In Vitro Metabolism of Six C-Glycosidic Flavonoids from Passiflora incarnata L.

Several medical plants, such as Passiflora incarnata L., contain C-glycosylated flavonoids, which may contribute to their efficacy. Information regarding the bioavailability and metabolism of these compounds is essential, but not sufficiently available. Therefore, the metabolism of the C-glycosylated flavones orientin, isoorientin, schaftoside, isoschaftoside, vitexin, and isovitexin was investigated using the Caco-2 cell line as an in vitro intestinal and epithelial metabolism model. Isovitexin, orientin, and isoorientin showed broad ranges of phase I and II metabolites containing hydroxylated, methoxylated, and sulfated compounds, whereas schaftoside, isoschaftoside, and vitexin underwent poor metabolism. All metabolites were identified via UHPLC-MS or UHPLC-MS/MS using compound libraries containing all conceivable metabolites. Some structures were confirmed via UHPLC-MS experiments with reference compounds after a cleavage reaction using glucuronidase and sulfatase. Of particular interest is the observed cleavage of the C-C bonds between sugar and aglycone residues in isovitexin, orientin, and isoorientin, resulting in unexpected glucuronidated or sulfated luteolin and apigenin derivatives. These findings indicate that C-glycosidic flavones can be highly metabolized in the intestine. In particular, flavonoids with ortho-dihydroxy groups showed sulfated metabolites. The identified glucuronidated or sulfated aglycones demonstrate that enzymes expressed by Caco-2 cells are able to potentially cleave C-C bonds in vitro.

New antifungal ent-labdane diterpenes against Candida glabrata produced by microbial transformation of ent-polyalthic acid.

Candida glabrata, the most common non-albicans Candida species and one of the primary causes of candidemia, exhibits decreased susceptibility to azoles and more recently to echinocandins. Polyalthic acid 1, a furan diterpene, has been shown promising biological potential and in this study ent-polyalthic acid derivatives with antifungal activity against Candida glabrata were produced by microbial transformation. Incubation of 1 with Aspergillus brasiliensis afforded two known (compounds 5 and 10) and eight new derivatives (compounds 2-4, 6-9 and 11). The most common reaction was hydroxylation, but isomerization of the double bond and acetylation were also detected. None of the tested compounds showed cytotoxicity against HeLa, MCF-7 and MCF-10A cell lines showing IC50 values ranging from 62.6 µM to > 500 µM. Compounds 1, 5, 6, 8 and 11 showed fungistatic effects (ranging from 34.1 µM to 39.5 µM) on C. glabrata at lower concentrations than fluconazole (163.2 µM). Compounds 1, 6 and 8 were more potent fungicides (ranging from 79.0 to 143.6 µM) than fluconazole, which showed fungicidal effect at concentrations higher than 163.2 µM. These results suggest that ent-polyalthic acid and some of its derivatives could be used as lead compounds to develop new antifungal agents.

Identification of phase-II metabolites from human serum samples after oral intake of a willow bark extract.

