Crosstalk between biological and chemical diversity with cytotoxic and cytostatic effects of Aphanothece halophytica in vitro.

Different solvent extracts from Aphanothece halophytica (A. halophytica) were evaluated for their cytotoxic effects against four human cancer cell lines. The samples demonstrated different percentages of cyanobacteria species populations. The samples containing 100% A. halophytica and 90% A. halophytica showed a significant cytotoxic effect in human breast cancer cells MDA231. The cytostatic effect was demonstrated in MDA231 and human glioblastoma T98G cells regardless of the treatment, resulting in a significant cell cycle arrest in the S phase. The chemical profiles of the extracts were proven to be diverse in qualitative and quantitative compositions. This variability was dependent on the A. halophytica´s abundance in each extract. The 100% A. halophytica extract induced cytotoxic and cytostatic effects in breast cancer cells, and those could be associated with the predominance of fatty acids, hydrocarbons and phthalates, indicating that A. halophytica is an interesting source of novel compound with anticancer effect.

Microwave irradiation is a suitable method for caulerpin extraction from the green algae Caulerpa racemosa (Chlorophyta, Caulerpaceae).

Caulerpin is a bisindolic alkaloid that has been obtained from many species of the genus Caulerpa. The main objective of this paper is to evaluate four extraction methods of caulerpin in the C. racemosa: maceration (DMA), Soxhlet extraction (SOX), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE). The methods were compared through caulerpin content quantified by Ultraviolet-visible (UV-vis) spectrophotometry. The highest extract yield was obtained by SOX but the highest contain of caulerpin was presented in the MAE extract. The caulerpin content was significant different within the extacts by MAE and UAE, it yielded by MAE more than three times as much as UAE. The most efficient caulerpin extraction method had the parameters solvent, temperature and time optimised. Thus, the best conditions were achieved with MAE in ethanol during 7 min at 90 °C. Therefore, this work suggests an improved routine analysis of caulerpin by the green chemistry concept.

Differential susceptibility of distinct parts of the aquatic plant Nymphoides humboldtiana to herbivory supports the optimal defense theory.

PREMISE: The optimal defense theory (ODT) predicts that the allocation of chemical defenses in plants will be concentrated in parts or tissues that are of higher fitness value for the individuals that produce them. Chemicals are known to be allocated to certain parts of aquatic plants, and the morphological architecture of Nymphoides humboldtiana, a species that exposes its parts to different environmental factors and consumers, may be an excellent model to evaluate within-plant susceptibility to consumers according to the ODT. METHODS: Under laboratory experimental conditions, we evaluated the defensive properties of extracts from vegetative (leaves, rhizomes, roots) and reproductive (long stem internodes, flowers, fruits) parts of N. humboldtiana against consumption by the generalist herbivorous gastropod Biomphalaria glabrata. Extracts were also subjected to chemical analysis by high-performance liquid chromatography, principal component analysis, and analysis of their relationships to defensive actions. RESULTS: Extracts of all vegetative and internode (reproductive) parts of N. humboldtiana exhibited defensive properties against B. glabrata, but the long stem internodes exhibited the highest percentage of inhibition. Chemical profiles of these parts were qualitatively and quantitatively different, but a major unidentified compound is presumably responsible for the higher defensive property found in internodes. CONCLUSIONS: Our results support the ODT, since chemical defense was more effective in long stem internodes, which have a high fitness value for N. humboldtiana to keep the flowers emerged on the water surface in response to the rapid and dynamic changes in water levels typical of freshwater environments.

The sea-hare Aplysia brasiliana promotes induction in chemical defense in the seaweed Laurencia dendroidea and in their congeneric neighbors.

