Azacoccones F-H, new flavipin-derived alkaloids from an endophytic fungus Epicoccum nigrum MK214079.

Three new flavipin-derived alkaloids, azacoccones F-H (1-3), along with six known compounds (4-9) were isolated from the endophytic fungus Epicoccum nigrum MK214079 associated with leaves of Salix sp. The structures of the new compounds were established by analysis of their 1D/2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS) data. The absolute configuration of azacoccones F-H (1-3) was determined by comparison of experimental electronic circular dichroism (ECD) data with reported ones and biogenetic considerations. Epicocconigrone A (4), epipyrone A (5), and epicoccolide B (6) exhibited moderate antibacterial activity against Staphylococcus aureus ATCC 29213 with minimal inhibitory concentration (MIC) values ranging from 25 to 50 µM. Furthermore, epipyrone A (5) and epicoccamide A (7) displayed mild antifungal activity against Ustilago maydis AB33 with MIC values of 1.6 and 1.8 mM, respectively. Epicorazine A (8) showed pronounced cytotoxicity against the L5178Y mouse lymphoma cell line with an IC50 value of 1.3 µM.

Anticancer potential of NF-κB targeting apoptotic molecule "flavipin" isolated from endophytic Chaetomium globosum.

BACKGROUND: Anticancer compounds from natural sources have drawn attention due to their structural diversity and relatively lesser side effects. Endophytic fungi are one such natural resource from, which plethoras of anticancerous compounds have been isolated. PURPOSE: The objective of the study was to isolate and characterize the bioactive metabolite from Chaetomium globosum that exhibits astonishing antiproliferative activity against cancerous cell lines. METHODS: Flavipin was isolated by bioassay-guided fractionation and identified using FT-IR, EI-MS and NMR studies. MTT assay was used to determine the cytotoxicity. Fluorescent staining (AO/EB) and DNA fragmentation studies confirmed the occurrence of apoptosis. Real time PCR and Western blotting were used to analyze the expression of apoptosis related genes and its proteins, respectively. RESULTS: Flavipin inhibited proliferation of A549, HT-29 and MCF-7 cancer cells in dose dependent manner with an IC50 concentration of 9.89 µg/ml, 18 µg/ml and 54 µg/ml, respectively, whereas it was comparatively less sensitive (IC50 = 78.89 µg/ml) against normal cell line (CCD-18Co). At IC50 concentration cancerous cells exhibited cell shrinkage and fragmentation of DNA, which indicated that flavipin induced apoptotic cell death. In treated cells there is an up-regulation of p53 gene and its associated protein, whereas reciprocal expression was observed in BCL-2 gene and its protein. Furthermore, western blotting results also showed down-regulation of NFκB. CONCLUSION: This is the first report on the antiproliferative activity of flavipin isolated from endophytic C. globosum and also proposed that interaction of flavipin with NFкB could be a possible mechanism for this activity. Flavipin induced apoptosis at low concentrations in cancer cell lines (A549, HT-29) and exhibited itself as a potential anticancer agent.

Photodecomposition of o-phthaldialdehyde-derivatized amino acids by the photodiode array detector during their high-performance liquid chromatographic analysis.

During the high-performance liquid chromatographic (HPLC) analysis of o-phthaldialdehyde (OPA)-derivatized amino acids such as arginine and homoarginine, we observed that the response of the fluorescence detection (FLD) system is decreased when the photodiode array detection (DAD) system (located before the FLD system) was simultaneously switched on. The decrease in the FLD response, i.e., the difference in the FLD peak area (DeltaA) obtained with DAD modes off (A(off)) and on (A(on)) was dependent upon the flow rate, but the relative FLD response decrease (DeltaA/A(off)) was practically independent of the amount of analyte injected. For example, decreasing the flow rate from 1 to 0.5 mL/min resulted in the relative decrease of FLD response from approximately 5% to 11%. It was concluded that DAD caused a photoinduced partial decomposition of the OPA-derivatized amino acids flowing through the cell. The photoinduced dissociation of OPA derivatives was independently supported by using off-line photodiode array spectrometric measurements with long and short irradiation pulses. Based on the experimental results, for description of the variation of FLD responses with the flow rate upon the irradiation by DAD a simple mathematical model is proposed and reported.

Advances in the o-phthalaldehyde derivatizations. Comeback to the o-phthalaldehyde-ethanethiol reagent.

The main aims of this work were (a) to present the characteristics and stability of the o-phthalaldehyde (OPA)-ethanethiol (ET) derivatives of 22 amino acids, including the believed-to-be less stable OPA derivatives providing glycine, gamma-aminobutyric acid, beta-alanine, histidine, ornithine, lysine and the C(1)-C(5) aliphatic amines; (b) to compare the stability properties of the most common amino acids and amines as OPA-ET-fluorenylmethyl chloroformate (FMOC) derivatives to the corresponding ones obtained from OPA reagents containing various (SH)-additives; (c) to show the molar responses of alanine and lysine depending on the OPA reagent's composition; as well as (d) to prove the practical utility of these basic researches, by the simultaneous HPLC separation of 22 amino acids and 15 amines as their OPA-ET-FMOC derivatives. Investigations have been carried out by varying the composition of the reagents, the molar ratios of reactants and the reaction time, applying diode array and fluorescence detections simultaneously. Average reproducibility of quantitations, characterized with the relative standard deviations (RSDs) based on the fluorescence intensities of derivatives, in the order of listing, proved to be 1.2-5.9% for amino acids and 1.1-8.7% for amines. The practical utility of the method is demonstrated by the analysis of the amino acid and amine contents of mouse tissues, with an average reproducibility of 3.5%.

A high-performance liquid chromatography method for the detection of diaminopimelic acid in urine.

We describe a quantitative assay for diaminopimelic acid (DAP) in urine. It involves (i) hydrolysis of urine samples, (ii) purification by several different liquid chromatography steps, and (iii) analysis by high-performance liquid chromatography on a reversed-phase C18 column. Tritiated-DAP, the internal standard, allows one to precisely follow the DAP-containing fractions and to determine the yield during purification. Sensitive and relatively accurate quantification of DAP, with a threshold of 50 fmol, is based on ion-pairing properties of eluants and ortho-phthaldialdehyde derivatization. The presence of DAP in relevant fractions was confirmed by combined gas chromatography and mass spectrometry. The DAP concentration in adult human urine pooled over 24 h ranges from 0.69 to 2.01 microM, a result in fair agreement with previously published values obtained by ninhydrin derivatization or gas chromatography.

Separation strategies for o-phthalaldehyde-mercaptoethanol derivatives of amino acids for reversed-phase high-performance liquid chromatography.

Twenty-three o-phthalaldehyde-mercaptoethanol derivatives of primary amino acids in serum were separated with Waters C18 5-microns Radial-Pak Resolve columns, using aqueous phosphate-methanol mobile phase with acetate and tetrahydrofuran as modifiers. Resolution is critical in this system for glutamine/histidine, citrulline/glycine/threonine/3-methylhistidine, and tryptophan/methionine derivatives, and is affected by small changes in column properties or mobile phase. However, pre-run adjustments in gradient scheme and/or mobile phase composition can usually be used to obtain chromatograms in which all derivatives can be quantitated. For this purpose, and for general method development, we have written two very fast, interactive programs for the Zenith Z-100 Microsoft BASIC compiler: RTGRAPH, for retention, separation, and resolution plots; and LCSIM for simulations of multisegment binary gradient elution. These programs are shown here to be useful and accurate in most aspects required for developing strategies for this method.