The Synthesis and Absolute Configuration of Enantiomeric Pure (R)- and (S)-3-(piperidin-3-yl)-1H-Indole Derivatives.

This article describes the synthesis of new chiral 3-(piperidin-3-yl)-1H-indole derivatives (R)-10a-c and (S)-11a-c from the corresponding diastereomers: (3R, 2R) and (3S, 2R)-2-[3-(1H-indol-3-yl)-1-piperidyl]-2-phenyl-acetamides (3R, 2R)-4a, (3R, 2R)-6b, (3R, 2R)-8c and (3S, 2R)-5a, (3S, 2R)-7b, (3S, 2R)-9c. Diastereomers were obtained by N-alkylation of derivatives of racemic 3-(piperidin-3-yl)-1H-indoles 1a-c using (S)-2-(4-toluenesulfonyloxy)-phenylacetic amide (S)-II. The same method was applied to obtain (3R, 2S)-methyl-2-[3-(1H-indole-3-yl)-1-piperidyl]-2-phenylacetate (3R, 2S)-2a and (3S, 2S)-methyl-2-[3-(1H-indole-3-yl)-1-piperidyl]-2-phenylacetate (3S, 2S)-3a diastereomers by treating amine 1a with (R)-2-(4-toluenesulfonyloxy)-phenylacetic acid methylester (R)-I. Systematic studies via single crystal X-ray crystallography were used to determine the molecular structure of the racemates 1a-c and the absolute configuration of the enantiomers. The solid racemates 1b and 1c were "true racemates" crystallizing in a centrosymmetric space group, while 1a formed a racemic conglomerate of homoenantiomeric crystals. The absolute configuration was determined for the enantiomeric pairs (R)-10a/(S)-11a, (R)-10b/(S)-11b, and (R)-12c/(S)-13c, as well as for (3S,2S)-3a. Spectra of 1H, 13CNMR, HPLC, and HRMS for diastereomers and enantiomers were consistent with the determined structures.

Synthesis of Novel Pyrido[1,2-c]pyrimidine Derivatives with 6-Fluoro-3-(4-piperidynyl)-1,2-benzisoxazole Moiety as Potential SSRI and 5-HT1A Receptor Ligands.

Two series of novel 4-aryl-2H-pyrido[1,2-c]pyrimidine (6a-i) and 4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine (7a-i) derivatives were synthesized. The chemical structures of the new compounds were confirmed by 1H and 13C NMR spectroscopy and ESI-HRMS spectrometry. The affinities of all compounds for the 5-HT1A receptor and serotonin transporter protein (SERT) were determined by in vitro radioligand binding assays. The test compounds demonstrated very high binding affinities for the 5-HT1A receptor of all derivatives in the series (6a-i and 7a-i) and generally low binding affinities for the SERT protein, with the exception of compounds 6a and 7g. Extended affinity tests for the receptors D2, 5-HT2A, 5-HT6 and 5-HT7 were conducted with regard to selected compounds (6a, 7g, 6d and 7i). All four compounds demonstrated very high affinities for the D2 and 5-HT2A receptors. Compounds 6a and 7g also had high affinities for 5-HT7, while 6d and 7i held moderate affinities for this receptor. Compounds 6a and 7g were also tested in vivo to identify their functional activity profiles with regard to the 5-HT1A receptor, with 6a demonstrating the activity profile of a presynaptic agonist. Metabolic stability tests were also conducted for 6a and 6d.

Understanding performance of 3D-printed sorbent in study of metabolic stability.

Metabolic stability tests are one of the fundamental steps at the preclinical stages of new drug development. Microsomes, used as a typical enzymatic model of liver biotransformation, can be a challenging matrix for analytical scientists due to a high concentration of cellular proteins and membrane lipids. In the work, we propose a new procedure integrating biotransformation reaction with SPME-like protocol for sample clean-up. It is beneficial to increase the overall quality of results in contrary to the typical protein precipitation approach. A set of ten arylpiperazine analogs, six of which are considered promising drug candidates (and four are accepted drugs) were used as a probe to assess the goodness of the newly proposed approach. In order to promote an efficient extraction protocol, a new, miniaturized shape of a sorbent, suitable to perform the extraction in 100 µL of the sample has been designed. Termination of the biotransformation process by protein denaturation with hot water was additionally evaluated. A quantitative structure-property relationship (QSPR) study using Orthogonal Partial Least Squares (OPLS) technique to reveal insights to the sorption mechanism was also performed. The obtained results showed the new 3D-printed sorbent can be an attractive basis for the new sample preparation approach for metabolic stability studies and an alternative for commercially available protocols based on solid-phase microextraction (SPME) or solid-phase extraction (SPE) principles.

