Naphthoquinones and triterpenoids from Arnebia euchroma (Royle) Johnst and their hypoglycemic and lipid-lowering effects.

A new pentacyclic triterpenoid, 2-hydroxy-1-ene-hydroxyhopanone (19), and a new benzoxepin-5-one, 3-(4-methyl-3-penten-1-yl)-6-hydroxy-9-methoxy-2H-1-benzoxepin-5-one (25), along with 26 known compounds (1-18, 20-24, 26-28), were isolated from the roots of Arnebia euchroma (Royle) Johnst. The structures of the new compounds were elucidated by extensive spectroscopic analyses. The absolute configurations of shikonofurans 9-13 were determined by quantum chemical ECD calculations and CD spectra comparison for the first time. Pharmacological study revealed that naphthoquinones 1-5, 7, and 8 had obvious cytotoxicity toward human lung adenocarcinoma A549 cell line. Meanwhile, the hypoglycemic and lipid-lowering effects of isolated compounds were assessed by checking their inhibitory effects on key enzymes regulating glucose and lipid metabolism. Results showed that compounds 1, 3, 5, 6, 8, 18, and 19 could inhibit the activity of ATP-citrate lyase (ACL); compound 7 could inhibit the activity of acetyl-CoA carboxylase (ACC1); while compounds 8 and 19 showed inhibitory effects on protein tyrosine phosphatase 1B (PTP1B). Among them, the naphthoquinone 6, steroid 18, and triterpenoid 19 showed moderate inhibitory effects on ACL and PTP1B, but didn't exhibit obvious cytotoxicity. This study demonstrated that compounds 6, 18, and 19 show great promising for the development of new agents for the treatment of metabolic diseases.

Nomilin and Its Analogues in Citrus Fruits: A Review of Its Health Promotion Effects and Potential Application in Medicine.

Nomilin is one of the major limonoids, which are plant secondary metabolites also known as tetranortriterpenoids. Nomilin is found mostly in common edible citrus fruits including lemons, limes, oranges, grapefruits, mandarins, along with traditional Chinese medicines derived from citrus fruits, such as tangerine seed, tangerine peel, fructus aurantii immaturus, etc. A number of studies have demonstrated that nomilin and its analogues exhibit a variety of biological and pharmacological activities. These include anti-cancer, immune-modulatory, anti-inflammatory, anti-obesity, anti-viral, anti-osteoclastogenic, anti-oxidant, and neuro-protective effects. Thus, nomilin and its analogues have emerged as a potential therapy for human diseases. The purpose of this review is to chronicle the evolution of nomilin research from examining its history, structure, occurrence, to its pharmacological and disease-preventing properties as well as its potential utilization in medicine and food science.

Cytochrome P450 Can Epoxidize an Oxepin to a Reactive 2,3-Epoxyoxepin Intermediate: Potential Insights into Metabolic Ring-Opening of Benzene.

Dimethyldioxirane epoxidizes 4,5-benzoxepin to form the reactive 2,3-epoxyoxepin intermediate followed by very rapid ring-opening to an o-xylylene that immediately isomerizes to the stable product 1H-2-benzopyran-1-carboxaldehyde. The present study demonstrates that separate incubations of 4,5-benzoxepin with three cytochrome P450 isoforms (2E1, 1A2, and 3A4) as well as pooled human liver microsomes (pHLM) also produce 1H-2-benzopyran-1-carboxaldehyde as the major product, likely via the 2,3-epoxyoxepin. The reaction of 4,5-benzoxepin with cerium (IV) ammonium nitrate (CAN) yields a dimeric oxidized molecule that is also a lesser product of the P450 oxidation of 4,5-benzoxepin. The observation that P450 enzymes epoxidize 4,5-benzoxepin suggests that the 2,3-epoxidation of oxepin is a major pathway for the ring-opening metabolism of benzene to muconaldehyde.

Synthesis and Antifungal Activity of Chromones and Benzoxepines from the Leaves of Ptaeroxylon obliquum.

