Kinetics and inhibition study of tyrosinase by pressure mediated microanalysis.

In the present study, pressure mediated microanalysis (PMMA), a fast, convenient and efficient capillary electrophoresis (CE) method was developed for studying enzyme kinetics of tyrosinase and inhibition kinetics of kojic acid, a model inhibitor of tyrosinase. The enzymatic reaction conditions and CE conditions were optimized in order to obtain high enzyme activity and short analysis time. By PMMA, only the product could be detected at 475 nm, and no voltage was applied to separate the product from the reaction mixture thus greatly simplifying the optimization procedure. The spectrophotometric assay and electrophoretically mediated microanalysis (EMMA) were also performed to validate the developed method. With the present method, the Michaelis-Menten constant (Km) was calculated to be 1.347 mM for tyrosinase. The inhibition constant of kojic acid to free tyrosinase (KI) and kojic acid to tyrosinase/L-DOPA complex (KIS) were calculated to be 36.64 and 74.35 µM, respectively, and the half-maximal inhibitory concentration (IC50) was determined to be 46.64 µM for kojic acid. The developed method is fast and convenient for studying enzyme kinetics, inhibition kinetics and further screening enzyme inhibitors.

New pseudopterosin and seco-pseudopterosin diterpene glycosides from two Colombian isolates of Pseudopterogorgia elisabethae and their diverse biological activities.

As part of an ongoing program to explore the chemical constituents of Caribbean marine invertebrates, a family of 13 new diterpene glycosides, pseudopterosins P-Z (1-11) and seco-pseudopterosins H (12) and I (13), have been isolated from the organic extracts of two collections of the sea whip Pseudopterogorgia elisabethae procured near the Colombian Southwestern Caribbean Sea. The structures of compounds 1-13, including absolute stereochemistry, have been proposed on the basis of comprehensive spectral analyses, chemical transformations, specific rotation, and TLC chromatographic analyses. Pseudopterosin Q (2) inhibited thromboxane B2 (TXB2) (IC50 = 4.7 microM) and superoxide anion (O2-) (IC50 = 11.2 microM) generation from E. coli lipopolysaccharide (LPS) activated rat neonatal microglia in vitro. In contrast, pseudopterosins P (1), U (6), V (7), W (8), and X (9) as well as seco-pseudopterosins H (12) and I (13) demonstrated minimal effects on both TXB2 and O2- release. In addition, some of the new compounds displayed strong antituberculosis, antiviral, antimalarial, and anticancer activity.

New terpenoid constituents from Eunicea pinta.

The secondary metabolite composition of the gorgonian octocoral Eunicea pinta from San Andrés Island, Colombia, is described for the first time. This investigation has resulted in the isolation of eight new gamma-cembranolide-type diterpenes, namely, compounds 3-10, and a new saponin, 13, possessing a pregnene-derived aglycon. In addition, two previously known alpha-methylene-gamma-lactone cembranolides, euniolide (1) and succinolide (2), were also isolated. Their structures were determined on the basis of the results of spectroscopic analysis, X-ray diffraction analysis, and chemical conversions. The discovery of cembranolides 3-10 has prompted us to conclude that the structures proposed earlier for several uprolide-type cembranolides need revision and that a minor stereochemical correction is also in order for succinolide. Cytotoxicity of compounds 3, 4, and 13 toward various cancer cell lines and results of an antimycobacterial screening for 2, 4, and 13 also are described.