Synthesis and biological evaluation of new multi-target 3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives with potential antidepressant effect.

A series of novel 3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives were synthesised and evaluated for their 5-HT1A/D2/5-HT2A/5-HT6/5-HT7 receptor affinity and serotonin reuptake inhibition. Most of the evaluated compounds displayed high affinities for 5-HT1A receptors (e.g., 4cKi = 2.3 nM, 4lKi = 3.2 nM). The antidepressant activity of the selected compounds was screened in vivo using the forced swim test (FST). The results indicate that compound MW005 (agonist of the pre- and postsynaptic 5-HT1A receptor) exhibited promising affinities for the 5-HT1A/SERT/D2/5-HT6/5-HT7 receptors and showed an antidepressant-like activity in the FST model.

Synthesis of new 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives with rigidized tryptamine moiety as potential SSRI and 5-HT1A receptor ligands.

Extended studies in the 4-aryl-pyrido[1,2-c]pyrimidine group resulted in 27 new compounds (10.1-10.27), 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine derivatives. In vitro tests (RBA) were carried out for 10.1-10.27 compounds in order to determine their affinity to 5-HT1A receptor and SERT protein. 10.1-10.3, 10.6, 10.7, 10.16 and 10.27 compounds had high binding ability to both molecular targets (5-HT1A Ki = 8-87 nM; SERT Ki = 8-52 nM). For these compounds (10.1-10.3, 10.6, 10.7, 10.16, 10.27) further in vitro, in vivo and metabolic stability tests were performed. In vitro studies in the extended receptor profile (D2, 5-HT2A, 5-HT6 and 5-HT7) showed their selectivity towards 5-HT1A receptor and SERT protein. In vivo tests revealed that compounds 10.7 and 10.16 had the properties of presynaptic antagonists of the 5-HT1A receptor. The redesign of the 2H-pyrido[1,2-c]pyrimidine residue present in the terminal part towards 5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine resulted in the improved metabolic stability and enhanced affinity to both molecular targets (5-HT1A-R and SERT) compared to the precursors.

Molecular Docking Supplements an In vitro Determination of the Leading CYP Isoform for Arylpiperazine Derivatives.

BACKGROUND: Molecular docking has often been used before to calculate in silico affinity of drugs towards their molecular target, but not to estimate leading CYP isoform responsible for metabolism of studied compounds. OBJECTIVE: The aim of this study is to present molecular docking as a valid alternative for costly in vitro studies resulting in estimation of leading CYP isoform. METHODS: In vitro part was based on incubations of studied compounds with isolated CYP3A4 isoform followed by LC-MS analysis. The in silico stage consisted of docking three-dimensional models of the studied compounds with a three-dimensional model of the leading metabolizing isoform (CYP3A4), which was designated during the in vitro part of the study. XenoSite P450 metabolism prediction was also used to predict sites of metabolism and calculate probability values. RESULTS: The calculated affinities showed a clear similarity when the in vitro results were compared with the calculated in silico affinity values. XenoSite CYP3A4 metabolism probability values also confirm significant participation of CYP3A4 in metabolism of studied compounds. CONCLUSION: Both molecular docking and XenoSite P450 metabolism prediction provide data that stands in agreement with in vitro studies, granting a more detailed spectrum on predicting CYP3A4 metabolism, and presenting molecular docking as a promising tool to cut costs and increase effectiveness in early drug development stages.

Synthesis of novel pyrido[1,2-c]pyrimidine derivatives with rigidized tryptamine moiety as potential SSRI and 5-HT1A receptor ligands.

