OBJECTIVETo screen the antimalarial compounds of daphnetin derivatives against Plasmodium falciparum in vitro.

METHODPlasmodium faciparum (FCC1) was cultured in vitro by a modified method of Trager and Jensen. Antimalarial compounds were screened by microscopy-based assay and microfluorimetric method.

RESULTSDA79 and DA78 showed potent antimalarial activity against Plasmodium falciparum cultured in vitro.

CONCLUSIONThough the relationship between the structures of daphnetin derivatives and their antimalarial activities has not been clarified yet, this study may provide a new direction for discovery of more potential antimalarial compounds.

Animals ; Antimalarials ; chemistry ; pharmacokinetics ; pharmacology ; Plasmodium falciparum ; drug effects ; Umbelliferones ; chemistry ; pharmacokinetics ; pharmacology

BACKGROUNDAlthough there are several drugs and drug combinations for the treatment of Pneumocystis carinii (P. carinii) pneumonia, all drugs have the toxicity as well as low efficacy. Iron chelators have been proposed as a source of new drugs for combating these infections. We hypothesized that iron chelators would suppress the growth of P. carinii by deprivation of the nutritional iron required for growth. In this study, a short-term axenic culture system of P. carinii was established. Daphnetin (7,8-dihydroxycoumarin), a known iron chelator, was demonstrated to exhibit in vitro activity against P. carinii in this system.

METHODSP. carinii organisms were obtained from the lungs of immunosuppressed rats. The culture system consisted of Iscove Dulbecco Eagle's Minimum Essential Medium (IMDM), supplemented with S-adenosyl-L-methionine, N-acetylglucosamine, putrescine, L-cysteine, L-glutamine, 2-mercaptoethanol, and fetal bovine serum, and was maintained at 37 degrees C, in 5% CO(2), 95% O(2), at the optimal pH of 8.0. The culture system was used to assess the effect of daphnetin on the proliferation of P. carinii organisms. The ultrastructures of the treated organisms were observed by transmission electron microscopy.

RESULTSThe number of cysts and trophozoites increased 8- to 9-fold and 11- to 12-fold, respectively, after 10 days of culture. Daphnetin was found to suppress the growth of P. carinii in a dose-dependent manner at concentrations between 1 micromol/L and 20 micromol/L. The inhibitory activity was suppressed by the chelation of daphnetin with ferrous sulfate in a 2:1 molar ratio, but it was not suppressed by mixing the culture medium with magnesium sulfate. Reduction of P. carinii numbers after treatment with daphnetin correlated with morphological changes in the organisms, as determined by transmission electron microscopy.

CONCLUSIONSDaphnetin can suppress the growth of P. carinii in vitro. The efficacy of daphnetin in suppressing the the growth of P. carinii in vitro is related to its ability to chelate iron.

Iron ; physiology ; Iron Chelating Agents ; pharmacology ; Microscopy, Electron ; Pneumocystis carinii ; drug effects ; growth & development ; ultrastructure ; Umbelliferones ; pharmacology

OBJECTIVETo research the influence of glycyrrhiza extract on the pharmacokinetics characteristic parameters of daphnetin, which was aimed to explore the rationality of concert application of drugs.

METHODThe rats received intragastric administration of daphnetin and glycyrrhiza extract containing the same daphnetin respectively. The blood concentration of daphnetin was assayed by LC-MS. The data was processed by program DAS2.1.1.

RESULTGlycyrrhiza extract can reduce the t(1/2), tmax and Ke of daphnetin, while increased the Ka and AUC(0-infinity).

CONCLUSIONGlycyrrhiza extract promoted the oral absorption of daphnetin, slowed down the elimination and increased the biological availability.

