Química y actividad biológica en Huperzia saururus. Chemistry and biological activity in Huperzia saururus.

El presente proyecto apunta a buscar una posible solución a dos problemáticas de salud que afectan a un importante grupo de la población mundial y son los asociados a la enfermedad de Alzheimer (EA) y a la disfunción eréctil. La EA es una enfermedad neurodegenerativa progresiva caracterizada por la pérdida de memoria a corto plazo, entre otras manifestaciones. En la actualidad la terapia más utilizada son los inhibidores de la acetilcolinesterasa (AChE), ya que acetilcolina es el principal neurotransmisor afectado. No obstante, los agentes terapéuticos utilizados presentan efectos no deseados a nivel gastrointestinal, lo que motiva la búsqueda de nuevos agentes. A partir del estudio de la especie autóctona Huperzia saururus aislamos hasta el presente 10 alcaloides, algunos de los cuales resultaron activos sobre la retención de memoria, investigación realizada mediante ensayos tanto in vitro como in vivo. A partir de los promisorios resultados obtenidos se continuarán profundizando estos estudios y también se buscará el farmacóforo, la parte de la estructura responsable de la actividad. Para ello, se realizarán modificaciones químicas de los grupos funcionales y los productos obtenidos serán igualmente evaluados por su actividad sobre memoria. Los que resulten activos serán investigados en relación a su toxicidad, en la búsqueda de potenciales agentes terapéuticos. Paralelamente se estudiarán las diferentes familias de compuestos presentes en la especie, dado que el uso popular proclama su efecto afrodisíaco. La búsqueda de un afrodisíaco perfecto que incremente el deseo sexual, el placer y el desempeño, ha sido una constante desde los tiempos remotos. En todos los casos, se ha buscado mejorar el rendimiento sexual o combatir la disfunción eréctil (DE). Este término se describe como “la incapacidad constante para alcanzar o mantener una erección que es satisfactoria para el rendimiento sexual”. Para investigar la actividad biológica de H. saururus, los extractos y compuestos aislados serán evaluados por la generación de NO que produzcan, la que será estimada mediante la acumulación de nitrito, metabolito estable del NO, en sobrenadantes de cultivo de células endoteliales (HUVEC). Los que resulten efectivos serán evaluados mediante ensayos in vivo tales como, el de conducta sexual en machos y de comportamiento de monta. Del mismo modo que para los alcaloides, se estudiará la toxicidad de los compuestos que resulten activos a los fines de proseguir la metodología en la investigación preclínica de potenciales medicamentos. The present Project aims to look for a possible solution for two health problems that affect an important group of world population and they are those associated to Alzheimer Disease (AD) and Erectile Dysfunction. AD is a neurodegenerative progressive disease characterized by short term memory loss, among other signs. Nowadays the most used therapy are the acethylcholinesterase inhibitors (AChE), in view that the acethylcholine is the main neurotransmitter affected. Nevertheless, the therapeutic agents used present collateral effects at gastrointestinal level, so it motivates the search for new agents. From the autochthonous species Huperzia saururus we have isolated 10 alkaloids until the present, some of which were active on memory retention, investigation developed in vitro and in vivo. After these promising results, these studies will be continued, as well as the search for the pharmacophore. For this reason, chemical modifications on functional groups will be done and the obtained products will be evaluated on their activity on memory as well. In line with this, the different families of compounds present in H. saururus will be studied in view that the popular use claims its aphrodisiac effect. The search for a perfect aphrodisiac that increase the sexual desire, the pleasure and performance was a constant since the old times. In all the cases it was looked a better sexual performance or fight against the erectile dysfunction (DE). For investigate the biological activity of H. saururus, the extracts and the isolated compounds will be evaluated on the NO generation, which will be estimated by means of nitrite accumulation, stable metabolite of NO, in supernatants of endothelial cell cultures (HUVEC). The compounds that appear effectives will be evaluated by in vivo assays such as sexual male behavior, and mount behavior. As for alkaloids, toxicity of active compounds will be studied to follow the pre-clinic methodology research of potential medicines.