Inducible chemical defenses are more common in temperate seaweeds than tropical ones, and are directly detected by increase of chemical contents, or indirectly by differential consumption of live seaweed tissues or artificial food with algal extracts by herbivores. In general, seaweed-induced chemical defense occur between 11 and 20 days after both simulated/artificial or direct herbivory. Here, we used experimental procedures to assess induced chemical defense in the tropical red seaweed Laurencia dendroidea as response to direct grazing, chemical cues from grazed conspecific neighbors and only presence of herbivores. Chemical defenses were analyzed by detecting the palatability of artificial food containing L. dendroidea extracts offered to Aplysia brasiliana and by comparative analyses of extracts from this seaweed by Gas Chromatography/Mass Spectroscopy, as well as metabolomic data analysis by Principal Component Analysis. Our results revealed that direct grazing by A. brasiliana induced a rapid (after 48 h) response among individuals of L. dendroidea, as did waterborne chemical cues from grazed conspecifics, but the presence of sea hare alone did not elicit a response. Increased resistance to grazing was accompanied by significative changes in sesquiterpene metabolomic chemical profile, revealing that induced defense: may be more widespread among seaweeds, independent of latitude; can involve changes in other classes of substances besides phlorotannins or not only the increase in the content of a single compound; and may be a rapid and ecologically coherent response to consumers. In addition, the importance of incorporating the metabolomic approach when examining inducible chemical defense in seaweeds is also emphasized.

Geographic Distribution of Natural Products Produced by the Red Alga Laurencia dendroidea J. Agardh.

In order to evaluate the chemical diversity of Laurencia dendroidea J. Agardh, a widely distributed seaweed in Brazilian coast, a phytochemical study was carried out with algae collected from six different locations along the Southeast Brazilian coast. Purified compounds were identified by MS and NMR techniques. The chemical profiles of lipophilic extracts were obtained by GC/MS for each population. In total, 15 compounds were described. The sesquiterpene composition accounted for 49 - 63% of the GC/MS chromatogram area. The discrimination of three chemotypes was done by the use of HCA on GC/MS chromatograms. They were also analyzed by the PCA and, together with peak area analysis, it was possible to discriminate all populations by the main variation of elatol, obtusol, rogiolol, and triquinane. The results revealed the high diversity of sesquiterpene composition among populations of L. dendroidea. Curiously, the within and among population variation of elatol and obtusol suggested a biochemical interplay on the content of these compounds. More studies are necessary to understand the patterns of chemical diversity and compound variation within and among populations of L. dendroidea.

Intraspecific variation of meroditerpenoids in the brown alga Stypopodium zonale guiding the isolation of new compounds

Abstract Intraspecific variation on meroditerpenoids production by the brown marine alga Stypopodium zonale at four different populations along the Brazilian coast was analyzed using Principal Component Analysis over high-performance liquid chromatography profiles from algae extracts. The ordination of the samples by the similarities of their chromatographic traits showed the existence of three chemotypes: (i) the populations Búzios and Abrolhos which were characterized by the presence of atomaric acid (1), (ii) the population Atol das Rocas which contained the compound stypoldione (2), and (iii) the population Marataízes which was characterized by other peaks that guided the isolation of three new meroditerpenoids stypofuranlactone (3), 10,18-dihydroxy-5′a-desmethyl-5′-acetylatomaric acid (4), and the 10-keto-10-deisopropyliden-5′a-desmethyl-5′-acetylatomaric acid (5) together with the known compound the 10-keto-10-deisopropyliden-atomaric acid (6). The structures and relative stereochemistry of 3, 4 and 5 were elucidated by NMR and MS techniques. The observed chemical differences among populations of S. zonale can be related to its geographic distribution and can open an avenue to the discovery of new compounds in algae.

Chemical diversity and antileishmanial activity of crude extracts of Laurencia complex (Ceramiales, Rhodophyta) from Brazil

Chemical profiles of extracts of four species from Laurencia complex (Ceramiales, Rhodophyta) from different populations collected along Southeast Brazilian coast were assessed by High Performance Liquid Chromatography coupled with a Diode Array Detector in order to observe geographic chemical variability. Aiming to evaluate the impact of chemical diversity on potential pharmaceutical uses, the extracts were tested against the promastigote form of Leishmania amazonensis. The most active extracts were submitted to anti-amastigote and cytotoxicity assays. Principal Component Analysis of the chromatograms resulted in four major groups of chemical profiles according to the presence of leishmanicidal chamigranes (-)-elatol and obtusol. The existence of chemotypes, displaying variable pharmacological action, is proposed for the differences observed in L. dendroidea samples. Although all extracts were found active against promastigote form of L. amazonensis, their efficacy was remarkably different and not related to the variation of (-)-elatol and obtusol, which indicates the presence of additional compounds with antileishmanial activity. Moreover, the active extracts also displayed anti-amastigote activity and none of them were considered cytotoxic. The results highlight that the knowledge of chemical geographic variability can be valuable in the search of new antileishmanial compounds from marine sources.

