Simultaneous determination of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in human serum and plasma by high-performance liquid chromatography

A HPLC method was developed for the determination of the metabolites of coumarin and 7-hydroxycoumarin in plasma and serum. Separation was based on gradient elution of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, coumarin and finally 4-hydroxycoumarin (which is used as an internal standard). Standards, prepared in plasma or serum, and samples were treated with trichloroacetic acid, mixed and centrifuged. The supernatant was removed and analyzed by reversed-phase high-performance liquid chromatography on a C₁₈ column. The limit of detection was 50 ng/ml for 7-hydroxycoumarin and 200 ng/ml for coumarin and 7-hydroxycoumarin glucuronide. The linear range was 0.5–100 μg/ml for each of the analytes. The percentage relative standard deviation about the mean measured concentrations were all below 10%. There was no statistical difference between the standard curves prepared in plasma or serum. The method developed was applied to the determination of each of the three compounds in serum, after the administration of 7-hydroxycoumarin, and in plasma after the administration of coumarin. The concentrations of total 7-hydroxycoumarin in the serum samples were also determined by another HPLC method and the results were compared. There was no statistical difference between the results determined.     

Foetidin,a sesquiterpenoid coumarin from Ferula assa-foetida

A new sesquiterpenoid coumarin, foetidin, has been isolated from the roots of Ferula assa-foetida.     

Promiscuous enzyme-catalyzed cascade reaction in water: Synthesis of dicoumarol derivatives

Lipase RMIM was firstly used as a promiscuous biocatalyst to catalyze the Knoevenagel-Michael cascade reactions of 4-hydroxycoumarin with aromatic, heterocyclic or aliphatic aldehydes to synthesize dicoumarol derivatives in water. Results showed that the adopted methodology could offer many advantages, such as mild reaction conditions, pure aqueous reaction system, wide substrate applicability, recyclable catalyst, excellent yields (81–98%), operational simplicity, and environmentally friendly reactions.     

Synthesis and biological evaluation of novel 3-substituted amino-4-hydroxylcoumarin derivatives as chitin synthase inhibitors and antifungal agents

A series of novel 3-substituted amino-4-hydroxycoumarin derivatives have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their CHS inhibition activity and antimicrobial activity in vitro. The enzymatic assay indicated that most of the compounds have good inhibitory activity against CHS, in which compound 6o with IC₅₀ of 0.10?mmol/L had stronger activity than that of polyoxins B, which acts as control drug with IC₅₀ of 0.18?mmol/L. As far as the antifungal activity is concerned, most of the compounds possessed moderate to excellent activity against some representative pathogenic fungi. Especially, compound 6b was found to be the most potent agent against Cryptococcus neoformans with minimal inhibitory concentration (MIC) of 4?μg/mL. Moreover, the results of antibacterial screening showed that these compounds have negligible actions to some tested bacteria. Therefore, these compounds would be promising to develop selective antifungal agents.     

Synthesis of 2-acylated and sulfonated 4-hydroxycoumarins: In vitro urease inhibition and molecular docking studies

Sixteen 4-hydroxycoumarin derivatives were synthesized, characterized through EI-MS and ¹H NMR and screened for urease inhibitory potential. Three compounds exhibited better urease inhibition than the standard inhibitor thiourea (IC₅₀ = 21 ± 0.11 μM) while other four compounds exhibited good to moderate inhibition with IC₅₀ values between 29.45 ± 1.1 μM and 69.53 ± 0.9 μM. Structure activity relationship was established on the basis of molecular docking studies, which helped to predict the binding interactions of the most active compounds.     

The effects of tannic acid and other phenolics on the growth of the fungus cultivated by the leaf-cutting ant, Myrmicocrypta buenzlii

Using a liquid defined medium for the bioassays, the effects of tannic acid, naringin and 4-hydroxycoumarin on the growth of an ant-cultivated fungus were determined. The results with tannic acid indicated greater growth retardation than was obtained during bioassays on solid agar media. The reasons for this discrepancy are attributed to the undesirable properties of solid agar media for conducting tannin bioassays.     

