Utilization of Vibrio cholerae as a Model Organism to Screen Natural Product Libraries for Identification of New Antibiotics.

The development of antibiotic-resistant bacteria requires increasing research efforts in drug discovery. Vibrio cholerae can be utilized as a model gram-negative enteric pathogen in high- and medium-throughput screening campaigns to identify antimicrobials with different modes of action. In this chapter, we describe methods for the optimal growth of V. cholerae in 384-well plates, preparation of suitable microtiter natural product sample libraries, as well as their screening using measurements of bacterial density and activity of type II secretion-dependent protease as readouts. Concomitant LC-MS/MS profiling and spectral data networking of assay sample libraries facilitate dereplication of putative known and/or nuisance compounds and efficient prioritization of samples containing putative new natural products for further investigation.

Hydroxyalkenylresorcinols from Stylogyne turbacensis.

Two new compounds, 5-(11'(S)-hydroxy-8'-heptadecenyl)resorcinol (3) and 5-(12'(S)-hydroxy-8',14'-heptadecadienyl)resorcinol (4), were isolated from the leaves of Stylogyne turbacensis together with the known analogue metabolites 1 and 2. Compounds 3 and 4 showed the strongest activity in the leishmania assay, 7 and 3 microM, respectively, while compounds 1, 2, and 4 showed moderate activity against a drug-resistant strain of Trypanosoma cruzi with IC(50) values of 30, 25, and 22 microM, respectively. Additional testing in MCF-7 and NCI-H460 was performed for compounds 3 and 4. The structures of compounds 1-4 were elucidated using NMR, MS, and other spectroscopic data. The absolute stereochemistry of compounds 3 and 4 was also investigated using the Mosher ester approach. Peracetylated derivatives of these four metabolites were produced and their activities determined in the Trypanosoma cruzi assay.

Weakly antimalarial flavonol arabinofuranosides from Calycolpus warszewiczianus.

Three new flavonol arabinosides (2-4) were isolated from the young leaves of Calycolpus warszewiczianus. The structures were determined as myricetin-3-O-alpha-L-3' '-acetylarabinofuranoside (2), myricetin-3-O-alpha-L-3' ',5' '-diacetylarabinofuranoside (3), and 5-galloylquercetin-3-O-alpha-L-arabinofuranoside (4). Molecular structures were elucidated using NMR spectroscopy in combination with IR and MS data. Two known compounds, myricetin-3-O-alpha-L-arabinofuranoside (1) and (-)-epi-catechin (5), were also isolated. The compounds were tested in vitro against a chloroquine-resistant strain of Plasmodium falciparum, Leishmania mexicana, and Trypanosoma cruzi parasites. Compound 4 demonstrated weak activity against a chloroquine-resistant strain of P. falciparum (14.5 microM), whereas none of the compounds demonstrated activity against L. mexicana and T. cruzi at the concentrations of 40 and 50 microg/mL, respectively, and no cytotoxicity was detected against mammalian cells below 100 microg/mL.

Novel cassane and cleistanthane diterpenes from Myrospermum frutescens: absolute stereochemistry of the cassane diterpene series.

Four new diterpenes (1-4) were isolated from the leaves of Myrospermum frutescens as minor constituents. Chagresnol (1), 6beta,18-diacetoxycassan-13,15-diene (2), and chagreslactone (3) possess cassane skeletons, while chagresnone (4) exhibits a cleistanthane skeleton. Molecular structures and their relative stereochemistries were elucidated using NMR spectroscopy in combination with UV, IR, and MS spectral data. Although compound 2 was previously reported as a synthetic product, we report its first isolation as a natural product. Derivative products (10-13) were obtained to test their activities against Chagas's disease. In addition, the absolute stereochemistry of the previously isolated cassane diterpene 5 from M. frutescens is presented.

Evaluation of the efficiency of three different solvent systems to extract triterpene saponins from roots of Panax quinquefolius using high-performance liquid chromatography.

Despite the wide availability of liquid herbal extracts using mixtures of alcohol, glycerin, and water, or glycerin and water as solvents, no data on the chemical composition of such extracts is readily available. In this study, the amount and the stability of the major saponins in Panax quinquefolius root extracts, made either with 50% (v/v) aqueous ethanol, a mixture (v/v/v) of 20% ethanol, 40% glycerin, and 40% water, or with 65% (v/v) aqueous glycerin, were evaluated by HPLC-UV analysis. The amount of total saponins was highest in the 50% aqueous ethanol extract (61.7 +/- 0.1 mg/g dry root), although similar to the ethanol-glycerin-water extract (59.4 +/- 0.5 mg/g dry root). Saponins were significantly lower in the 65% aqueous glycerin extract (51.5 +/- 0.2 mg/g dry root). Interestingly, the amounts of individual saponins were quite variable depending on the solvent. This is in part due to enzymatic cleavage of ginsenosides in the glycerin containing extracts during the maceration process. Storage of the extracts at 25 degrees C over the period of a year led to a 13-15% loss of saponins with all three types of extractions.