Animals ; Drug Interactions ; Drugs, Chinese Herbal ; pharmacology ; Glycyrrhiza ; chemistry ; Rats ; Rats, Sprague-Dawley ; Tissue Distribution ; drug effects ; Umbelliferones ; pharmacokinetics

This study aimed to investigate the effects of hypoxia on RhoA/Rho-kinase (ROCK) signaling pathway and autophagy in pulmonary artery smooth muscle cells (PASMCs), and to explore the underlying mechanism of Umbelliferone (Umb) in ameliorating chronic hypoxic pulmonary hypertension. PASMCs were cultured from Sprague-Dawley (SD) rats and randomly divided into control group, hypoxia group, hypoxia + Umb intervention group and normoxia + Umb intervention group. Alpha smooth muscle actin (α-SMA) and LC3 were assessed by immunofluorescence staining. Protein expression of RhoA, ROCK2, p-MYPT1, LC3-II, Beclin-1, p62, C-Caspase 3, Bax and Bcl-2 was analyzed by Western blotting. In in vivo study, SD rats were divided into control group, hypoxia group and hypoxia + Umb intervention group. Weight ratio of the right ventricle (RV)/left ventricle plus septum (LV+S) was detected, and pulmonary arterial morphological features were examined by HE staining. The results indicated that compared with the control group, the LC3-II/LC3-I ratio and expression of Beclin-1 were significantly increased, while p62 expression was significantly decreased, and the expressions of RhoA, ROCK2 and p-MYPT1 were significantly increased in PASMCs of hypoxia group (P < 0.05). The changes of LC3-II/LC3-I ratio, the expressions of Beclin-1, p62, RhoA, ROCK2 and p-MYPT1 in PASMCs were reversed by Umb treatment (P < 0.05). Consistently, the pulmonary arterial wall was thickened and the RV/(LV+S) ratio was increased in hypoxic rats, which were significantly improved by Umb treatment (P < 0.05). These results suggest that Umb can improve hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy in PASMCs.
Animals ; Autophagy ; Beclin-1/pharmacology* ; Hypertension, Pulmonary/etiology* ; Hypoxia/complications* ; Myocytes, Smooth Muscle/metabolism* ; Pulmonary Artery ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Umbelliferones/pharmacology* ; rho-Associated Kinases/pharmacology*

OBJECTIVETo compare the schizontocidal activity of recrystallized or crude daphnetin against malaria parasites in vivo.

METHODSSchizontocidal activity of recrystallized or crude daphnetin at various dosages was assessed in mice infected with Plasmodium berghei ANKA using a "4-day suppress assay".

RESULTSThe comparison of the reduction rate of parasitemia caused by either recrystallized or crude dephnetin showed that ED(50) of crude daphnetin was 18.36 mg/kg, with 95% confidence limit of 5.96-56.54 mg/kg while ED50 of recrystallized daphnetin was 11.46 mg/kg, with 95% confidence limit of 8.63-15.22 mg/kg.

CONCLUSIONThe results indicate that the efficacy of recrystallized daphnetin is 37.6% higher than that of crude daphnetin.

Animals ; Antimalarials ; pharmacology ; Chromatography, High Pressure Liquid ; Dose-Response Relationship, Drug ; Malaria ; drug therapy ; parasitology ; Mice ; Parasitic Sensitivity Tests ; Plasmodium berghei ; drug effects ; Umbelliferones ; pharmacology

OBJECTIVETo investigate phase I and phase II enzyme activities in drug metabolism with tissue-like cultures of rat hepatocytes.

METHODSThe gel entrapment and spheroid culture of hepatocytes were used as tissue-like cultures and the monolayer culture was used as a control. The metabolism of phenacetin and 7-hydroxycoumarin (7-HC) was evaluated as the activities of phase I and phase II enzymes after incubated in medium for a period of time. The metabolites were assayed by HPLC. The hepatocytes were exposed to beta-naphthoflavone (BNF, 50 micromol x L(-1)) before the phase I and phase II enzyme activities were analyzed.

RESULTIn monolayer culture, phase I parameters decreased quickly and did not detected at d 5, and the phase II enzyme activities were not detected at d 7. In other two models of tissue-like cultures, the activities of phase I and phase II enzyme maintained at 32%-50% of the initial value at d 7. Paracetamol formation rates in spheroid culture maintained at 96% of that at d 1. The phase I enzyme activities of the spheroid culture were maintained from d 1 to d 3 at a level of 2.7-3.9-fold higher than the monolayer culture. After exposure to BNF the activities on phase I enzyme increased by about 2.5-fold (P <0.05) in all three culture models, while the increase in phase II enzyme was not significant.

