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.

Molecular docking study of GSK-3ß interaction with nomilin, kihadanin B, and related limonoids and triterpenes with a furyl-δ-lactone core.

Glycogen synthase kinase-3ß (GSK-3ß) is a target enzyme considered for the treatment of multiple human diseases, from neurodegenerative pathologies to viral infections and cancers. Numerous inhibitors of GSK-3ß have been discovered but thus far only a few have reached clinical trials and only one drug, tideglusib (1), has been registered. Natural products targeting GSK-3ß have been identified, including the two anticancer limonoids obacunone (5) and gedunin (4), both presenting a furyl-δ-lactone core. To help identifying novel GSK-3ß ligands, we have performed a molecular docking study with 15 complementary natural products bearing a furyl-δ-lactone unit (such as limonin (6) and kihadanins A (8) and B (9)) or a closely related structure (such as cedrelone (10) and nimbolide (11)). The formation of GSK-3ß-binding complexes for those natural products was compared to reference GSK-3ß ATP-competitive inhibitors LY2090314 (3) and AR-A014418 (2). Our in silico analysis led to the identification of two new GSK-3ß-binding natural products: kihadanin B (9) and nomilin (7). The latter surpassed the reference compounds in terms of calculated empirical energy of interaction (ΔE). Nomilin (7) can possibly bind to the active site of GSK-3ß, notably via the furyl-δ-lactone core and its 1-acetyl group, implicated in the protein interaction. Compound structure-binding relationships are discussed. The study should help the discovery of novel natural products targeting GSK-3ß.

New cytotoxic obacunone-type limonoid and others constituents from the stem bark of Carapa procera DC (Meliaceae).

The phytochemical study of the CH2Cl2- MeOH (1:1, v/v) extract of the stem bark of Carapa procera DC (Meliaceae) led to the isolation and characterisation of a new natural limonoid 7ß-obacunol (6), along with seven known compounds. Their structures were elucidated by spectroscopic means, including 1 D and 2 D NMR, HRESI-MS and by comparison with published data. The cytotoxicity of compounds 1-6 was assessed in vitro by the WST-1 assay on human lung adenocarcinoma A549 and Raw 264.7 mouse macrophage cell lines. Results suggested that obacunone (3) exhibited the most potent cytotoxic effect against A549 and Raw 264.7 cells with respective IC50 values of 25.24 µM and 29.14 µM, while the new natural limonoid 7ß-obacunol (6) exhibited 32.75 µM and 39.19 µM, respectively on both cell lines. Therefore, limonoid derivatives might be promising sources of natural bioactive metabolites against cancer.

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.

Limonoid Triterpene, Obacunone Increases Runt-Related Transcription Factor 2 to Promote Osteoblast Differentiation and Function.

Root bark of Dictamnus dasycarpus Turcz. has been widely used as a traditional medicine and is a well-known anti-inflammatory agent. We isolated limonoid triterpene, obacunone (Obac) from the dried root bark of D. dasycarpus. Obac has been reported to exhibit varieties of biological activities including anti-inflammatory, anti-cancer, and anti-oxidant effects. This study aimed to investigate the beneficial effects and biological mechanisms of Obac in osteoblast differentiation and bone matrix mineralization. In the present study, Obac at concentrations ranging from 1 to 100 µM showed no proliferation effects in MC3T3-E1. The treatment of Obac (1 and 10 µM) increased wound healing and migration rates in a dose-dependent manner. Alkaline phosphatase (ALP) staining and activity showed that Obac (1 and 10 µM) enhanced early osteoblast differentiation in a dose-dependent manner. Obac also increased late osteoblast differentiation in a dose-dependent manner, as indicated by the mineralized nodule formation of ARS staining. The effects of Obac on osteoblast differentiation was validated by the levels of mRNAs encoding the bone differentiation markers, including Alp, bone sialoprotein (Bsp), osteopontin (Opn), and osteocalcin (Ocn). Obac increased the expression of bone morphogenetic protein (BMP), and the phosphorylation of smad1/5/8, and the expression of runt-related transcription factor 2 (RUNX2); Obac also inhibited GSK3ß and upregulated the protein level of ß-catenin in a dose-dependent manner during osteoblast differentiation. Obac-mediated osteoblast differentiation was attenuated by a BMP2 inhibitor, Noggin and a Wnt/ß-catenin inhibitor, Dickkopf-1 (Dkk1) with the abolishment of RUNX2 expression and nuclear accumulation by Obac. Taken together, the findings of this study demonstrate that Obac has pharmacological and biological activates to promote osteoblast differentiation and bone mineralization through BMP2, ß-catenin, and RUNX2 pathways, and suggest that Obac might be a therapeutic effect for the treatment and prevention of bone diseases such as osteoporosis and periodontitis.