This study reports the first total synthesis of the bioactive oxepinochromones 12-O-acetyleranthin (8) (angular isomer) and 12-O-acetylptaeroxylinol (9) (linear isomer). The antifungal activity of these compounds and their derivatives was determined against Candida albicans and Cryptococcus neoformans. Most compounds had good selectivity between the two fungi and showed moderate to good activity. 12-O-Acetyleranthin (8) had the highest activity against C. albicans, with an MIC value of 9.9 µM, while 12-O-acetylptaeroxylinol (9), the compound present in Ptaeroxylon obliquum, had the highest activity against C. neoformans, with an MIC value of 4.9 µM.

Identification of key amino acid residues in the hTGR5-nomilin interaction and construction of its binding model.

TGR5, a member of the G protein-coupled receptor (GPCR) family, is activated by bile acids. Because TGR5 promotes energy expenditure and improves glucose homeostasis, it is recognized as a key target in treating metabolic diseases. We previously showed that nomilin, a citrus limonoid, activates TGR5 and confers anti-obesity and anti-hyperglycemic effects in mice. Information on the TGR5-nomilin interaction regarding molecular structure, however, has not been reported. In the present study, we found that human TGR5 (hTGR5) shows higher nomilin responsiveness than does mouse TGR5 (mTGR5). Using mouse-human chimeric TGR5, we also found that three amino acid residues (Q77ECL1, R80ECL1, and Y893.29) are important in the hTGR5-nomilin interaction. Based on these results, an hTGR5-nomilin binding model was constructed using in silico docking simulation, demonstrating that four hydrophilic hydrogen-bonding interactions occur between nomilin and hTGR5. The binding mode of hTGR5-nomilin is vastly different from those of other TGR5 agonists previously reported, suggesting that TGR5 forms various binding patterns depending on the type of agonist. Our study promotes a better understanding of the structure of TGR5, and it may be useful in developing and screening new TGR5 agonists.

Versicones E-H and arugosin K produced by the mangrove-derived fungus Aspergillus versicolor HDN11-84.

Four new xanthonoids, versicones E-H (1-4), and a biogenetically related new derivative arugosin K (5), were isolated from a culture extract of the mangrove-derived fungus Aspergillus versicolor HDN11-84. Their structures were elucidated on the basis of extensive NMR spectroscopic data analysis. Versicone E (1) represents the first example of naturally occurring xanthonoids containing a 2-butenamide moiety. Among them, 5 showed the best cytotoxicity against Hela cell line with an IC50 value of 9.2 µm.

Temporal and spatial variations on accumulation of nomilin and limonin in the pummelos.

Limonoids are the important secondary metabolites in the citrus. In this study, the accumulation of limonoids at different fruit developmental stages and distribution among different genotypes, tissues and developmental stages were investigated in 12 pummelo varieties. The large variations on limonoids concentration were found among different varieties, which ranged from 233.78 mg/kg FW to 4090.41 mg/kg FW in the seeds at full color stage of the fruit. Classification of pummelos based on the limonoids content divided 12 varieties into three groups. It was matched well with the geographic origination of the pummelo varieties, suggesting that the accumulation of limonoids was mainly determined by the genotype of the pummelo. Accumulation of the limonoids in different tissues was highly variable, and in a tissue specific fashion. The trend of the change on the levels of nomilin and limonin in the seeds and segment membrane were corresponded to the physiological development of the fruit. The rapid accumulation of nomilin and limonoids was observed from the physiological ripening of the seeds. It suggested that physiological maturation of the seeds is a key point that the seeds accelerate the accumulation of nomilin and limonin. In most of pummelo varieties, 10% color break of the fruit was a phenotypic landmark associated with the maximum level of nomilin accumulated in the seeds.

The Rational Design of Selective Benzoxazepin Inhibitors of the α-Isoform of Phosphoinositide 3-Kinase Culminating in the Identification of (S)-2-((2-(1-Isopropyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)oxy)propanamide (GDC-0326).

