography-mass spectrometry. The studies were aimed at the diversity of defense-related compounds from the glucosinolate-myrosinase system - the isothiocyanates and nitriles. Metabolites from methionine, leucine and phenylalanine-derived glucosinolates were most abundant (4-methylthiobutyl, 4-methylpentyl, 2-phenylethyl). In addition, 24 monoterpenes, 26 sesquiterpenes and 12 aromatic structures, predominantly observed in inflorescenses, are described. Excluding the vast group of straight chain aliphatic structures, a total of 102 volatile compounds were detected, of which 59 are reported in Arabidopsis thaliana for the first time, thus emphasizing the sensitivity and applicability of solid-phase microextraction for volatile profiling of plant secondary metabolites.

Effect of harvest time and drying method on biomass production, essential oil yield, and quality of peppermint (Mentha x piperita L.).

In the period from 2000 to 2002, studies on peppermint (Mentha x piperita) herb and essential oil (EO) production have been conducted at Planteforsk, Apelsvoll Research Centre Div. Kise in Norway. The trials were aimed at finding the optimal harvest date and suitable drying methods to maximize EO yield and to obtain a desirable oil quality. Peppermint plants from the first production year (2000 and 2001) and the second production year (2002) were harvested during flowering at three developmental stages (early, full, and late bloom). Biomass and leaf production were recorded, and the water content of the plant material was detected after the application of different drying methods: instantaneous drying at 30, 50, and 70 degrees C and prewilting (ground drying) for 1 or 5 days followed by final drying at 30 degrees C. Finally, plant samples were transferred to The Plant Biocentre at NTNU, Trondheim, Norway, for hydrodistillation and gas chromatography-mass spectrometry (GC-MS) analyses of the EOs. Peppermint oil yield increased from early to full bloom and late bloom (average of all years and drying methods except for 50 and 70 degrees C: 2.95, 4.13 and 4.20 L/daa, respectively) as an effect of biomass production and leaf growth. The flavor-impact compounds, menthol and menthone, reached their optimum at full bloom (43-54 and 12-30%, respectively). Prewilting led to slight decreased EO levels after 1 day (7.7%) and 5 days of ground drying (1.5%) and no EO quality changes, compared to direct drying at 30 degrees C. The plant weight (H2O content) was drastically decreased to the average under 80 and 45% in all years, thus reducing the energy supply and costs for the necessary final drying step.

Chemotypical variation of tansy (Tanacetum vulgare L.) from 40 different locations in Norway.

Between 2001 and 2002, plant collections from wild populations of Norwegian tansy (Tanacetum vulgare L.) were studied with a focus on essential oil (EO) yield and composition in order to characterize the chemotypical EO variability. Tansy collections of 40 different locations from North, Mid-, and South Norway were transplanted to the Apelsvoll Research Centre Div. Kise in 2000 and grown for 2 years before the aerial parts (leaves and flower buds) were harvested in June 2002. The EO from individual plants was isolated from dried plant material by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS) on a DB5 column at the Plant Biocenter. The EO yield ranged between 0.35 and 1.90% (v/w) (average: 0.81%); the most abundant thujone plants were especially rich in EO volatiles (0.95%). On the basis of GC-MS data, seven chemotypes could be identified as follows: A, alpha-thujone (two individuals); B, beta-thujone (22); C, camphor (six); D, chrysanthenyl acetate/chrysanthenol (three); E, chrysanthenone (two); F, artemisia ketone/artemisia alcohol (three); and G, 1,8-cineole (two). The thujone chemotype was dominated by beta-thujone (81%) associated with alpha-thujone, but tansy plants rich in alpha-thujone were also detected (61%). The chemotypical classification of Norwegian tansy genotypes was underscored by preliminary studies from 2001, indicating the genetic uniformity and biochemical stability of the domesticated plants.

Essential oil composition of sachalinmint from Norway detected by solid-phase microextraction and gas chromatography-mass spectrometry analysis.

The essential oil of leaves and flowers of sachalinmint [Mentha sachalinensis (Briq.) Kudô] grown in Norway (Trondheim) has been studied by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry analysis (GC-MS). The essential oil content increased linearly in acropetal direction from 1.08% (0-20 cm plant height) to 1.75% (60-80 cm; young leaves and flowers). The steam-distilled samples showed a minor complex matrix with a very high menthol and a much lower menthone content (87.89 and 4.05%, respectively). From testing of HS-SPME unequilibrated exposure times ranging from 10 s to 5 min, an extraction time of 30 s was found to be sufficient to detect both low- and high-eluting compounds. Comparison of HS-SPME and steam-distilled samples established that the same tendencies of increasing menthol/menthone content in the basipetal/acropetal direction could be detected by both analysis methods. With regard to the extraction efficiency, HS-SPME gave additional detailed information about less important terpenic compounds.