Effect of Aloe vera whole leaf extract on short chain fatty acids production by Bacteroides fragilis, Bifidobacterium infantis and Eubacterium limosum.

AIMS: To investigate the effect of Aloe vera whole leaf extract on pure and mixed human gut bacterial cultures by assessing the bacterial growth and changes in the production of short chain fatty acids. METHODS AND RESULTS: Bacteroides fragilis, Bifidobacterium infantis, and Eubacterium limosum were incubated with Aloe vera extracts [0%, 0.5%, 1%, 1.5% and 2%; (w/v)] for 24 and 48 h. Short chain fatty acids production was measured by gas chromatography/mass spectrometry analyses. A significant linear increase in growth response to Aloe vera supplementation was observed at 24 h for each of the bacterial cultures; however, only B. infantis and a mixed bacterial culture showed a significant positive linear dose response in growth at 48 h. In pure bacteria cultures, a significantly enhanced dose response to Aloe vera supplementation was observed in the production of acetic acid by B. infantis at 24 h and of butyric acid by E. limosum at 24 and 48 h. In the mixed bacterial culture, the production of propionic acid was reduced significantly at 24 and 48 h in a dose-dependent fashion, whereas butyric acid production showed a significant linear increase. CONCLUSIONS: The results indicated that Aloe vera possessed bacteriogenic activity in vitro and altered the production of acetic, butyric and propionic acids by micro-organisms selected for the study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the study suggest that consumption of a dietary supplement, Aloe vera, may alter the production of short chain fatty acids by human intestinal microflora.

Identification of metabolites produced from N-phenylpiperazine by Mycobacterium spp.

Mycobacterium sp. 7E1B1W and seven other mycobacterial strains known to degrade hydrocarbons were investigated to determine their ability to metabolize the piperazine ring, a substructure found in many drugs. Cultures were grown at 30 degrees C in tryptic soy broth and dosed with 3.1 mM N-phenylpiperazine hydrochloride; samples were removed at intervals and extracted with ethyl acetate. Two metabolites were purified from each of the extracts by high-performance liquid chromatography; they were identified by mass spectrometry and (1)H nuclear magnetic resonance spectroscopy as N-(2-anilinoethyl)acetamide and N-acetyl-N'-phenylpiperazine. The results show that mycobacteria have the ability to acetylate piperazine rings and cleave carbon-nitrogen bonds.

Degradation of biphenyl by Mycobacterium sp. strain PYR-1.

The metabolism of biphenyl by Mycobacterium sp. PYR-1 was investigated. The Mycobacterium sp. degraded >98% of the biphenyl added within 72 h. Analysis of ethyl acetate extracts of the culture medium by HPLC indicated that benzoic acid was the major metabolite. Other products were 4-hydroxybiphenyl, 4-hydroxybenzoic acid, and 5-oxo-5-phenylpentanoic acid. The metabolites were characterized by mass and 1H NMR spectrometry. Identification of benzoic acid and 5-oxo-5-phenylpentanoic acid indicates that biphenyl degradation by Mycobacterium sp. PYR-1 is generally similar to known pathways. A novel alternative metabolic pathway consisted of monooxygenation at C-4 of biphenyl to give 4-hydroxybiphenyl, with subsequent degradation via ring cleavage to 4-hydroxybenzoic acid.

Metabolism of anthracene by a Rhodococcus species.

A Rhodococcus sp. isolated from contaminated river sediment was investigated to determine if the isolate could degrade high molecular mass polycyclic aromatic hydrocarbons. The Rhodococcus sp. was able to utilize anthracene (53%), phenanthrene (31%), pyrene (13%), and fluoranthene (5%) as sole source of carbon and energy, but not naphthalene or chrysene. In a study of the degradation of anthracene by a Rhodococcus sp., the identification of ring-fission products indicated at least two ring-cleavage pathways. One results in the production of 6,7-benzocoumarin, previously shown to be produced chemically from the product of meta cleavage of 1,2-dihydroxyanthracene, a pathway which has been well established in Gram-negative bacteria. The second is an ortho cleavage of 1,2-dihydroxyanthracene that produces 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, a dicarboxylic acid ring-fission product. This represents a novel metabolic pathway only identified in Gram-positive bacteria.

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.

Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. strain PYR-1.

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, (1)H and (13)C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2'-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.

Use of 13C NMR spectrometric data to produce a predictive model of estrogen receptor binding activity.

We have developed a spectroscopic data-activity relationship (SDAR) model based on 13C NMR spectral data for 30 estrogenic chemicals whose relative binding affinities (RBA) are available for the alpha (ERalpha) and beta (ERbeta) estrogen receptors. The SDAR models segregated the 30 compounds into strong and medium binding affinities. The SDAR model gave a leave-one-out (LOO) cross-validation of 90%. Two compounds that were classified incorrectly in the SDAR model were in the transition zone between classifications. Real and predicted 13C NMR chemical shifts were used with test compounds to evaluate the predictive behavior of the SDAR model. The 13C NMR SDAR model using predicted 13C NMR data for the test compounds provides a rapid, reliable, and simple way to screen whether a compound binds to the estrogen receptors.