Inhibitors of the class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have received substantial attention for their potential use in cancer therapy. Despite the particular attraction of targeting PI3Kα, achieving selectivity for the inhibition of this isoform has proved challenging. Herein we report the discovery of inhibitors of PI3Kα that have selectivity over the other class I isoforms and all other kinases tested. In GDC-0032 (3, taselisib), we previously minimized inhibition of PI3Kß relative to the other class I insoforms. Subsequently, we extended our efforts to identify PI3Kα-specific inhibitors using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors with selectivity for PI3Kα through interactions with a nonconserved residue. Several molecules selective for PI3Kα relative to the other class I isoforms, as well as other kinases, were identified. Optimization of properties related to drug metabolism then culminated in the identification of the clinical candidate GDC-0326 (4).

Synthesis of targeted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines as potential lead molecules with promising anti-breast cancer activity.

A targeted library of substituted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines (prototypes I, II and III), and structurally analogous to tamoxifen have been synthesized as a new class of anti-breast cancer agents. All the prototype molecules exhibited potential antiproliferative activity against ER + ve and ER-ve breast cancer cell lines. Dibenzo[b,f]thiepine prototypes were found to be more active. Of all the compound tested, 14b exhibited potent in-vitro antiproliferative activity at 1.33 µM and 5 µM concentration in MCF-7 and MDA-MB-231 cell lines and was devoid of any cytotoxicity in normal HEK cells even at 50 µM. Cell cycle analysis showed that the compound 14b inhibited cell proliferation due to G0/G1 arrest in MCF-7 cells. Annexin-V FITC and PI staining experiments confirmed that the cell inhibition was primarily due to apoptosis and not by necrosis, which was also supported by LDH release assay experiment. Molecular docking studies showed better binding interaction of the new dibenzo[b,f]thiepine analogue 14b with the estrogen receptor (ER) as compared to 4-hydroxy-tamoxifen and this enhanced binding might be responsible for its estrogen antagonistic activity that induces cell cycle arrest, apoptosis and inhibition of breast cancer cells.

Species-related difference between limonin and obacunone among five liver microsomes and zebrafish using ultra-high-performance liquid chromatography coupled with a LTQ-Orbitrap mass spectrometer.

RATIONALE: Limonin and obacunone are two major limonoids distributed in the Rutaceae and Meliaceae families. Their defined anti-tumor activity is closely connected with the furan ring and the multi-carbonyls in their structures. In vivo and in vitro biotransformations may influence their structures and further change their effects. The metabolic profiles of limonin and obacunone have not been studied previously. In order to clarify their in vivo and in vitro metabolism, a comparative investigation of their metabolic pathways in five different species of liver microsomes and zebrafish was carried out. METHODS: In the present study, ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC/HRMS) and related electrospray ionization (ESI) tandem mass spectrometric (MS/MS) dissociation of limonin and obacunone were applied for the analysis. Each metabolite was identified by its accurate mass data. Human liver microsomes (HLMs), monkey liver microsomes (MLMs), dog liver microsomes (DLMs), rat liver microsomes (RLMs), mice liver microsomes (XLMs) and zebrafish were included in the biotransformations. RESULTS: One phase I metabolite of limonin (M1-1) and two phase I metabolites of obacunone (M2-1, M2-2) were identified by accurate mass measurement and MS/MS fragmentation behaviors. A reduction reaction was regarded as the major metabolic pathway of limonoids in liver microsomes. The reduction reaction site of M1-1 and M2-1 was at the C-16 carbonyl, while for M2-2 it was at C-7. M1-1 was the major and unique metabolite of limonin and the metabolic rate of limonin varied from 11.5% to 17.8% in liver microsomes (LMs). M2-2 was the main metabolite of obacunone in LMs and zebrafish. M1-1 and M2-1 were only detected in LMs while M2-2 was found in both LMs and zebrafish incubation systems. The metabolic rate of obacunone varied from 2.5% to 19.1% and the content of M2-2 was about five times higher than that of M2-1. CONCLUSIONS: The ESI-HR-MS/MS fragmentation behaviors of limonin and obacunone were investigated for the first time. A qualitative and semi-quantitative method was developed for the in vivo and in vitro metabolic analysis of limonin and obacunone. The results demonstrated that the metabolic processes of limonin and obacunone were different between LMs and zebrafish. However, both of these two parent compounds presented similar metabolic processes in five species of LMs. This was caused by the metabolic difference between mammals and fish or because limonin probably cannot be absorbed in zebrafish.

