Evaluation of novel soya-lecithin formulations for dermal use containing ketoprofen as a model drug.

In this study soya-lecithin aggregates, prepared by a technique using compressed gas, are used to formulate new dermal preparations. Ketoprofen (KP), a nonsteroidal anti-inflammatory drug (NSAID) is included as a model drug. The technique offers the possibility of incorporating auxiliary agents, such as penetration enhancers, anti-irritants and moisturisers together with the drug in one process. Apparent partition coefficients for n-octanol-phosphate buffer were determined for each of the lecithin aggregates. In general, soya-lecithin improves the partition of KP into n-octanol. The resulting products were included in widely used hydrophilic and hydrophobic vehicles. After 24 h, the cumulative amount of drug released through an artificial membrane was higher from the hydrophilic gels (2.6-4.3 mg) and the hydrophobic creams (0.23-0.392 mg) than from the control preparations (control hydrogel: 1.3 mg; control hydrophobic cream: 0.141 mg). However, the cumulative amount released from the hydrophobic vehicles was generally lower than from the hydrophilic matrices. Cumulative amounts such as those released from the hydrophilic preparations can also be achieved using supersaturated formulations based solely on the drug-loaded lecithin aggregates and a suitable oily component (4.07 mg). Results from the diffusion studies using artificial membranes were confirmed by permeation studies using excised rat skin. The improvement in skin permeation is related to both the solubilising effect of the lecithin matrix and the penetration enhancing effect of lecithin itself. The novel soya-lecithin aggregates are promising candidates for new drug delivery systems in dermatology and cosmetology. Lecithin aggregates loaded with drugs are multifunctional carriers that also act as penetration enhancers.

Purification and properties of two oxidoreductases catalyzing the enantioselective reduction of diacetyl and other diketones from baker's yeast.

The NADPH-linked diacetyl reductase system from the cytosolic fraction of Saccharomyces cerevisiae has been resolved into two oxidoreductases catalyzing irreversibly the enantioselective reduction of diacetyl (2,3-butanedione) to (S)- and (R)-acetoin (3-hydroxy-2-butanone) [so-called (S)- and (R)-diacetyl reductases] (EC 1.1.1.5) which have been isolated to apparent electrophoretical purity. The clean-up procedures comprising streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B column chromatography, affinity chromatography on Matrex Gel Red A and Superose 6 prep grade filtration led to 120-fold and 368-fold purifications, respectively. The relative molecular mass of the (R)-diacetyl reductase, estimated by means of HPLC filtration on Zorbax GF 250 and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was 36,000. The (R)-enzyme was most active at pH 6.4 and accepted in addition to diacetyl C5-, C6-2,3-diketones, 1,2-cyclohexanedione, 2-oxo aldehydes and short-chain 2- and 3-oxo esters as substrates. The enzyme was characterized by high enantioselectivity and regiospecificity. The Km values for diacetyl and 2,3-pentanedione were determined as 2.0 mM. The Mr of the (S)-diacetyl reductase was determined as 75,000 by means of HPLC filtration of Zorbax GF 250. The enzyme decomposed into subunits of Mr 48,000 and 24,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The optimum pH was 6.9. The purified (S)-enzyme reduced stereospecifically a broad spectrum of substrates, comprising 2,3-, 2,4- and 2,5-diketones, 2-oxo aldehydes, 1,2-cyclohexanedione and methyl ketones as well as 3-, 4- and 5-oxo esters. The 2,3- and 2,4-diketones are transformed to the corresponding (S)-2-hydroxy ketones; 2,5-hexanedione, however, was reduced to (S,S)-2,5-hexanediol. The Km values for diacetyl and 2,3-pentanedione were estimated as 2.3 and 1.5 mM, respectively. Further characterization of the (S)-diacetyl reductase revealed that it is identical with the so-called '(S)-enzyme', involved in the enantioselective reduction of 3-, 4- and 5-oxo esters in baker's yeast.

Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae.

A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diastereomeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C5-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (Mr) of the enzyme was estimated as 140,000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of Mr 35,000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast.

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.

[Aroma of the papaya fruit (Carica papaya, L.): indication of volatile precursors of terpene compounds]. / Uber das Aroma der Papaya-Frucht (Carica papaya, L.): Hinweise auf Vorstufen flüchtiger Terpenverbindungen.

In fruit pulp of papaya (Carica papaya, L.) the enzymes were inhibited by Hg2+. These sample were compared with others, in which the enzymes had not been inhibited. After separation and prefractionation of volatiles by means of high vacuum distillation/solvent extraction and subsequent adsorption chromatography on silicagel, capillary gas chromatography-mass spectrometry showed that in the experiment with Hg2+ the formation of terpene alcohols and hydrocarbons as well as benzylisothiocyanate was totally inhibited. The formation of linalooloxides and related compounds was inhibited to about 60%. From these results the occurrence of bound terpene precursors in papaya fruit may be postulated, from which the free volatile terpenes are enzymatically liberated after decompartmentalization of tissue during fruit pulp preparation.