New labdane diterpenes from Araucaria cooki

Eight new and seven known labdane diterpenes were identified in the oleoresin of Araucaria cooki.     

Neutral diterpenes from Araucaria bidwilli

Five ent-labdane diterpenes were isolated from the neutral fraction of the resin of Araucaria bidwilli. Three of them, previously unknown, were     

New diterpenes from Araucaria cunninghami

Eleven labdane diterpenes, nine of which are new compounds, were identified in the oleoresin of Araucaria cunninghami.     

Rodent handling of Araucaria araucana seeds

In an intermast year of very low seed production, we studied seed handling in an Araucaria araucana (Araucariaceae) forest in Neuquén Province, Argentina. Rodents identified in 844 photographs from automatic cameras removed 589 seeds marked with small embedded magnets. Within 12 days of removal, 460 were recovered using a magnetic field locator; 79% of recoveries were from burrows and caches in litter or soil. Rodents exhibited a diversity of seed‐handling strategies and differed in their potential as dispersal agents. Seed removal was most likely between 15.00 hours and 21.00 hours, but diurnal and nocturnal visits were recorded for all species. Oligoryzomys longicaudatus (Cricetidae) was a seed predator that took 8% of marked seeds and left none whole. Chelemys macronyx (Cricetidae) and Rattus norvegicus (Muridae) removed 25% and 19% of marked seeds respectively. These two species deposited the majority of seeds in groups of 10 or more in burrow larders that were unfavourable seedling establishment sites far from daylight. Abrothrix longipilis (Cricetidae) removed 43% of seeds, scatter‐hoarded the largest percentage of whole seeds (37%), moved some seeds farther than 40 m, and left them in favourable seedling establishment sites near daylight. For all species, the number and proportion of seeds cached whole increased as more seeds were removed. Rodents, especially A. longipilis, may play an important role in regeneration of A. araucana. Seed‐handling strategies and potentially effective dispersal are discussed in terms of masting seed production.     

Minor labdane diterpenes from Marrubium thessalum

Five new labdane diterpenes, thessalines A, B, and D, 14β-hydroxythessaline A, and 14β-hydroxythessaline B (1-5, resp.) were isolated from the aerial parts of Marrubium thessalum, along with the known labdane diterpene deacetylvitexilactone (6) and the methoxylated flavones 4',7-dimethylapigenin and salvigenin. (3S,5R)-Loliolide was also found in the same source. Their structures were established by 1D- and 2D-NMR (COSY, HSQC, HMBC, NOESY, and ROESY) and MS analyses. The plant produces a great variety of labdane-type diterpenes, with variations in functionalities, particularly in the side chain. Their structures could be of chemotaxonomic significance for the genus Marrubium.     

Three new labdane diterpenes from Loxocalyx urticifolius

Three new labdane diterpenes, namely loxocalyxin D (1), loxocalyxin E (2) and 13-epiloxocalyxin E (3), were isolated from the whole plants of Loxocalyx urticifolius Hemsl. Their structures were elucidated by extensive spectral analysis and chemical methods. The absolute configuration of loxocalyxin D (1) was confirmed by X-ray crystallographic analysis. The cytotoxic activities of the isolated labdane diterpenes were evaluated against the four human cancer cell lines A549, HepG2, HL-60 and MCF-7. 13-epiloxocalyxin E (3) exhibited selective cytotoxicity against A549 and MCF-7 with the IC₅₀ values of 22.4 and 47.3 μM, respectively.     

