Search

Spindle Tree (Euonymus europaea L.) has long been used in folk medicine for the treatment of dermatomycoces and as an antiparasitic and anthelmintic agent. However, with what substances it is associate this activity, it is not known for certain. In a previous work, a was developed extracting scheme from the bark of Spindle Tree (Euonymus europaea L.) to obtain water-soluble and lipophilic substances and the composition of the lipophilic fraction was studied using the GC-MS method. The purpose of this work was to obtain lipophilic substances from the flowers and leaves of Spindle Tree and to compare their chemical composition with the composition of substances obtained from the bark of the plant. Flowers and leaves were dried in a drying oven at a temperature of 52 °C, after which a fraction of lipophilic substances was obtained by extraction with petroleum ether. This fraction was analyzed using gas chromatography-mass spectrometry (GC-MS). For this, a chromatographic column was used, which was washed with methanol (model NR-5ms, length 30 m, diameter 0.25 mm, filler: 95% dimethylpolysiloxane + 5% diphenylpolysiloxane; carrier gas ‒ helium with a constant flow of 1.5 ml/min) and 6C / MS Agilent Technologies 6890 N / 5975 B mass spectrometer (USA). The etxtraction by petroleum ether was obtained fraction of lipophilic substances in the amount of 0.83% of the taken weight of dried flowers and 0.62% by weight of dried leaves. In total, 28 compounds were detected in flowers, and 19 in leaves. It was established that flowers and leaves of Spindle Tree, like bark, are characterized by a high content of paraffinic carbohydrates (from C20H42 to C31H64). Their content is higher in flowers (more than 50% of the composition of the fraction), and in leaves and bark ≈ 35%. Steroid and terpene compounds in the lipophilic fractions of the leaves and bark of the plant are respectively 28.26% and 24.32%. Among them quantitatively dominated by freedeen. A rather high content of squalene (2.06%) was found in the leaves of the plant. Among other compounds, a significant proportion of fatty acids and their esters, of which 24.59% by weight of the lipophilic fraction of flowers and 14.74% by weight of the lipophilic fraction of leaves. An analysis of the results indicates a greater promise for the use in medicine of lipophilic substances of the bark and leaves of Spindle Tree than of flowers.

The aim of this investigation was to study chemical composition and biological effects of substances present in methanol extract of dried basidiocarps of Lactarius quetus and L. volemus and the action of substances of milky juice isolated from these fungi on the mammalian cells and invertebrate organisms. It was found that fresh juice of Lactarius quetus mushrooms reduced the viability of transformed mouse fibroblasts of L929 line and caused the death of the crustaceans (Cyclops) plankton. The extract from dried basidiocarps was fractionated by various organic solvents under the scheme described above for Lactarius pergamenus mushrooms. Obtained fractions were analyzed by gas chromatography combined with mass spectrometry. The largest range of compounds was present in the first (hexane) fraction containing substances whose analysis required additional separation on a silica gel column. The obtained fractions contained large amount of saturated and unsaturated higher fatty acids and their esters, phthalates and sesquiterpene compounds. Their composition and content in studied mushroom species differed significantly. The results of chemical analysis of mushroom basidiocarps allowed one to conclude that primary protective function in L. quetus, probably, was dependent on several sesquiterpene compounds of the azulene series - lactarorufin A and B. Since in L. volemus fungus the content of azulene and its derivatives is rather low, the biological activity of its milky juice towards the mammalian cells and small crustaceans is poorly studied.

Fresh juice of basidiocarps of Lactarius pergamenus Fr. (Fr.) fungi was subjected to ion exchange chromatography with used DEAE-toyopearl and CM-cellulose columns, as well as preparative electrophoresis in 7.5% polyacrylamide gels (pH 8.6). Three isoforms of polyphenol oxidase (PPO) were discovered and two isoforms (1-1 and 1-2) were purified with a release of protein 0.42 mg/kg and 0.15 mg/kg of basidiocarps, respectively. These isoforms differ in the mobility at disc-electrophoresis in 7.5% PAGE in alkaline buffer system (pH 8.6). Specific activity of isoform 1-2 is 4.8 times higher than that of the isoforms 1-1. The molecular weight determination by gel chromatography on the Toyopearl HW-55 demonstrated that both isoforms 1-1 and 1-2 have the same 64 ± 2 kDa molecular mass. Electrophoresis in 15% PAGE in the presence of sodium dodecylsulphate and β-mercaptoethanol revealed one band with molecular mass of 64 ± 1 kDa which suggests the presence of one polypeptide chain in the molecule of the enzyme. The enzyme has demonstrated the highest activity at pH 6.0 and temperature +10 ºC, and at +70 ºC the enzyme was inactivated. The PPO activity was the highest in young mushrooms and it decreased with their age and positively correlated with the content of the milky juice. Ortho-aminophenol was most effective among all the tested substrates to determine the activity of PPO (o-, m– and p-aminophenol, catechol, tyrosine, resorcinol, phloroglucinol) and its relative activity was 129% of the activity of catechol. Ascorbic acid was the most effective inhibitor of the polyphenol oxidase activity which was completely blocked at 1 mM concentration, whereas the same concentration of thiourea and sodium sulphite decreased the enzymatic activity by 40-45%. The PPO in L. pergamenus fungi basidiocarps was mainly localized in the mushroom milky juice where its high activity may be associated with protection of basidiocarps against various pathogens.