The study enabled obtaining a number of new derivatives of 4-aryl-pyrido[1,2-c]pyrimidine 9.1-9.27 having conformationally restricted tryptamine moiety. In vitro studies (RBA) have shown that derivatives 9.1, 9.2, 9.4, 9.7, 9.9, 9.14 and 9.27 exhibit high affinity to molecular targets 5-HT1A receptor and SERT protein. In general, compounds with an unsubstituted or a para-substituted benzene ring of the pyrido[1,2-c]pyrimidine residue in the terminal part were characterized by higher binding ability, which can be justified by the greater flexibility of the structure. For the selected compounds 9.1, 9.7, 9.9 and 9.27, further in vitro, in vivo and metabolic stability tests were performed. The in vitro studies in the extended receptor profile (D2, 5-HT2A, 5-HT6 and 5-HT7) indicated their selectivity toward the 5-HT1A receptor and SERT protein. The in vivo studies (8-OH-DPAT-induced hypothermia in mice, FST) revealed that the compound 9.1 has the properties of presynaptic agonist of the 5-HT1A receptor, and compound 9.7 demonstrated the properties of a presynaptic antagonist of the 5-HT1A receptor. Metabolic stability studies, in turn, showed that compounds 9.1, 9.7 and 9.9, having an unsubstituted indole residue, were more resistant to biotransformation reactions of the first pass phase than was compound 9.27 containing a 5-methoxy-substituted indole residue. The obtained results allowed further optimization of the structure.

Synthesis and biological investigation of new equatorial (ß) stereoisomers of 3-aminotropane arylamides with atypical antipsychotic profile.

A series of novel 3ß-aminotropane derivatives containing a 2-naphthalene or a 2-quinoline moiety was synthesised and evaluated for their affinity for 5-HT1A, 5-HT2A and D2 receptors. Their affinity for the receptors was in the nanomolar to micromolar range. p-Substitution (6c, 6f, 6i, 6l, 6o), as well as substitution with chlorine atoms (6g, 6h, 6i), led to a significant increase in binding affinity for D2 receptors with compounds 6f (Ki=0.6nM), 6c and 6i (Ki=0.4nM), having the highest binding affinities. m-Substituted derivatives were the most promising ligands in terms of 5-HT2A receptor binding affinity whereas 2-quinoline derivatives (10a, 10b) displayed the highest affinity for 5-HT1AR and were the most selective ligands with Ki=62.7nM and Ki=30.5nM, respectively. Finally, the selected ligands 6b, 6d, 6e, 6g, 6h, 6k, 6n and 6o, with triple binding activity for the D2, 5-HT1A and 5-HT2A receptors, were subjected to in vivo tests, such as those for induced hypothermia, climbing behaviour and the head twitch response, in order to determine their pharmacological profile. The tested ligands presented neither agonist nor antagonist properties for the 5-HT1A receptors in the induced hypothermia and lower lip retraction (LLR) tests. All tested compounds displayed antagonistic activity against 5-HT2A, with 6n and 6o being the most active. Four (6b, 6k, 6n and 6o) out of eight tested compounds could be classified as D2 antagonists. Additionally, evaluation of metabolic stability was performed for selected ligands, and introduction of halogen atoms into the benzene ring of 6h, 6k, 6n and 6o improved their metabolic stability. The project resulted in the selection of the lead compounds 6n and 6o, which had antipsychotic profiles, combining dopamine D2-receptor and 5-HT2A antagonism and metabolic stability.

Novel 4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT(1A) activity. Part 5.

A series of novel 4-aryl-pyrido[1,2-c]pyrimidine derivatives containing a 1-(2-quinoline)piperazine moiety was synthesized. The chemical structure of new compounds was confirmed by FT-IR, (1)H NMR, (13)C NMR and HRMS spectra as well as elemental analysis. Affinity of the novel pyrido[1,2-c]pyrimidine derivatives for 5-HT1A, 5-HT2A receptors and serotonin transporter (SERT) was evaluated in an in vitro radioligand binding assay. Tested compounds showed moderate to high affinity for 5-HT1AR and SERT and low affinity for 5-HT2AR. Selected ligands were subjected to in vivo tests, such as induced hypothermia and the forced swimming test in mice, which determined presynaptic agonistic activity of the ligands 8d, 8e, 9d and 9e and presynaptic antagonistic activity of the ligands 8a, 8b, 9a, 9b. Additionally, metabolic stability evaluation was performed for selected ligands, proving that a para-substitution in the 4-aryl-pyrido[1,2-c]pyrimidine moiety leads to an increase in stability, whereas a substitution in the ortho-position lowers the stability.

Prediction of overall in vitro microsomal stability of drug candidates based on molecular modeling and support vector machines. Case study of novel arylpiperazines derivatives.