Tree-ring isotopes from Araucaria araucana as useful proxies for climate reconstructions

Tree-ring width (TRW) chronologies have been widely and long-time used to reconstruct past climate variations in the Andes in South America. The use of tree-ring isotopic chronologies is still not widespread in this region although they have proved to be very efficient climate proxies. Araucaria araucana (Molina) K. Koch is a conifer tree species with some multi-century-old individuals that offers an excellent opportunity to measure stable carbon (δ¹³C) and oxygen (δ¹⁸O) isotopes in cellulose from long tree-ring records. Here, we explore whether current or stored carbohydrates are used for A. araucana radial growth and we assess the potential of a tree-ring isotopic record of to study past climate variability. Eleven A. araucana cores from a dry and high-elevation forest at the northern border of Patagonia, Argentina (38°55’S, 70°44’W) were selected for stable isotopes analyses. The strong correlation between the isotopic composition of the first and second parts of the same ring, but also the strong relationships between δ¹³C and δ¹⁸O records with climate parameters of the current growing season such as temperature, show that tree-rings are built mostly with carbohydrates produced during the current growing season with little or no supply from storage or reserves. This finding leads to reconsidering the interpretation of the legacy effect (i.e. ecological memory effects) based on the previously described strong negative correlation between A. araucana TRW chronologies and previous growing season temperature and suggests a dependence of radial tree growth on the level of development of organs. Regarding climate sensitivity, the A. araucana tree-ring δ¹³C chronology is strongly related to current summer temperature (r = 0.82, p < 0.001), vapour pressure deficit (VPD; r = 0.79, p < 0.001), precipitation (r = −0.53, p < 0.001) and SPEI2 (r = −0.73, p < 0.001). These strong relationships support the use of δ¹³C of A. araucana tree-ring cellulose to reconstruct past temperature variations at regional scale in relation with large-atmospheric drivers of climate variability such as the Southern Annular Mode. The A. araucana tree-ring δ¹⁸O chronology is also correlated with temperature (r = 0.42, p < 0.01) and VPD (r = 0.45, p < 0.01) of the winter preceding the growing season. This suggests that trees are using water from precipitation infiltrated in the soil during the previous recharge period (autumn-winter). The weak correlations of δ¹⁸O with current summer atmospheric conditions and the decoupling between δ¹⁸O and δ¹³C, may be due to a high rate of oxygen exchange between sugars and xylem water (P_ex) during cellulose synthesis, which dampens evaporative isotopic fractionation.     

Resilience capacity of Araucaria araucana to extreme drought events

Ongoing climate change has induced modification in the frequency and intensity of extreme climatic events, with consequent impact on tree and forest growth resilience. Araucaria araucana is an endangered Patagonian conifer, which provides several ecosystem services to local human societies and plays fundamental ecological roles in natural communities. These woodlands have historically suffered different types of anthropogenic disturbance, such as fire, logging and grazing, nevertheless the species resilience to extreme drought events remains still poorly understood. To fill this gap of knowledge, we applied dendrochronological methods to several A. araucana stands distributed along a steep bioclimatic gradient in order to reconstruct resilience capacity, in term of stem growth resistance and recovery, to three successive extreme spring-early summer droughts which occurred during the 20th century. Results showed an increase in the species recovery along the considered dry spells, whereas no clear trend emerged for resistance, suggesting no cumulative effect of drought upon resilience. Both resistance and recovery presented different values depending on bioclimatic settings, being xeric stands more sensitive to extreme episodes with respect to mesic woodlands, particularly during the more recent drought event when trees growing in drier environments were not able to reach pre-drought stem growth rates. Tree-level characteristics, such as age and growth trends prior to drought, modulated the species resilience, suggesting that future dry spells would possibly induce shifts in population dynamics, and furthermore be detrimental for fast-growing trees. Our analysis highlighted the response of a key Patagonian tree species to extreme drought events, providing bioclimatic-specific useful information for conservation plans of this natural resource.     

Two new cytotoxic labdane diterpenes from the rhizomes of Hedychium coronarium

The phytochemical investigation of the hexane extract of the Hedychium coronarium led to the isolation and identification of two new labdane diterpenes (1 and 2) along with 10 known metabolites (3-12). The structures of the new compounds were established on the basis of spectroscopic data analysis (1D and 2D NMR and MS). Cytotoxic activities of the isolates were studied against the A-549 (lung cancer), SK-N-SH (human neuroblastoma), MCF-7 (breast cancer) and HeLa (cervical cancer) cell lines.     