Obacunone protects retinal pigment epithelium cells from ultra-violet radiation-induced oxidative injury.

Ultra-violet (UV) radiation (UVR) causes significant oxidative injury to retinal pigment epithelium (RPE) cells. Obacunone is a highly oxygenated triterpenoid limonoid compound with various pharmacological properties. Its potential effect in RPE cells has not been studied thus far. Here in ARPE-19 cells and primary murine RPE cells, obacunone potently inhibited UVR-induced reactive oxygen species accumulation, mitochondrial depolarization, lipid peroxidation and single strand DNA accumulation. UVR-induced RPE cell death and apoptosis were largely alleviated by obacunone. Obacunone activated Nrf2 signaling cascade in RPE cells, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation. It promoted transcription and expression of antioxidant responsive element-dependent genes. Nrf2 silencing or CRISPR/Cas9-induced Nrf2 knockout almost reversed obacunone-induced RPE cytoprotection against UVR. Forced activation of Nrf2 cascade, by Keap1 knockout, similarly protected RPE cells from UVR. Importantly, obacunone failed to offer further RPE cytoprotection against UVR in Keap1-knockout cells. In vivo, intravitreal injection of obacunone largely inhibited light-induced retinal damage. Collectively, obacunone protects RPE cells from UVR-induced oxidative injury through activation of Nrf2 signaling cascade.

Obacunone Attenuates Liver Fibrosis with Enhancing Anti-Oxidant Effects of GPx-4 and Inhibition of EMT.

Obacunone, a limonin triterpenoid extracted from Phellodendronchinense Schneid or Dictamnus dasycarpusb Turcz plant, elicits a variety of pharmacological effects such as anti-inflammatory, anti-neoplastic, anti-oxidation, and anti-lung-fibrosis ones. However, the anti-fibrotic effect of obacunone and the detailed underlying mechanism in liver fibrosis remain unclear. Liver fibrosis is a debilitating disease threatening human health. Transforming growth factor (TGF)-ß/P-Smad is a major pathway of fibrosis featured with epithelia mesenchymal transformations (EMT) and collagen depositions, accompanying with excessive oxygen-free radicals. Nrf-2 acts as a key anti-oxidative regulator driving the expressions of various antioxidant-related genes. Glutathionperoxidase-4 (GPx-4) is a member of the glutathione peroxidase family that directly inhibits phospholipid oxidation to alleviate oxidative stress. In the present study, we aimed to explore the role of obacunone in mouse liver fibrosis model induced by carbon tetrachloride (CCl4) and in hepatic stellate cells (LX2 cell line) challenging with TGF-ß. Obacunone demonstrated potent ameliorative effects on liver fibrosis both in activated LX2 and in mice liver tissues with reduced levels of α-SMA, collagen1, and vimentin. Obacunone also remarkably suppressed the TGF-ß/P-Smad signals and EMT process. Meanwhile, obacunone exerted a potent anti-oxidation effect by reducing the levels of reactive oxygen species (ROS) in both models. The antioxidant effect of obacunone was attributed to the activation of GPx-4 and Nrf-2. In addition, the therapeutic effect of obacunone on LX2 cells was significantly removed in vitro plus with GPx-4 antagonist RSL3, in parallel with the re-elevated levels of ROS. Thus, we demonstrate that obacunone is able to attenuate liver fibrosis via enhancing GPx-4 signal and inhibition of the TGF-ß/P-Smad pathway and EMT process.