Obacunone represses Salmonella pathogenicity islands 1 and 2 in an envZ-dependent fashion.

Obacunone belongs to a class of unique triterpenoids called limonoids, present in Citrus species. Previous studies from our laboratory suggested that obacunone possesses antivirulence activity and demonstrates inhibition of cell-cell signaling in Vibrio harveyi and Escherichia coli O157:H7. The present work sought to determine the effect of obacunone on the food-borne pathogen Salmonella enterica serovar Typhimurium LT2 by using a cDNA microarray. Transcriptomic studies indicated that obacunone represses Salmonella pathogenicity island 1 (SPI1), the maltose transporter, and the hydrogenase operon. Furthermore, phenotypic data for the Caco-2 infection assay and maltose utilization were in agreement with microarray data suggesting repression of SPI1 and maltose transport. Further studies demonstrated that repression of SPI1 was plausibly mediated through hilA. Additionally, obacunone seems to repress SPI2 under SPI2-inducing conditions as well as in Caco-2 infection models. Furthermore, obacunone seems to repress hilA in an EnvZ-dependent fashion. Altogether, the results of the study seems to suggest that obacunone exerts an antivirulence effect on S. Typhimurium and may serve as a lead compound for development of antivirulence strategies for S. Typhimurium.

Antioxidative and cardiovascular-protective activities of metabolite usnic acid and psoromic acid produced by lichen species Usnea complanata under submerged fermentation.

CONTEXT: Lichens have been used for various purposes such as dyes, perfumes and remedies in folk medicine indicating the pharmaceutical potential of lichens. OBJECTIVE: Lichen growth in nature is very slow. To overcome this major drawback, we standardized the culture media to culture the lichen Usnea complanata (Müll.Arg.) Motyka (Parmeliaceae) for (1) in vitro synthesis of natural lichen substances, and (2) determination of antioxidative and cardiovascular-protective activity of usnic acid and psoromic acid. MATERIALS AND METHODS: Lichen U. complanata has been cultured in fermentor under submerged condition. Antioxidative and cardiovascular-protective activity of the extract and the purified lichen substances usnic and psoromic acid have been determined. RESULTS: Except methanol, all other extracts exhibited antioxidative action in terms of free radical scavenging activity (FRSA) with a half-inhibiting concentration (IC50) value of 22.86 to 25.0 µg/mL, nitric oxide radical scavenging activity (NORSA) 141.3 to 149.1 µg/mL and for lipid peroxidation inhibition (LPI) 125 to 157.9 µg/mL. Usnic acid or psoromic acid showed antioxidative action with IC50 values ranging from 0.174 to 0.271 mg/mL. Methanol and ethyl acetate extract showed hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) inhibition of 65.18 to 74.81%. Only 43.47% inhibition of angiotensin converting enzyme (ACE) was shown by methanol extract. Usnic acid showed noncompetitive type of HMGR inhibition and uncompetitive type of ACE inhibition. Psoromic acid exhibited competitive type of HMGR inhibition and mixed type of ACE inhibition. DISCUSSION: U. complanata showed both cardiovascular-protective and antioxidant properties. The lichen species U. complanata may be a natural bioresource for possible pharmaceutical applications.

Calorimetric evidence of differentiated transport of limonin and nomilin through biomembranes.

