12α-hydroxy-N-demethyl-sauroxine, a lycodane type alkaloid from Phlegmariurus saururus.

12α-hydroxy-N-demethyl-sauroxine (1), another new Lycopodium alkaloid from the Lycodane group, was isolated from Phlegmariurus saururus (Lam.) B. Øllg. (Lycopodiaceae). Elucidation of the chemical structure and relative stereochemistry were stated by spectroscopic data and chemical correlation. In addition, the inhibitory activity on acetylcholinesterase for 1 was determined as well as for N-methyllycodine (2), a derivative with the same nucleus, previously identified in P. saururus (IC50 = 33.8 ± 0.8 µM and 547.5 ± 0.5 µM, respectively) and N-demethylsauroxine (3) whose inhibition in the actual conditions was better than the previously informed.

An alkaloid extract obtained from Phlegmariurus Saururus induces neuroprotection after status epilepticus.

BACKGROUND: The brain is exposed to many excitotoxic insults that can lead to neuronal damage. Among these, Epilepsy is a neurological disease that affects a large percentage of world population and is commonly associated with cognitive disorders and excitotoxic neuronal death. Most experimental strategies are focused on preventing Status Epilepticus (SE), but once it has already occurred, the key question is whether it is possible to save neurons. PURPOSE: The aim of this study was to determine if a purified alkaloid extract (AE) obtained from Phlegmariurus saururus, a genus of Lycophyte plants (sometimes known as firmossesor fir club mosses) could induce neuroprotection following SE. METHODS: In vitro and in vivo techniques were applied for this purpose. Protein levels were measured by western blotting procedures. Neuronal death analysis was performed by calcein-ethidium staining and the presence of the NeuN protein as a marker for presence or absence of cells (in vitro experiments) and by Fluoro Jade B staining for the in vivo experiments. RESULTS: The effect of AE in the hippocampal neurons culture was the first determination, where we found an increase in neuronal survival and in the level of pErk and TrkB activation, 24 h after the addition of AE. In a well-established in vitro model of SE, we found that 24 h after being added to the hippocampal neuron-astrocyte co-culture, the AE induces a significant increase in neuronal survival. In addition to this, in the in vivo Li-pilocarpine model of SE, the AE induced a remarkable neuroprotection in areas such as the entorhinal cortex and hippocampal CA1 area. CONCLUSION: These results make the AE an excellent candidate for potential clinical use in neurological disorders where memory impairment and neuronal death occurs.

Sauroxine reduces memory retention in rats and impairs hippocampal long-term potentiation generation.

In the present paper it was investigated the role of sauroxine, an alkaloid of Phlegmariurus saururus, as a modulator of some types of learning and memory, considering the potential nootropic properties previously reported for the alkaloid extract and the main alkaloid sauroine. Sauroxine was isolated by means of an alkaline extraction, purified by several chromatographic techniques, and assayed in electrophysiological experiments on rat hippocampus slices, tending towards the elicitation of the long-term potentiation (LTP) phenomena. It was also studied the effects of intrahippocampal administration of sauroxine on memory retention in vivo using a Step-down test. Being the bio distribution of a drug an important parameter to be considered, the concentration of sauroxine in rat brain was determined by GLC-MS. Sauroxine blocked LTP generation at both doses used, 3.65 and 3.610-2µM. In the behavioral test, the animals injected with this alkaloid (3.6510-3nmol) exhibited a significant decrease on memory retention compared with control animals. It was also showed that sauroxine reached the brain (3.435µg/g tissue), after an intraperitoneal injection, displaying its ability to cross the blood-brain barrier. Thus, sauroxine demonstrated to exert an inhibition on these mnemonic phenomena. The effect here established for 1 is defeated by other constituents according to the excellent results obtained for P. saururus alkaloid extract as well as for the isolated alkaloid sauroine.

Purification of substances from Achyrocline satureioides with inhibitory activity against Paenibacillus larvae, the causal agent of American foulbrood in honeybees' larvae.