Cytotoxicity of botanicals and isolated phytochemicals from Araliopsis soyauxii Engl. (Rutaceae) towards a panel of human cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Araliopsis soyauxii Engl. (Rutaceae) is a Cameroonian medicinal plant traditionally used to treat lung diseases, malaria, and gonorrhea. It has been demonstrated that infectious disease contribute to about 20% of all human tumours. AIMS OF THE STUDY: (1) To perform a phytochemical investigation of the dichloromethane-methanol 1:1 extracts of the bark (ASB), roots (ASR), and leaves (ASL) from Araliopsis soyauxii; (2) to evaluate the cytotoxicity of extracts and isolated compounds; (3) to determine the mode of induction of apoptosis of ASB and kihadanin B (12). MATERIALS AND METHODS: Fourteen constituents of the crude extracts were isolated by column chromatography, while spectroscopic techniques were used for structural elucidation. The resazurin reduction assay (RRA) was applied to determine the cytotoxicity of samples towards a panel of 9 cancer cell lines. For caspases activity, the Caspase-Glo assay was used; flow cytometry was applied to investigate the cell cycle distribution (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP; JC-1 staining), and the reactive oxygen species (ROS; H2DCFH-DA staining). RESULTS: Phytochemical investigations of botanicals (ASB, ASR, and ASL) led to the isolation of 14 compounds. Extract ASB, obacunone (11), kihadanin B (12) as well as doxorubicin (control drug) revealed cytotoxicity towards the 9 cancer cell lines tested. The IC50 values ranged from 11.11 µg/mL (against CCRF-CEM leukemia cells) to 28.18 µg/mL (against HCT116 p53+/+ colon adenocarcinoma cells) for ASB; from 28.25 µM (against MDA-MB-231-pcDNA breast adenocarcinoma cells) to 65.13 µM (against HepG2 hepatocarcinoma cells) for compound 11, and from 5.77 µM (against CCRF-CEM cells) to 43.56 µM (against U87.MGΔEGFR glioblastoma cells) for compound 12. ASB and compound 12 induced apoptosis in CCRF-CEM cells. ASB induced the apoptotic process mediated by MMP alteration and enhanced ROS production, while compound 12 induced apoptosis by caspases activation, MMP alteration, and enhanced ROS production. CONCLUSION: This study demonstrated that Araliopsis soyauxii is a potential source of cytotoxic phytochemicals such as kihadanin B and that ASB and compound 12. Extract and compounds will be explored further to develop anticancer drugs.

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.

Obacunone reduces inflammatory signalling and tumour occurrence in mice with chronic inflammation-induced colorectal cancer.

CONTEXT: Obacunone, a limonoid abundantly found in Citrus fruits, exhibits a variety of bioactivities. OBJECTIVE: To investigate the effects of obacunone on a colorectal cancer (CRC) mouse model, and clarify its potential molecular mechanisms. MATERIALS AND METHODS: The male Balb/c mice were induced with azoxymethane and dextran sulfate sodium for 12 weeks. Obacunone (50 mg/kg) was administered via oral gavage three times every week until the end of the experiment. Disease indexes including body weight, spleen weight, bloody diarrhea, colon length, histopathological score, and tumor size were measured. The anti-proliferation activities of obacunone were analyzed by MTT or flow cytometry. The expression of protein and mRNA related to cell proliferation or inflammatory cytokines was determined by Western blot, q-PCR and IHC. RESULTS: Obacunone significantly alleviated bloody diarrhea, colon shortening (7.35 ± 0.2128 vs. 8.275 ± 0.2169 cm), splenomegaly, histological score (9 ± 0.5774 vs. 6 ± 0.5774) and reduced tumor size (4.25 ± 0.6196 vs. 2 ± 0.5669). Meanwhile, the expression of protein and mRNA related to cell proliferation or inflammatory cytokines was remarkably decreased in tumor tissue. Obacunone inhibited the proliferation activities of colorectal cancer cells. Moreover, obacunone induced colorectal cancer cells G1 and G2 phases arrest, and suppressed the expression of cell cycle genes. CONCLUSIONS: Obacunone could alleviate CRC via inhibiting inflammatory response and tumor cells proliferation. The results may contribute to the effective utilization of obacunone or its derivatives in the treatment of human CRC.

Sudachinoid- and Ichangensin-Type Limonoids from Citrus junos Downregulate Pro-Inflammatory Cytokines.

Limonoids, a dominant group of phytochemicals in the Rutaceae family, are known to exhibit several pharmacological activities. To identify natural products having efficacy against inflammatory bowel disease (IBD), we isolated 13 limonoids including a new compound, methyl sudachinoid A, from the seeds of Citrus junos and investigated their anti-inflammatory effects by assessing the expression of pro-inflammatory cytokines in lipopolysaccharide-stimulated RAW 264.7 mouse macrophages and HT-29 human colon epithelial cells. Our findings revealed that limonoids significantly downregulated the pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-α, and nuclear transcription factor κB. In particular, sudachinoid-type compounds, methyl sudachinoid A and sudachinoid B, and ichangensin-type compound, 1-O-methyichangensin downregulated the expression of pro-inflammatory cytokines more potently than other limonoids, nomilin and limonin, which have been previously reported to exhibit anti-inflammatory activities in other cells; nomilin and limonin were therefore employed as positive controls in this study. Herein, we reveal that the anti-inflammatory activities of limonoids including a new compound methyl sudachinoid A from C. junos were mediated via the downregulation of pro-inflammatory cytokines and these limonoids can be employed as potential therapeutic phytochemicals for IBD.