BACKGROUND: Willow bark (Salicis cortex) is a herbal medicinal drug used to treat fever and pain, such as headaches and lower back pain. Until now, it has not been fully understood which compounds are responsible for the efficacy of the drug. PURPOSE: Although salicylic acid is known as a metabolite of salicylic alcohol derivatives of willow bark in vivo, it has been shown in previous studies that its concentration is too low to account for the overall efficacy of Salicis cortex. The aim this study was to broaden the knowledge regarding phenolic phase-II metabolites after oral intake of a willow bark extract. STUDY DESIGN/METHODS: Serum samples of a human pharmacokinetic study (Salicis cortex extract intake corresponding to 240 mg of total salicin, 10 volunteers, 12 h fasting time, controlled diet low in phenolics, and 12 blood withdrawals over a period of 24 h) were analyzed by LC-ESI-MS. A library of 142 possible metabolites associated with salicylic alcohol derivatives, flavonoids, and proanthocyanidins was used to characterize possible metabolization products. Their structures were confirmed by LC-ESI-MS experiments with reference compounds after a cleavage reaction using glucuronidase and sulfatase as well as by LC-MS/MS experiments. RESULTS: In the serum samples, phase-II metabolites of naringenin (2x glucuronides, 2x sulfates, 2x mixed glucuronide-sulfates), eriodictyol (3x glucuronides, 1x sulfate), taxifolin (1x sulfate), catechin (1x sulfate, 1x mixed glucuronide sulfate), ferulic acid (1x sulfate), hydroxyphenyl-propionic acid (1x sulfate), dihydroxyphenyl-valerolactone (1x sulfate), saligenin (1x glucuronide, 1x sulfate), salicylic acid (1x sulfate, 1x unconjugated, 1x salicyluric acid), and catechol (1x glucuronide, 1x sulfate) were characterized. Because taxifolin, dihydroxyphenyl-valerolactone, ferulic acid, and hydroxyphenyl-propionic acid could not be detected in the willow bark preparation, they could be metabolization products of genuine flavanones and flavan-3-ols as well as coumaric acid or C-ring cleavage products of flavonoids, which were present in the extract. No phase-II metabolites of procyanidins and no genuine flavonoid glycosides were detected in all serum samples. CONCLUSION: This is the first study to identify human metabolites of flavonoids, proanthocyanidins and salicylic alcohol derivatives of Salicis cortex beside salicylic acid or catechol. For the most characterized metabolites, anti-inflammatory activity has been described in the literature, and the present results are an important step in understanding the anti-inflammatory efficacy of willow bark in vivo.

Isomer-Selective Detection of Aromatic Molecules in Temperature-Programmed Desorption for Model Catalysis.

Based on three different molecules dosed on a Pt(111) single crystal the selectivity and sensitivity of REMPI-TPD in UHV is investigated for a potential application in heterogeneous catalysis. It is shown that the two structural isomers ethylbenzene and p-xylene can be discriminated by REMPI in a standard TPD experiment. The latter is not possible for the ionization with electrons in a Q-MS. It is further demonstrated by benzene TPD studies that the sensitivity of the REMPI-TOF-MS is comparable to commercial EI-Q-MS solutions and enables the detection of less than 0.6% molecules of a monolayer.

Ethanol photocatalysis on rutile TiO2(110): the role of defects and water.

In this work we present a stoichiometric reaction mechanism for the photocatalytic ethanol oxidation on TiO2(110). The reaction products are analyzed either under reaction conditions or after irradiation at lower temperatures. Water is identified as a quantitative by-product, which resides in a defect site. These water molecules cause a blocking of the defect sites which results in poisoning of the catalyst. By different preparation techniques of the TiO2(110) surface, the role of surface defects is further elucidated and the role of molecular oxygen is investigated. Based on the investigation, a complete photochemical reaction mechanism is given, which provides insights into general photon driven oxidation mechanisms on TiO2.

Development of a spray congealing process for the preparation of insulin-loaded lipid microparticles and characterization thereof.

A spray congealing process for the preparation of protein-loaded microparticles was developed. The influence of the process parameters atomization pressure and spraying temperature on particle size and process yield was investigated by experimental design. The employed spray congealing technique enabled the production of microparticles with yields ranging from 79% to 95% and median particle sizes (d(0.5)) from 182.2 to 315 microm. Insulin lipid microparticles could be prepared without any loss of insulin during the preparation process and the protein stability was not affected by the spray congealing process as investigated by HPLC-MS analysis. The stability of insulin encapsulated in lipid microparticles under release conditions over 28 days was assessed by investigating the residual insulin content. Starting after 3 days of release, a continuous increase of desamidoinsulin in the remaining particles of up to 7.5% after 28 days was observed. An additional degradation product was detected by HPLC and HPLC-MS analysis and identified as a covalent insulin dimer by MALDI-ToF. The microparticles did not show a burst release and testing the insulin lipid microparticles in a fibrin gel chondrocyte culture revealed that the released insulin was bioactive and had a significant effect on chondrocyte extracellular matrix production.

Micronization of insulin by high pressure homogenization.