Achyrocline satureioides extracts were tested in vitro against the growth of Paenibacillus larvae. Four different extracts were obtained by liquid-liquid extraction from an aqueous-ethyl alcohol macerate of the aerial parts of the plant. The biological activity was tested by the broth microdilution technique. Hexane extract showed the highest activity (minimum inhibitory concentration = 0.060 ± 0.037 mg/mL). Transmission electron microscopy experiments showed that the main effect exerted by the hexane extract on the cell was at the cellular membrane level. The hexane extract was analyzed by thin-layer chromatography, and the activity of its components was tested by bioautography. Four growth inhibition zones were observed in the bioautographic experiments (using hexane-acetone (7:3) as mobile phase), with that at Rf = 0.57 showing the largest zone of inhibition. High-performance liquid chromatographic experiments, using ultraviolet and electrospray ionization coupled to tandem mass spectrometric detection, showed the presence of one compound with a m/z ratio of 442, which may be related to phloroglucinols α-pyrone compounds recently discovered. The high antibacterial activity of the hexane extract and of the isolated compound determined in this work may be useful for the development of future new alternatives for the treatment of American foulbrood.

Mass spectrometry studies of lycodine-type Lycopodium alkaloids: sauroxine and N-demethylsauroxine.

RATIONALE: Sauroxine and N-demethylsauroxine are lycodine-type Lycopodium alkaloids. In recent years, Lycopodium alkaloids have gained significant interest due to their unique skeletal characteristics as well as due to their acetylcholinesterase activity. It is known that drugs that inhibit acetylcholinesterase can be used to treat the early stages of Alzheimer's disease. METHODS: Sauroxine and N-demethylsauroxine were isolated from the aerial parts of Huperzia saururus (Lam.) Trevis. Electron ionization mass spectrometry (EI-MS) (low resolution) and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) fragmentation was conducted using an ion trap, GCQ Plus mass spectrometer with MS/MS. Electron ionization high-resolution mass spectrometry (EI-HRMS) was performed in a magnetic sector mass spectrometer (Micromass VG). RESULTS: Using GC/EI-CID-MS/MS we obtained different fragmentation routes that connect all the ionic populations. In addition, the use of EI-HRMS allowed us to measure the exact masses of all the fragment ions, and, with all this information gathered, we tried to establish a fragmentation scheme concordant with the ascendant and descendant species. CONCLUSIONS: The mass spectrometry studies presented in this work complete our mass studies of Lycopodium alkaloids. The mass spectrometry work presented has been very useful to confirm the structures as well as to support the biogenetic relationships between the lycodine-type Lycopodium alkaloids: sauroxine and N-demethylsauroxine.

Estudio descriptivo sobre la utilización de plantas como alternativas terapeuticas / No disponible

Objetivo: Conocer el grado de utilización, Prevalencia (P) de uso, de Plantas Medicinales (PM) como alternativa terapéutica en la ciudad de Córdoba, Argentina. Métodos: Se realizó un estudio Transversal aplicando una encuesta autoadministrada en 12 barrios de la ciudad de Córdoba elegidos aleatoriamente, 4 de cada clase social. El estudio se llevó a cabo entre mayo y agosto de 2009. Los datos fueron procesados utilizando el programa Epi info 3.5.1 y cuando fue factible se aplicó el test Chi cuadrado (χ2) para determinar asociación entre variables. Resultados: La P de uso de PM como alternativa terapéutica fue de 53,30% (IC95%= 49,10% - 57,40%). Luego del análisis del χ2 se determinó que las PM son más usadas por el sexo femenino (54,60%), las personas de clase media (56,85%) y en forma similar por los grupos etarios correspondientes a Senectud (67,59%) y Adultez Media (62,50%). El 69,36% de los consumidores fueron inducidos por familiares o conocidos y sólo el 23,12%, por un profesional del equipo de salud. El 86,07% (IC95%= 83,57% - 88,57%) de las personas que consumieron PM refieren haber obtenido un buen resultado; sólo el 4,9% (IC95%= 2,87% - 6,87%) de los encuestados respondieron haber padecido efectos adversos luego de la utilización de diversas PM. Conclusiones: Si se suma al 53,30% de personas que usan PM un 29,17% que aunque respondió que no usa PM afirma que consume diversas especies para “ayudar a la digestión”, la P asciende a 81,90%, resultado coherente con el mencionado por la OMS (AU)