In Vitro and In Vivo Metabolic Activation of Obacunone, A Bioactive and Potentially Hepatotoxic Constituent of Dictamni Cortex.

Obacunone is one of the major bioactive constituents from Dictamni cortex, a traditional Chinese medicine widely used in China. Oral administration of obacunone or Dictamni cortex extract has been shown to cause liver injury in rats. Given that obacunone contains a furan ring, which is a structural alert, metabolic activation might be responsible for obacunone-induced liver injury. In this study, bioactivation pathways of obacunone in rat and human liver microsomes were investigated. Obacunone was first metabolized into cis-butene-1,4-dial, and then cis-butene-1,4-dial was captured by glutathione, N-acetyl-cysteine, and N-acetyl-lysine in the microsomal incubation system. A total of 13 adducts derived from the reaction of cis-butene-1,4-dial with glutathione and/or N-acetyl-lysine were detected and structurally identified by liquid chromatography coupled to high-resolution tandem mass spectrometry. The major metabolite (M7) was identified to be the cyclic mono-glutathione conjugate of cis-butene-1,4-dial, which was detected in bile and urine of obacunone-treated rats. M9 and M10, obacunone-derived glutathione-cis-butene-1,4-dial-NAL conjugates, were detected in the microsomal incubations of obacunone fortified with glutathione and N-acetyl-lysine as trapping agents. M3 and M4, pyrroline-2-one derivatives, were also detected in microsomal incubations. Further phenotyping studies indicated that ketoconazole showed a strong inhibitory effect on the production of cis-butene-1,4-dial in a concentration-dependent manner. CYP3A4 was demonstrated to be the primary enzyme responsible for the bioactivation of obacunone by using individual recombinant human CYP450 enzymes. The current study provides an overview of CYP450-dominated bioactivation of obacunone and contributes to the understanding of the role of bioactivation in obacunone-induced liver injury.

Identification of the perturbed metabolic pathways associating with prostate cancer cells and anticancer affects of obacunone.

Functional metabolomics could bring correlative information about specific cell types under different conditions for exploring cell properties and functions. In this study, we adopt a non-targeted cell metabolomics strategy to reveal the proliferation inhibition mechanism of obacunone on 22RV1 prostate cancer cells. Using high-throughput liquid chromatography-high definition mass spectrometry combined with pattern recognition methods was performed to analyze the cell metabolic profiles and pathway of obacunone on prostate cancer. A total of twenty one proposed metabolites in prostate cancer cell and nine vital metabolic pathways such as nicotinate and nicotinamide metabolism, phenylalanine metabolism as well as tryptophan metabolism were identified from large amounts of data. Then, we have built an overall metabolic description network of obacunone to defense prostate cancer. In addition, morphological observation, cell proliferation and apoptosis analysis of 22RV1 human prostate cancer cells were performed to better understand physiopathologic changes after obacunone treatment. Functional metabolomics is a valuable tool that insight into the natural product mechanisms and contributes to new drug discovery. SIGNIFICANCE: In this study, we probe into the proliferation inhibition effect of obacunone on 22RV1 prostate cancer cells by differentiating metabolic changes of cell sample in control and obacunone administration. Using the non-targeted and targeted cell metabolomics approaches, our findings were manifested that obacunone effectually control proliferation and promote apoptosis in 22RV1 prostate cancer cells, which were related to twenty one proposed metabolites, and nicotinate and nicotinamide metabolism, phenylalanine metabolism, tryptophan metabolism as well as ascorbate metabolism. These data were suggested that functional metabolomics analysis have potential to explore the pharmacodynamic mechanism through resolving metabolic changes in cancer cells that possesses higher clinical application value. The advances in the molecular understanding of the roles of metabolomic pathway concerned with particular metabolites in obacunone administration attract more attention in favor of burgeoning therapeutic measures resisting prostate cancer.

Subchronic Toxicity Studies of Cortex Dictamni Extracts in Mice and Its Potential Hepatotoxicity Mechanisms in Vitro.