The effect exerted by two structurally similar limonoids possessing antifeedant and anticancer activity, limonin and nomilin, on the thermotropic behavior of model membranes constituted by dimyristoylphosphatidylcholine (DMPC) vesicles was studied by differential scanning calorimetry. Attention was directed to evaluate modifications in phytochemical-lipid interaction induced by compound structure and lipophilicity and to evidence their different membrane penetration. The two examined compounds, when dispersed in liposomes during their preparation, were found to exert a very different action on the L(beta)-L(alpha) gel-to-liquid crystal phase transition of DMPC multilamellar vesicles. Nomilin caused a detectable effect on the transition temperature (T(m)), shifting it toward lower values with a concomitant small decrease of the associated enthalpy (DeltaH) changes, while limonin was not able to modify the lipid vesicles thermotropic behavior. Modifications induced by nomilin were a function of phytochemical concentration, while the different behavior of limonin can be due to the different polarity induced by the presence of the single A ring in nomilin that possesses an acetyl group versus the A,A' ring system of limonin. Solid limonoids and aqueous dispersions of multilamellar (MLVs) or unilamellar vesicles (LUVs) (limonoids molar fraction 0. 045, 0.12, and 0.18) were left in touch for long incubation times at temperatures higher than T(m) to detect their spontaneous transfer through the medium. By following this procedure, no interaction was detected for limonin with lipid vesicles. The rate of transfer and interaction of nomilin was a function of the kind of vesicle species (faster for LUV, slower for MLV). The interaction, monitored by compound transfer from the solid phytochemical to the lipidic species after several periods of incubation, was on the same order as that detected by preparation carried out in organic solvent. The obtained results can be explained in terms of compound hydrophobicity, and a relation between compound structure and membrane interaction can be suggested. This allows the membrane interaction with nomilin, but the low water solubility of limonin hinders or totally blocks its transfer through the aqueous medium.

Effects of KC 2450 on the lower esophageal sphincter in vivo and in vitro.

The effect of KC 2450 (racemic 3,5-cis-3-methylamino-2,3,4,5-tetrahydro-1-benzoxepine-5-ol hydrochloride) on lower esophageal sphincter pressure in pentobarbital-anesthetized dogs was determined and compared to the effect of metoclopramide. The ED20 value (i.e. the dose that increased lower esophageal sphincter pressure 20 mm Hg) was 0.72 (0.45-1.04) mg/kg i.v. for KC 2450, significantly different from 2.18 (1.30-3.42) mg/kg i.v. for metoclopramide (P less than 0.01). The superior potency of KC 2450 over metoclopramide also was demonstrated at a dose of 2 mg/kg i.v.; KC 2450 produced an increase in sphincter pressure of 43.2 +/- 4.4 mm Hg and metoclopramide produced an increase in sphincter pressure of only 28.5 +/- 5.4 mm Hg (P less than 0.05). Intraduodenally administered KC 2450 increased lower esophageal sphincter pressure at a threshold dose of 2 mg/kg with 10 mg/kg producing an increase in pressure of 53.2 +/- 9.9 mm Hg. KC 2450-induced increases in sphincter pressure were not affected by bilateral cervical vagotomy or ketanserin, but were eliminated by atropine and reduced by neuronal blockade using tetrodotoxin (TTX). KC 2450 effects also were determined in isolated circular strips of lower esophageal sphincter muscle. KC 2450 produced a concentration-related increase in canine (EC50 = 27 microM) and opossum (EC50 = 199 microM) sphincter muscle strip tension. The KC 2450 concentration-response curve was antagonized by atropine in canine and opossum sphincter muscle strips. Neuronal blockade of canine sphincter muscle with TTX antagonized the KC 2450 concentration-response curve in a non-competitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological modification of trichothecene mycotoxins: acetylation and deacetylation of deoxynivalenols by Fusarium spp.

Attempts were made to elucidate the acetyl transformation of novel trichothecene mycotoxins, 3a,7a,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one (deoxynivalenol) and its derivatives, by trichothecene-producing strains of Fusarium nivale, F. roseum, and F. solani. In the peptone-supplemented Czapek-Dox medium, F. roseum converted 3a-acetoxy-7a,15-dihydroxy-12,13-epoxytrichothec-9-en-8-one (3-acetyldeoxynivalenol) to deoxynivalenol. 3-Acetyldeoxynivalenol was also deacetylated by intact mycelia of the three strains in sugar-free Czapek-Dox medium. The growing F. nivale acetylated deoxynivalenol to afford a small amount of 3-acetyldeoxynivalenol. 3a,7a,15-Triacetoxy-12,13-epoxytrichothec-9-en-8-one (7,15-diacetyl-deoxynivalenol), which was then deacetylated to give 7a-acetoxy-3a,15-dihydroxy-12,13-epoxytrichothec-9-en-8-one (7-acetyldeoxynivalenol). It was noted that the ester at C-7 was not hydrolyzed by the fungal mycelium.