Genetic diversity of Escherichia coli isolated from ice cube production sites.

OBJECTIVE: The prevalence of Escherichia coli including from ice cubes in Indonesia is quite high. Unfortunately, little is known about the genetic diversity of E. coli from ice cube production site. Genotypic variation in E. coli populations is a major barrier to control public health risk associated with foodborne pathogen. The aims of this study were to analyze the genotypic diversity of E. coli strains isolated from various samples in order to determine the genetic relationship between those strains. This study is also important to understand the occurrence, prevalence and profile picture of different pathogenic E. coli in various sources which potentially cause disease. RESULTS: Enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic polymerase chain reaction (REP-PCR) dendrogram showed high genetic diversity of 120 E. coli isolates in majority of sampling sites. DNA fingerprint patterns showed 26 and 21 clusters with 11 and 3 fingerprints individual lineages for ERIC and REP-PCR respectively. There was no correlation observed between phylogenetic relationship and virulence genes. The result indicated a variation of E. coli isolates in ice cube manufacturers. ERIC-PCR method is more discriminative compared with REP-PCR to analyze the genetic diversity of E. coli from ice cubes production sites.

Potent water extracts of Indonesian medicinal plants against PTP1B

Objective: To examine the potent of water as a solvent agent in the preparation of traditional herbal medicine. Methods: Water extracts of 18 plants were prepared through reflux and examined (25 mg/mL) to evaluate their possibility for inhibiting protein tyrosine phosphatase 1B (PTP1B). The determination of IC50 values was performed for the samples possessing more than 80% inhibition. Meanwhile, those exhibiting IC50 values more than 7.0 mg/mL were further profiled for their chemical constituents through nuclear magnetic resonance (NMR) measurement. Results: About 44% (8) of the examined samples showed more than 80% inhibition against PTP1B. The water extracts of Elephantopus scaber, Helicteres isora aerial parts, Elaeocarpus grandiflorus (E. grandiflorus) fruits, Melaleuca leucadendron leaves, and Quercus infectoria gum had IC50 values ranging from 2.05 to 6.90 mg/mL. Meanwhile, Andropogon nardus and Centella asiatica were at the area of d 3.0–4.0 ppm. Further, the 13C NMR observation of samples possessing the most intensive signals in their proton NMR Cinnamomum burmannii and E. grandiflorus showed the peaks at the area of d 60–90 ppm as the supportive evidence for sugar group signals. Intriguingly, a disaccharide from E. grandiflorus could be an active inhibitor towards PTB1B. Conclusions: In contrast to the mainstream solvents currently used in modern herbal manufactures especially Jamu medicine in Indonesia, pure-water-extracted materials should be reconsidered and could be reemerged for future studies and for the manufacture of herbal medicines. In addition, the activity of Jamu components should be confirmed that their antidiabetes and antiobesity activities could be through the inhibition of PTP1B.

Mechanism-based inhibition of human liver microsomal cytochrome P450 2D6 (CYP2D6) by alkamides of Piper nigrum.

Nineteen alkamides isolated from Piper nigrum L. were tested for their mechanism-based inhibition on human liver microsomal dextromethorphan O-demethylation activity, a prototype marker for cytochrome P450 2D6 (CYP2D6). All compounds increased their inhibitory activity with increasing preincubation time. Among them, 15 and 17 showed more than 50 % decrease of the CYP2D6 residual activity after 20 min preincubation. Further investigations on 15 and 17 showed that the characteristic time- and concentration-dependent inhibition, which required a catalytic step with NADPH, was not protected by nucleophiles, and was decreased by the presence of a competitive inhibitor. The kinetic parameters for inactivation (kinact and KI) were 0.028 min-1 and 0.23 microM for 15 and 0.064 min-1 and 0.71 microM for 17, respectively, which were stronger than the known mechanism-based inhibitor, paroxetine (a positive control). Thus, 15 and 17 are potent mechanism-based inhibitors of CYP2D6.

Mechanism-based inhibition of CYP3A4 and CYP2D6 by Indonesian medicinal plants.

Thirty samples of Indonesian medicinal plants were tested for their mechanism-based inhibition on cytochrome P450 3A4 (CYP3A4) and CYP2D6 via erythromycin N-demethylation and dextromethorphan O-demethylation activities in human liver microsomes. From screening with 0 and 20min preincubation at 0.5mg/ml of methanol extracts, five plants (Cinnamomum burmani bark, Foeniculum vulgare seed, Strychnos ligustrina wood, Tinospora crispa stem, and Zingiber cassumunar rhizome) showed more than 30% increase of CYP3A4 inhibition, while three (Alpinia galanga rhizome, Melaleuca leucadendron leaf, and Piper nigrum fruit) showed more than 30% increase of CYP2D6 inhibition. In these eight plants, Foeniculum vulgare seed, Cinnamomum burmani bark, and Strychnos ligustrina wood showed time-dependent inhibition on CYP3A4 and Piper nigrum fruit and Melaleuca leucadendron leaf on CYP2D6. Among these, four plants other than Melaleuca leucadendron revealed NADPH-dependent inhibition. Thus, Foeniculum vulgare, Cinnamomum burmani, and Strychnos ligustrina should contain mechanism-based inhibitors on CYP3A4 and Piper nigrum contain that on CYP2D6.