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.

Producing 13C NMR, infrared absorption, and electron ionization mass spectrometric data models of the monodechlorination of chlorobenzenes, chlorophenols, and chloroanilines.

We have developed four spectroscopic data-activity relationship (SDAR) models of monodechlorination of 32 chlorinated benzene compounds in anaerobic estuarine sediment. The SDAR models were based on combinations of 13C nuclear magnetic resonance (NMR), infrared absorption (IR), and electron ionization mass spectrometric (EI MS) data. The SDAR models segregated the 32 compounds into 17 readily monodechlorinated compounds and 15 not readily monodechlorinated compounds. The SDAR model based on 13C NMR, IR, and EI MS data gave a leave-one-out cross-validation of 93.8%. The SDAR model based on a composite of 13C NMR and IR data gave a leave-one-out cross-validation of 90.6%. The SDAR model based on a composite of IR and EI MS data gave a leave-one-out cross-validation of 84.4%. The SDAR model based on a composite of 13C NMR and EI MS data gave a leave-one-out cross-validation of 84.4%. These reliable SDAR models provide a rapid and simple way to predict whether a chlorinated benzene compound will readily go through monodechlorination. The FDA has filed a patent application on methods of using any combination of spectral data (NMR, MS, UV-vis, IR, and fluorescence, phosphorescence) to model a chemical, physical, or biological endpoint.

(13)C NMR and electron ionization mass spectrometric data-activity relationship model of estrogen receptor binding.

Two Spectroscopic Data-Activity Relationship (SDAR) models based on (13)C nuclear magnetic resonance (NMR) and electron ionization mass spectra (EI MS) data were developed for 108 compounds whose relative binding affinities (RBA) to the estrogen receptor are known. The (13)C NMR and EI MS data were used as spectrometric digital fingerprints to reflect the electronic and structural characteristics of the compounds. Both SDAR models segregated the 108 compounds into 20 strong, 15 medium, and 73 weak relative binding classifications. The first SDAR model, based on (13)C NMR data alone, gave a leave-one-out (LOO) cross-validation of 75.0%. The second SDAR model, based on a composite of (13)C NMR and EI MS data, gave a LOO cross-validation of 82.4%. Many of the misidentifications from the cross-validations were between medium and weak classifications, where there were fewer specific spectrometric characteristics to identify the relationship of spectra to estrogen receptor binding. Real and predicted (13)C NMR chemical shifts were used to test the predictive behavior of both SDAR models. The ease of use and speed of SDAR modeling may facilitate their use with other toxicological endpoints.

Evaluation of major active components in St. John's Wort dietary supplements by high-performance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation.

A RP-HPLC method with photodiode array detection and LC-electrospray ionization (ESI) MS confirmation was established for the determination of major active components in St. John's Wort dietary supplement capsules. The samples alternatively were extracted with ethanol-acetone (2:3) using a 55 degrees C water-bath shaker or an ambient temperature ultrasonic bath. Extracts were separated by RP-C18 chromatography using a 95-min water-methanol-acetonitrile-trifluoroacetic acid gradient. The major components were identified by photodiode array detection and then confirmed by LC-ESI-MS. The quantification of components was performed using an internal standard (luteolin). This method may serve as a valuable tool for the quality evaluation of St. John's Wort dietary supplement products.

Potential clinical use of butyric acid derivatives to induce antigen-specific T cell inactivation.

Compounds with the capacity to induce antigen-specific unresponsiveness in CD4(+) T cells can in some clinical situations be more beneficial than general immune suppressants. Newly synthesized ester, ester/amide, and amide derivatives of butyrate with the capacity to induce antigen-specific T cell unresponsiveness in vivo and in vitro were tested here. The ester and ester/amide derivatives of butyrate were shown to block proliferation by interleukin-2-stimulated murine Th1 cells in vitro. A 3-day treatment with these same two derivatives also suppressed a primary antibody response to a thymus-dependent antigen in mice. In addition, even a single injection of the ester derivative of n-butyrate 2-(4-morpholinyl)ethyl butyrate hydrochloride (MEB) on day 2 or 3 after immunization suppressed the generation of memory T cells capable of proliferating to antigen or of promoting a secondary antigen-specific antibody response. MEB also induced antigen-specific unresponsiveness in antigen-activated, but not resting or interleukin-2-activated, T cells in vitro. DNA analysis showed that regardless of when MEB was added to the cultures, it induced the eventual G(1) sequestration of essentially all activated Th1 cells. Because G(1) blockade is associated with Th1 cell anergy, this finding suggests that MEB has the potential to induce anergy in already-activated CD4(+) T cells. Taken together, the results presented here establish MEB as a novel means of inducing anergy in CD4(+) T cells both in vitro and in vivo and underscore the likelihood that MEB and/or other butyrate derivatives can be used as immunotherapeutic reagents.

