Jadwiga Turło

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.

Enhancement of tacrolimus productivity in Streptomyces tsukubaensis by the use of novel precursors for biosynthesis.

In this report the optimization of biosynthesis of tacrolimus, the immunosupressant widely used in transplantology and dermatology was described. The enhancement of the productivity of Streptomyces tsukubaensis strain was achieved by development of new precursors of tacrolimus biosynthesis, which should allow to reduce the costs of the process. The enrichment of the fermentation medium in pyridine-2-carboxylic acid (picolinic acid), piperidine-2-carboxylic acid (pipecolic acid), pyridine-3-carboxylic acid (nicotinic acid) or pyridine-3-carboxylic acid amide (nicotinamide) caused significant growth of the productivity of tacrolimus: 7-fold, 6-fold, 3-fold and 5-fold, respectively. The optimum concentration of the precursors in medium was 0.0025-0.005%. The investigation of the kinetics of tacrolimus biosynthesis together with the analysis of the impact of tested compounds on the culture growth and NAD (nicotinamide adenine dinucleotide) concentration in S. tsukubaensis cells enables to put forward a hypothesis concerning the mechanism of action of tested culture medium additives. The compounds active as tacrolimus precursors (pipecolic and picolinic acids) are more effective than these active mainly as the growth promoters (nicotinamide and nicotinic acid). Nicotinamide and nicotinic acid--vitamin B₃ components--promote S. tsukubaensis growth most probably due to the stimulation of NAD/NADP biosynthesis.

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

Derivatives of 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine were synthesized. These compounds contain the 3-(4-piperidyl)-1H-indole residue or its 5-methoxy or 2-methyl derivative. In vitro binding tests were performed to determine the affinity of the compounds for the 5-HT(1A) receptor and serotonin transporter (SERT) proteins in the rat brain cortex. In vivo studies, particularly the inducible hypothermia test and forced swimming test, were conducted to determine agonistic/antagonistic activity with pre- and postsynaptic 5-HT(1A) receptors. Molecular modeling techniques were used to determine the binding modes of the selected compounds at the 5-HT(1A) receptor and SERT. The SAR analysis showed that the presence of the 3-(4-piperidyl)-1H-indole group or its 5-methoxy derivative, as well as a para substitution with -OCH(3) or -F in the aryl ring of 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidine, results in an increased affinity for both the 5-HT(1A) receptors and SERT. In contrast, the presence of the 2-methyl-3-(4-piperidyl)-1H-indole group resulted in a considerable decrease in binding affinity.

Optimization of Selenium-Enriched Mycelium of Lentinula edodes (Berk.) Pegler as a Food Supplement

Our goal was to optimize the growth conditions of submerged mycelial cultures of Lentinula edodes (Shiitake mushroom) in order to obtain a new dietary supplement enriched in selenium We designed a process technology in which mycelial cultures were cultivated in media composed of beet molasses, 10%; liquid stillage, 5%; corn steep liquor, 0.15%; and KH2PO4, 0.3%, enriched with selenium in concentrations ranging from 0 to 100 μg/mL by the addition of sodium selenite of selenic acid. Se concentrations in mycelial dry mass rose from 0.001 mg/g (mycelia cultivated in media containing no selenium) to 50 mg/g (mycelia cultivated in medium containing 100 μg Se/mL). The highest mycelial specific growth rate (0.46/day) was recorded when the concentration of selenium in the medium was lower than 20 μg/mL. In vitro, estimated Se bioavailabilities from selenized mycelium strongly depended on its preparation in a proper manner and were 60% and 82% for dried mycelium and mycelial lyophilizate, respectively. Our results suggest that these optimized culture conditions could be applied to obtain a new Se-enriched dietary supplement.

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 D2 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 μg/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 μg/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.

Expanding the substrate scope of ugi five-center, four-component reaction U-5C-4CR): ketones as coupling partners for secondary amino acids

Various symmetrical and unsymmetrical ketones were successfully coupled with secondary amino acids in the course of Ugi five-center, four-component reaction (U-5C-4CR), thus expanding the molecular diversity possible to be achieved by the reaction. The chemical yields depended on the degree of hindrance of the components employed and were satisfactory in view of possible steric interactions in the U-5C-4CR zwitterionic intermediate. The sense of diastereoinduction for reactions employing unsymmetrical ketones was examined by converting the resulting Ugi adducts into the corresponding rigid 2,6-diketopiperazine derivatives.

Use of three-carbon chain compounds as biosynthesis precursors to enhance tacrolimus production in Streptomyces tsukubaensis.

Tacrolimus, a 23-membered polyketide macrolide, is isolated as a metabolite from the whole fermentation broth of Streptomyces species. This potent immunosuppressive calcineurin inhibitor has been widely used in various fields of medicine, including transplantology, dermatology and pharmacotherapy of autoimmune diseases. The current study was focused on optimisation of tacrolimus biosynthesis by Streptomyces tsukubaensis through the use of culture media supplements. Enrichment of the fermentation medium with propionic acid, propylene glycol or propanol resulted in a 5.5-, 3.5- and 1.8-fold, respectively, increase in tacrolimus production. The optimal concentration of the precursors was 0.25% for both propanol and propionic acid and 0.75% for propylene glycol. The mode of action of each media supplement tested was unique. For instance, propionic acid acted as a tacrolimus biosynthesis precursor while propylene glycol induced mycelial growth of S. tsukubaensis. Results from the current study clearly demonstrate that a set of novel culture medium supplements considerably increased tacrolimus production. Application of such promoters of tacrolimus biosynthesis may lead to a substantial improvement in the production of tacrolimus by S. tsukubaensis in industrial fermentation processes.

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

A series of novel 3β-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.

Biological Availability and Preliminary Selenium Speciation in Selenium-Enriched Mycelium of Lentinula edodes (Berk.)

Our goal is to obtain a new food supplement — a chemopreventive preparation derived from selenium (Se)-enriched Lentinula edodes mycelium. In the present study the bioavailability of selenium from Se-enriched mycelium preparations was tested in vitro and in vivo. Three different preparations of selenated mycelium were compared: dried mycelium, lyophilized mycelium, and lyophilized-autolyzed mycelium. In vitro, estimated Se bioavailabilities were 60%, 82%, and 98%, respectively. In vivo bioavailability was determined in rats. As reference, sodium selenite and Se yeast formulations were used at an Se-equivalent dose. The pharmacokinetic data for tested mycelial preparations suggest a rapid but incomplete Se absorption, and rapid elimination, without risk of accumulation. The speciation of selenium in Se-enriched mycelial cultures was carried out by specific oxido-reduction reactions. For tested mycelium the main part of Se was in the 0 and IV oxidation states in inorganic form or combined in a lipid or carbohydrate structure, about 47% was in the –II state in Se-amino acids or in other undefined water- or alcohol soluble organic compounds. Differences in pharmacokinetic data for Se yeast and L. edodes mycelial formulations probably arise from differences in Se speciation. Our data suggest that formulations of selenized Lentinula edodes mycelium could be used in chemoprevention as food supplements.

Synthesis of N-(4-aryl-1-piperazinylbutyl)-substituted 7,8-benzo-1,3-diazaspiro[4,5]decane-2,4-dione derivatives with potential anxiolytic activity

Continuing our studies connected with the design of new anxiolytics we have now synthesized a series of new compounds, derivatives of 7,8-benzo-1,3-diazaspiro[4,5]decane-2,4-dione bearing a 4-aryl-1-piperazinylbutyl group attached to the imide nitrogen. One single compound was submitted to the 5-HT1A receptor binding assay and found to display the expected--though rather weak--receptorial affinity.