Other than efficacy of interaction with the molecular target, metabolic stability is the primary factor responsible for the failure or success of a compound in the drug development pipeline. The ideal drug candidate should be stable enough to reach its therapeutic site of action. Despite many recent excellent achievements in the field of computational methods supporting drug metabolism studies, a well-recognized procedure to model and predict metabolic stability quantitatively is still lacking. This study proposes a workflow for developing quantitative metabolic stability-structure relationships, taking a set of 30 arylpiperazine derivatives as an example. The metabolic stability of the compounds was assessed in in vitro incubations in the presence of human liver microsomes and NADPH and subsequently quantified by liquid chromatography-mass spectrometry (LC-MS). Density functional theory (DFT) calculations were used to obtain 30 models of the molecules, and Dragon software served as a source of structure-based molecular descriptors. For modeling structure-metabolic stability relationships, Support Vector Machines (SVM), a non-linear machine learning technique, were found to be more effective than a regression technique, based on the validation parameters obtained. Moreover, for the first time, general sites of metabolism for arylpiperazines bearing the 4-aryl-2H-pyrido[1,2-c]pyrimidine-1,3-dione system were defined by analysis of Q-TOF-MS/MS spectra. The results indicated that the application of one of the most advanced chemometric techniques combined with a simple and quick in vitro procedure and LC-MS analysis provides a novel and valuable tool for predicting metabolic half-life values. Given the reduced time and simplicity of analysis, together with the accuracy of the predictions obtained, this is a valid approach for predicting metabolic stability using structural data. The approach presented provides a novel, comprehensive and reliable tool for investigating metabolic stability, factors that affect it, and the proposed structures of metabolites at the same time. The performance of the DFT-SVM-based approach provides an opportunity to implement it in a standard drug development pipeline.

Novel 4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT1A activity. Part 4.

This project describes the synthesis, pharmacological and pharmacodynamic tests on two series of novel derivatives of 2H-pyrido[1,2-c]pyrimidine with potential binary binding to 5-HT1A receptors and SSRI + serotonin transporters. The influence of piperidinyl-indole (8.1-8.7) and tetrahydropyridinyl-indole (8.8-8.32) residues and indole 5-position substituents (R3 = Br, Cl, F) present in the pharmacophore element of ligands on their binding to both molecular targets was tested. A considerable impact of piperidinyl-indole residue on binding to both targets was confirmed and compounds with a high binding affinity were identified: Ki 5-HT1A = 12.4 nM; Ki SERT = 15.6 nM 8.1; Ki 5-HT1A = 5.6 nM; Ki SERT = 20.7 nM 8.7, while the presence of a tetrahydropyridinyl-indole residue was found to reduce the affinity of ligands to 5-HT1AR. The presence of chlorine (R3) in this series resulted in a notable reduction in binding to both targets (5-HT1A and SERT). Selected compounds had their metabolic stability in a first-pass test (human liver microsomes, NADPH) determined in vitro, and R1 and R2 substituents present on the terminal residue of pyrido[1,2-c]pyrimidine were recognized as having an impact on stability.

A method for rapid screening of interactions of pharmacologically active compounds with albumin.

We determine the association constants for ligand-protein complex formation using the flow injection method. We carry out the measurements at high flow rates (F=1 mL min(-1)) of a carrier phase. Therefore, determination of the association constant takes only a few minutes. Injection of 1 nM of the ligand (10 µL of 1 µM concentration of the ligand solution) is sufficient for a single measurement. This method is tested and verified for a number of complexes of selected drugs (cefaclor, etodolac, sulindac) with albumin (BSA). We obtain K=4.45×10(3) M(-1) for cefaclor, K=1.00×10(5) M(-1) for etodolac and K=1.03×10(5) M(-1) for sulindac in agreement with the literature data. We also determine the association constants of 20 newly synthesized 3ß- and 3α-aminotropane derivatives with potential antipsychotic activity--ligands of 5-HT1A, 5-HT2A and D2 receptors with the albumin. Results of the studies reported here indicate that potential antipsychotic drugs bind weakly to the transporter protein (BSA) with K≈10(2)-10(3) M(-1). Our method allows measuring K in a wide range of values (10(2)-10(9) M(-1)). This range depends only on the solubility of the ligand and sensitivity of the detector.

Model structure-activity relationship studies of potential tropane 5HT1A, 5HT2A, and D2 receptor ligands.

