Ken-ichi Nakashima

Berberine Is a Novel Type Efflux Inhibitor Which Attenuates the MexXY-Mediated Aminoglycoside Resistance in Pseudomonas aeruginosa.

The emergence and spread of multidrug-resistant P. aeruginosa infections is of great concern, as very few agents are effective against strains of this species. Methanolic extracts from the Coptidis Rhizoma (the rhizomes of Coptis japonica var. major Satake) or Phellodendri Cortex (the bark of Phellodendron chinense Schneider) markedly reduced resistance to anti-pseudomonal aminoglycosides (e.g., amikacin) in multidrug-resistant P. aeruginosa strains. Berberine, the most abundant benzylisoquinoline alkaloid in the two extracts, reduced aminoglycoside resistance of P. aeruginosa via a mechanism that required the MexXY multidrug efflux system; berberine also reduced aminoglycoside MICs in Achromobacter xylosoxidans and Burkholderia cepacia, two species that harbor intrinsic multidrug efflux systems very similar to the MexXY. Furthermore this compound inhibited MexXY-dependent antibiotic resistance of other classes including cephalosporins (cefepime), macrolides (erythromycin), and lincosamides (lincomycin) demonstrated using a pseudomonad lacking the four other major Mex pumps. Although phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux inhibitor, antagonized aminoglycoside in a MexXY-dependent manner, a lower concentration of berberine was sufficient to reduce amikacin resistance of P. aeruginosa in the presence of PAβN. Moreover, berberine enhanced the synergistic effects of amikacin and piperacillin (and vice versa) in multidrug-resistant P. aeruginosa strains. Thus, berberine appears to be a novel type inhibitor of the MexXY-dependent aminoglycoside efflux in P. aeruginosa. As aminoglycosides are molecules of choice to treat severe infections the clinical impact is potentially important.

Identification of a naturally occurring retinoid X receptor agonist from Brazilian green propolis.

BACKGROUND Brazilian green propolis (BGP), a resinous substance produced from Baccharis dracunculifolia by Africanized honey bees (Apis mellifera), is used as a folk medicine. Our present study explores the retinoid X receptor (RXR) agonistic activity of BGP and the identification of an RXR agonist in its extract. METHODS RXRα agonistic activity was evaluated using a luciferase reporter gene assay. Isolation of the RXRα agonist from the ethanolic extract of BGP was performed using successive silica gel and a reversed phase column chromatography. The interaction between the isolated RXRα agonist and RXRα protein was predicted by a receptor-ligand docking simulation. The nuclear receptor (NR) cofactor assay was used to estimate whether the isolated RXRα agonist bound to various NRs, including RXRs and peroxisome proliferator-activated receptors (PPARs). We further examined its effect on adipogenesis in 3T3-L1 fibroblasts. RESULTS We identified drupanin as an RXRα agonist with an EC50 value of 4.8 ± 1.0 μM. Drupanin activated three RXR subtypes by a similar amount and activated PPARγ moderately. Additionally, drupanin induced adipogenesis and elevated aP2 mRNA levels in 3T3-L1 fibroblasts. CONCLUSIONS Drupanin, a component of BGP, is a novel RXR agonist with slight PPARγ agonistic activity. GENERAL SIGNIFICANCE This study revealed for the first time that BGP activates RXR and drupanin is an RXR agonist in its extract.

Xanthones from the roots of Maclura cochinchinensis var. gerontogea and their retinoic acid receptor-α agonistic activity.

A new xanthone (1) and a new naturally occurring xanthone (2) were isolated from the roots of Maclura cochinchinensis (Lour.) Corner var. gerontogea (Sieb. et Zucc.) Ohashi together with 10 known xanthones (3-12). Their structures were established by spectroscopic analyses including 1D and 2D NMR. Their retinoic acid receptor-α agonistic activity was evaluated using a luciferase reporter assay. Compound 2, gerontoxanthone A (3), gerontoxanthone B (4), and cudraxanthone I (11) showed moderate concentration-dependent activity. Furthermore, these four xanthones synergistically increased transcriptional activity in this assay in the presence of bexarotene, an RXR agonist.

