11-Hydroxylation of Protoberberine by the Novel Berberine-Utilizing Aerobic Bacterium Sphingobium sp. Strain BD3100.

Protoberberine alkaloids, including berberine, palmatine, and berberrubine, are produced by medicinal plants and are known to have various pharmacological effects. We isolated two berberine-utilizing bacteria, Sphingobium sp. strain BD3100 and Rhodococcus sp. strain BD7100, from soil collected at a natural medicine factory. BD3100 had the unique ability to utilize berberine or palmatine as the sole carbon and energy source. BD3100 produced demethyleneberberine in berberine-supplemented medium. In a resting-cell incubation with berberine, BD3100 produced 11-hydroxyberberine; the structure of 11-hydroxyberberine was determined by detailed analysis of NMR and MS spectroscopic data. α-Naphthoflavone, miconazole, and ketoconazole, which are known inhibitors of cytochrome P450, interfered with BD3100 metabolism of berberine in resting cells. Inhibition by miconazole led to the production of a new compound, 11-hydroxydemethyleneberberine. In a resting-cell incubation with palmatine, BD3100 generated 11-hydroxypalmatine. This work represents the first report of the isolation and characterization of novel berberine-utilizing aerobic bacteria for the production of 11-hydroxylation derivatives of berberine and palmatine.

Inhibition of cytochrome P450s enhances (+)-usnic acid cytotoxicity in primary cultured rat hepatocytes.

(+)-Usnic acid (UA) is consumed as a dietary supplement to promote weight loss; however, dietary supplements containing UA have been associated with clinical cases of severe liver injury. UA has been shown to be hepatotoxic in rats and is extensively metabolized by hepatic cytochrome P450s (CYPs); therefore, we examined if UA metabolism results in the formation of cytotoxic metabolites or if metabolism is a detoxification process in primary rat hepatocytes. When CYP activity was suppressed by the non-isoenzyme-selective inhibitor SKF-525A (20 µM), or the CYP1A inhibitor alpha-naphthoflavone (10 µM), or the CYP3A inhibitor ketoconazole (25 µM), the cytotoxicity of UA at 3~6 µM after 3~20 h of exposure was significantly increased as measured by lactate dehydrogenase (LDH) leakage. At 2 h after UA exposure, an earlier time point prior to LDH release, these CYP inhibitors potentiated UA-induced inhibition of cellular respiration as determined by the Clark type oxygen electrode. Cellular adenosine triphosphate (ATP) depletion by UA was also exacerbated by these CYP inhibitors. The CYP2B/2C inhibitor, ticlopidine at 20 µM, showed no effects in parallel experiments. These data demonstrate that UA is bio-transformed to less toxic metabolites in rat primary hepatocytes, probably mainly by CYP1A and 3A, but not 2B/2C. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

The binding of lignans, flavonoids and coumestrol to CYP450 aromatase: a molecular modelling study.

Androgens are transformed into aromatic estrogens by CYP450 aromatase in a three-step reaction consuming three equivalents of oxygen and three equivalents of NADPH. Estrogens are substrates for nuclear estrogen receptors (ERs) and play a key role in estrogen-dependent tumour cell formation and proliferation. Natural phytoestrogens are proved to be competitive inhibitors of aromatase enzyme at IC(50) values in micromolar levels. In order to understand the mechanisms involved in the binding of various phytoestrogens, we used our model of CYP450 aromatase to study the binding of phytoestrogens using molecular dynamics simulations with a bound phytoestrogen. The simulation trajectory was analysed to find the essential interactions which take place upon binding and a representative structure of the trajectory was minimized for docking studies. Sets of phytoestrogens, such as lignans, flavonoids/isoflavonoids and coumestrol, were docked into the aromatase active site and the binding modes were studied.

Development of a novel cytochrome p450 bioaffinity detection system coupled online to gradient reversed-phase high-performance liquid chromatography.

A high-resolution screening platform, coupling online affinity detection for mammalian cytochrome P450s (Cyt P450s) to gradient reversed-phase high-performance liquid chromatography (HPLC), is described. To this end, the online Cyt P450 enzyme affinity detection (EAD) system was optimized for enzyme (beta-NF-induced rat liver microsomes), probe substrate (ethoxyresorufine), and organic modifier (methanol or acetonitrile). The optimized Cyt P450 EAD system has first been evaluated in a flow injection analysis (FIA) mode with 7 known ligands of Cyt P450 1A1/1A2 (alpha-naphthoflavone, beta-naphthoflavone, ellipticine, 9-hydroxy-ellipticine, fluvoxamine, caffein, and phenacetin). Subsequently, IC50 values were online in FIA-mode determined and compared with those obtained with standardmicrosomal assay conditions. The IC50 values obtained with the online Cyt P450 EAD system agreed well with the IC50 values obtained in the standard assays. For high affinity ligands of Cyt P450 1A1/1A2, detection limits of 1 to 3 pmol injected (n=3; signal to noise [S/N]=3) were obtained. The individual inhibitory properties of ligands in mixtures of the ligands were subsequently investigated using an optimized Cyt P450 EAD system online coupled to gradient HPLC. Using the integrated online gradient HPLC Cyt P450 EAD platform, detection limits of 10 to 25 pmol injected (n=1; S/N=3) were obtained for high-affinity ligands. It is concluded that this novel screening technology offers new perspectives for rapid and sensitive screening of individual compounds in mixtures exhibiting affinity for liver microsomal Cyt P450s.