The two-stages studies of structure-activity relationship for model ligands of 5HT1A, 5HT2A, and D2 receptors were performed. On the first stage, the pharmacophores of two potential ligands of known in vitro binding to 5HT1A, 5HT2A, D2 receptors and model pharmacophore of strongly interacting D2 receptor ligands were found and their parameters were related to affinity data. The analyzed parameters were hydrophobic, hydrophilic, aromatic, donor and acceptor of proton centers. The geometry of spatial distribution of these properties was also investigated in comparative analysis. The studied, model compounds were two 3ß-acylamine derivatives of tropane. The second stage includes docking of studied compounds to D2 receptor model and the comparison of its quality with in vivo binding data. The obtained results are consistent with in vitro binding data and applied procedure accurate estimates the affinity of potential ligands to D2 receptors.

Synthesis and biological evaluation of novel pyrrolidine-2,5-dione derivatives as potential antidepressant agents. Part 1.

A series of 3-(1H-indol-3-yl)pyrrolidine-2,5-dione derivatives was synthesized and their biological activity was evaluated. The chemical structures of the newly prepared compounds were confirmed by (1)H NMR, (13)C NMR and ESI-HRMS spectra data. All tested compounds proved to be potent 5-HT1A receptor and serotonin transporter protein (SERT) ligands. Among them, compounds 15, 18, 19 and 30 showed significant affinity for 5-HT1A and SERT. Computer docking simulations carried out for compounds 15, 31 and 32 to models of 5-HT1A receptor and SERT confirm the results of biological tests. Due to high affinity for the 5-HT1A receptor and moderate affinity for SERT, compounds 31, 32, 35, and 37 were evaluated for their affinity for D2L, 5-HT6, 5-HT7 and 5-HT2A receptors. In vivo tests, in turn, resulted in determining the functional activity of compounds 15, 18, 19 and 30 to the 5-HT1A receptor. The results of these tests indicate that all of the ligands possess properties characteristic of 5-HT1A receptor agonists.

Synthesis and anticonvulsant activity of novel 2,6-diketopiperazine derivatives. Part 2: Perhydropyrido[1,2-a]pyrazines.

A new series of chiral pyrido[1,2-a]pyrazine derivatives was synthesised and evaluated in in vivo animal models of epilepsy. A significant influence of the stereochemistry of the pyrido[1,2-a]pyrazine framework on the pharmacological activity was observed. Compounds with (4R,9aS) absolute configuration proved inactive, whereas other stereoisomers exhibited markedly dissimilar spectra of anti-seizure efficacy in the maximal electroshock seizure (MES), subcutaneous Metrazol seizure (scMET) and Pilocarpine-induced status prevention (PISP) tests. Importantly, the investigated agents revealed high potency in the 6Hz model, with the ED(50) values comparable to the reference drug Levetiracetam. Derivatives (4S,9aR)-6 and (4R,9aR)-6 emerged as promising new lead structures, the former having a broad spectrum of anticonvulsant activity and the latter showing high potency in 6Hz and PISP models.

Synthesis and anticonvulsant activity of novel 2,6-diketopiperazine derivatives. Part 1: perhydropyrrole[1,2-a]pyrazines.

A number of novel pyrrole[1,2-a]pyrazine derivatives were synthesized and evaluated in in vivo animal models of epilepsy. Among them, several compounds displayed promising seizure protection in the maximal electroshock seizure (MES), subcutaneous metrazol seizure (scMET), 6 Hz and pilocarpine-induced status prevention (PISP) tests, with ED(50) values comparable to the reference anticonvulsant drugs (AEDs). A critical influence of the stereochemistry and conformational preferences of the pyrrole[1,2-a]pyrazine core on in vivo pharmacological activity was observed. The mechanism of the anticonvulsant action of the agents synthesized is most probably not via inhibition of the voltage-dependent sodium (Na(+)) currents.

Synthesis and biological investigation of potential atypical antipsychotics with a tropane core. Part 1.

The synthesis, structure, in vitro and in vivo pharmacological activities of 3ß-acylamine derivatives of tropane (4a-n, 5a-g, 6a,b, 8a-c) are described. Among the investigated compounds, several displayed very high (in nM) affinity for the monoamine receptors 5-HT(1A), 5-HT(2A,) and D(2). The most interesting agent 6b revealed very high affinity for the 5-HT(2A) and D(2) receptors and high affinity for the 5-HT(1A) receptor. The in vivo head twitch model was used to demonstrate antagonism of the 5-HT(2A) receptor subtype by this compound. In another test, 6b caused hypothermia in mice, which was not attenuated by WAY 100635. In the climbing test, the compound did not significantly modify climbing behaviour following apomorphine administration. Moreover, 6b significantly reduced locomotor activity in mice. Molecular docking studies using a homology model of the 5-HT(1A) receptor revealed a significant role of the N-8 atom of the tropane core in stabilising the ligand-receptor complex due to strong hydrogen bonding with Asp116 located in the TMH 3 helix. Analogically, in a homology model of the 5-HT(2A) receptor, the N-8 atom formed a hydrogen bond with Gly369. In another homology model of the D(2) receptor, strong hydrogen bonding of the amide moiety in the 3ß position of the tropane nucleus with Asp85 was observed. Compound 6b displayed a favourable Meltzer index (1.21) which is a feature of atypical antipsychotic agents.