Cortex Dictamni is a commonly-used traditional Chinese herbal medicine for the treatment of skin inflammation, tinea, and eczema. Recently, some studies reported that Cortex Dictamni might induce liver injury, suggesting more attention to its safety. The current study was designed to investigate subchronic toxicity of Cortex Dictamni aqueous extract (CDAE) and ethanol extract (CDEE) in mice and the potential hepatotoxicity mechanisms in vitro. Firstly, CDAE or CDEE groups were administrated with varying dosages (2.3, 4.6, or 9.2 g/kg/day, p.o.) in mice for 28 days in subchronic toxicity studies. General clinical signs and biochemical parameters were examined, and morphological analyses were conducted. Secondly, we identified the different constituents of CDAE and CDEE using HPLC-MS/MS and chose major components for further study. In order to determine the toxic components, we investigated the cytotoxicity of extracts and chosen components using CCK-8 assay in HepG2 cells. Furthermore, we explored the possible hepatotoxicity mechanisms of Cortex Dictamni using a high content analysis (HCA). The results showed that no significant differences of general clinical signs were observed in mice. Aspartate alanine aminotransferase (ALT) and aminotransferase (AST) were significantly increased in the high-dose CDAE and CDEE groups compared to the control group. Meanwhile, the absolute and relative liver weights and liver/brain ratio were significantly elevated, and histological examination of liver demonstrated cellular enlargement or nuclear shrinkage. In UPLC analysis, we compared the chemical constituents between CDAE and CDEE, and chose dictamnine, obakunone, and fraxinellone for hepatotoxicity evaluation in the in vitro studies. In the CCK-8 assay, CDAE, CDEE, dictamnine, obakunone, and fraxinellone decreased the cell viability in a dose-dependent manner after treatment for 48 h. Furthermore, the cell number decreased, while the nuclear intensity, cell membrane permeability, and concentration of reactive oxygen species were shown to increase, meanwhile, mitochondrial membrane potential was also changed in HepG2 cells following 48 h of compounds treatment using HCA. Our studies suggested that CDAE and CDEE have potential hepatotoxicity, and that the alcohol extraction process could increase toxicity. Dictamnine, obakunone, and fraxinellone may be the possible toxic components in Cortex Dictamni with dictamnine as the most potentially hepatotoxic component, whose potential hepatotoxicity mechanism may be associated with cell apoptosis. Moreover, this study could provide valuable data for clinical drug safety research of Cortex Dictamni and a good example for safety study of other Chinese herbal medicines.

Lead Optimization of Benzoxepin-Type Selective Estrogen Receptor (ER) Modulators and Downregulators with Subtype-Specific ERα and ERß Activity.

Estrogen receptor α (ERα) is an important target for the design of drugs such as tamoxifen (2a) and fulvestrant (5). Three series of ER-ligands based on the benzoxepin scaffold structure were synthesized: series I containing an acrylic acid, series II with an acrylamide, and series III with a saturated carboxylic acid substituent. These compounds were shown to be high affinity ligands for the ER with nanomolar IC50 binding values. Series I acrylic acid ligands were generally ERα selective. In particular, compound 13e featuring a phenylpenta-2,4-dienoic acid substituent was shown to be antiproliferative and downregulated ERα and ERß expression in MCF-7 breast cancer cells. Interestingly, from series III, the phenoxybutyric acid derivative compound 22 was not antiproliferative and selectively downregulated ERß. A docking study of the benzoxepin ligands was undertaken. Compound 13e is a promising lead for development as a clinically relevant SERD, while compound 22 will be a useful experimental probe for helping to elucidate the role of ERß in cancer cells.

Obacunone causes sustained expression of MKP-1 thus inactivating p38 MAPK to suppress pro-inflammatory mediators through intracellular MIF.

Obacunone (OBA) is a highly oxygenated triterpenoid with various pharmacological activities. In this study, we explored its anti-inflammatory effect and underlying mechanisms in LPS-activated macrophages. Our data showed that OBA potently decreased pro-inflammatory mediators (eg, NO, IL-6, IL-1ß, and MCP-1) at the transcriptional and translational levels without cytotoxicity. A mechanism study showed that OBA significantly suppressed p38-mediated AP-1 signaling by stabilizing the mRNA of mitogen-activated protein kinase phosphatase 1 (MKP-1), thus prolonging the expression time of the MKP-1 protein. Next, we used computational target-fishing technology to predict the possible target of OBA. Only one potential target, macrophage migration inhibitory factor (MIF), was presented. Experimentally, the interaction between OBA and MIF was also confirmed. By using an anti-mouse MIF antibody, extracellular MIF (exMIF) was neutralized. Our results showed that autocrine MIF had slight influence on the pro-inflammatory mediator production. Correspondingly, the anti-inflammatory activity of OBA was also not affected. Accordingly, we knocked down the MIF gene in RAW 264.7 cells and obtained stable MIF deficient cells MIF(-), in which the effects of OBA on p38 phosphorylation, AP-1 activation, and pro-inflammatory mediator production in response to LPS nearly disappeared. In contrast to MIF(+) cells, the MKP-1 protein expression time of the MIF(-) cells was markedly prolonged. We conclude that OBA exerts its anti-inflammatory effect by targeting intracellular MIF (inMIF) inhibition to regulate the MKP-1/p38/AP-1 pathway. Our findings also provide a chain of evidence that the inhibition of inMIF, rather than exMIF, may become a novel target for inflammation.

