Antagonistic effects of indoloquinazoline alkaloids on antimycobacterial activity of evocarpine.

AIMS: The interaction of quinolone and indoloquinazoline alkaloids concerning their antimycobacterial activity was studied. METHODS AND RESULTS: The antimycobacterial and modulating activity of evodiamine (1), rutaecarpine (2) and evocarpine (3) was tested on mycobacteria including three multidrug-resistant (MDR) clinical isolates of Mycobacterium tuberculosis. Antagonistic effects were concluded from fractional inhibitory concentration (FICI) values. Interaction energies of the compounds were calculated using GLUE docking module implemented in GRID. 1 and 2 exhibited weak inhibition of rapidly growing mycobacteria, however, 1 was active against Myco. tuberculosis H37Rv (MIC = 10 mg l(-1) ) while 2 was inactive. Both 1 and 2 showed a marked antagonistic effect on the susceptibility of different mycobacterial strains to 3 giving FICI values between 5 and 9. The interaction energies between compounds 1 and 2 with compound 3 suggested the possibility of complex formation in solution. CONCLUSIONS: Indoloquinazoline alkaloids markedly reduce the antimycobacterial effect of the quinolone alkaloid evocarpine. Complex formation may play a role in the attenuation of its antimycobacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives a striking example of antagonism between compounds present in the same plant extract which should be considered in natural product based screening projects.

[A simple method of synthesis of triterpene glycosides similar to glycyrrhizic acid and their hepatoprotective activity in vitro].

A simplified method of synthesis of triterpene l,2-trans-glycosides was developed using the glycosylation of glycyrrhetic acid (GLA) and 18,19-dehydro-GLA by beta-pyranose peracetates in the presence of SnCl(4) and molecular sieves 4 A. The synthesized glycosides exhibited hepatoprotective activity toward the human hepatoma HepG2 cell line on the model of alcohol hepatitis and decreased the level of TNF-alpha protein.

Phytochemical composition and in vitro pharmacological activity of two rose hip (Rosa canina L.) preparations.

The aim of the present study was to compare powdered rose hip with and without fruits (Rosae pseudofructus cum/sine fructibus, Rosa canina L., Rosaceae) with regard to their phytochemical profile and their in vitro anti-inflammatory and radical-scavenging properties. The two powders were subsequently extracted with solvents of increasing polarity and tested for inhibition of cyclooxygenase (COX-1, COX-2) and of 5-LOX-mediated leukotriene B(4) (LTB(4)) formation as well as for DPPH-radical-scavenging capacity. While the water and methanol extracts were inactive in the COX-1, COX-2 and LTB(4) inhibition assays, the n-hexane and the dichloromethane extracts inhibited all three enzymes. In the active extracts, the triterpenoic acids ursolic acid, oleanolic acid and betulinic acid were identified, although only in minute amounts. Furthermore, oleic, linoleic and alpha-linolenic acid were identified apart from several saturated fatty acids. Even though unsaturated fatty acids are known to be good inhibitors of COX-1, COX-2 and LT formation, no clear correlation between their concentration in the extracts and their activity was found. We suggest that other, yet unidentified, lipophilic constituents might play a more important role for the observed in vitro inhibitory activity on arachidonic acid metabolism. Some of the extracts also showed considerable DPPH radical scavenging activity, the methanolic extracts being most potent. The radical scavenging activity of the extracts correlated very well with their total phenolic content, while ascorbic acid contributes only little to the radical-scavenging activity due to its low concentration present in the extracts. In summary, extracts derived from powdered rose hip without fruits were more effective in all assays carried out compared with extracts derived from powdered rose hip with fruits.

Hydroxymethylfurfural: an enemy or a friendly xenobiotic? A bioanalytical approach.

Hydroxymethylfurfural (HMF), a well-known heterocyclic Maillard reaction product, has often been studied for its potential toxic, mutagenic, and carcinogenic effects. Recent clinical studies, however, have strongly suggested that HMF might have exciting antitumor potential. We report on the development and validation of a bioanalytical assay for HMF that could be suitable as a basis for pharmacokinetic models in cancer patients. Two strategies were tested, i.e., direct and indirect methodologies. A direct isocratic LC determination at 283 nm was designed. Two indirect attempts involved derivatization coupled to HPLC-UV. It was possible to resolve the stereoisomers of the HMF derivative, and factors influencing their equilibrium ratio are discussed. HMF was extracted from the biomatrix by solid-phase extraction using different cartridges. A comparative study was made of the implemented methods as well as the extraction protocols. Both indirect assays proved to be more sensitive and were used to assess HMF quantitatively in human plasma. However, the newly introduced derivatization conditions led to the highest sensitivity with a LOD (S/N ratio = 3) of at least 2 pmol analyte on column. The assay selectivity was satisfactory in pre- and post-dose real samples. The mean recoveries of the assays were 79% and 89%, with acceptable accuracies and reproducibilities. Figure Schematic representation of hydroxymethylfurfural (HMF) in human plasma.