Leptodactylone,a yellow coumarin from Leptodactylon and Linanthus species

A new phenol in Leptodactylon and Linanthus leaf has been identified by spectral procedures as 5,7-dimethoxy-8-hydroxycoumarin. This structure has been confirmed by synthesis. Leptodactylone is unique among simple hydroxycoumarins in its yellow colour.     

Induction of 4-Hydroxycinnamate Decarboxylase in Klebsiella oxytoca Cells Exposed to Substrates and Non-substrate 4-Hydroxycinnamate Analogs

The 4-hydroxycinnamate decarboxylase (4-HCD)-inducing activity of several substrate analogs toward Klebsiella oxytoca was investigated. Four E-cinnamateclass compounds, E-4-hydroxycinnamic acid (1), caffeic acid (2), ferulic acid (3) and E-2,4-dihydroxycinnamic acid (4), all of which were accepted as substrates, all of which were accepted as substrates of 4-HCD, enable K. oxytoca cells to induce the decarboxylase at a 2.0 mM concentration, while five non-substrate compounds of the E-cinnamate class so far tested were completely in-active. However, 6-hydroxy-2-naphthoic acid (11) and 7-hydroxycoumarin 3-carboxylic acid (14), both of which are non-cinnamate-class analogs of the substrate, acted as strong 4-HCD inducers, even at a 0.5 mM concentration. The 4-HCD-inducing activities of compounds 11 and 14 at 0.5 mM were 10-12-fold higher than that of substrate 1. Compound 11 maintained its 4-HCD-inducing activity toward cultured cells through the late-log and stationary phases, unlike 1 that induced 4-HCD only in the early log phase. SDS-PAGE electrophoresis of protein mixtures from the cultured cells exposed to any 4-HCD inducer indicated that the 21.5 kDa protein was always present.     

Quantitative comparison of rat hepatocyte functions in two improved culture systems with or without rat liver epithelial cell line

We quantitatively evaluated two recently-developed novel techniques for hepatocyte cultivation in a dish level; that is, spheroid culture and membrane-supported collagen (CN) gel sandwich culture, in terms of cellular maintenance, albumin secretion and 7-ethoxycoumarin (7EC) metabolism to 7-hydroxycoumarin (7HC) as a marker for cytochrome P450 IA1 activity in the presence and absence of rat liver epithelial cell line (RLEC) during one month of culture, together with conventional coculture with RLEC in CN-coated dishes as a control. RLEC prevented spheroid loss caused by its detachment from the culture dishes often occurring in pure culture. CN-gel sandwich by itself improved remarkably hepatocyte maintenance when compared with CN-gel free systems, thereby resulting in enhancement of overall functional expressions as compared with CN-gel free systems. RLEC in CN-gel sandwhich, however, reduced cellular sustainment probably due to its suppression of hepatocyte growth. Although there were no significant differences in albumin secretion per cell among the five cultures examined, CN-gel sandwich expressed markedly higher 7EC metabolizing activity per cell, where RLEC presence had a preferable influence. Consequently, membrane-supported CN-gel sandwich was the most superior technique for hepatocyte cultivation from the standpont of both cellular maintenance and its functional expressions per cell.     

