Discovery of a naturally occurring broad-spectrum inhibitor against gut bacterial ß-glucuronidases from Ginkgo biloba.

Gut bacterial ß-glucuronidases (GUS) play an important role in deconjugation of various O-glucuronides, which are tightly linked with the drug-induced intestinal toxicity. Increasing evidence has indicated that inhibition of bacterial GUS could alleviate GUS-associated intestinal toxicity, but the potent and broad-spectrum inhibitors against multiple bacterial GUS have been rarely reported. This study aimed to find potent and broad-spectrum GUS inhibitors from Ginkgo biloba. It was found that amentoflavone displayed relatively strong inhibition on three GUS including CpGUS, SpasGUS and EcGUS. Further investigations demonstrated that amentoflavone could inhibit GUS-mediated PNPG hydrolysis in a dose-dependent manner with IC50 values of 2.36 µM, 2.88 µM and 3.43 µM for CpGUS, SpasGUS and EcGUS, respectively. Inhibition kinetic studies showed that amentoflavone functioned as a non-competitive inhibitor against all tested GUS with Ki values of less than 2 µM. Docking simulations indicated that amentoflavone could tightly bind on allosteric sites of three GUS mainly via hydrogen bonding interactions, and the number of hydroxyl groups of amentoflavone played crucial roles in these interactions. Collectively, our findings suggested that amentoflavone was a potent broad-spectrum inhibitor against bacterial GUS, which can be used as a promising lead compound for developing novel agents to alleviate GUS-associated intestinal toxicity.

Pancreatic lipase inhibitory constituents from Fructus Psoraleae.

Pancreatic lipase (PL), a crucial enzyme in the digestive system of mammals, has been proven as a therapeutic target to prevent and treat obesity. The purpose of this study is to evaluate and characterize the PL inhibition activities of the major constituents from Fructus Psoraleae (FP), one of the most frequently used Chinese herbs with lipid-lowering activity. To this end, a total of eleven major constituents isolated from Fructus Psoraleae have been obtained and their inhibition potentials against PL have been assayed by a fluorescence-based assay. Among all tested compounds, isobavachalcone, bavachalcone and corylifol A displayed strong inhibition on PL (IC50 < 10 µmol·L-1). Inhibition kinetic analyses demonstrated that isobavachalcone, bavachalcone and corylifol A acted as mixed inhibitors against PL-mediated 4-methylumbelliferyl oleate (4-MUO) hydrolysis, with the Ki values of 1.61, 3.77 and 10.16 µmol·L-1, respectively. Furthermore, docking simulations indicated that two chalcones (isobavachalcone and bavachalcone) could interact with the key residues located in the catalytic cavity of PL via hydrogen binding and hydrophobic interactions. Collectively, these finding provided solid evidence to support that Fructus Psoraleae contained bioactive compounds with lipid-lowering effects via targeting PL, and also suggested that the chalcones in Fructus Psoraleae could be used as ideal leading compounds to develop novel PL inhibitors.

Structure-activity relationships of flavonoids as natural inhibitors against E. coli ß-glucuronidase.

Bacterial ß-glucuronidases play key roles in the deconjugation of a variety of endogenous and drug glucuronides, thus have been recognized as important targets to modulate the enterohepatic circulation of various glucuronides. In this study, more than 30 natural flavonoids were collected and their inhibitory effects against E. coli ß-glucuronidase (EcGUS) were assayed. The results demonstrated that some flavonoids including scutellarein, luteolin, baicalein, quercetin and scutellarin displayed strong to moderate inhibitory effects against EcGUS, with the IC50 values ranging from 5.76 µM to 29.64 µM, while isoflavones and dihydroflavones displayed weak inhibitory effects against EcGUS. Further investigation on inhibition kinetics revealed that scutellarein and luteolin functioned as potent competitive inhibitors against EcGUS-mediated PNPG hydrolysis, with the Ki values less than 3.0 µM. Molecular docking simulations demonstrated that scutellarein and luteolin could be well-docked into the catalytic site of EcGUS, while the binding areas of these two natural inhibitors on EcGUS were highly overlapped with that of PNPG on EcGUS. Additionally, the structure-inhibition relationships of natural flavonoids against EcGUS are also summarized, which will be very helpful for the medicinal chemists to design and develop more potent flavonoid-type inhibitors against EcGUS.