Sulfation of resveratrol in human liver: evidence of a major role for the sulfotransferases SULT1A1 and SULT1E1.

Sulfation of resveratrol, a polyphenolic compound present in grapes and wine with anticancer and cardioprotective activities, was studied in human liver cytosol. In the presence of 3'-phosphoadenosine-5'-phosphosulfate, three metabolites (M1-3) whose structures were identified by mass spectrometry and NMR as trans-resveratrol-3-O-sulfate, trans-resveratrol-4'-O-sulfate, and trans-resveratrol-3-O-4'-O-disulfate, respectively. The kinetics of M1 formation in human liver cytosol exhibited an pattern of substrate inhibition with a K(i) of 21.3 +/- 8.73 microM and a V(max)/K(m) of 1.63 +/- 0.41 microLmin(-1)mg(-1) protein. Formation of M2 and M3 showed sigmoidal kinetics with about 56-fold higher V(max)/K(m) values for M3 than for M2 (2.23 +/- 0.14 and 0.04 +/- 0.01 microLmin(-1)mg(-1)). Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 is almost exclusively catalysed by SULT1A1 and only to a minor extent by SULT 1A2, 1A3 and 1E1, whereas M2 is selectively formed by SULT1A2. M3 is mainly catalysed by SULT1A2 and 1A3. In conclusion, the results elucidate the enzymatic pathways of resveratrol in human liver, which must be considered in humans following oral uptake of dietary resveratrol.

Measurement of fractional whole-body gluconeogenesis in humans from blood samples using 2H nuclear magnetic resonance spectroscopy.

Several problems limit quantification of gluconeogenesis. We applied in vitro 2H-nuclear magnetic resonance (NMR) spectroscopy to simultaneously measure 2H in all glucose carbons for direct assessment of gluconeogenesis. This method was compared with 2H measurement in carbons 5 and 2 using gas chromatography-mass spectrometry (hexamethylenetetramine [HMT]) and with in vivo 13C magnetic resonance spectroscopy (MRS). After 14 h of fasting, and following 2H2O ingestion, blood was obtained from nine healthy and seven type 2 diabetic subjects. Glucose was purified, acetylated, and analyzed for 2H in carbons 1-6 with 2H-NMR. Using 5:2 ratios, gluconeogenesis increased (P < 0.05) over time and mean gluconeogenesis was lower in control subjects than in type 2 diabetic patients (63 +/- 3 vs. 75 +/- 2%, P < 0.01). 13C-MRS revealed higher hepatic glycogenolysis in control subjects (3.9 +/- 0.4 vs. 2.3 +/- 0.2 micromol.kg(-1).min(-1)) yielding mean contribution of gluconeogenesis of 65 +/- 3 and 77 +/- 2% (P < 0.005). Measurement of gluconeogenesis by 2H-NMR correlated linearly with 13C-MRS (r = 0.758, P = 0.0007) and HMT (r = 0.759, P = 0.0007). In an additional protocol, 2H enrichments demonstrated a fast decline of gluconeogenesis from approximately 100 to approximately 68% (P < 0.02) within 4 h of galactose infusion after 40-44 h of fasting. Thus, in vitro 2H-NMR offers an alternative approach to determine fractional gluconeogenesis in good agreement with standard methods and allows monitoring of rapid metabolic alterations.

In vitro glucuronidation of the cyclin-dependent kinase inhibitor flavopiridol by rat and human liver microsomes: involvement of UDP-glucuronosyltransferases 1A1 and 1A9.

