Cinnamate of inulin as a vehicle for delivery of colonic drugs.

Colon diseases are difficult to treat because oral administrated drugs are absorbed at the stomach and intestine levels and they do not reach colon; in addition, intravenous administrated drugs are eliminated from the body before reaching colon. Inulin is a naturally occurring polysaccharide found in many plants. It consists of ß 2-1 linked D-fructose molecules having a glucosyl unit at the reducing end. Various inulin and dextran hydrogels have been developed that serve as potential carrier for introduction of drugs into the colon. Because inulin is not absorbed in the stomach or in the small intestine, and inulin is degraded by colonic bacteria, drugs encapsulated in inulin-coated vesicles could be specifically liberated in the colon. Therefore, the use of inulin-coated vesicles could represent an advance for the treatment of colon diseases. Here, we study the use of a cinnamoylated derivative of chicory inulin as a vehicle for the controlled delivery of colonic drugs. The encapsulation of methotrexate in inulin vesicles and its release and activity was studied in colon cancer cells in cultures.

Production of o-diphenols by immobilized mushroom tyrosinase.

The o-diphenols 4-tert-butyl-catechol, 4-methyl-catechol, 4-methoxy-catechol, 3,4-dihydroxyphenylpropionic acid and 3,4-dihydroxyphenylacetic acid were produced from the corresponding monophenols (4-tert-butyl-phenol, 4-methyl-phenol, 4-methoxy-phenol, p-hydroxyphenylpropionic acid and p-hydroxyphenylacetic acid) using immobilized mushroom tyrosinase from Agaricus bisporus. In all cases the yield was R(diphenol)> or =88-96%, which, according to the literature, is the highest yield so far, obtained using tyrosinase. The reaction was carried out in 0.5M borate buffer pH 9.0 which was used to minimize the diphenolase activity of tyrosinase by complexing the o-diphenols generated. Hydroxylamine and ascorbic acid were also present in the reaction medium, the former being used to reduce mettyrosinase to deoxytyrosinase, closing the catalytic cycle, and the latter to reduce the o-quinone produced to o-diphenol. Inactivation of the tyrosinase by ascorbic acid was also minimized due to the formation of an ascorbic acid-borate complex. Concentrations of the o-diphenolic compounds obtained at several reaction times were determined by gas chromatography-mass spectrometry (GC-MS) and UV-vis spectroscopy. The experimental results are discussed.

Immobilization of recombinant invertase (re-INVB) from Zymomonas mobilis on D-sorbitol cinnamic ester for production of invert sugar.

The recombinant invertase (re-INVB) from Zymomonas mobilis was immobilized by adsorption onto the totally cinnamoylated derivative of D-sorbitol. The polymerization and cross-linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of re-INVB on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. Enzyme concentration, immobilization time, and irradiation time were important parameters affecting the immobilization efficiency. The optimum reaction pH of immobilized enzyme was 5, and the optimal reaction temperature was 40 degrees C. The apparent Michaelis constant and the apparent catalytic constant of re-INVB immobilized on the SOTCN derivative acting on sucrose was 78+/-5 mM and 5x10(4)+/-3x10(2) s(-1), respectively, while for the free enzyme, it was 98.0+/-4 mM and 1.2x10(4)+/-2.5x10(2) s(-1), respectively, suggesting a better apparent affinity of the enzyme for the substrate and a better hydrolysis rate when immobilized than when in solution. Immobilized re-INVB also showed good thermal stability and good operational stability (40% of the initial activity remaining after 45 cyles of 1 min duration and 90.6 mg of sucrose being hydrolyzed in 45 min per 2.5 mg of immobilized protein). The results showed that cinnamic carbohydrate esters of D-sorbitol are an appropriate support for re-INVB immobilization and the production of invert sugar.

Effects of the immobilization supports on the catalytic properties of immobilized mushroom tyrosinase: a comparative study using several substrates.