PURPOSE: The aim of this study was to establish the high pressure homogenization of proteins in non-aqueous suspension as an alternative method for classical micronization strategies and to investigate the effect of high pressure on protein stability and bioactivity. METHODS: The influence of drug loading, homogenization pressure and cycles on particle size reduction was investigated by experimental design using a Box Behnken matrix with insulin as a model compound. Particle size measurements were performed by laser light scattering. Protein stability was investigated by HPLC and HPLC-MS analysis and the bioactivity of insulin was tested in a chondrocyte proliferation assay. For investigations into the effect of temperature on protein stability, insulin was micronized in molten lipid at 75 degrees C in one cycle at 1,000 bar. RESULTS: Within one homogenization cycle at 1,500 bar, the particle size of insulin could be reduced from 15.8 to 7.3 microm, six cycles resulted in a particle size of 3.7 microm d(0.5) (50% of the particles are smaller than the indicated value). Evaluation of the response surface diagram revealed that the homogenization pressure had the highest impact on micronization efficiency, followed by the number of homogenization cycles. Protein stability was maintained during the micronization process as well as bioactivity. Micronization at elevated temperature (75 degrees C) had no effect on protein stability. CONCLUSION: High pressure homogenization of protein suspensions can be used as an alternative method for the micronization of proteins without affecting protein stability or bioactivity.

Bioavailability of opipramol from a film-coated tablet, a sugar-coated tablet and an aqueous solution in healthy volunteers.

Opipramol (4-[3-(5H-dibenz[b,f]-azepine-5-yl)-propyl]-1-piperazine-ethanol dihydrochloride, CAS 315-72-0) is regarded as an anxiolytic compound with antidepressant properties, and it is one of the most frequently prescribed psychotropic drugs in Germany. In two open, randomized cross-over studies in 20 (study 1) and 18 (study II) healthy volunteers, the relative bioavailability of 50 mg opipramol-2HCl from a sugar-coated tablet was compared with an aqueous solution, and of 100 mg opipramol-2HCl from a newly developed film-coated tablet was compared with the sugar-coated tablet. The concentrations of opipramol were determined in plasma by high-performance liquid chromatography (HPLC) with photometric detection. The mean dose corrected kinetic parameters of opipramol were similar after administration of all formulations. The peak concentrations of opipramol were 13-15 ng ml-1 (study I) and 28 ng ml-1 (study II). They were achieved after 3 h. The area under the plasma concentration-time curve was about 170 ng ml-1 h (study I) and about 320 ng ml-1 h (study II). The terminal plasma half-life was 11 h. Bioequivalence was proven between sugar-coated tablet and aqueous solution, and between film-coated tablet and sugar-coated tablet, respectively. In addition, in study II the plasma concentrations and pharmacokinetic parameters of the metabolites opipramol N-oxide and deshydroxyethyl opipramol were determined.

Peptide acylation by poly(alpha-hydroxy esters).

PURPOSE: Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) microspheres were investigated concerning the possible acylation of incorporated peptides. METHODS: Atrial natriuretic peptide (ANP) and salmon calcitonin (sCT) were encapsulated into PLA and PLGA microspheres. Peptide integrity was monitored by HPLC-MS analysis during microsphere degradation for four weeks. sCT fragmentation with endoproteinase Glu-C was used for identifying modified amino acids. Peptide stability in lactic acid solutions was investigated to elucidate possible mechanisms for preventing peptide acylation. RESULTS: Both peptides were acylated by lactic and glycolic acid units inside degrading microspheres in a time-dependent manner. After 21 days, 60% ANP and 7% sCT inside PLA microspheres were acylated. Fragmentation of sCT with endoproteinase Glu-C revealed that besides the N-terminal amine group, lysine, tyrosine or serine are further possible targets to acylation. Stability studies of the peptides in lactic acid solutions suggest that oligomers are the major acylation source and that lower oligomer concentration and higher pH substantially decreased the reaction velocity. CONCLUSIONS: The use of PLA and PLGA for drug delivery needs substantially more circumspection. As, according to FDA standards. the potential hazards of peptide acylation products need to be assessed, our findings may have significant implications for products already on the market. Techniques to minimize the acylation reaction are suggested.