The metabolism of flavopiridol (FLAP), a novel anticancer drug currently undergoing clinical development, was investigated in rat and human liver microsomes. In the presence of uridine 5'-diphosphoglucuronic acid, two biotransformation products (M1 and M2) could be detected. Formation of metabolite M1 and M2 in terms of enzymatic efficacy (Vmax/K(M)) was about 50- and 5-fold higher in rat (1.58 +/- 2.23 and 7.22 +/- 1.17 microl/min/mg) as compared with human liver microsomes (0.032 +/- 0.016 and 1.52 +/- 0.93 microl/min/mg), indicating species-related differences in FLAP glucuronidation. Incubation in the presence of human recombinant UDP-glucuronosyltransferases (UGTs) demonstrated that M1 is almost exclusively catalyzed by UGT1A1, whereas M2 is formed by UGT1A9 and only to a minor extent by UGT1A1 and UGT1A10. Chemical inhibition experiments further prove the involvement of UGT1A1 and UGT1A9 in the formation of M1 and M2, as the UGT1A1 substrate bilirubin preferably inhibited M1 over M2 (K(i): 36 and 258 microM, respectively), whereas the UGT1A9 substrate propofol showed a more pronounced decrease in M2 but not in M1 formation (K(i): 47 and 142 microM, respectively). Both conjugates were purified from rat liver microsomes and analyzed by mass spectrometry, NMR, and UV experiments. On the basis of these results, M1 was identified as 5-O-beta-glucopyranuronosyl-flavopiridol and M2 as 7-O-beta-glucopyranuronosyl-flavopiridol. In conclusion, our results elucidate the enzymatic pathways of FLAP in rat and human liver, which must be considered during cancer therapy of patients.

Triterpenoid saponins and sapogenin lactones from Albizia gummifera.

The structures of two new monodesmosidic and bisdesmosidic triterpenoid saponins (1 and 2) and the known compound delta 5-stigmasterol-3-O-beta-D-glucopyranoside (3) as well as two new oleanane type triterpene lactone glycosides 4, 5 and a new sapogenin lactone 6 isolated from the stem bark of Albizia gummifera C.A. Smith (Mimosaceae) have been elucidated as 3-O-¿beta-D-glucopyranosyl(1-->2)-[alpha-L-arabinopyranosyl(1-->6) ]-beta-D- glucopyranosyl¿-oleanolic acid (1), beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl 3-O-¿beta-D-glucopyra-nosyl(1-->2)-[alpha-L-arabinopyrano syl(1-->6)]-beta-D- glucopyranosyl¿-oleanolate (2), 3 beta-¿O-D-glucopyranosyl-(1-->2)-[O-alpha-L-arabinopyranosyl(1-->6 )] beta-D-glucopyranosyloxy¿-machaerinic acid gamma-lactone (4), 3 beta-O-beta-D-glucopyranosiduronic acid (1-->2)-beta-D-glucopyranosyloxy]-machaerinic acid gamma-lactone (5), and A-homo-3a-oxa-5 beta-olean-12-en-3-one-28-oic acid (6), respectively. The complete assignment of the 1H and 13C resonances of 1, 2, 4 and 6 and of the peracetate of 5 were achieved by means of 2D-NMR studies.

Steroidal saponins from Asparagus africanus.

The structures of two new monodesmosidic spirostanosides and a new bisdesmosidic furostanol glycoside isolated from the roots of Asparagus africanus Lam. (Liliaceae) have been elucidated as (25R)-3 beta-hydroxy-5 beta-spirostan-12-one 3-O-{beta-D-glucopyranosyl-(1-->2)-[alpha-1-arabinopyranosyl-(1--> 6)]-beta- D-glucopyranoside} (1), (25R)-5 beta-spirostan-3 beta-ol 3-O-{beta-D-glucopyranosyl-(1-->2)-[alpha-L-arabinopyranosyl-(1--> 6)]-beta- D-glucopyranoside} (2) and 26-O-beta-D-glucopyranosyl]-22 alpha-methoxy-(25R)-furostan-3 beta,26-diol 3-O-{beta-D-glucopyranosyl-(1-->2)-[beta-D-glucopyranoside} (3), respectively, by the combined use of one and two dimensional NMR experiments. The complete 13C and 1H assignments of the peracetyl spirostanosides and the furostanol oligoside were derived. The interconversions between the methoxyl and hydroxyl group at C-22 of the furostanol glycoside was investigated and the genuine furostanol oligoside of A. africanus appears to be the hydroxyl type based on the comparative study of the methanol, pyridine and dioxane extracts.

Solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24.