Cytotoxicity and immunomodulating characteristics of labdane diterpenes from Marrubium cylleneum and Marrubium velutinum

From the aerial parts of Marrubium cylleneum, one labdane nor-diterpene has been isolated together with two labdane diterpenes, hitherto not known as natural products. The structures of the isolated compounds were established by means of NMR [(1)H-(1)H-COSY, (1)H-(13)C-HSQC, HMQC-TOCSY, HMBC, NOESY] and MS spectral analyses. Several diterpenoids from M. cylleneum and M. velutinum were tested for their cytotoxic effect against various cancer cell lines and their immunomodulating potential in human peripheral blood mononuclear cells in standard in vitro assays. Our results show a differential cytotoxicity of some compounds as well as their ability to improve selected lymphocyte functions.     

New labdane diterpenes from the aerial parts of Scoparia dulcis L.

Three new labdane-derived diterpenes, dulcinodal (1), dulcinodiol (2), and scopadiol decanoate (3) were isolated from the aerial parts of Scoparia dulcis. The structures were determined by extensive NMR studies and comparison of their spectral data with related compounds.     

Biochemical and immunological characterization of α-amylase isoenzymes of Araucaria araucana

Seven α-amylase isoenzymes present in quiescent seeds of the South American conifer Araucaria araucana were purified by affinity chromatography and partially characterized. The molecular masses of these isoenzymes were 45.7, 47.0, 50.2, 51.2, 52.0, 53.5 and 55.2 kDa. The two main isoforms were separated from each other and from the rest of the isoenzymes by anion-exchange chromatography using a linear gradient of 0 to 0.6 M NaCl and slightly different CaCl₂ concentrations. All isoenzyme bands stained with periodic acid/dansylhydrazine, suggesting that they are glycoproteins. Electroblotting of the isoenzymes onto polyvinylidene difluoride membranes allowed determination of the amino acid composition and NH₂-terminal sequence of the 53.5-, 50.2-and 47.0-kDa isoenzymes. Amino acid compositional analysis demonstrated that these enzymes are rich in glycine, aspartic acid/asparagine, alanine, serine, proline and glutamic acid/glutamine. The NH₂-terminal sequences of the three isoenzymes are identical. Comparison of the amino acid compositions and the NH₂-terminal sequence of these isoenzymes with the cereal and Vigna radiata α-amylases demonstrated that there is no relation between them. However, polyclonal antibodies generated against barley α-amylase cross-reacted with all the A . araucana α-amylases. Peptide mapping analysis of the isoenzymes using cyanogen bromide suggests that there are genetic differences between them.     

New labdane resin acids from Pinus elliottii

Slash pine needles and cortex oleoresin have been found to contain a new major diterpene constituent, imbricataloic acid. The closely related imbricatoloic acid, previously reported only in Araucaria imbricata, was found to be present in small amounts in slash pine needle extract. Spectral data are given for an unidentified diterpene alcohol isolated from the cortex oleoresin.     

Phytochemical investigation of labdane diterpenes from the rhizomes of Hedychium spicatum and their cytotoxic activity

A comprehensive reinvestigation of chemical constituents from the rhizomes of Hedychium spicatum led to the isolation of two new labdane-type diterpene (1, 2), together with six known compounds (3–8). Their structures were established on the basis of extensive spectroscopic (IR, MS, 2D NMR) data analysis and by comparison with the spectroscopic data reported in the literature. In addition, all the isolates were tested for their cytotoxicity against the THP-1 (human acute monocytic leukemia), HL-60 (human promyelocytic leukemia), A-375 (human malignant melanoma) and A-549 (human lung carcinoma) cancerous cell lines.     

