Purification and characterization of malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase (Dp21MaT) from leaves of Digitalis purpurea L.

With respect to the cardenolide pathway and the characterization of enzymes involved in the formation of cardenolides, a malonyltransferase, termed malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase (Dp21MaT) has been purified. The enzyme catalyses the transfer of the malonyl moiety from malonyl-coenzyme A to 21-hydroxypregnane substrates. Malonyltransferase activity was checked in several potential starting materials including fresh leaves and cell suspension cultures from different plants. Fresh Digitalis purpurea L. leaves turned out to be the best enzyme source. The purification protocol included ammonium sulphate precipitation, hydrophobic interaction chromatography on Phenylsepharose 6 FF, ion exchange chromatography on Source 30 Q, affinity chromatography on Cibacron Blue 3GA and gel filtration on Superdex 75. Gel filtration and native SDS-PAGE analysis showed that Dp21MaT exists as a monomer with a molecular mass of 27kDa. Its pI, as determined by isoelectric focusing, was 4.66. The enzyme showed maximal activity at pH 6.5 when incubated at 42 degrees C. The energy of activation was 29.28kJmol(-1), whereas that of inactivation was 48.57kJmol(-1). Dp21MaT was purified 252-fold with a yield of about 1%. Hanes plots of kinetic data indicated K(m) values of 99microM (V(max) 47.57microkatkg(-1)) and 28.44microM (V(max) 39.4microkatkg(-1) protein) for 3beta-benzoyloxy-5beta-pregnane-14beta,21-dihydroxy-20-one and malonyl-CoA, respectively.

A simple chemical method for synthesizing malonyl hemiesters of 21-hydroxypregnanes, potential intermediates in cardenolide biosynthesis.

A simple and versatile method for the chemical synthesis of 21-hydroxypregnane 21-O-malonyl hemiesters which may be important intermediates of cardenolide biosynthesis is described. Starting from commercial beta-methyldigitoxin, acid hydrolysis followed by 3beta-O-acetylation and ozonolysis with reductive cleavage of the ozonides afforded 3beta-acetoxy-5beta-pregnane-14beta,21-diol-20-one which was finally converted into the target compound by treatment with malonyl chloride. The malonylation protocol was optimized using deoxycorticosterone (DOC) as the pregnane educt.

Synthesis of a putative substrate for malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase and development of an HPLC method for the quantification of the enzyme reaction.

The butenolide ring is the main common characteristic of all cardenolides. Its formation is supposed to be initiated by the transfer of a malonyl moiety from malonyl-coenzyme A to an appropriate 21-hydroxypregnane. A new, reliable, fast and sensitive method to determine malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase activity had to be developed since previous attempts employing HPLC, TLC or GC did not prove successful. A surrogate substrate was synthesized containing a side chain resembling the sugar side chain attached to C-3 of putative cardenolide precursors and containing a chromophor allowing UV detection. 3beta-benzoyloxy-5beta-pregnane-14beta,21-dihydroxy-20-one and its 21-O-malonylated derivative were synthesized, the latter being the expected product of the enzyme reaction. The new substrate was well accepted by the enzyme. An HPLC method has been established to detect and quantify 3beta-benzoyloxy-5beta-pregnane-14beta,21-dihydroxy-20-one and its 21-O-malonylated derivative, 3beta-benzoyloxy-5beta-pregnane-14beta-hydroxy-20-one 21-O-malonylhemiester. The method was validated.

Chemical composition and antidermatophytic properties of volatile fractions of hexanic extract from leaves of Cupressus lusitanica Mill. from Cameroon.

The chemical composition of five column fractions of hexanic leaf extract of Cupressus lusitanica were analysed by gas chromatography and gas chromatography-mass spectrometry and then tested for their antidermatophytic activities using the agar dilution method. The first fraction (F(1)) has only hydrocabon monoterpenes with alpha-pinene (80.0%) as major component. The main constituents of the second fraction (F(2)) were epi-bicyclosesquiphellandrene (35.3%), epi-zonarene (10.3%), 1S, cis-calamenene (13.1%) and beta-himachalene (10.4%). The third fraction (F(3)) was rich in hydrocarbon sesquiterpenes (45.4%) and a relatively high amount of diterpenes (29.8%) with epi-bicyclosesquiphellandrene (14.3%), pimaric acid (7.5%), kaurenoic acid (6.9%) and 8-beta-hydroxysandaracopimarane (3.5%) as main components. The last two fractions contain high molecular weight aliphatic hydrocarbons, their main constituents been eicosane (41.1%) and tricosane (37.3%) and heptacosane (22.1%). The agar dilution method was used to evaluate the antifungal properties of the crude extract and its fractions. These fractions showed several degrees of antidermatophytic activities against Microsporum audouinii, Microsporum Langeronii, Microsporum canis, Trichophyton rubrum and Trichophyton tonsurans. Fractions F(1) and F(3) exhibited the highest antidermatophytic activities with repective MICs of 250 and 125 mug/ml while the fractions F(4) and F(5) did not prevent the growth of the tested fungi up to dose 2,500 mug/ml.

Potential of the Desert Locust Schistocerca gregaria (Orthoptera: Acrididae) as an Unconventional Source of Dietary and Therapeutic Sterols.

Insects are increasingly being recognized not only as a source of food to feed the ever growing world population but also as potential sources of new products and therapeutic agents, among which are sterols. In this study, we sought to profile sterols and their derivatives present in the desert locust, Schistocerca gregaria, focusing on those with potential importance as dietary and therapeutic components for humans. Using coupled gas chromatography-mass spectrometry (GC-MS), we analyzed and compared the quantities of sterols in the different sections of the gut and tissues of the locust. In the gut, we identified 34 sterols which showed a patchy distribution, but with the highest composition in the foregut (55%) followed by midgut (31%) and hindgut (14%). Fed ad libitum on wheat seedlings, five sterols unique to the insect were detected. These sterols were identified as 7-dehydrocholesterol, desmosterol, fucosterol, (3ß, 5α) cholesta-8, 14, 24-trien-3-ol, 4, 4-dimethyl, and (3ß, 20R) cholesta-5, 24-dien-3, 20-diol with the first three having known health benefits in humans. Incubation of the fore-, mid- and hindgut with cholesterol-[4-13C] yielded eight derivatives, three of these were detected in the gut of the desert locust after it had consumed the vegetative diet but were not detected in the diet. Our study shows that the desert locust ingests phytosterols from a vegetative diet and, amplifies and metabolizes them into derivatives with potential salutary benefits and we discuss our findings in this context.