Two-dimensional-NMR and three-dimensional-NMR experiments were performed to determine the solution structure of the DNA-binding domain of the tomato heat-stress transcription factor HSF24. Samples of uniformly 15N-labeled and 15N, 13C-labeled recombinant proteins were used in the investigation. A near-complete assignment of the backbone 1H, 15N, and 13C resonances was obtained by three-dimensional triple-resonance experiments, whereas three-dimensional 15N-TOCSY-heteronuclear-single-quantum-correlation-spectroscopy, HCCH-COSY and HCCH-TOCSY spectra were recorded for side-chain assignments, 885 non-redundant distance constraints from two-dimensional-homonuclear and three-dimensional-15N-edited and 13C-edited NOESY spectra and 40 hydrogen-bond constraints from exchange experiments were used for structure calculations. The resulting three-dimensional structure contains a three-helix bundle and a small four-stranded antiparallel beta-sheet that forms a hydrophobic core. The two C-terminal helices are parts of a highly conserved helix-turn-helix motif that is probably involved in DNA recognition and binding. In contrast to heat-stress factors from yeast and animals, the plant heat-stress factors lack a loop of 11 amino acid residues inserted between beta3 and beta4. This leads to a tight turn between these beta-strands.

Solution structure of the hydrophilic region of HIV-1 encoded virus protein U (Vpu) by CD and 1H NMR spectroscopy.

The HIV-1 specific Vpu is a class I oligomeric membrane phosphoprotein of unknown structure and mechanism. The first experimental evidence for the position of secondary structural elements present in the hydrophilic C-terminal region of Vpu under various solution regimes is reported. CD data for nine overlapping 15 amino-acid fragments and 3 longer fragments indicate the presence of only transitory amounts of stable structure in aqueous solution alone, while with increasing trifluoroethanol content limiting structures were found indicating two helical segments in the hydrophilic region of Vpu. These limiting structures were more precisely defined from a detailed study of Vpu41-58, Vpu52-74 and Vpu63-81, by a combination of 2D 1H NMR spectroscopy, distance geometry, and restrained molecular dynamics and energy minimization calculations. Sets of low-energy conformations compatible with the quantitative NOE data indicate that Vpu41-58 has an alpha-helix from residues 42 to 50 while a second helix is found for Vpu52-74 from residues 57 to 69. Vpu63-81 shows only the presence of a single reverse turn at residues 74 to 77, without any evidence of helix, under the same conditions. From CD measurements the first helix extends back to residue 30 and is connected to the N-terminal anchor of Vpu. Thus the hydrophilic region of Vpu consists of two alpha-helices joined by a flexible region of 6 or 7 residues, which contains the phosphoacceptor sites of Vpu at positions 52 and 56. The second helix is followed by a single reverse turn and a flexible C-terminus.

Synthesis and characterization of the hydrophilic C-terminal domain of the human immunodeficiency virus type 1-encoded virus protein U (Vpu).

The HIV-1-specific virus protein U (Vpu) is an amphipathic membrane-associated phosphoprotein that probably possesses an N-terminal hydrophobic membrane anchor and a hydrophilic moiety. Because of an as yet undefined cytotoxic effect and the low concentration of Vpu in host cells, it has not been possible to obtain sufficient quantities of this protein for biochemical and structural investigations. We describe the synthesis, in two forms, of 50-residue peptides representing the polar cytoplasmic domain of Vpu: pVpu, comprising the wild-type Vpu sequence of HIV-1, strain HTLVIIIB, from position Ile32 to Leu81, and a mutant, pVpum2/6, with replacement of Ser52-56 with Asn. This mutation affects the two phosphorylation sites of Vpu for casein kinase-2 (CK-2), the enzyme that phosphorylates Vpu in HIV-1-infected cells. The peptides were purified to homogeneity and characterized by N-terminal sequencing, mass spectrometry and SDS-PAGE. Furthermore, both peptides were immunologically characterized by Western blot and ELISA using Vpu-specific monoclonal and polyclonal antibodies. Recombinant human CK-2 caused in vitro phosphorylation of pVpu, but had no effect on the mutant pVpum2/6. Investigation of the peptides by circular dichroism showed that addition of trifluoroethanol stabilized the alpha-helical secondary structure. Preliminary 1H NMR spectroscopic data indicated that, in the presence of trifluoroethanol, both peptides in solution have stable secondary structures with considerable alpha-helical content.