Novel 4-aryl-pyrido[1,2-c]pyrimidines with dual SSRI and 5-HT(1A) activity. part 3.

A number of 4-aryl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidine with 3-(1,2,3,6-tetrahydro-pyridin-4-yl)-1H-indole or 2-methyl-3-(1,2,3,6-tetrahydro-pyridin-4-yl)-1H-indole residues were synthesized for further investigation of SAR in a group of pyrido[1,2-c]pyrimidine derivatives with dual 5-HT(1A)/SERT activity. Compounds 8a-8p were found to be potent ligands for both 5-HT(1A) and SERT with K(i) ranging from 28,3 to 642 nM and 42,4 nM-1,8 µM, respectively. Moreover compounds 8a, 8b, 8c, 8d, 8e and 8g were found to be selective agonists, while 8i as an antagonist of 5-HT(1A) presynaptic receptors in the inducible hypothermia test in mice.

Relationship between selenium accumulation and mycelial cell composition in Lentinula edodes (Berk.) cultures.

It was postulated that fractions enriched in selenium (Se) isolated from Lentinula edodes mycelium polysaccharide might possess higher biological activity than the non-enriched fractions currently used to treat cancer. In order to obtain Se-enriched mycelial preparations, L. edodes cultures were cultivated in media enriched with sodium selenite. In order to determine whether the concentration of Se in the culture medium affected the biosynthesis and composition of cell wall and cell membrane, concentrations of the exopolysaccharide (EPS), chitin, and sterol (ergosterol) were measured in harvested mycelia. In addition, the relationship between Se accumulation and content of polyphenols and vitamin D(2) in L. edodes mycelium was examined. The effects of Se levels on the mycelium cell composition were determined in culture media enriched with Se at concentrations ranging from 0 to 30 microg/ml. In each culture mycelial growth, total Se and Se distribution were determined between mycelial fractions of different polarity. The EPS, polyphenolics, and ergosterol content in harvested mycelia rose in proportion to Se concentration in the culture medium. The chitin content in mycelia increased with Se concentrations in the range 0-5 microg/ml, but at higher concentrations chitin levels decreased. Data showed that Se in culture medium exerted potent effects on the composition of the mushroom cell wall and semipermeable membrane, and on the content of polyphenolics that are involved in detoxification processes. Our findings indicate the optimal concentration of Se required in the culture medium for maximal yield of immunostimulatory-active selenated exopolysaccharides.

Effect of selenium enrichment on antioxidant activities and chemical composition of Lentinula edodes (Berk.) Pegl. mycelial extracts.

Preparations derived from Lentinula edodes (Berk.) Pegl. mycelium are worldwide used as dietary supplements containing compounds active as immune system enhancers, demonstrating chemopreventive and anticancer activity. L. edodes mycelium enriched with organic forms of selenium like selenized yeast possess putative, higher cancer preventive properties. The objective of this study was to test the effect of enrichment in selenium on antioxidant, reducing and free radical scavenging activity of water and alcohol extracts from mycelium of L. edodes (Berk.). To elucidate the cause of enhanced antioxidant activity of extracts, a preliminary selenium speciation by specific oxido-reduction reaction was performed. Se-enrichment enhanced antioxidant activity, reducing power and free radical scavenging effect of mycelial extracts by almost 100-400%. Increase of activity was particularly high for diluted extracts (concentrations 0.1-0.5 mg/ml). The chemical composition of extracts from both Se-enriched and non-enriched mycelium was compared by determination of polyphenols, proteins, carbohydrates and lipids. Results showed that Se-enrichment enhanced antioxidant activities of mycelial extracts, likely by high amounts of organic Se-compounds (-II oxidation state) and elemental red selenium, and by increased polyphenols content. Our results suggest that Se-enrichment is a good method for enhancement of important activities of human dietary supplements, including Shiitake preparations.