Tree-ring responses in Araucaria araucana to two major eruptions of Lonquimay Volcano (Chile)

Palaeoclimate proxies have demonstrated links between climate changes and volcanic activity. However, not much is known about the impact of volcanic eruptions on forest productivity. Here we used tree-ring width and annually resolved carbon and oxygen isotopic records from tree rings of Araucaria araucana (Molina) K. Koch, providing a centennial-scale reconstruction of tree ecophysiological processes in forest stands nearby the Lonquimay Volcano (Chile). We observed a mean decrease in tree-ring width following the major eruption of 1988–1990 (with aerosol emission), most probably caused by the modified ecological conditions due to acid rain and ash deposition, while a generally negative relationship between δ¹³C and δ¹⁸O would point to a decline in humidity and precipitation. More negative δ¹³C and lower δ¹⁸O values (positive correlation) following the major eruption of 1887–1890 (without aerosol emission) would suggest high stomatal conductance and moisture availability, though tree-ring width (and probably photosynthetic rate) was unaltered. At least for this sample of trees, in the case of eruption with large tephra emission, the beneficial effect of aerosol light scattering on tree productivity appears to be outweighed by the detrimental effect of eruption-induced toxic deposition. Signals of the two major eruptions of the past 200?years at Lonquimay were present in tree rings of nearby A. araucana. No unique response of tree functions to volcanic eruptions can be expected, but rather (1) the variable volcanic properties and (2) the complex interplay of diffuse light increase (aerosol scattering), air temperature decrease (cloud shading), and toxic deposition impact (volcanic ash), makes any prediction of tree growth and ecophysiological response very challenging.     

Antifungal and antibacterial activities of Araucaria araucana (Mol.) K. Koch heartwood lignans

Five lignans (secoisolariciresinol, pinoresinol, eudesmin, lariciresinol, and lariciresinol-4-methyl ether) were isolated from an MeOH extract from Araucaria araucana (Mol.) K. Koch wood for the first time in this species and their structures determined with spectroscopic methods. The antimicrobial activities of these compounds were determined for the bacteria Citrobacter sp., Bacillus subtilis, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, and Pseudomonas aeruginosa, and for the white rooting and staining fungi Mucor miehei, Paecilomyces variotii, Ceratocystis pilifera, Trametes versicolor, and Penicillium notatum, and in addition, the MeOH extract was evaluated against Aspergillus niger, Candida albicans, Fusarium moniliforme, F. sporotrichum and Trichophyton mentagrophytes. The most sensitive bacteria against pinoresinol were the Gram-positive. However, secoisolariciresinol exhibited a significant antifungal activity on fungi of white rooting and wood staining and this compound completely inhibited the mycelial growth of T. versicolor and C. pilifera at 300 and 400 microg per disc, respectively, whereas pinoresinol showed a moderate inhibitory activity. On the other hand, the MeOH extract had the highest activity against rooting and staining and pathogenic fungi as well as T. versicolor, Fusarium spp. and Trichophyton mentagrophytes, inhibiting completely the growth at 400 microg per disc.     

New insights into the genetic structure of Araucaria araucana forests based on molecular and historic evidences

The conservation of genetic resources is a prerequisite for the maintenance of long-lived forest species. Araucaria araucana (Mol.) K. Koch is one of the oldest conifers in South America and a representative symbol of Chilean forest due to its endemicity and longevity. The aim of this study was to evaluate the genetic structure of the current A. araucana populations in Chile, to verify the possible genetic divergence between Coastal and Andean populations and to assess whether bottleneck events have influenced habitat fragmentation and threaten the genetic resources and evolutionary potential of the species. Twelve natural populations, nine from the Andes Cordillera and three from the Coast Cordillera were analysed by means of eight genomic microsatellite markers developed in A. araucana. Results of analysis of molecular variance (AMOVA) highlighted significant differentiation between Coastal and Andean populations (16 %; P?=?0.004), detecting one significant barrier that separated populations from both Cordilleras as maximally differentiated areas. At local scale, both ranges revealed significant inter-population differentiation, with higher values for Coastal populations compared with Andean populations. These results suggested the presence of four gene pools (three in the Andes and one in the Coast Cordilleras) and one population (VIL) in the Coast Cordillera that differed to the rest. The differentiation between the Andean and Coastal populations may provide important baseline data that should allow further studies of landscape genetics in the species and that can contribute to develop conservation strategies for its genetic resources.     