Morfologia externa e interna das folhas vegetativas, esporofilos e esporângios de espécies de Huperzia Bernh. (Lycopodiaceae - Pteridophyta) do Brasil

Lycopodiaceae está inserida em Lycopsida, cuja autapomorfia é a presença de folhas microfilas. Foram estudadas 10 espécies, sete delas endêmicas do Brasil: Huperzia christii (Silveira) Holub, H. friburgensis (Nessel) B. Øllg., H. hemleri (Nessel) B. Øllg., H. intermedia Trevis., H. reflexa (Lam.) Trevis. e H. treitubensis (Silveira) B. Øllg., estas terrestres; H. fontinaloides (Spring) Trevis., H. heterocarpon (Fée) Holub, H. hexasticha B. Øllg. & P.G. Windisch e H. quadrifariata (Bory) Rothm., estas epífitas. As características anatômicas encontradas, que podem ser adaptativas ao ambiente com escassez hídrica são a presença de células epidérmicas com paredes anticlinais sinuosas, invaginações na face interna da parede periclinal externa das células epidérmicas e folhas anfiestomáticas. As espécies estudadas foram reunidas em dois grupos, corroborando classificações existentes. Os grupos foram definidos com base nas seguintes características: a filotaxia, se helicoidal ou oposta, a posição das folhas em relação ao caule, se reflexas ou adpressas, e o número de camadas de células do esporângio, se três ou quatro. Foi elaborada uma chave de identificação.

ESTRATEGIA QUÍMICA Y FARMACOLÓGICA PARA LA BÚSQUEDA DE PRODUCTOS BIOACTIVOS DE PLANTAS AUTÓCTONAS Y MEDICINALES

A partir de especies autóctonas bioactivas, se pretende obtener metabolitos químicos con potenciales actividades biológicas. Esta metodología de trabajo es válida para el descubrimiento de nuevas drogas de origen natural y que podrían ser de aplicación en diversas áreas de la farmacología experimental. Dentro de los diferentes metabolitos químicos que produce el reino vegetal, tanto la familia de flavonoides como la de alcaloides, presentan importantes características farmacológicas, haciéndolos interesantes para su estudio. El presente proyecto contempla la búsqueda de principios activos de origen natural orientados a su evaluación con potenciales aplicaciones terapéuticas: A) Drogas con actividad inhibidora de Acetilcolinesterasa (AcHE) Se evaluará el contenido químico en alcaloides de Lycopodium de especies sudamericanas de Huperzia:Huperzia quadrifariata, Huperzia reflexa y Huperzia acerosa, estudiando sus propiedades como agentes con actividad anticolinesterasa y su relación sobre la memoria y el aprendizaje. B) Drogas con actividad inhibidora sobre sistemas enzimáticos relacionados a eventos inflamatorios: La óxido nítrico sintasa inducilbe (iNOS) es la enzima responsable de la sobreproducción patológica del óxido nítirico (NO), un mediador citotóxico en procesos inflamatorios. Así, evaluaremos si flavonoides obtenidos de Dalea elegans, Dalea boliviana y Prosopis strombulifera, podrían tener algún efecto inhibitorio tanto sobre la producción de NO, como en la expresión de la iNOS, utilizando línea celular derivada de macrófago: J774. C) Drogas con actividad inhibidora de la enzima tirosinasa Tirosinasa es la enzima encargada de la formación de melanina y su inhibición, permitiría la obtención de nuevos agentes terapéuticos y cosméticos que serían útiles para tratar trastornos de hiperpigmentación. De este modo, se estudiará la actividad de flavonoides prenilados obtenidos de especies de Dalea (Dalea elegans y Dalea boliivana) sobre la inhibición de tirosinasa de hongos "in vitro". De este modo, el presente plan de trabajo tiene como propósito el estudio de flavonoides y alcaloides, obtenidos de especies sudamericanas, no solo en su aspecto químico sino también su aplicación en estudios biológicos que permitan asignar actividades, evaluando potenciales efectos farmacológicos y sus posibles mecanismos de acción.

