Mariana Vallejo

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 alkaloids 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.

Huperzia saururus Lam. Trevis. (Lycopodiaceae) facilitates ejaculation in spinal cord transected male rats.

ETHNOPHARMACOLOGICAL RELEVANCE Huperzia saururus (Lam.) Trevis. has an extensive ethnopharmacological use, mainly because of its aphrodisiac properties. The species is consumed as decoctions or infusions in traditional medicine. The purpose of the present research was to determine if Huperzia saururus is able to increase sexual potency by evaluating the ejaculatory response, in the presence of a decoction in spinal cord transected male rats. MATERIALS AND METHODS The fictive ejaculation model to record the rhythmic contractions of the bulbospongiosus muscles that accompany ejaculation as an indicator of ejaculation occurrence was used. Sexually experienced male Wistar rats were used. The activation of the fictive ejaculation by the i.v. administration of a decoction was tested, as well as the effects of the oxytocinergic, cholinergic, adrenergic and nitrergic antagonism upon the pro-ejaculatory activity of Huperzia saururus. RESULTS Decoction (3µg/animal) was able to activate the fictive ejaculation in spinal male rats, producing a statistically significant diminution on the latency of discharge parameter and a statistically significant augment for the number of discharges. Moreover, when sequential treatments using antagonists plus decoction were administered, the effects produced showed that prazosin prevent the pro-ejaculatory effect of the decoction and that the four antagonists assayed blocked the facilitatory effect of Huperzia saururus since the facilitation in the latency of response was prevented, and the number of discharges was reduced. Together these findings support the notion that the decoction exerts an aphrodisiac effect influencing the ejaculatory potency which is partially mediated by oxytocinergic, cholinergic, adrenergic and nitrergic spinal mechanisms. CONCLUSION In agreement to the ethnopharmacological uses, Huperzia saururus decoction has aphrodisiac properties by influence on the ejaculatory potency.

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 Alzheimers 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.

A new semisynthetic derivative of sauroine induces LTP in hippocampal slices and improves learning performance in the Morris Water Maze.

Two semisynthetic acetyl derivatives of the alkaloid sauroine from Huperzia saururus, monoacetyl sauroine, and diacetyl sauroine (DAS) were obtained and their chemical structures were analyzed by NMR. While monoacetyl sauroine is the typical product of acetylation, DAS is an unexpected derivative related to the keto‐enol formation of sauroine. Recordings of field excitatory post‐synaptic potentials from the CA1 region of rat hippocampal slices showed that only DAS acutely applied induced chemical long‐term potentiation (LTP) in a dose‐dependent manner with an EC50 of 1.15 ± 0.09 μM. This effect was blocked by 10 μM D(‐)‐2‐amino‐5‐phosphonopentanoic acid (AP5), suggesting dependence on the NMDA receptor. DAS significantly increased NMDA receptor‐dependent excitatory post‐synaptic currents without affecting α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor‐dependent currents. Repetitive administration of DAS improved visuo‐spatial learning in the Morris Water Maze. In slices from rats tested in the Morris Water Maze, LTP resulting from electrical synaptic stimulation was 2.5 times larger than in controls. Concentration of DAS measured in the brain after repetitive administration was 29.5 μM. We conclude that slices perfused with DAS display a robust NMDA receptor‐dependent chemical LTP. During chronic treatment, DAS enhances learning abilities through a metaplastic mechanism as revealed by the augmentation of LTP in slices. DAS, therefore, may be a promising compound as a nootropic therapeutic drug.

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

Abstract 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.

Aphrodisiac activity of Phlegmariurus saururus in copulating and noncopulating male rats

BACKGROUND Phlegmariurus saururus is popularly known in Argentina as aphrodisiac. For this reason, it was previously investigated and determined that the decoction of this plant elicits pro-ejaculatory activity and increases the ejaculatory potency in the Fictive Ejaculation Model. HYPOTHESIS/PURPOSE: the decoction of P. saururus facilitates sexual behavior in sexually experienced male rat and induces copulatory behavior in non-copulating male rats. METHODS The extraction method (decoction) was validated through Selectivity, Accuracy and Precision, by identification of the majority alkaloids, expressed as sauroxine. Male (sexually experienced and noncopulating) and female (receptive) Wistar rats were used to determine sexual behavior. Sildenafil was used as positive control. The following variables were evaluated: Mount Latency, Intromission Latency, Ejaculation Latency, Post Ejaculatory Interval, as well as the Mounts and Intromissions Number. RESULTS In sexually experienced male rats, P. saururus decoction stimulates sexual arousal and facilitates sexual execution. In noncopulating male rats, this decoction induces copulation with behavioral characteristics similar to sexually experienced animals. CONCLUSION P. saururus possesses aphrodisiac activity in copulating and noncopulating male rats.

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.