Rexinoids Isolated from Sophora tonkinensis with a Gene Expression Profile Distinct from the Synthetic Rexinoid Bexarotene

The retinoid X receptor (RXR) plays a critical role in transcriptional regulation via formation of an RXR homodimer or heterodimers with partner nuclear receptors. Despite the numerous beneficial effects, only a limited number of naturally occurring RXR agonists are known. In this report, two prenylated flavanones (1 and 2) isolated from Sophora tonkinensis were identified as new rexinoids that preferentially activated RXRs, relative to the retinoic acid receptor. The activities of 1 and 2 were the most potent among naturally occurring rexinoids, yet 2 orders of magnitude lower than the synthetic rexinoid bexarotene. Compounds 1 and 2 activated particular RXR heterodimers in a manner similar to bexarotene. A microarray assay followed by quantitative real-time polymerase chain reaction analyses on RNAs isolated from C2C12 myotubes treated with 1 or 2 demonstrated that they significantly increased mRNA levels of lipoprotein lipase, angiopoietin-like protein 4, and heme oxygenase-1. In contrast, bexarotene preferentially potentiated transcription of genes involved in lipogenesis and lipid metabolism such as sterol regulatory element-binding protein-1, fatty acid synthase, and apolipoprotein D by a liver X receptor agonist. In this study, we have demonstrated that two newly identified naturally occurring rexinoids, 1 and 2, possess properties different from bexarotene.

Atranorin and lecanoric acid antagonize TCDD-induced xenobiotic response element-driven activity, but not xenobiotic response element-independent activity

Lichens are symbiotic organisms that consist of fungi and photosynthetic symbionts (algae and/or cyanobacteria). Previous studies of their constituents suggested lichens produce many kinds of aromatic secondary metabolites, such as depsides, quinones, and dibenzofurans. In this study, we evaluated the aryl hydrocarbon receptor (AhR) antagonistic activity of 17 lichen substances and demonstrated that atranorin (1) and lecanoric acid (2), isolated from Parmotrema tinctorum Hale, showed an inhibitory effect on luciferase activity increased by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), using an XRE-driven pX4TK-Luc reporter gene assay. In addition, CYP1A1 mRNA and protein levels increased by TCDD were also suppressed by 1 and 2. Conversely, neither 1 nor 2 antagonized the suppressive effect of TCDD on interleukin (IL)-1β-induced acute-phase response (APR) gene expression. Thus, we concluded that 1 and 2 were selective AhR modulators that antagonize XRE-dependent activity, but not XRE-independent activity. However, 1 has different characteristics to 2 in that 1 alone showed a suppressive effect on IL-1β-induced APR gene expression in a similar fashion to TCDD.

Gerontoxanthone B from Maclura cochinchinensis var. gerontogea exhibits anti-inflammatory potential as an aryl hydrocarbon receptor agonist

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcriptional factor belonging to the basic helix-loop-helix-Per-Ahr/Arnt-Sim family. In this study, we evaluated the AhR agonistic activities of 12 xanthones isolated from the roots of M. cochinchinensis var. gerontogea using HepG2 cells transfected with pX4TK-Luc reporter plasmids. Gerontoxanthone B (GXB) showed the most potent activity at a concentration of 10μM, and the activity was inhibited by AhR antagonists such as GNF-351. GXB also increased cytochrome P450 1A1 mRNA and protein levels in HepG2 cells. Similar to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin, however, GXB suppressed the IL-1β-induced mRNA level of SAA1, an acute-phase response gene that is up-regulated AhR-dependently but XRE-independently. Thus, GXB shows XRE-dependent transcriptional activity and XRE-independent activity involving AhR.

Yuccalides A–C, three new phenolic compounds with spiro-structures from the roots of Yucca gloriosa

Three new phenolic compounds, yuccalides A-C (1-3), were isolated from the roots of Yucca gloriosa L., along with four known compounds (4-7). The structures of the new compounds were established by extensive NMR spectroscopic analyses. Inducible nitric oxide synthase (iNOS) mRNA levels induced by lipopolysaccharide (LPS) in mouse macrophage-like RAW 264.7 cells were effectively suppressed by compounds 2, 4, and 6, all of which had the (2R*, 3R*)-configuration. IL-1β and IL-6 mRNA levels induced by LPS were significantly attenuated by compounds 4, 5, and 6, but not by 2.