CYP1A1 and CYP1B1 expressions in medulloblastoma cells are AhR-independent and have no direct link with resveratrol-induced differentiation and apoptosis.

Resveratrol induces apoptosis and regulates CYP1A1 and CYP1B1 expression in human medulloblastoma cells. To elucidate the potential correlation of their expressions with the anti-medulloblastoma effects of resveratrol, human medulloblastoma cells, UW228-3, were treated with CYP1A1 selective inhibitor (alpha-naphthoflavone, alpha-NF), selective CYP1A1/1A2 inducer (beta-naphthoflavone, beta-NF) and their combination with resveratrol, respectively. The influences of those treatments on the expressions of CYP1A1, 1A2 and 1B1 as well as the cell growth, differentiation and death were analyzed. It was found that neither alpha-NF nor beta-NF had any effect on cell growth. alpha-NF inhibited resveratrol-induced CYP1A1 expression without interfering cell differentiation and apoptosis. beta-NF could up-regulate resveratrol-induced CYP1A1 expression but not enhance the anti-cancer effects of resveratrol. CYP1A2 was undetectable in the cells irrespective to the treatments. Aryl hydrocarbon receptor (AhR) was absent in UW228-3 cells under normal culture and treated with resveratrol but induced by both alpha- and beta-NF. Immunohistochemical examination performed on 11 pairs of human medulloblastoma and noncancerous cerebellar tissues revealed that AhR was undetectable in either of them, whereas CYP1A1 was expressed in cerebellum but down-regulated or diminished in their malignant counterparts. Our data suggest for the first time that CYP1A1 and 1B1 expressions in human medulloblastoma cells are AhR-independent and have no direct links with resveratrol-induced differentiation and apoptosis. Appearance of CYP1A1 expression may reflect a more maturated status and a better prognosis of medulloblastomas.

Resveratrol promotes differentiation and induces Fas-independent apoptosis of human medulloblastoma cells.

Resveratrol has tumor-suppressive activities in some in vitro and in vivo experimental systems but its effect on medulloblastoma cells is still unknown. In this study, resveratrol was used to treat four human medulloblastoma cell lines (Med-3, UW228-1, -2 and -3) and its effects on cell growth, differentiation and death were examined by multiple approaches. Expression of Fas, FasL and caspase-3 in the cells without and with resveratrol treatments was examined by immunocytochemical staining and mRNA in situ hybridization and the influence of anti-Fas antibody (200 ng/ml) in cell growth and survival was determined as well. The results demonstrated that resveratrol could suppress growth, promote differentiation and commit its target cells to apoptosis in time- and dose-related fashions. Fas was constitutively expressed but FasL was undetectable in the four lines in spite of resveratrol treatment. Anti-Fas antibody (200 ng/ml) neither inhibited growth nor induced apoptosis of those cell lines. Up-regulated caspase-3 was found in resveratrol-treated populations and appearance of its cleaved form was closely associated with the apoptotic event. These findings suggest for the first time that resveratrol is an effective anti-medulloblastoma agent that kills medulloblastoma cells through a Fas-independent pathway.

Differential regulation of CYP1A1 and CYP1B1 expression in resveratrol-treated human medulloblastoma cells.

Resveratrol induces differentiation and Fas-independent apoptosis of medulloblastoma cells by a largely unknown mechanism. CYP1A1 and 1B1 are involved in resveratrol-mediated tumor suppression but their expression in medulloblastoma cells and their relevance to anti-medulloblastoma activity of resveratrol have not been described. The statuses of CYP1A1 and 1B1 in UW228-3 medulloblastoma cells without and with resveratrol treatments were elucidated in this study with ethoxyresorufin O-deethylation assay, followed by RT-PCR, immunocytochemical staining and Western blot hybridization. CYP1A1/1B1 enzymatic activity was low in UW228-3 cells but became several folds higher upon resveratrol treatments. CYP1A1 was undetectable and CYP1B1 was expressed in normally cultured cells. Accompanied by the increased fraction of apoptosis, enhanced CYP1A1 and downregulated CYP1B1 were observed in resveratrol-treated cells in time- and dose-related fashions. Our results demonstrate for the first time that in the medulloblastoma cell system, CYP1A1 upregulation is paralleled with resveratrol-induced differentiation and apoptosis, while CYP1B1 may not be an essential element in metabolic activation of resveratrol in those cells. CYP1A1 and 1B1 are resveratrol response genes and potential chemosensitive markers of medulloblastoma cells.