Development of natural products as drugs acting on central nervous system

We have recenty studied several natural product constituents which have effects on the CNS. (1) Tetrahydropalmatine (THP) and its analogues were isolated from Corydalis ambigua and various species of Stephania. (+)-THP and (-)-THP posses not only analgesic activity, but also exert sedative-tranquillizing and hypnotic actions. Results of receptor binding assay and their pre-and post-synaptic effects on dopaminergic system indicate that (-)-THP and (-)-stepholidine are dopamine receptor antagonists while (+)-THP is a selective dopamine depletor. (2) 3-Acetylaconitine (AAC) is an alkaloid isolated from Aconitum flavum. The relative potency of analgesic action of AAC was 5.1-35.6 and 1250-3912 times that of morphine and aspirin, respectively. The analgesic effect of AAC was antagonized by naloxone, but was eliminated by reserpine. In monkeys, after AAC was injected for 92 days, no abstinence syndrome was seen after sudden AAC withdrawal or when challenged with nalorphine. (3) Huperzine A (Hup-A) is an alkaloid isolated from Huperzia serrata which was found to be a selective ChE inhibitor and could improve learning and retrieval process. Preliminary clinical studies showed that Hup-A improve short-and long-term memory in patients of cerebral arteriosclerosis with memory impairment. (4) Ranamargarin is a new tetradecapeptide isolated from the skin of the Chines frog Rana margaratae. This peptide may mainly act on NK-1 receptor.

Patterns of nucleotide substitution in angiosperm cpDNA trnL (UAA)-trnF (GAA) regions

Patterns of substitution in chloroplast encoded trnL_F regions were compared between species of Actaea (Ranunculales), Digitalis (Scrophulariales), Drosera (Caryophyllales), Panicoideae (Poales), the small chromosome species clade of Pelargonium (Geraniales), each representing a different order of flowering plants, and Huperzia (Lycopodiales). In total, the study included 265 taxa, each with > 900-bp sequences, totaling 0.24 Mb. Both pairwise and phylogeny-based comparisons were used to assess nucleotide substitution patterns. In all six groups, we found that transition/transversion ratios, as estimated by maximum likelihood on most-parsimonious trees, ranged between 0.8 and 1.0 for ingroups. These values occurred both at low sequence divergences, where substitutional saturation, i.e., multiple substitutions having occurred at the same (homologous) nucleotide position, was not expected, and at higher levels of divergence. This suggests that the angiosperm trnL-F regions evolve in a pattern different from that generally observed for nuclear and animal mtDNA (transitional/transversion ratio > or = 2). Transition/transversion ratios in the intron and the spacer region differed in all alignments compared, yet base compositions between the regions were highly similar in all six groups. A>-C transversions were significantly less frequent than the other four substitution types. This correlates with results from studies on fidelity mechanisms in DNA replication that predict A<->T and G<->C transversions to be least likely to occur. It therefore strengthens confidence in the link between mutation bias at the polymerase level and the actual fixation of substitutions as recorded on evolutionary trees, and concomitantly, in the neutrality of nucleotide substitutions as phylogenetic markers.

Pollen records and age determinations from 2 profiles at Lake Nikolay, Lena Delta

Radiocarbon-dated pollen, rhizopod, chironomid and total organic carbon (TOC) records from Nikolay Lake (73°20'N, 124°12'E) and a pollen record from a nearby peat sequence are used for a detailed environmental reconstruction of the Holocene in the Lena Delta area. Shrubby Alnus fruticosa and Betula exilis tundra existed during 10,300-4800 cal. yr BP and gradually disappeared after that time. Climate reconstructions based on the pollen and chironomid records suggest that the climate during ca. 10,300-9200 cal. yr BP was up to 2-3 °C warmer than the present day. Pollen-based reconstructions show that the climate was relatively warm during 9200-6000 cal. yr BP and rather unstable between ca. 5800-3700 cal. yr BP. Both the qualitative interpretation of pollen data and the results of quantitative reconstruction indicate that climate and vegetation became similar to modern-day conditions after ca. 3600 cal. yr BP. The chironomid-based temperature reconstruction suggests a relatively warm period between ca. 2300 and 1400 cal. yr BP, which corresponds to the slightly warmer climate conditions reconstructed from the pollen. Modern chironomid and rhizopod assemblages were established after ca. 1400 cal. yr BP.

Mean cover and frequencies of vascular plant species during three decades around Abisko Scientific Research Station

Plant species distributions are expected to shift and diversity is expected to decline as a result of global climate change, particularly in the Arctic where climate warming is amplified. We have recorded the changes in richness and abundance of vascular plants at Abisko, sub-Arctic Sweden, by re-sampling five studies consisting of seven datasets; one in the mountain birch forest and six at open sites. The oldest study was initiated in 1977-1979 and the latest in 1992. Total species number increased at all sites except for the birch forest site where richness decreased. We found no general pattern in how composition of vascular plants has changed over time. Three species, Calamagrostis lapponica, Carex vaginata and Salix reticulata, showed an overall increase in cover/frequency, while two Equisetum taxa decreased. Instead, we showed that the magnitude and direction of changes in species richness and composition differ among sites.