Aim: To known the degree of utilization, Prevalence (P) of use, of Medicinal Plants (MP) as therapeutic alternative in Córdoba city, Argentina. Material and methods: It was carried out a Transversal study applying an auto administered survey in 12 randomly selected neighborhoods in Córdoba city, being 4 of each social class. The study was performed between May and August 2009. Data were processed by using the Epi info Program version 3.5.1, and when it was possible, Chi Square Test (χ2) was calculated in order to determine association among variables. Results: MP P of use as therapeutic alternative was 53.30% (CI95%= 49.10% - 57.40%). After χ2 analysis it was determined that MP are mostly used by Female sex (54.60%), Middle Class persons (56.85%) and in a similar proportions by the etary groups correspondent to Senescence (67.59%) and Middle Adulthood (62.50%). The 69.36% of the MP consumers were induced by relatives or acquaintances and only the 23.12% was advised by a professional of the health team. The 86.07% (CI95%= 83.57% - 88.57%) of persons that consumed MP refer to have obtained a good result; only the 4.90% (CI95%= 2.87% - 6.87%) of the polled answered to have suffered from adverse effects after diverse MP use. Conclusions: If it is added to the 53.30% of the persons that use MP a 29.17% of those people that even when declare that don’t consume MP affirm that consume diverse species to “help the digestion”, the P increase to 81.90%, coherent result with that mentioned by the WHO (AU)

Study of the interaction of Huperzia saururus Lycopodium alkaloids with the acetylcholinesterase enzyme.

In the present study, we describe and compare the binding modes of three Lycopodium alkaloids (sauroine, 6-hydroxylycopodine and sauroxine; isolated from Huperzia saururus) and huperzine A with the enzyme acetylcholinesterase. Refinement and rescoring of the docking poses (obtained with different programs) with an all atom force field helped to improve the quality of the protein-ligand complexes. Molecular dynamics simulations were performed to investigate the complexes and the alkaloid's binding modes. The combination of the latter two methodologies indicated that binding in the active site is favored for the active compounds. On the other hand, similar binding energies in both the active and the peripheral sites were obtained for sauroine, thus explaining its experimentally determined lack of activity. MM-GBSA predicted the order of binding energies in agreement with the experimental IC50 values.

Amino acid content and acetylcholinesterase inhibition of Huperzia saururus infusion and decoction.

UNLABELLED: CONTEXT. Huperzia saururus (Lam.) Trevis. (Lycopodiaceae), an autochthonous plant species in Argentina, is used as a memory improver in traditional medicine. It was studied for this reason and the purified alkaloid extract did show significant activity on learning and memory. The species is mostly consumed in the form of infusions and decoctions. OBJECTIVES: To evaluate the activity of the H. saururus infusion and decoction as inhibitors of acetylcholinesterase (AChE) and to determine the amino acid content in both extracts. MATERIAL AND METHODS: Infusion and decoction were purified by ionic exchange chromatography and analyzed by high-performance liquid chromatography HPLC-UV, and the AChE inhibition of these extracts was evaluated by using the Ellman method. RESULTS: Both infusion and decoction exerted strong AChE inhibitory activities (IC50=7.2 ± 0.4 and 22.7 ± 5.6 µg/mL, respectively). Among nine amino acids, arginine (Arg) was identified in a concentration greater than 9 mg/100 g of dried aerial parts in both extracts. DISCUSSION AND CONCLUSION: This high content of Arg could be considered a contributing factor to the activity on memory previously demonstrated for the H. saururus alkaloid extract, since Arg is implicated indirectly in mnemonic processes as a precursor in nitric oxide synthesis. Thus, the central effect of H. saururus could involve two different mechanisms, the cholinergic mechanism and the nitric oxide pathway.

Estudio descriptivo sobre la utilización de plantas como alternativas terapéuticas / Descriptive study on the use of plants as therapeutic alternatives

Objetivo: Conocer el grado de utilización, Prevalencia (P) de uso, de Plantas Medicinales (PM) como alternativa terapéutica en la ciudad de Córdoba, Argentina. Métodos: Se realizó un estudio Transversal aplicando una encuesta autoadministrada en 12 barrios de la ciudad de Córdoba elegidos aleatoriamente, 4 de cada clase social. El estudio se llevó a cabo entre mayo y agosto de 2009. Los datos fueron procesados utilizando el programa Epi info 3.5.1 y cuando fue factible se aplicó el test Chi cuadrado (χ2 ) para determinar asociación entre variables. Resultados: La P de uso de PM como alternativa terapéutica fue de 53,30% (IC95%= 49,10% - 57,40%). Luego del análisis del χ2 se determinó que las PM son más usadas por el sexo femenino (54,60%), las personas de clase media (56,85%) y en forma similar por los grupos etarios correspondientes a Senectud (67,59%) y Adultez Media (62,50%). El 69,36% de los consumidores fueron inducidos por familiares o conocidos y sólo el 23,12%, por un profesional del equipo de salud. El 86,07% (IC95%= 83,57% - 88,57%) de las personas que consumieron PM refieren haber obtenido un buen resultado; sólo el 4,9% (IC95%= 2,87% - 6,87%) de los encuestados respondieron haber padecido efectos adversos luego de la utilización de diversas PM. Conclusiones: Si se suma al 53,30% de personas que usan PM un 29,17% que aunque respondió que no usa PM afirma que consume diversas especies para "ayudar a la digestión", la P asciende a 81,90%, resultado coherente con el mencionado por la OMS.