7,8-Benzoflavone: a phytotoxin from root exudates of invasive Russian knapweed.

Root exudates from Acroptilon repens (Russian knapweed) were found to be phytotoxic and the phytotoxin in the exudate was identified as 7,8-benzoflavone (alpha-naphthoflavone), (1), not previously known as a natural product. In tests on growing seedlings both 1 and its isomer 5,6-benzoflavone (2) were phytotoxic. Flavone, a structural analog of 1 and a known granular leaf and stem exudate of other plant species, was also phytotoxic and more potent than 1 or 2.

Prevention of chronic alcohol and nicotine-induced azospermia, sterility and decreased libido, by a novel tri-substituted benzoflavone moiety from Passiflora incarnata Linneaus in healthy male rats.

Excessive long term consumption of alcohol and nicotine have serious detrimental effects upon the libido, fertility, and sperm count in male species. The present work describes the beneficial effects of a novel tri-substituted benzoflavone moiety (BZF) isolated from Passiflora incarnata Linneaus, the phyto-chemical isolation, spectroscopic elucidation, and multifarious biological activities of which have recently been reported by the authors. The BZF moiety has been reported to increase libido, sperm count, and sexual fertility in 2 years old male rats at 10 mg/kg, po dose, in the one of our previous studies. Presently, the BZF moiety has been evaluated against chronic ethanol- and nicotine-induced decrease in libido, sexual fertility and mating efficiency in healthy male rats. The male rats were given ethanol (3 g/kg, po) A, nicotine (2 mg/kg, sc) N, alcohol-nicotine combinations (AN) alone, and also with 10 mg/kg po dose of BZF (concurrent administrations). These treatments were given for 30 days. At the end of treatments, it was observed that rat groups A, N, and AN had no libido (evaluated by mounting behaviour), declined sperm count, and consequently no mating efficiency or fertility (upon pairing with pro-estrus female rats). However, the rats which were given 10 mg/kg BZF along-with nicotine (NP group), alcohol (AP group), and alcohol-nicotine combination (ANP) exhibited significant libido-oriented mounting behaviour, increased sperm count (significantly comparable to the control group), and increased fertilization potential. The rats having decreased sperm count, libido and fertilization potential due to chronic administration of alcohol, nicotine and alcohol-nicotine combinations, i.e., rats of A, N, and AN groups were again subdivided and were given 10 mg/kg BZF for 7 days. This treatment confirmed that BZF speeds up the restoration of sexuality in rats upon cessation of the administration of substances like alcohol, nicotine and alcohol-nicotine combinations, which have severe detrimental effects upon male sexuality, fertility and vigour. BZF, the strongest inhibitor of aromatase enzyme, when administered concurrently with substances like alcohol and nicotine restores sexual virility, libido and vigour in male rats by maintaining the blood-testosterone levels to be high.

Suppression of cell cycle progression by flavonoids: dependence on the aryl hydrocarbon receptor.

Some flavonoids are ligands of the aryl hydrocarbon receptor (AHR) and cause cell cycle arrest. The dependency of the cytostatic effects of five flavonoids (flavone, alpha-naphthoflavone, apigenin, 3'-methoxy-4'-nitroflavone and 2'-amino-3'-methoxyflavone) on a functional AHR was examined in AHR-containing rat hepatoma 5L cells and an AHR-deficient cell line (BP8) derived from the 5L line. The potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was cytostatic to the 5L line due to the induction of a G(1) arrest and dramatically elevated steady-state levels of CYP1A1 mRNA. TCDD affected neither the proliferation nor CYP1A1 mRNA contents of BP8 cells. With the exception of apigenin, the flavonoids under study induced G(1) arrest in both 5L and BP8 cells when used at concentrations at which they functioned as AHR agonists, but not antagonists. Apigenin-treated 5L and BP8 cultures primarily arrested in G(2)/M. The AHR-containing murine hepatoma cell line 1c1c7 arrested following exposure to AHR agonist concentrations of flavone and alpha-naphthoflavone, but not TCDD. Unlike the G(1) arrest observed in 5L cultures, the latter two flavonoids caused principally G(2)/M arrest in 1c1c7 cells. These studies demonstrate that the cytostatic activities of flavonoids do not require the AHR and the site of checkpoint arrest with a specific flavonoid can vary with cell type.

[Chemical constituents of Stellera chamaejasme L].

Twelve compounds were isolated from the roots of Stellera chamaejasma. Five of them were elucidated by spectroscopic and chemical methods as 3', 14-dimethyl-4', 11-dimethoxy-5, 7-dihydroxybenzoflavanone (10), daphnetin (1), umbelliferone (2), daucosterol (5) and beta-sitosterol (3). Compound 10 is new. Compounds 1, 2, 5, 10 were isolated for the first time from S. chamaejasme.