Pollen record and age determination of a sediment profile from Lake Teletskoye, Russia

A high-resolution pollen record from Lake Teletskoye documents the climate-related vegetation history of the northern Altai Mountain region during the last millennium. Siberian pine taiga with Scots pine, fir, spruce, and birch dominated the vegetation between ca. AD 1050 and 1100. The climate was similar to modern. In the beginning of the 12th century, birch and shrub alder increased. Lowered pollen concentrations and simultaneous peaks in herbs (especially Artemisia and Poaceae), ferns, and charcoal fragments point to colder and more arid climate conditions than before, with frequent fire events. Around AD 1200, regional climate became warmer and more humid than present, as revealed by an increase of Siberian pine and decreases of dry herb taxa and charcoal contents. Climatic conditions were rather stable until ca. AD 1410. An increase of Artemisia pollen may reflect slightly drier climate conditions between AD 1410 and 1560. Increases in Alnus, Betula, Artemisia, and Chenopodiaceae pollen and in charcoal particle contents may reflect further deterioration of climate conditions between AD 1560 and 1810, consistent with the Little Ice Age. After AD 1850 the vegetation gradually approached the modern one, in conjunction with ongoing climate warming.

Pollen records of the Bykovsky Peninsula

New pollen and radiocarbon data from the Bykovsky Peninsula document the Late Pleistocene and Holocene environmental history of the Laptev Sea coast. More than 60 AMS-14C and conventional 14C dates indicate that the deposits accumulated during the last 60,000 radiocarbon yr BP. High concentration of green alga colonies (Pediustrum and Botryococcus) in the investigated sediment show that sedimentation was mostly in shallow water environments. Scarce grass and sedge communities dominated the vegetation 53-60 kyr BP. Climate was cold and dry. Open Poaceae and Cypcraccae associations with Asteraceae, Ranunculaceae, and Cichoriaceac, dominated in the area about 48-42.5 kyr BP. Steppic communities with Artemisia and shrubby tundra communities with Salix and Betula sect. Nanae were also present. Climate was dry, but relatively warm. Vegetation cover became denser about 42.5-33.5 kyr BP, reflecting more favorable climate conditions. Scarce Poaceae communities with some Caryophyllaceae, Asteraceae, Cichoriaceae, and Selaginella rupestris covered the Bykovsky Peninsula area during the Sartan (Late Weichselian) stage about 26-16 kyr BP. Disturbed, uncovered soils were very common in the area. Climate was extremely cold and dry. Poaceae and Cyperaceae associations with Caryophyllaceae, Asteraceae, Cichoriaceae dominated the vegetation in the late Sartan, ca 16-12.2 kyr BP. Climate was significantly warmer than in the early Sartan time. The lee Complex sedimentation was interrupted about 12 kyr BP; most likely it was connected with the beginning of the Allerod warnring. Shrubby (Betula sect. Nanae, Alnusfnuicosa, Salix, Ericales) tundra was widely distributed on the Bykovsky Peninsula during the early-middle Holacene. Climate was most favorable between 8200 and 4500 yr BP. Vegetation became similar to modern after 4500 yr BP, suggesting a deterioration of climate.

