Isolation and determination of ginsenosides in American ginseng leaves and root extracts by LC-MS.

Ginseng saponins (ginsenosides) were extracted from the root and leaves of locally cultivated American ginseng (Panax quinquefolium L.). For the isolation of compounds from plant samples three different extraction methods were utilized: accelerated solvent extraction, the ultrasound-assisted solvent extraction and mechanical shaking assisted solvent extraction. The separation of compounds was achieved with a water-acetonitrile gradient system using a C18 reversed-phase column. Target compounds were identified in MS(2) and MS(3) experiments. The relative distribution of these ginsenosides in each root and leaf extract was established. The limit of detection of the method was less than 30 ng/ml. Recovery of ginseng saponins in spiked samples exceeded 80%, while the relative standard deviation ranged from 7.1 to 9.1%. The total concentrations of ginsenosides were 41 and 13 mg/g in root and leaves.

Simultaneous isolation of Rutin and Esculin from plant material and drugs using solid-phase extraction.

Simultaneous isolation of Rutin and Esculin from pharmaceutical materials (plant--Flos hippocastani and drugs--Venescin, Venacorn) using solid-phase extraction (SPE) have been made. For this investigation the Bakerbond SPE columns with different unpolar and polar chemically bonded phases were used. On the basis of isolation investigation the influence of SPE packing materials on the selectivity change and recovery of both extracted substances were studied.

[Effect of drugs inhibiting the formation of hydroxide radicals on healing of experimental corneal ulcer]. / Wplyw srodków hamujqcych powstawanie rodników wodorotlenowych na gojenie sie doswiadczalnego owrzodzenia rogówki.

The 60 per cent water solution of dimethyl sulfoxide (DMSO) was used in the investigations. It was instilled into the conjunctival sac of an eye with experimental corneal ulceration. The dimethyl sulfoxide is inhibiting the formation of hydroxide radicals which cause a damage of the tissue in the course of inflammation. Clinical observations as well as histochemical and histopathological examinations showed that ulcerations treated by dimethyl sulfoxide healed faster than those from the control group and reactions to acid mucopolysaccharides++ returned more rapidly to normal values.

Powdered diatrizoic acid for radiography of the respiratory tract. Part I. Experimental investigation.

Powdered diatrizoic acid as a contrast medium administered by inhalation and insufflation for visualization of the airways was tested in vitro and in 21 dogs. Good radiopacity of the contrast medium and its antibacterial activity was found in vitro. In the majority of animal experiments visualization of the bronchial tree down to segmental and partially to subsegmental bronchi with only minimal agglomeration of contrast medium and with good or very good demonstration of anatomic details was achieved. In the majority of dogs contrast medium was eliminated from the lungs within 18 hours. Arterial blood gases tested on 5 dogs showed only unimportant changes after contrast medium administration. No adverse reactions were observed. Histologic and ultrastructural examinations after contrast studies showed phagocytic reaction to diatrizoic acid, transient impairment of production of surfactant, reactive changes of bronchial mucosa, and no fibrotic changes in the long-term observation.

Screening the keratinolytic activity of dermatophytes in vitro.

Sixteen strains out of 12 species dermatophytes were examined in respect to their ability of utilizing keratin substrates as the only sources of C and N. The employed keratin substrates included a solubilized preparation of feather keratin (KS) and native keratin, guinea pig hair and chicken feathers. It has been shown that the preparation KS constitutes a convenient model for a preliminary estimation of fungal keratinolytic activity and it can be a source of information about the localization of these enzymes. It has been found that, among the 16 fungal strains, 13 strains synthesize mainly intracellular keratinases while 3 strains of T. verrucosum release enzymes mainly to the medium. Native keratin from hair and feathers was degraded only by some of the examined strains which, under the experimental conditions, developed characteristic spore forms. Keratin of guinea pigs hair was attacked only by the T. mentagrophytes strains, T. verrucosum and K. ajelloi, and only T. gallinae grew on native keratin from chicken feathers.

Feather keratin as a ligand in an affinity chromatographic technique for isolation of protease from Trichophyton verrucosum.

A technique of affinity chromatography was developed and optimized for proteases from the postculture fluid of Trichophyton verrucosum. The technique employs porous glass with adsorbed feather keratin or keratin covalently bound to the glass. Modifications of the amounts of proteases introduced into the columns and the manner of elution (pH gradient, buffer concentration, EDTA) made it possible to achieve yields of the isolated enzymes of the order of 80%. The degree of purification of four fractions isolated by the proposed technique was about 6-fold and allowed electrophoretically almost homogeneous enzymatic forms to be isolated. Substrate and inhibition tests on the four chromatographically purified proteolytic enzymes indicated a specifically keratinolytic nature of the enzymes studied.

Influence of immobilization on properties of polyphosphate glucokinase.

Polyphosphate glucokinase from Mycobacterium tuberculosis H37Ra was covalently bound to the glutaraldehyde-activated collagen coating silica gel, to the nylon, and to the CNBr-activated Sepharose. After immobilization kinetic properties of the enzyme were altered.

Comparative characterization of proteolytic enzymes from Trichophyton gallinae and Trichophyton verrucosum.