Linoleic acid diols are novel substrates for human UDP-glucuronosyltransferases.

Linoleic acid diol glucuronides have been isolated previously from urine of patients suffering from generalized peroxisomal disorders. Glucuronidation of linoleic acid and linoleic acid diols by human liver microsomes was studied to investigate the role of glucuronide conjugation in the metabolism of linoleic acid diols. Glucuronide products were isolated and analyzed by TLC and HPLC-MS. HPLC-MS showed ions with (m/z) corresponding to singly glucuronidated linoleic acid diols while TLC revealed that the glucuronidation was at a hydroxyl position. Kinetic analysis gave apparent K(m) values in the range of 50-200 microM and V(max) rates from 5 to 12 nmol/mg x min. These rates are substantially higher than activities seen for most endogenous hydroxylated substrates. Assays using each of the four individually purified linoleic acid diol enantiomers suggest that glucuronidation occurs at only one of the two hydroxyl groups of each enantiomer. These results show for the first time that hydroxylated fatty acids are actively glucuronidated by human liver microsomes and suggest that glucuronidation may play a significant role in the biotransformation of linoleic acid diols in humans.

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%).

Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin.

Fecal bacteria from a healthy individual were screened for the specific bacteria involved in the metabolism of dietary isoflavonoids. Two strains of bacteria capable of producing primary and secondary metabolites from the natural isoflavone glycosides daidzin and genistin were detected. The metabolites were identified by comparison of their HPLC/mass, 1H NMR and UV spectra with those of standard and synthetic compounds. Both Escherichia coli HGH21 and the gram-positive strain HGH6 converted daidzin and genistin to the their respective aglycones daidzein and genistein. Under anoxic conditions, strain HGH6 further metabolized the isoflavones daidzein and genistein to dihydrodaidzein and dihydrogenistein, respectively. The reduction of a double bond between C-2 and C-3 to a single bond was isoflavonoid-specific by strain HGH6, which did not reduce a similar bond in the flavonoids apigenin and chrysin. Strain HGH6 did not further metabolize dihydrodaidzein and dihydrogenistein. This is the first study in which specific colonic bacteria that are involved in the metabolism of daidzin and genistin have been detected.

Biotransformation of vinclozolin by the fungus Cunninghamella elegans.

This study investigated the biotransformation of the dicarboximide fungicide vinclozolin [3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione] by the fungus Cunninghamella elegans. Experiments with phenyl-[U-ring-14C]vinclozolin showed that after 96 h incubation, 93% had been transformed to four major metabolites. Metabolites were separated by HPLC and characterized by mass and NMR spectroscopy. Biotransformation occurred predominantly on the oxazolidine-2,4-dione portion of vinclozolin. The metabolites were identified as the 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide, N-(2-hydroxy-2-methyl-1-oxobuten-3-yl)-3,5-dichlorophenyl-1-carbamic acid, and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide. The enanilide compound has been reported previously as a plant and mammalian metabolite and is implicated to contain antiandrogenic activity. The 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide are novel metabolites.

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%).

Syntheses of benzoquinolizidine and benzoindolizidine derivatives as anti-amnesic agents.

Tacrine, one of the drugs available for Alzheimer's disease based on the cholinergic approach, suffers from considerable toxicity. Many analogues of tacrine have been prepared which retain the pharmacologically rich aminopyridine or aminoquinoline motifs. The current research was undertaken to produce an acetylcholinesterase inhibitor by employing 11-aminobenzoquinolizidines (4) and 10-aminobenzoindolizidines (5) as templates. Thus, we aimed to achieve three goals relative to tacrine: eliminate the pyridine and quinoline moieties and render the molecule less flat. Overall, the compounds we prepared were poorer inhibitors of acetylcholinesterase compared to tacrine. The single exception was compound 6f which exhibited an effect comparable to that of tacrine, but only at a dose of the order of 10(-3) M. However, despite the poor acetylcholinesterase inhibition by 6b, this compound proved to be an effective anti-amnesic agent at 45 mg/kg dose.

Syntheses of 1,2-diamino and 1,2-aminoalcohol derivatives in the piperidine and pyrrolidine series as anti-amnesic agents.

Tacrine, one of the drugs available for Alzheimer's disease based on the cholinergic approach, suffers from considerable toxicity. Many analogues of tacrine has been prepared which retain the pharmacologically rich aminopyridine or aminoquinoline motifs. The current research is a continuation of our efforts in the area of 11-aminobenzoquinolizidines (4) and 10-aminobenzoindolizidines (5) (cf. ref9). A serendipitous discovery led us to the biologically active open chain analogue 9, and we proceeded to elaborate on this molecule. Overall, the compounds we prepared were poor inhibitors of acetylcholinesterase as compared to tacrine. The single exception was compound 20 which exhibited an effect comparable to that of tacrine, but only at a dose in the order of 10(-3) M. However, despite the poor acetylcholinesterase inhibition by 9, this compound was found to be an effective antiamnesic agent.