Schistosomicidal activity of kaurane,labdane and clerodane-type diterpenes obtained by fungal transformation

Schistosomiasis continues to be a huge challenge for researchers, pharmaceutical companies, and governments in developing countries. Diterpene compounds are good source for the development of novel potential leading compounds to treat schistosomiasis. We are reporting herein the schistosomicidal activity of ent-kaurenoic acid, ent-copalic acid, ent-hardwickiic acid, isolated from oleoresins of Copaifera spp, and of their derivatives obtained by fungal transformation with strains of Aspergillus fumigatus, A. terreus, A. phoenicis and A. ochraceus, and of Cunninghamella echinulata e C. elegans. The in vitro antiparasitical assays were performed using adult worm pairs of Schistosoma mansoni for the evaluation of the worm pairing, egg production, and eggs development. Ten kaurane, labdane and clerodane-type diterpenes were obtained by fungal transformation and 7α-acetoxy-ent-kaur-16-en-19-oic acid and ent-hardwickiic acid were the most active ones by causing mortality of 100 % of the parasites within 24 h (concentrations of 100.0 and 200.0 μM) and displaying respective IC₅₀ values for 24, 48 and 72 h of 56.7, 37.6 and 29.2 μM and 29.6, 30.8 and 25.7 μM. Additionally, 7α-acetoxy-ent-kaur-16-en-19-oic acid and ent-hardwickiic acid highly reduced the number of laid eggs at 6.25 and 12.5 μM, respectively. These diterpenes should be further investigated as potential candidates for antiparasitic drug discovery.     

Patterns and mechanisms of masting in the large‐seeded southern hemisphere conifer Araucaria araucana

Abstract Masting, the intermittent and synchronous production of large seed crops, may result from either of two major processes: resource matching and economy of scale. Components of cone production in Araucaria araucana were partitioned among populations and trees to ascertain the existence of masting and the processes involved. Cone production data from seven populations were obtained during a 9‐year period and seed gathering data were available for an 18‐year time series from six sites in an area of more than 7600 km². Araucaria araucana showed environmentally triggered, intermittent, moderately fluctuating, and highly regionally synchronous reproduction. The mean pairwise correlations of cones production among populations and seed gathering sites were 0.89 and 0.74, respectively, suggesting synchrony in reproduction. Among trees we observed a mean correlation of 0.74 with values ranging from 0.66 to 0.81 for the analysed populations. The existence of negative autocorrelation in seed production between year 0 and year ?2 at the individual tree level suggests the presence of ‘switching’ or internal resource allocation, thus discarding the Resource Matching hypothesis. Mean coefficient of variation (CV_p) among populations was moderate (0.95) and similar to the modal CV_p values reported in the published reports. Mean CV_i among individual trees was 1.16, suggesting a large number of equally and synchronously fluctuating trees, rather than a few largely fluctuating individuals. These results suggest that pollination efficiency and/or predator satiation hypotheses could be responsible for the masting cycles in this conifer. Ancillary data about limitation of airborne pollen dispersion and temporal variation in the amount of seeds per cone and about seed predator satiation, also support both proposed mechanisms.     

The effect of beta-endorphin on natural cytotoxicity and antibody dependent cellular cytotoxicity

The ability of the central nervous system to modulate immune responsiveness has received increasing attention. A potential mechanism that would allow the central nervous system to alter the immune system is the release of neuroendocrine and neurotransmitter polypeptides into the peripheral circulation with subsequent modulation of immunocyte function. In this report, we demonstrate that the neuropeptide, beta-[D-ALA2]-endorphin augments natural cytotoxicity but does not effect antibody-dependent cellular cytotoxicity. The observations are discussed in relation to the mechanisms for natural cytotoxicity and antibody dependent cellular cytotoxicity.