蔓生卷柏的化学成分研究

采用Diaion HP-20、Toyopearl HW-40、Sephadex LH-20和硅胶等多种柱色谱对蔓生卷柏的化学成分进行分离纯化,结合现代波谱技术分离鉴定了5个化合物,分别为邻羟基苯丙酸(1)、邻羟基肉桂酸(2)、5-羟基香豆素(3)、阿曼托双黄酮(4)和(4E,6Z)-2,7-二甲基-8-羟基-辛二烯酸(5)。这些成分均为首次从该植物中分离得到。     

DFT-based molecular modeling and vibrational study of the La(III) complex of 3,3′-(benzylidene)bis(4-hydroxycoumarin)

Molecular modeling of the La(III) complex of 3,3′-(benzylidene)bis(4-hydroxycoumarin) (PhDC) was performed using density functional theory (DFT) methods at B3LYP/6-31G(d) and BP86/TZP levels. Both Stuttgart-Dresden effective core potential and ZORA approximation were applied to the La(III) center. The electron density distribution and the nucleophilic centers of the deprotonated ligand PhDC^2- in a solvent environment were estimated on the basis of Hirshfeld atomic charges, electrostatic potential values at the nuclei, and Nalewajski-Mrozek bond orders. In accordance with the empirical formula La(PhDC)(OH)(H₂O), a chain structure of the complex was simulated by means of two types of molecular fragment: (1) two La(III) cations bound to one PhDC^2- ligand, and (2) two PhDC^2- ligands bound to one La(III) cation. Different orientations of PhDC^2-, OH^- and H₂O ligands in the La(III) complexes were investigated using 20 possible [La(PhDC^2-)₂(OH)(H₂O)]^2- fragments. Energy calculations predicted that the prism-like structure based on “tail-head” cis-LML2 type binding and stabilized via HO...HOH intramolecular hydrogen bonds is the most probable structure for the La(III) complex. The calculated vibrational spectrum of the lowest energy La(III) model fragment is in very good agreement with the experimental IR spectrum of the complex, supporting the suggested ligand binding mode to La(III) in a chain structure, namely, every PhDC^2- interacts with two La(III) cations through both carbonylic and both hydroxylic oxygens, and every La(III) cation binds four oxygen atoms of two different PhDC^2-. Figure Low energy prism-like model fragment, [La(PhDC^2-)₂(OH)(H₂O)]^2-, optimized at BP86/TZP level of theory     

Hematoporphyrin photosensitization of epidermal microsomes results in destruction of cytochrome P-450 and in decreased monooxygenase activities and heme content

Epidermal microsomal cytochrome P-450 was rapidly degraded when microsomes were aerobically exposed to ultraviolet light in the presence of hematoporphyrin derivative (HPD). Destruction of microsomal cytochrome P-450 was accompanied by loss of heme content, and inhibition of catalytic activity of the monooxygenases, including aryl hydrocarbon hydroxylase and 7-ethoxycoumarin-O-deethylase. Destruction of cytochrome P-450 by photosensitized HPD was oxygen dependent. Quenchers of singlet oxygen, including 2,5 dimethylfuran, histidine, and B-carotene, largely pre- vented photodestruction of cytochrome P-450. Inhibitors of hydroxyl radical including benzoate and mannitol, protected microsomal cytochrome P-450 from destruction. Superoxide dismutase and catalase, scavengers of superoxide anion and hydrogen peroxide, respectively, had no protective effect. These results indicate that generation of singlet oxygen and hydroxyl radicals during hematoporphyrin photosensitization is associated with rapid degradation of cytochrome P-450 and heme in epidermal microsomes, and suggest a novel target for this type of tissue damage in the skin.     

Antioxidant aryl-prenylcoumarin, flavan-3-ols and flavonoids from Eysenhardtia subcoriacea

Antioxidant activity (AOA) assay-guided chemical analysis, using a rat pancreas homogenate model, of aerial parts from Eysenhardtia subcoriacea, led to isolation of the new compound subcoriacin (3-(2'-hydroxy-4',5'-methylendioxyphenyl)-6-(3'-hydroxymethyl-4'-hydroxybut-2'-enyl)-7-hydroxycoumarin) together with the known substances: (+)-catechin, (-)-epicatechin, (+)-afzelechin, eriodictyol, (+)-catechin 3-O-beta-D-galactopyranoside and quercetin 3-O-beta-D-galactopyranoside as bioactive constituents. The structure of the compound was determined from 1D and 2D NMR spectroscopic analyses. Additional known constituents were characterized. The bioactive compounds showed also moderate to strong radical scavenging properties against diphenylpicrylhydrazyl radical (DPPH). In addition, subcoriacin, (+)-catechin, (-)-epicatechin and (+)-afzelechin improved the reduced glutathione levels in rat pancreatic homogenate.     

