Characterization of an endophytic whorl-forming Streptomyces from Catharanthus roseus stems producing polyene macrolide antibiotic.

An endophytic whorl-forming Streptomyces sp. designated as TS3RO having antifungal activity against a large number of fungal pathogens, including Sclerotinia sclerotiorum, Rhizoctonia solani, Colletotrichum gloeosporioides, Cryphonectria parasitica, Fusarium oxysporum, Pyrenophora tritici-repentis, Epidermophyton floccosum, and Trichophyton rubrum, was isolated from surface-sterilized Catharanthus roseus stems. Preliminary identification showed that Streptomyces cinnamoneus subsp. sparsus was its closest related species. However, strain TS3RO could readily be distinguished from this species using a combination of phenotypic properties, 16S rDNA sequence similarity, and phylogenetic analyses. Thus, the whorl-forming Streptomyces sp. strain TS3RO is likely a new subspecies within the Streptomyces cinnamoneus group. Direct bioautography on a thin-layer chromatography plate with Cladosporium cucumerinum was conducted throughout the purification steps for bioassay-guided isolation of the active antifungal compounds from the crude extract. Structural elucidation of the isolated bioactive compound was obtained via LC-MS spectrometry, UV-visible spectra, and nuclear magnetic resonance data. It revealed that fungichromin, a known methylpentaene macrolide antibiotic, was the main antifungal component of TS3RO strain, as shown by thin-layer chromatography bioautography. This is the first report of an endophytic whorl-forming Streptomyces isolated from the medically important plant Catharanthus roseus.

Application of design of experiments and design space methodology for the HPLC-UV separation optimization of aporphine alkaloids from leaves of Spirospermum penduliflorum Thouars.

Spirospermum penduliflorum Thouars (Menispermaceae) is an endemic species of Madagascar traditionally used as vasorelaxant. Recently, two aporphine alkaloids known to possess antihypertensive activity (dicentrine and neolitsine) were isolated and identified from the leaves of this plant. In the present study, a HPLC-UV method allowing the separation of all alkaloids and the quantification of dicentrine in the alkaloidic extract of leaves was developed using design of experiments and design space methodology. Three common chromatographic parameters (i.e. the mobile phase pH, the initial proportion of methanol and the gradient slope) were selected to construct a full factorial design of 36 experimental conditions. The times at the beginning, the apex (i.e. the retention time) and the end of each peak were recorded and modelled by multiple linear equations. The corresponding residuals were normally distributed which confirmed that the models can be used for the prediction of the retention times and to optimize the separation. The optimal separation was predicted at pH 3, with a gradient starting at 32% of methanol and a gradient slope of 0.42%/min. Good agreement was obtained between predicted and experimental chromatograms. The method was also validated using total error concept. Using the accuracy profile approach, validation results gave a LOD and LOQ for dicentrine of 3 µg/ml and 10 µg/ml, respectively. A relative standard deviation for intermediate precision lower than 10% was obtained. This method was found to provide accurate results in the concentration range of 10-75 µg/ml of dicentrine and is suitable for routine analysis.

Screening for anti-infective properties of several medicinal plants of the Mauritians flora.

Several plants of the Mauritian flora alleged to possess anti-infective properties were studied against different strains of pathogenic bacteria and fungi. The grounded dried plant materials were extracted with different extractants and screened for anti-microbial activity using the disk diffusion and the micro-dilution techniques. Preliminary screening revealed that the methanol extracts were most active. Salmonella enteritidis, Enterobacter cloacae and Bacillus subtilis were the three test organisms, which were found to be susceptible to all the crude methanolic extracts of the different plants investigated (100% susceptibility), followed by Escherichia coli (57.1%) and Pseudomonas aeruginosa (57.1%), and Staphylococcus aureus (28.6%). The lowest minimum inhibitory concentration recorded for the different crude methanol extracts against Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, Enterobacter cloacae, Bacillus subtilis and the mould fungus Candida albicans were 500, 1000, 125, 250, 1000 and 125 micro g/ml, respectively. Bioautography using Cladosporium cucumerinum revealed that dichloromethane (DCM) extracts had the highest activity against the phytopathogenic fungus. It was also noted that the DCM extracts of Michelia champaca and Antidesma madagascariense yielded the maximum number of growth inhibiting compounds against Cladosporium cucumerinum. Activity of the different crude extracts was also investigated against several phytopathogenic filamentous fungi, Colletotrichum glocosporoides, Rhizoctonia solani, Sclerotinia sclerotium, Guignardia sp. and Fusarium oxysporum. It was found that crude hexane extracts as well as crude DCM extracts exhibited marked activity against several strains of fungi, especially Colletotrichum glocosporoides, Sclerotinia sclerotium and Guignardia sp.

An LC/DAD-UV/MS method for the rapid detection of aristolochic acid in plant preparations.

A new method using direct on-line coupling between HPLC and UV-DAD/MS was developed for the rapid detection of aristolochic acid I in plant preparations. The use of both UV-DAD and MS detectors enables this method to be very selective. A ng range detection limit was determined for both UV and MS detections. A quantitative determination of aristolochic acid I in plant preparations was achieved using UV and MS detection. A thin layer chromatography assay was also developed for the preliminary detection of aristolochic acid I in complex mixtures.