Cytochrome P450 2D6 (CYP2D6) inhibitory constituents of Catharanthus roseus.

The MeOH-soluble fraction of the water extract of Catharanthus roseus from Indonesia, having shown potent inhibitory activity on the metabolism mediated by CYP2D6, was subjected to activity-guided isolation to yield two triterpenes, ursolic acid (1) and oleanolic acid (2), and three alkaloids, vindoline (3), ajmalicine (4), and serpentine (5). The isolated compounds were tested for their inhibitory activity on the metabolism mediated by CYP3A4 or CYP2D6 using [N-methyl-14C]erythromycin or [O-methyl-14C]dextromethorphan as a substrate, respectively. Ajmalicine (4) and serpentine (5) showed very potent inhibitory activity against CYP2D6 with IC50 values of 0.0023 and 3.51 microM, respectively. All isolated compounds showed weak or no inhibition against CYP3A4. On time-, concentration-, and NADPH-dependent assay, serpentine (5) appear to be the mechanism-based inhibitor for CYP2D6 enzyme in which the inhibition was irreversible and driven by catalytic process. K(I) and k(inact) values for serpentine (5) were 0.148 microM and 0.090 min-1, respectively. On the other hand, ajmalicine (4) showed no time-dependent inhibition or reversible inhibition, and thus appear to be not mechanism-based inhibitor.

Mechanism-based inhibition of CYP3A4 by constituents of Zingiber aromaticum.

Sixteen compounds isolated from Zingiber aromaticum and showing concentration-dependent inhibition with IC50 values less than 100 microM, were analyzed for their possibility of time-, concentration-, and NADPH-dependent inhibition of CYP3A4 and four were analyzed for CYP2D6. All seven kaempferol glycosides and two kaempferol derivatives (4, 5, 8-14) appear to be the mechanism-based inhibitors of CYP3A4 enzyme in which the inhibition is irreversible and driven by the catalytic process. The other compounds showed no NADPH-dependent inhibition or reversible inhibition, and thus do not appear to be mechanism-based inhibitors. K(I) values for compounds 4, 5, 8-14 were in the range of 2.21-27.01 microM, whereas the k(inact) values were 0.23-0.65 min(-1). Kaempferol-3-O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranoside) (5) was found to be the most potent CYP3A4 inactivator with K(I) and k(inact) values of 2.21 microM and 0.45 min(-1), respectively.

Constituents of Zingiber aromaticum and their CYP3A4 and CYP2D6 inhibitory activity.

A new sesquiterpene 2,9-humuladien-6-ol-8-one (1) was isolated from methanol extract of Zingiber aromaticum, along with 15 known compounds. The structures of the isolated compounds were elucidated on the basis of spectroscopic analyses. The isolated compounds were tested for their inhibitory activity on the metabolism mediated by cytochrome P450 3A4 (CYP3A4) and CYP2D6.

Metabolite-cytochrome P450 complex formation by methylenedioxyphenyl lignans of Piper cubeba: mechanism-based inhibition.

Five methylenedioxyphenyl lignans, (-)-clusin (1), (-)-dihydroclusin (2), (-)-yatein (3), (-)-hinokinin (4), and (-)-dihydrocubebin (5), were isolated from Piper cubeba as potent and selective inhibitors against cytochrome P450 3A4 (CYP3A4). In this study, we investigated the mechanism of inhibition of CYP3A4 by these lignans and the possibility of their mechanism-based inhibition. Using [N-methyl-14C]erythromycin as a substrate, all lignans appear to be showed mixed-type of inhibition with apparent Ki of 1.96-4.07 microM. Furthermore, all lignans (1-5) inhibited CYP3A4 in a time-, concentration-, and NADPH-dependent manners and thus appear to be the mechanism-based inhibitors of CYP3A4. The apparent inactivation parameter, K(I) for these compounds were in the range of 0.054-0.373 microM, whereas the k(inact) values were 0.225-0.320 min-1. Among them, (-)-clusin (1) and (-)-dihydroclusin (2) were found to be the most potent CYP3A4 inactivator with apparent K(I) and k(inact) values of 0.082, 0.054 microM and 0.253, 0.310 min-1, respectively. Spectral scanning of microsomes with these lignans yielded an absorbance at 455 nm, suggesting that all of them appear to inactivate the cytochrome P450 via the formation of a metabolite intermediate complex. This pattern is consistent with the metabolism of the methylenedioxyphenyl compounds. These results indicate that (-)-clusin (1), (-)-dihydroclusin (2), (-)-yatein (3), (-)-hinokinin (4), and (-)-dihydrocubebin (5) are effective mechanism-based inhibitors of CYP3A4.