Thallium(III)-mediated changes in membrane physical properties and lipid oxidation affect cardiolipin-cytochrome c interactions

Trivalent thallium (Tl(III)) is a highly toxic heavy metal through not completely understood mechanisms. Previously, we demonstrated that Tl(III) causes mitochondrial depolarization in PC12 cells leading to a decrease in cell viability. Given the role of the phospholipid cardiolipin (CL) in mitochondrial events, we evaluated in vitro the short- (2 min) and long- (60 min) time effects of Tl(III) (1-75 μM) on CL-containing membranes physical properties, and the consequences on cytochrome c binding to CL. After 2 min of incubation, Tl(III) significantly decreased liposome surface potential, lipid packing, and hydration of phosphatidylcholine:CL liposomes, while CL pK₂ decreased from 9.8 to 8.2. The magnitude of these changes was even higher after 60 min of incubation. While no Tl(III) was found bound to membranes, Tl(I) was present in the samples. Accordingly, significant oxidative damage to both CL fatty acids and polar headgroup was observed. Cytochrome c binding to CL was decreased in Tl(III)-treated liposomes. The present results indicate that Tl(III) interaction with CL-containing membranes affected their physical properties, caused lipid oxidation and CL hydrolysis, and resulted in a decrease of cytochrome c binding. If occurring in vivo, these effects of Tl(III) could partially account for mitochondrial dysfunction in cells exposed to this metal.     

Effect of “helper lipid” on lipoplex electrostatics

Lipoplexes, which are complexes between cationic liposomes (L⁺) and nucleic acids, are commonly used as a nucleic acid delivery system in vitro and in vivo. This study aimed to better characterize cationic liposome and lipoplex electrostatics, which seems to play a major role in the formation and the performance of lipoplexes in vitro and in vivo. We characterized lipoplexes based on two commonly used monocationic lipids, DOTAP and DMRIE, and one polycationic lipid, DOSPA—each with and without helper lipid (cholesterol or DOPE). Electrical surface potential (Ψ₀) and surface pH were determined using several surface pH-sensitive fluorophores attached either to a one-chain lipid (4-heptadecyl hydroxycoumarin (C17HC)) or to the primary amino group of the two-chain lipids (1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein (CFPE) and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-7-hydroxycoumarin) (HC-DOPE). Zeta potentials of the DOTAP-based cationic liposomes and lipoplexes were compared with Ψ₀ determined using C17HC. The location and relatively low sensitivity of fluorescein to pH changes explains why CFPE is the least efficient in quantifying the differences between the various cationic liposomes and lipoplexes used in this study. The fact that, for all cationic liposomes studied, those containing DOPE as helper lipid have the least positive Ψ₀ indicates neutralization of the cationic charge by the negatively-charged phosphodiester of the DOPE. Zeta potential is much less positively charged than Ψ₀ determined by C17HC. The electrostatics affects size changes that occurred to the cationic liposomes upon lipoplex formation. The largest size increase (based on static light scattering measurements) for all formulations occurred at DNA⁻/L⁺ charge ratios 0.5-1. Comparing the use of the one-chain C17HC and the two-chain HC-DOPE for monitoring lipoplex electrostatics reveals that both are suitable, as long as there is no serum (or other lipidic assemblies) present in the medium; in the latter case, only the two-chain HC-DOPE gives reliable results. Increasing NaCl concentrations decrease surface potential. Neutralization by DNA is reduced in a NaCl-concentration-dependent manner.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.