Induction of beige adipocytes by naturally occurring β3-adrenoceptor agonist p-synephrine

Abstract The prevalence of obesity and its associated diseases is increasing worldwide, and the therapeutic potential of increasing energy expenditure through differentiation or activation of beige adipocytes has attracted much interest. Therefore, we explored naturally occurring compounds that induce beige adipocytes by screening for activity to induce mRNA expression of uncoupling protein 1 (UCP1) in stromal vascular fraction (SVF) cells cultured in beige adipocyte differentiation medium. Through screening, p‐synephrine, a compound isolated from Citrus unshiu Marcov., was found to be an active compound that increased UCP1 mRNA expression in a dose‐dependent manner from a concentration of 3.12 &mgr;M, which induced morphological changes specific for beige adipocytes. Similar effects were also observed in SVF cells prepared from db/db obese mice. While investigating the underlying mechanism of p‐synephrine‐induced beige adipocyte differentiation, we found that the effects of p‐synephrine were abolished by the &bgr;3‐adrenoceptor antagonist SR58894. Intriguingly, p‐synephrine increased UCP1 mRNA levels in SVF cells cultured in beige adipocyte differentiation medium lacking insulin to an extent different from those by the &bgr;‐agonist isoprenaline. Furthermore, phosphatidylinositol 3‐kinase inhibitor LY294002 decreased isoprenaline‐induced UCP1 mRNA levels in the early phase of beige adipocyte differentiation and p‐synephrine‐induced UCP1 mRNA levels in fully differentiated beige adipocytes. Thus, p‐synephrine appears to elicit signals via &bgr;3‐adrenoceptor combined with some part of the insulin signaling pathway, finally resulting in efficient stimulation of beige adipocyte differentiation with the support of certain beige adipocyte differentiation‐inducing factors. The present results suggest the potential of p‐synephrine for prophylaxis and treatment of obesity and its associated diseases.

Ameliorative effect of panaxynol on the reduction in high-molecular-weight adiponectin secretion from 3T3-L1 adipocytes treated with palmitic acids

ABSTRACT Reduced plasma levels of the high‐molecular weight (HMW) form of adiponectin, rather than total adiponectin levels, have been shown to be closely associated with various metabolic diseases including insulin resistance, type 2 diabetes, and cardiovascular disease. Therefore, we sought to explore active, naturally occurring compounds that promote the recovery of HMW adiponectin secretion suppressed by palmitic acid in our model. A total of 90 crude drug extracts were screened for the ability to augment HMW adiponectin secretion from 3T3‐L1 adipocytes treated with palmitic acid. Panaxynol was isolated from Saposhnikovia divaricata as an active compound with HMW adiponectin promoting properties. Peroxisome proliferator‐activated receptor‐&ggr; (PPAR&ggr;) agonists are reported to increase the secretion of HMW adiponectin, although the effects of panaxynol were found to be independent of PPAR&ggr; activation. When the underlying mechanisms were further examined, panaxynol was found to inhibit the palmitic‐acid‐induced downregulation of forkhead box O1 (FoxO1) protein, and the anti‐lipotoxic effects were abolished by a FoxO1 inhibitor. Furthermore, CCAAT/enhancer‐binding protein‐&agr; (C/EBP&agr;) mRNA levels were also increased by panaxynol. Reactive oxygen species have critical roles in the reduction in HMW adiponection secretion by palmitic acid; however, panaxynol reduced this increase in reactive oxygen species generation, followed by reductions in markers of endoplasmic reticulum stress and inflammation. Taken together, these findings suggest that panaxynol ameliorates the impaired HMW adiponection secretion in adipocytes treated with palmitic acid by restoring FoxO1 expression, owing to inhibition of reactive oxygen species generation, in a PPAR&ggr;‐independent manner.

Pteleifolols A–E, acetophenone di-C-glycosides and a benzopyran dimer from the leaves of Melicope pteleifolia

Four new acetophenone di-C-glycosides, pteleifolols A–D (1–4) and a new dimeric benzopyran, pteleifolol E (5), were isolated from the leaves of Melicope pteleifolia. Seven known compounds, including 2,4,6-trihydroxyacetophenone-3,5-di-C-glucopyranoside (6), were also isolated. Structures of the new compounds (1–5) were established by using spectroscopic and spectrometric techniques, including 1D and 2D nuclear magnetic resonance (NMR), UV, and high-resolution electrospray ionization mass spectrometry (HR-ESI–MS) data. Pteleifolols A–D (1–4) were E-p-coumaroyl, Z-p-coumaroyl, E-feruloyl, and benzoyl esters of 6, respectively. Pteleifolol E (5) was a dichromene dimerized through a C2 unit.