Prenylated flavanones with anti-tyrosinase activity from Dalea boliviana.

Three new prenylated flavanones, (2S)-5,7,2'-trihydroxy-5'-(1''',1'''-dimethylallyl)-8-prenylflavanone (1), (2S)-5,7,2'-trihydroxy-8,3'-diprenylflavanone (2), and (2S)-5,2'-dihydroxy-6'',6''-dimethylchromeno-(7,8:2'',3'')-3'-prenylflavanone (3), and a known chromeno (dimethylpyrano) flavanone, obovatin (4), were isolated from the n-hexane extract of Dalea boliviana roots. The compounds were evaluated in vitro in relation to their inhibitory effect on the tyrosinase activity by using a spectrophotometric method.

Two new alkamides from roots of Acmella decumbens.

From the roots of Acmella decumbens (Sm) R.K. Jansen three compounds belonging to the alkamide family were isolated: (2E, 4E)-N-hydroxiphenylethyl-2,4-decadien-9-inamide (1); (4E, 6E)-N-isobutyl-4,6-undecen-10-inamide; (2) and (2Z)-N-phenylethyl-2-nonane-6,8-diinamide (3). The structures were determined by means of IR, MS, (1)H NMR, (13)C NMR, DEPT 135 and COSY. Compounds 1 and 2 are reported for the first time, while 3 was previously isolated from Spilanthes acmella L.

Anthraquinone derivatives from Heterophyllaea pustulata.

From the leaves of Heterophyllaea pustulata two new monomeric anthraquinones, heterophylline (1,6-dihydroxy-7-methoxy-2-methylanthraquinone, 1) and pustuline (2-hydroxy-3-methoxy-7-methylanthraquinone, 2), and one new bianthraquinone, (S)-5,5'-bisoranjidiol [(S)-5,5'-bis(1,6-dihydroxy-2-methylanthraquinone), 3], were isolated. Furthermore, the iridoid glycoside asperuloside and three known flavonoids, quercetin, isoquercitrin, and quercetin-3-O-beta-d-glucosyl-6' '-acetate, were obtained. The structures were determined by analysis of their spectroscopic data and chemical evidence.

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation.

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3',4'-tetrasulphate (QTS) and quercetin 3-acetyl-7,3',4'-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 microM, final concentration), and the platelet aggregation was induced by using adenosine 5'-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 microM) and Qc (250 microM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 microM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA2) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B2 (TxB2) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA2, as judged by TxB2 formation, in stimulated WP and a marked interference on the TxA2 receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.

Anticoagulant effect and action mechanism of sulphated flavonoids from Flaveria bidentis.

Quercetin 3-acetyl-7,3',4'-trisulphate (ATS) and quercetin 3,7,3',4'-tetrasulphate (QTS) obtained from Flaveria bidentis (Asteraceae) were investigated in vitro for anticoagulant activity. Three different concentrations of each flavonoid were assayed at different incubation times, showing at 1 mM significant prolongation on the activated partial thromboplastin time (APTT), less on the prothrombin time (PT), and no effect on the thrombin time (TT). In order to define the action mechanism of the anticoagulant activity, all coagulation factors were evaluated and no important activity decrease was observed, indicating that another mechanism is involved. Thus, thrombin inhibition mediated by antithrombin III (ATIII) and heparin cofactor II (HCII) activation was investigated in comparison to the physiological activators, heparin and dermatan sulphate (DS), respectively. As a conclusion, no activation on ATIII for neither flavonoids was observed. On the contrary, QTS much more than ATS produced an activation on HCII comparable to the one of DS, indicating that these flavonoids act as agonists of this inhibitor. A plausible explanation of the effects of both flavonoids could be due to the different degree of sulphation of these molecules. According to the results obtained, and taking in account the high solubility of these natural products in aqueous media and the nontoxic nature of this family of compounds, further investigation on the antithrombotic effects of these flavonoids are merited.