Lateral diffusion of CO2 in leaves of the crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perrier.

Dynamic patchiness of photosystem II (PSII) activity in leaves of the crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana Hamet et Perrier, which was independent of stomatal control and was observed during both the day/night cycle and circadian endogenous oscillations of CAM, was previously explained by lateral CO2 diffusion and CO2 signalling in the leaves [Rascher et al. (2001) Proc Natl Acad Sci USA 98:11801-11805; Rascher and Luttge (2002) Plant Biol 4:671-681]. The aim here was to actually demonstrate the importance of lateral CO2 diffusion and its effects on localized PSII activity. Covering small sections of entire leaves with silicone grease was used for local exclusion of a contribution of atmospheric CO2 to internal CO2 via transport through stomata. A setup for combined measurement of gas exchange and chlorophyll fluorescence imaging was used for recording photosynthetic activity with a spatiotemporal resolution. When remobilization of malic acid from vacuolar storage and its decarboxylation in the CAM cycle caused increasing internal CO2 concentrations sustaining high PSII activity behind closed stomata, PSII activity was also increased in adjacent leaf sections where vacuolar malic acid accumulation was minimal as a result of preventing external CO2 supply due to leaf-surface greasing, and where therefore CO2 could only be supplied by diffusion from the neighbouring malic acid-remobilizing leaf tissue. This demonstrates lateral CO2 diffusion and its effect on local photosynthetic activity.

Perturbations of malate accumulation and the endogenous rhythms of gas exchange in the Crassulacean acid metabolism plant Kalanchoë daigremontiana: testing the tonoplast-as-oscillator model.

In continuous light, leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier exhibit a circadian rhythm of CO2 uptake, stomatal conductance and leaf-internal CO2 pressure. According to a current quantitative model of CAM, the pacemaking mechanism involves periodic turgor-related tension and relaxation of the tonoplast, which determines the direction of the net flux of malate between the vacuole and the cytoplasm. Cytoplasmic malate, in turn, through its inhibitory effect on phospho enolpyruvate carboxylase, controls the rate of CO2 uptake. According to this mechanism, when the accumulation of malate is disrupted by removing CO2 from the ambient air, the induction of a phase delay with respect to an unperturbed control plant is expected. First, using the mathematical model, such phase delays were observed in numerical simulations of three scenarios of CO2 removal: (i) starting at a trough of CO2 uptake, lasting for about half a cycle (ca. 12 h in vivo); (ii) with the identical starting phase, but lasting for 1.5 cycles (ca. 36 h); and (iii) starting while CO2 increases, lasting for half a cycle again. Applying the same protocols to leaves of K. daigremontiana in vivo did not induce the predicted phase shifts, i.e. after the end of the CO2 removal the perturbed rhythm adopted nearly the same phase as that of the control plant. Second, when leaves were exposed to a nitrogen atmosphere for three nights prior to onset of continuous light to prevent malate accumulation, a small, 4-h phase advance was observed instead of a delay, again contrary to the model-based expectations. Hence, vacuolar malic acid accumulation is ruled out as the central pacemaking process. This observation is in line with our earlier suggestion [T.P. Wyka, U. Lüttge (2003) J Exp Bot 54:1471-1479] that in extended continuous light, CO2 uptake switches gradually from a CAM-like to a C3-like mechanism, with oscillations of the two CO2 uptake systems being tightly coordinated. It appears that the circadian rhythm of gas exchange in this CAM plant emerges from one or several devices that are capable of generating temporal information in a robust manner, i.e. they are protected from even severe metabolic perturbations.