CONCLUSIONThe gel entrapment culture and spheroid culture are superior to the monolayer culture in maintenance of drug metabolic enzyme activities.

Animals ; Cells, Cultured ; Cytochrome P-450 Enzyme System ; metabolism ; Enzyme Activation ; Female ; Hepatocytes ; cytology ; Male ; Phenacetin ; metabolism ; Rats ; Rats, Sprague-Dawley ; Umbelliferones ; metabolism ; beta-Naphthoflavone ; pharmacology

OBJECTIVETo investigate the chemical constituents from the branch of Fraxinus sieboldiana, and evaluate their antioxidative activity.

METHODThe chemical constituents were isolated and purified by chromatographic techniques over silica gel, macroporous adsorbent resin, Sephadex LH-20, and preparative HPLC. Structures of the compounds were identified by spectroscopic methods. The antioxidant activity was evaluated by Fe(+2)-cystine induced rat liver microsomal lipid peroxidation.

RESULTEight coumarins were obtained and their structures were elucidated as esculin (1) , esculetin (2), fraxin (3), fraxetin (4), 6, 7-di-O-beta-D-glucopyranosylesculetin (5), scopoletin (6), cleomiscosin D (7) and cleomiscosin B (8). At a concentration of 10(-6) mol x L(-1), compound 4 showed antioxidative activity inhibiting Fe(+2)-cystine induced rat liver microsomal lipid peroxidation with inhibitory rate of 60%.

CONCLUSIONCompounds 5, 7 and 8 were obtained from the genus Fraxinus for the first time. Compound 4 showed remarkable antioxidative activity, which was higher than that of VE (35%).

Animals ; Antioxidants ; chemistry ; pharmacology ; Coumarins ; chemistry ; pharmacology ; Fraxinus ; chemistry ; Lipid Peroxidation ; drug effects ; Magnetic Resonance Spectroscopy ; Microsomes, Liver ; drug effects ; metabolism ; Rats ; Scopoletin ; chemistry ; pharmacology ; Spectrometry, Mass, Electrospray Ionization ; Umbelliferones ; chemistry ; pharmacology

OBJECTIVETo investigate the dual role of daphnetin in suppressing high mobility group box-1 protein (HMGB1) release and blocking HMGB1-induced inflammatory response.

METHODSMurine macrophage RAW264.7 cells were cultured in the presence of daphnetin, lipopolysaccharide (LPS), or both. HMGB1 release from the cells was determined using ELISA, and phosphorylations of JAK1/2 and of STAT1 were detected by Western blotting. Human monocytic THP-1 cells exposed to daphnetin, rhHMGB1, or both were examined for NO production using a NO detection kit, for the release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) using ELISA, and for expressions of iNOS, COX-2 and phosphorylated p38, ERK, and JNK with Western blotting.

RESULTSDaphnetin dose-dependently reduced the release of HMGB1 in RAW264.7 cells and suppressed rhHMGB1-induced iNOS and COX-2 expressions and release of TNF-α, IL-6, PGE2, and NO in THP-1 cells. Western blotting revealed that daphnetin significantly down-regulated the phosphorylations of JAK-STAT1 pathway in LPS-stimulated RAW264.7 cells but did not suppress the phosphorylations of MAPKs signaling pathway induced by rhHMGB1 in THP-1 cells.

CONCLUSIONDaphnetin can reduce the release of HMGB1 and suppress HMGB1-induced inflammatory response. In RAW264.7 cells, daphnetin inhibited LPS induced HMGB1 release is at least partly mediated by suppressing JAK-STAT1 signaling pathway activation.

Animals ; Cell Line ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; metabolism ; HMGB1 Protein ; metabolism ; Humans ; Inflammation ; metabolism ; Interleukin-6 ; metabolism ; Janus Kinase 1 ; metabolism ; Lipopolysaccharides ; Macrophages ; drug effects ; Mice ; Monocytes ; drug effects ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; RAW 264.7 Cells ; STAT1 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism ; Umbelliferones ; pharmacology