A new lectin with 6.5 mg/kg yield was purified from fresh bulb Hyacinthella аcutiloba K. Perss. by combination of affinity chromathography on yeast mannan and by ion exchange chromatography on DEAE-toyopearl. The best lectin inhibitor among tested mono- and disaccharides was D-turanose (Glcp ?1-3 Fruf). Very powerful inhibitor of lectin activity was yeast mannan. The lectin revealed weak affinity to ?-methyl-Dmannoside, D-fructose and 2-acetamido-D-galactopyranoside. The lectin interacted also with pig liver glycogen, starch and mannose-containing glycoproteins (ovoalbumin, ovomucoid and calf thyroglobulin), but don’t interacted with horsera dish peroxidase and calf intestine alkaline phosphatase. According to electrophoresis, in 20% SDS-PAAG containing SDS-Na the lectin consists with subunits of molecular weight 12 kDa. Molecular weight of the lectin is 48 kDa according to gelchromatography on Toyopearl HW-55. The lectin agglutinated best of all rabbit erythrocytes and worse agglutinated guinea-pig, very weak — rat erythrocytes and did not agglutinate human and goat erythrocytes. After dialysis against 1% EDTA sodium salt solution the lectin did not lose hemaglutinating activity and endured heating to +65 °C during one hour.

A new lectin was purified from the daylily (Hemerocallis fulva L.) with the yield of approximately 10 mg per kg of fresh plant rhizome. The purification procedure was based on application of the affinity chromathography on the column with yeast mannan and the ion-exchange chromatography on the column with DEAE-Toyopearl. The lectin possessed low affinity for α-methyl-D-mannopyranoside, D-fructose, D-turanose and 2-acetamido-D-galactopyranose and hight affinity for the yeast mannan. The lectin bound with greatly less affinity for the mannose-containig glycoproteins, such as ovoalbumin, ovomucoid and horseradish peroxidase. According to the results of electrophoresis in 20% DSNa-PAGE, the lectin consists of subunits of 12 kDa molecular weight. According to the results of gel-chromatography on the Toyopearl HW-55, the lectin’s molecular weight is 48 kDa. It agglutinated rabbit erythrocytes very well, while rat and guinea-pig erythrocytes were agglutinated worse, and human erythrocytes were not agglutinated at all. Lectin’s dialysis against 1% EDTA or heating to 60 ºC for 60 min did not stop its hemagglutinating activity.

Ergosterol (ergosta-5 ,7,22-trien-3β-ol ) is a compound found only in fungi. Ergosterol has medical interest, as it is easily converted into Vitamin D2. In the previous work we have studied 22 species of true fungi. The highest quantity of ergosterol was found in basidiomes of Clouded Agaric (Clitocybe nebularis (Fr.) Kumm). Meanwhile, there is no data on the purification of ergosterol from this fungus in the literature. The aim of research. The aim of research is to elaborate a rational method of obtaining ergosterol from basidiomes of Clouded Agaric and assess the perspectives of the raw material for its purification. Materials and methods. In order to choose the best method of ergosterol purification, it was obtained in two ways: 1 ) by alkaline hydrolisis that was used in the preparation of ergosterol from baker yeast and 2) by the extraction of raw matherial by methanol with following chromatography on silica gel. A substance with high ergosterol level was purified from marc basidiomes of Clouded Agaric by methanol extraction with following of freezing the methanol extract and the following chromatography on silica gel. This substance was dissolved in hexane and purified by chromatography on silica gel by washing the column sequentially with hexane, hexane - ethyl acetate (6:1 ), hexane -ethyl acetate -methanol 4:2:1. The obtained fractions were dried and weighed. The analysis of the obtained compounds was carried out by gas-liquid сhromatography - mass spectrometry , Lieberman-Burchard reaction and TLC on silica gel. Chromatograms were spraied with saturated sodium antimony in chloroform. Results and discussion Pure ergosterol was obtained from dried baker's yeast and basidiomes marc of Clouded Agaric by alkaline hydrolysis followed by methylene chloride extraction of raw material and crystallization from ethanol and mixtures of benzene with ethanol (yield 0.71% and 0.52% ). Methanol extraction of dried basidiomes marc of Clouded Agaric, concentration and freezing, helped to obtain the substance, which according to the GLC -MS contained up to 91.1% of ergosterol. Chromatography of the substance obtained on silica gel allowed to obtain pure ergosterol with minor losses and higher purity, than by the crystallization. Conclusions We found a high content of ergosterol in Clouded Agaric basidiomes using gas-liquid chromatography-mass spectrometry. Almost pure ergosterol was sedimented by freezing of methanol extract. Authentic sample of ergosterol obtained from baker's yeast by TLC confirmed the identification of Clitocybe nebularis ergosterol. It was shown that chromatographic purification on a column of silica gel had advantages over alkaline hydrolysis of raw matherial and allowed to obtain pure ergosterol in a single stage with a higher yield (2.54 % of pure ergosterol). This method of purification is also an opportunity to obtain other substances which are in the methanol extract.

Secretory immunoglobulin A (sIgA) contains glycoconjugates moieties which can be involved in humoral immunity providing antimicrobial defense. The aim of present work was to characterize carbohydrate moieties of sIgA isolated from human colostrum via their interaction with specific lectins. Preparations of sIgA were obtained from 23 colostrums samples of parturient women by 3-fold precipitation with in ammonium sulphate 50% saturation. In the study a panel of 44 lectins of known carbohydrate specificity was used immobilized onto nitrocellulose membrane. The panel was incu­bated with sIgА preparations and lectin-antibody complexes were detected by protein-A conjugated with horseradish peroxidase. Western-blot analysis was applied to cha­racterize glycosylated polypeptides of sIgA isolated from colostrum of parturient women. sIgA from the colostrum of clinically healthy parturient women contained mainly carbohydrate moieties consisting of N-acetyl-D-glucosamine, sialic acid and D-mannose. Substantial differences in interaction of carbohydrate determinants of slgA isolated from colostrum of individual parturient women with the lectines were found. It is proposed that such characteristics can define sensitivity of mother and child to the action of pathogenic microflora.