Pollen record and age determinations on four sites from Taymyr Peninsula, Russia

Pollen data from a Levinson-Lessing Lake sediment core (74°28'N, 98°38'E) and Cape Sabler, Taymyr Lake permafrost sequences (74°33'N, 100°32'E) reveal substantial environmental changes on the northern Taymyr Peninsula during the last c. 32 000 14C years. The continuous records confirm that a scarce steppe-like vegetation with Poaceae, Artemisia and Cyperaceae dominated c. 32 000-10 300 14C yr BP, while tundra-like vegetation with Oxyria, Ranunculaceae and Caryophyllaceae grew in wetter areas. The coldest interval occurred c. 18 000 yr BP. Lateglacial pollen data show several warming events followed by a climate deterioration c. 10 500 14C yr BP, which may correspond with the Younger Dryas. The Late Pleistocene/Holocene transition, c. 10 300-10 000 14C yr BP, is characterized by a change from the herb-dominated vegetation to shrubby tundra with Betula sect. Nanae and Salix. Alnus fruticosa arrived locally c. 9000-8500 14C yr BP and disappeared c. 4000-3500 14C yr BP. Communities of Betula sect. Nanae, broadly distributed at c. 10 000-3500 14C yr BP, almost disappeared when vegetation became similar to the modern herb tundra after 3500-3000 14C yr BP. Quantitative climate reconstructions show Last Glacial Maximum summer temperature about 4°C below the present and Preboreal (c. 10 000 14C yr BP) temperature 2-4°C above the present. Maximum summer temperature occurred between 10 000 and 5500 14C yr BP; later summers were similar to present or slightly warmer.

Pollen record and age determination of a sediment profile from Frengstadsetra, Norway

Innerdalen was once a mountain valley (ca. 780 m a.s.l.) with birch forests, bogs and several summer farms. Today it is a 6.5 km**2 artifical lake. In 1980 and 1981 archaeological and palynological investigations were carried out due to the hydroelectric power plans. Radiocarbon dated pollen diagrams from 9 different localities in Innerdalen provide information on a mountain environment which has been exploited to varying degrees by human groups for thousands of years. In the Birch Zone, ca. 9500-8500 years B.P., the deglaciated surface is vegetated by the normal sequence of pioneering species, first show-bed communities, then shrub/dwarf-shrub communities, and finally a birch forest community. In the Pine Zone, ca. 8500-7500 years B.P., the mixed Birch-Pine forest which prevailed at the end of the Birch Zone is replaced by a dense pine forest. The tree limit was higher than it is today. In the Alder Zone, ca. 7500-4000 years B.P., the newly arrived alder gradually succeeded pine, particularily on good soils. This alder forest has a modem analog in the pre-alpine gray alder forests in Norway. In the last part of the Alder Zone, ca. 6000-4000 years B.P., elm and hazel are nominally present on particularily rich soils, marking the edaphic and climatic optimum in Innerdalen. During this time the first evidence of human impact on the vegetation is apparent in the pollen diagrams. At both Sætersetra in the south of the valley and Liabekken in the north, forest clearance and the development of grazed grass meadows is documented, and human impact continues until the present. The Herb Zone, ca. 4000 years B.P. to 1600 A.D., is characterized by the rapid decline of alder. The forest is increasingly open, and bog formation is initiated. The sub-alpine belt of birch forest is established, probably due to the shift to a cooler, moister climate. Human activity can also have influenced the vegetational changes, although at 4 of the localities human activity also is first apparent after the alder decline. Some localities show measurably less human impact on the vegetation ca. 2600-2000 years B.P. Grazing intensity increases ca. 2000 years B.P. At the end of the Herb Zone rye and barley pollen is registered at Sætersetra and Flonan, indicating contact between the grazing activities of Innerdal and grain cultivation activities outside the valley. The Spruce Zone, ca. 1600 A.D. to the present, does not begin synchronously since the presence of long-distance transported spruce pollen at a locality is entirely dependent on the density of the vegetation ie. degree of human impact. The youngest spruce rise is ca. 1500 A.D. at Røstvangen, when summerfarming is initiated. Summerfarming activities in Innerdal produce an increasingly open landscape. Rye and barley pollen at several localities may indicate limited local cultivation, but is more likely long-distance transport via humans and domesticated animals from cultivated areas outside Innerdalen.

Palynology on a profile from the Krusné Hory Mountains

Site details: The raised bog Fláje-Kiefern (50°429N, 13°329 E; 760 m a.s.l.; size ca. 500x500 m) lies in the Krusné Hory Mountains (Erzgebirge), Czech Republic, about 10 km from Georgenfelder Moor in Germany. Hejny and Slavík (1988) described the phytogeographic region of the Krusne Hory Mountains as 'a region of mountain flora and vegetation, with thermophilous species largely missing. In the natural forests, conifers, especially spruce (Picea excelsa) prevail. The deforested areas have been converted into meadows and pastures'. The climate is cool with annual average temperatures of about 5°C and annual precipitation of about 900 mm. The bedrock is Precambrian crystallinicum.