The proteolytic activities of culture filtrates and cell homogenates of Trichophyton gallinae and Trichophyton verrucosum were compared when the fungi were grown in the presence of a readily assimilable source of C and N (Sabouraud's broth) and a poorly assimilable source (mineral medium containing soluble keratin (KS) protein prepared from either chicken feathers or guinea pig hair). The proteolytic activity of T. gallinae was found to be located predominantly in the cell homogenate although this depended somewhat on the nature of the C and N source in the medium. Enzyme activity in T. verrucosum on the other hand was located in the culture filtrate. The use of KS as a substrate revealed only one peak of enzymatic activity which occurred at pH 7.0 in both fungi. In the case of T. gallinae the total proteolytic activity was not related to the fungal biomass but did depend on the nature of the substrate; poorly assimilable substrates (native feathers and hair) stimulated a higher enzymatic activity per unit biomass than readily assimilable Sabouraud's broth. In T. verrucosum, proteolytic activity was related to the fungal biomass and was highest in Sabouraud's broth which proved most suitable for growth of the fungus. On the basis of the findings with six different inhibitors of protease activity, it is concluded that the proteolytic enzymes of T. gallinae differ from those of T. verrucosum in terms of their metal-dependence and contained serine as part of the active centre.

Intracellular keratinase of Trichophyton gallinae.

Trichophyton gallinae could grow on agar medium containing chicken feathers as sole sources of carbon and nitrogen, but it could not utilize human or guinea pig hair. The fungus grows rapidly when feather keratin solubilized in dimethyl sulphoxide was included in the growth medium. Keratinase activity could be detected by release of amino acids from solubilized keratin and by creation of zones of lysis in gel diffusion assay. However, the activity was observed only in homogenates of T. gallinae mycelia: it was not possible to detect it in culture filtrates.

New matrices for the purification of pectinases by affinity chromatography.

Polygalacturonic acid was used as a ligand in the affinity technique for pectinases purification from the filtrate of Aspergillus niger 71 culture. For this purpose four matrices were examined, namely, alkylamine controlled porous glass (CPG), alkylamine silica gel as well as keratin or polyamide coated silica gel. Good results of pectinase purification was obtained on silanized CPG or keratin coated silica gel supports.

Keratin and polyamide-coated inorganic matrices as supports for glucoamylase immobilization.

Previously solubilized feather keratin and polyamide were used for coating sand, glass beads and silica gel. These new seven supports were employed for comparative studies on pure glucoamylase / EC 3.2.1.3 / immobilization. The immobilization yield of glucoamylase on keratin and polyamide coated supports was comparable with conventional matrices used earlier. The highest activity per 1 g of support was shown by the enzyme bound to polyamide-coated CPG, and the bests operational stability by the enzyme immobilized on polyamide-coated CPG with keratin subsequently deposited on it.

Urogranoic acid as a radiopaque additive to the cyanoacrylic adhesive in transcatheter obliteration of renal arteries.

The purpose of the investigation was to study the application of radiopaque materials to the tissue adhesive base of the ester of n-butylic-alpha-cyanoacrylic acid for use in direct transcatheter renal artery obliteration. Lipiodol Ultra-fluid (Ethiodol) and urogranoic acid in quantities of 30--55% by weight of the adhesive were used as the radiopaque agents. The study was performed on seven dogs; obliterative material was administered through a telescopic, coaxial catheter system. Two dogs treated with the Lipiodol adhesive material died, which confirmed the inefficacy of Lipiodol for this purpose. In the other five dogs, treated by adding urogranoic acid to the adhesive, complete and permanent occlusion occurred and the adhesive remained radiopaque for 60--90 days. One of these dogs died of canine distemper infection. The complication of unpredictable embolization of the femoral artery appeared in a second dog. The same material has been used in two cases of nonoperative hypernephroma in humans. Permanent occlusion of the renal artery and elimination of hematuria were achieved. The obliterative material was sufficiently opaque for radiographic visualization.

Stability of equine hierarchies and the prevention of dominance related aggression.

The dominance hierarchy of a herd of 10 Thoroughbred mares was determined twice, at an interval of 18 months, using paired feeding tests. Each mare's rank was correlated significantly between the 2 tests. This indicated that the hierarchy within the herd was stable. The offspring of dominant and subordinate mares were also tested for dominance in their own age groups. The offspring of dominant mares tended to be near the top of the hierarchy while those of middle and low ranking mares were not consistently found in the middle or bottom of their own hierarchies. Paired feeding tests were carried out on 8 ponies. During tests the time that each pony spent eating and the ponies' aggressive interactions were recorded. Two situations were used. Each pony-pair was tested when both ponies were in the same paddock and also when they were separated by a rail fence. The subordinate ponies spent significantly more time eating and the domonant pony was significantly less aggressive, when the pony-pair was separated by a fence than when they were in one paddock. It was concluded that the dominance hierarchies of adult horse groups changed very little over time and that the foals of dominant mares will tend to be dominant in their own age groups. Management practices can be used to reduce aggression and consequent injury that may arise in group feeding situations.

The choice of medium suitable for the reaction of thiol groups with halogen derivatives in the synthesis of some purine and pyrimidine compounds. I. Synthesis of azathioprine.

Investigations were carried out using 6-mercaptopurine and 1-methyl-4-nitro-5-chloroimidazole as model compounds. The reaction kinetics were investigated by the UV spectroscopy at various time intervals. The investigations resulted in elaboration of an efficient synthesis of azathioprine which can be applied on an industrial scale in a yield of 80%.