Mushroom tyrosinase was immobilized from an extract onto glass beads covered with one of the following compounds: the crosslinked totally cinnamoylated derivatives of glycerine, D-sorbitol, D-manitol, 1,2-O-isopropylidene-alpha-D-glucofuranose, D-glucuronic acid, D-gulonic acid, sucrose, D-glucosone, D-arabinose, D-fructose, D-glucose, ethyl-D-glucopyranoside, inuline, dextrine, dextrane or starch, or the partially cinnamoylated derivative 3,5,6-tricinnamoyl-D-glucofuranose which was obtained by the acid hydrolysis of 1,2-O-isopropylidene-alpha-d-glucofuranose. The enzyme was immobilized by direct adsorption onto the support and the quantity of tyrosinase immobilized was found to increase with the hydrophobicity of the supports. The kinetic constants of immobilized tyrosinase acting on the substrates, 4-tert-butylcatechol, dopamine and DL-dopa, were studied. When immobilized tyrosinase acted on 4-tert-butylcatechol, the values of K(m)(app) were lower than these obtained for tyrosinase in solution while, when dopamine and DL-dopa were used, the K(m)(app) were higher. The order of the substrates as regards their ionizable groups, DL-dopa (two ionizable groups)>dopamine (one ionizable group)>4-tert-butylcatechol (no ionizable group) coincided with the order of the K(m)(app) values shown by tyrosinase immobilized on the hydrophobic supports, and was the inverse of that observed for tyrosinase in solution. The K(m)(app) values of immobilized tyrosinase were in all cases higher than those of soluble tyrosinase and depended on the nature of the support and the hydrophobicity of the substrate, meaning that it is possible to design supports with different degrees of selectivity towards a mixture of enzyme substrates in the reaction medium.

Stereospecificity of mushroom tyrosinase immobilized on a chiral and a nonchiral support.

Mushroom tyrosinase was immobilized from an extract onto glass beads covered with the cross-linked totally cinnamoylated derivates of d-sorbitol (sorbitol cinnamate) and glycerine (glycerine cinnamate). The enzyme was immobilized onto the support by direct adsorption, and the quantity of immobilized tyrosinase was higher for sorbitol cinnamate, the support with the higher number of esterified hydroxyls per unit of monosacharide, than for glycerine cinnamate. The results obtained from the stereospecificity study of the monophenolase and diphenolase activity of immobilized mushroom tyrosinase are reported. The enantiomers L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-alpha-methyldopa, DL-alpha-methyldopa, L-isoprenaline, DL-isoprenaline, L-adrenaline, DL-adrenaline, L-noradrenaline, and D-noradrenaline were assayed with tyrosinase immobilized on a chiral support (sorbitol cinnamate), whereas L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-alpha-methyldopa, and DL-alpha-methyldopa were assayed with tyrosinase immobilized on a nonchiral support (glycerine cinnamate). The same Vmax(app) values for each series of enantiomers were obtained. However, the Km(app) values were different, the l isomers showing lower values than the dl isomers, whereas the highest Km(app) value was obtained with d isomers. No difference was observed in the stereospecificity of tyrosinase immobilized on a chiral (sorbitol cinnamate) or nonchiral (glycerine cinnamate) support.

Direct immobilization of tyrosinase enzyme from natural mushrooms (Agaricus bisporus) on D-sorbitol cinnamic ester.

Mushroom tyrosinase was immobilized from an extract onto the totally cinnamoylated derivative of D-sorbitol by direct adsorption as a result of the intense hydrophobic interactions that took place. The immobilization pH value and mass of lyophilized mushrooms were important parameters that affected the immobilization efficiency, while the immobilization time and immobilization support concentration were not important in this respect. The extracted/immobilized enzyme could best be measured above pH 3.5 and the optimum measuring temperature was 55 degrees C. The apparent Michaelis constant using 4-tert-butylcatechol as substrate was 0.38+/-0.02 mM, which was lower than for the soluble enzyme from Sigma (1.41+/-0.20 mM). Immobilization stabilized the extracted enzyme against thermal inactivation and made it less susceptible to activity loss during storage. The operational stability was higher than in the case of the tyrosinase supplied by Sigma and immobilized on the same support. The results show that the use of p-nitrophenol as enzyme-inhibiting substrate during enzyme extraction and immobilization made the use of ascorbic acid unnecessary and is a suitable method for extracting and immobilizing the tyrosinase enzyme, providing good enzymatic activity and stability.

Molecular properties and prebiotic effect of inulin obtained from artichoke (Cynara scolymus L.).

A high molecular weight inulin has been prepared from artichoke (Cynara scolymus L.) agroindustrial wastes using environmentally benign aqueous extraction procedures. Physico-chemical analysis of the properties of artichoke inulin was carried out. Its average degree of polymerization was 46, which is higher than for Jerusalem artichoke, chicory, and dahlia inulins. GC-MS confirmed that the main constituent monosaccharide in artichoke inulin was fructose and its degradation by inulinase indicated that it contained the expected beta-2,1-fructan bonds. The FT-IR spectrum was identical to that of chicory inulin. These data indicate that artichoke inulin will be suitable for use in a wide range of food applications. The health-promoting prebiotic effects of artichoke inulin were demonstrated in an extensive microbiological study showing a long lasting bifidogenic effect on Bifidobacterium bifidum ATCC 29521 cultures and also in mixed cultures of colonic bacteria.