Design, Synthesis and Antibacterial Evaluation of Compounds Based on New Benzoxepine-oxime-1,2,3-triazole Hybrid.

BACKGROUND: A library of compounds related to the new benzoxepine-oxime-1,2,3-triazole hybrid was created as probable antibacterial agents. Their synthesis involved a Cu-catalyzed azidealkyne cycloaddition (CuAAC) as a key step to construct the desired 1,2,3-triazole ring. Thus the click reaction between the appropriate alkyne containing the benzoxepine-oxime framework with aryl azides afforded the target compounds in good yields. METHOD: To assess their antibacterial properties all the synthesized compounds were tested using four bacterial strains consisting of one Gram-positive and three Gram-negative species. RESULTS & CONCLUSION: These compounds were generally found to be effective towards gram -ve species and one of them showed selective cytotoxicity against lung cancer cell line.

Citrus nomilin down-regulates TNF-α-induced proliferation of aortic smooth muscle cells via apoptosis and inhibition of IκB.

Nomilin is a bitter compound present in citrus and has been demonstrated as useful for various disease preventions through anti-proliferative, anti-inflammatory, and pro-apoptotic activities. Although in vitro disease models have shown that certain limonoids in the p38 mitogen-activated protein kinase signal cascade, the downstream signaling pathways remain unclear. In this study, the effects of nomilin on the proliferation and apoptotic pathways of human aortic smooth muscle cells (HASMCs) that forms the basis of progression of atherosclerotic diseases and restenosis was tested for the first time. The cellular uptake level and stability of nomilin were determined by high-performance liquid chromatography and high-resolution mass spectra. Pretreatment of HASMCs with nomilin stimulated extrinsic caspase-8, intrinsic caspase-9, and apoptotic caspase-3 and resulted in significant inhibition of TNF-α-induced proliferation. Additionally, results showed a decreased ratio of anti-apoptotic Bcl-2 protein to pro-apoptotic Bax (Bcl2/Bax), indicating mitochondrial dysfunction consistent with apoptosis. Furthermore, nomilin significantly decreased the phosphorylation of IκBα, an inhibitor of NF-κB and subsequently, reduced the downstream inflammatory signaling in TNF-α treated HASMCs. Our findings indicate that the anti-proliferative activity of nomilin on TNF-α-induced HASMCs results from apoptosis through a mitochondrial-dependent pathway and suppression of inflammatory signaling mediated through NF-κB.

New Metabolites and Bioactive Chlorinated Benzophenone Derivatives Produced by a Marine-Derived Fungus Pestalotiopsis heterocornis.

Four new compounds, including two isocoumarins, pestaloisocoumarins A and B (1, 2), one sesquiterpenoid degradation, isopolisin B (4), and one furan derivative, pestalotiol A (5), together with one known isocoumarin, gamahorin (3), and three chlorinated benzophenone derivatives, pestalachloride B (6), pestalachloride E (7) and a mixture of pestalalactone atropisomers (8a/8b), were isolated from a culture of the fungus Pestalotiopsis heterocornis associated with sponge Phakellia fusca. These new chemical structures were established using NMR and MS spectroscopic data, as well as single-crystal X-ray crystallographic analysis and CD Cotton effects. All of the isolated compounds were evaluated for their antimicrobial and cytotoxic activities. Isocoumarins 1-3, showed antibacterial activities against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with MIC values ranging from 25 to 100 µg/mL and weak antifungal activities. Chlorinated benzophenone derivatives 6-8 exhibited antibacterial activities against S. aureus and B. subtilis with MIC values ranging from 3.0 to 50 µg/mL and cytotoxicities against four human cancer cell lines with IC50 values of 6.8-87.8 µM.

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.