Cytotoxicity of lapachol metabolites produced by probiotics.

UNLABELLED: Probiotics are currently added to a variety of functional foods to provide health benefits to the host and are commonly used by patients with gastrointestinal complaints or diseases. The therapeutic effects of lapachol continue to inspire studies to obtain derivatives with improved bioactivity and lower unwanted effects. Therefore, the general goal of this study was to show that probiotics are able to convert lapachol and are important to assess the effects of bacterial metabolism on drug performance and toxicity. The microbial transformations of lapachol were carried out by Bifidobacterium sp. and Lactobacillus acidophilus and different metabolites were produced in mixed and isolated cultures. The cytotoxic activities against breast cancer and normal fibroblast cell lines of the isolated metabolites (4α-hydroxy-2,2-dimethyl-5-oxo-2,3,4,4α,5,9ß-hexahydroindeno[1,2-ß]pyran-9ß-carboxilic acid, a new metabolite produced by mixed culture and dehydro-α-lapachone produced by isolated cultures) were assessed and compared with those of lapachol. The new metabolite displayed a lower activity against a breast cancer cell line (IC50 = 532.7 µmol l(-1) ) than lapachol (IC50 = 72.3 µmol l(-1) ), while dehydro-α-lapachone (IC50 = 10.4 µmol l(-1) ) displayed a higher activity than lapachol. The present study is the first to demonstrate that probiotics are capable of converting lapachol into the most effective cytotoxic compound against a breast cancer cell line. SIGNIFICANCE AND IMPACT OF THE STUDY: Probiotics have been used in dairy products to promote human health and have the ability to metabolize drugs and other xenobiotics. Naphthoquinones, such as lapachol, are considered privileged scaffolds due to their high propensity to interact with biological targets. The present study is the first to demonstrate that probiotics are capable of converting lapachol into the most effective cytotoxic compound against a breast cancer cell line. The developed approach highlights the importance of probiotics to assess the effects of bacterial metabolism on drug performance and toxicity.

Transformation of artemisinin by Cunninghamella elegans.

Semi-synthetic derivatives of the anti-malarial drug artemisinin hold great promise in the search for an effective and economical treatment of chloroquine-resistant forms of malaria. Unfortunately, synthetic functionalization of the artemisinin skeleton is often tedious and/or impractical. We seek to utilize 7beta-hydroxyartemisinin, obtained from microbial transformation, as a semi-synthetic precursor for the synthesis of novel 7beta-substituted artemisinin anti-malarial agents. Here we employ liquid cultures of Cunninghamella elegans as a means for the rational and economical bioconversion of artemisinin to 7beta-hydroxyartemisinin in 78.6% yield. In addition, there were three other bioconversion products: 7beta-hydroxy-9alpha-artemisinin (6.0%), 4alpha-hydroxy-1-deoxoartemisinin (5.4%), and 6beta-hydroxyartemisinin (6.5%).

The fungus Pestalotiopsis guepini as a model for biotransformation of ciprofloxacin and norfloxacin.

The metabolism of the fluoroquinolone drugs ciprofloxacin and norfloxacin by Pestalotiopsis guepini strain P-8 was investigated. Cultures were grown at 28 degrees C in sucrose/peptone broth for 18 days after dosing with ciprofloxacin (300 microM) or norfloxacin (313 microM). Four major metabolites were produced from each drug; and these were purified by high-performance liquid chromatography and identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Ciprofloxacin metabolites included N-acetylciprofloxacin (52.0%), desethylene-N-acetylciprofloxacin (9.2%), N-formylciprofloxacin (4.2%), and 7-amino-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.3%). Norfloxacin metabolites included N-acetylnorfloxacin (55.4%), desethylene-N-acetylnorfloxacin (8.8%), N-formylnorfloxacin (3.6%), and 7-amino-1-ethyl-6-fluoro4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2.1%). N-Formylciprofloxacin and the four transformation products from norfloxacin are all known to be mammalian metabolites.

Metabolism of the veterinary fluoroquinolone sarafloxacin by the fungus Mucor ramannianus.

To investigate the microbial biotransformation of veterinary fluoroquinolones, Mucor ramannianus was grown in sucrose/peptone broth with sarafloxacin for 18 days. Cultures were extracted with ethyl acetate and extracts were analyzed by liquid chromatography. The two metabolites (26% and 15% of the A280, respectively) were identified by mass and 1H nuclear magnetic resonance spectra as N-acetylsarafloxacin and desethylene-N-acetylsarafloxacin. The biological formation of desethylene-N-acetylsarafloxacin has not been previously observed.

Oxidation of phenothiazine and phenoxazine by Cunninghamella elegans.

1. To determine the ability of fungi to metabolize sulphur- and oxygen-containing azaarenes, Cunninghamella elegans ATCC 9245 was grown in 125-ml flasks containing fluid Sabouraud medium. The cultures and controls were incubated at 28 degrees C with shaking and dosed with 16.7 mM phenothiazine or phenoxazine. After incubation for 72h, the mycelia and filtrates were extracted with ethyl acetate and the combined residues analysed by high-performance liquid chromatography. Residual phenothiazine and phenoxazine were 21 and 22%, respectively, of the total UV absorbance at 254 nm. 2. The metabolites were identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. The fungus oxidized phenothiazine to phenothiazine sulphoxide, 3-hydroxyphenothiazine sulphoxide, phenothiazin-3-one, and 3-hydroxyphenothiazine and oxidized phenoxazine to phenoxazin-3-one. 3. Three of the four compounds produced by C. elegans from phenothiazine were identical to those produced by mammals, supporting the use of the fungus as a microbial model for drug metabolism.

Microbiological transformation of enrofloxacin by the fungus Mucor ramannianus.

Enrofloxacin metabolism by Mucor ramannianus was investigated as a model for the biotransformation of veterinary fluoroquinolones. Cultures grown in sucrose-peptone broth were dosed with enrofloxacin. After 21 days, 22% of the enrofloxacin remained. Three metabolites were identified: enrofloxacin N-oxide (62% of the total absorbance), N-acetylciprofloxacin (8.0%), and desethylene-enrofloxacin (3.5%).

Biotransformation of N-acetylphenothiazine by fungi.

Cultures of the fungi Aspergillus niger, Cunninghamella verticillata, and Penicillium simplicissimum, grown in a sucrose/peptone medium, transformed N-acetylphenothiazine to N-acetylphenothiazine sulfoxide (from 13% to 28% of the total) and phenothiazine sulfoxide (from 5% to 27%). Phenothiazin-3-one (4%) and phenothiazine N-glucoside (4%) were also produced by C. verticillata. The probable intermediate, phenothiazine, was detected only in cultures of P. simplicissimum (6%).

Regioselective transformation of ciprofloxacin to N-acetylciprofloxacin by the fungus Mucor ramannianus.

A strain of the saprobic fungus Mucor ramannianus, isolated from a forest, was used to demonstrate the potential for ciprofloxacin biotransformation by zygomycetes in the environment. The fungus carried out the regioselective N-acetylation of ciprofloxacin to a single product, which was purified from culture extracts by high-performance liquid chromatography. The metabolite was identified by mass and nuclear magnetic resonance spectrometry as 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(4-acetyl-1-piperazinyl)-3- quinolinecarboxylic acid.