Potent CYP3A4 inhibitory constituents of Piper cubeba.

The EtOAc-soluble fraction of the water extract of Piper cubeba, having shown potent inhibitory activity on the metabolism mediated by CYP3A4, was subjected to activity-guided isolation to yield two new lignans, (8R,8'R)-4-hydroxycubebinone (1) and (8R,8'R,9'S)-5-methoxyclusin (2), and two new sesquiterpenes, (5 alpha,8 alpha)-2-oxo-1(10),3,7(11)-guaiatrien-12,8-olide (3) and (1 alpha,2 beta,5 alpha,8 alpha 10 alpha)-1,10-epoxy-2-hydroxy-3,7(11)-guaiadien-12,8-olide (4), along with 16 known compounds (5-20). The structures of the isolated compounds were elucidated on the basis of spectroscopic and chemical analyses. The isolated compounds were tested for their inhibitory activity on the metabolism mediated by CYP3A4 or CYP2D6 using [N-methyl-(14)C]erythromycin or [O-methyl-(14)C]dextromethorphan as a substrate, respectively. The compounds (8R,8'R,9'S)-5-methoxyclusin (2), (-)-clusin (10), (-)-yatein (13), ethoxyclusin (15), and (-)-dihydroclusin (17), having one methylenedioxyphenyl moiety in their structures, showed very potent and selective inhibitory activity against CYP3A4 with IC(50) values (0.44-1.0 microM) identical to that of the positive control, ketoconazole (IC(50), 0.72 microM).

Sesquiterpenes and flavonol glycosides from Zingiber aromaticum and their CYP3A4 and CYP2D6 inhibitory activities.

Three new sesquiterpenes, (2R,3S,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one (1), (2R,3R,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one (2), and (5R)-2,6,9-humulatrien-5-ol-8-one (3), and two new flavonol glycosides, kaempferol-3-O-(2,3-di-O-acetyl-alpha-l-rhamnopyranoside) (4) and kaempferol-3-O-(2,3,4-tri-O-acetyl-alpha-l-rhamnopyranoside) (5), were isolated from the EtOAc-soluble fraction of the water extract of Zingiber aromaticum, along with 13 known compounds (6-18). The structures of the isolated compounds were elucidated on the basis of spectroscopic and chemical analyses. The isolated compounds were tested for their inhibitory activity on the metabolism mediated by CYP3A4 or CYP2D6 using [N-methyl-(14)C]erythromycin or [O-methyl-(14)C]dextromethorphan as a substrate, respectively. Kaempferol-3-O-(2,3,4-tri-O-acetyl-alpha-l-rhamnopyranoside) (5) showed the most potent inhibitory activity (IC(50), 14.4 microM) on the metabolism mediated by CYP3A4, and kaempferol-3-O-methyl ether (14) inhibited CYP2D6 most potently (IC(50), 4.63 microM).

Three new C-14 oxygenated taxanes from the wood of Taxus yunnanensis.

Three new C-14 oxygenated taxane-type diterpenes, hongdoushans A-C (1-3), were isolated from the wood of Taxus yunnanensis together with four known diterpenes and two lignans. The absolute stereochemistry of the 2-methylbutyryloxy group attached at C-14 of the taxane skeleton was determined to be S by GC analysis of the methyl ester of 2-methylbutyric acid obtained after alkaline hydrolysis of 1 and 4 followed by treatment with CH(2)N(2). The complete stereostructure of the known compound 2alpha,5alpha,10beta-triacetoxy-14beta-[(S)-2-methylbutyryloxy]-4(20),11-taxadiene (4) was established for the first time. The isolates obtained were evaluated for their antiproliferative activity toward murine colon 26-L5 carcinoma and human HT-1080 fibrosarcoma cell lines.

Constituents of Chinese propolis and their antiproliferative activities.

Two new flavonoids, 3-O-[(S)-2-methylbutyroyl]pinobanksin (1) and 6-cinnamylchrysin (2), were isolated from the EtOAc-soluble fraction of the MeOH extract of Chinese propolis, along with 12 known compounds (3-14). The structures of the isolated compounds were elucidated on the basis of spectroscopic and chemical analyses. The isolated compounds were tested for their antiproliferative activity toward five different cancer cell lines. Benzyl caffeate (13) and phenethyl caffeate (14) showed potent antiproliferative activity toward tested cell lines with a selective activity toward colon 26-L5 carcinoma cell line (EC(50) values: 13, 1.01; 14, 0.30 microM).