Benzopyran Derivatives and an Aliphatic Compound from a Mangrove Endophytic Fungus Penicillium citrinum QJF‐22

Two new benzopyran derivatives, (2R,4S)‐5‐methoxy‐2‐methyl‐3,4‐dihydro‐2H‐1‐benzopyran‐4‐ol and (2S,4R,2′S,4′R)‐4,4′‐oxybis(5‐methoxy‐2‐methyl‐3,4‐dihydro‐2H‐1‐benzopyran), and a new aliphatic compound, (3E,5Z,8S,10E)‐8‐hydroxytrideca‐3,5,10,12‐tetraen‐2‐one, together with three known benzopyran derivatives, were obtained from a mangrove endophytic fungus Penicillium citrinum QJF‐22 collected in Hainan island. Their structures were determined by analysis of spectroscopic data and the relative configuration of (2R,4S)‐5‐methoxy‐2‐methyl‐3,4‐dihydro‐2H‐1‐benzopyran‐4‐ol was also confirmed by single‐crystal X‐ray diffraction. The absolute configurations of four compounds were established by comparison of ECD spectra to calculations. The configuration of (3E,5Z,8S,10E)‐8‐hydroxytrideca‐3,5,10,12‐tetraen‐2‐one was confirmed by comparison of optical value to the similar compound. The configurations of the compounds (2S,4S)‐5‐methoxy‐2‐methyl‐3,4‐dihydro‐2H‐1‐benzopyran‐4‐ol and (2R,4R)‐5‐methoxy‐2‐methyl‐3,4‐dihydro‐2H‐1‐benzopyran‐4‐ol were first determined. (3R,4S)‐3,4,8‐Trihydroxy‐3,4‐dihydronaphthalen‐1(2H)‐one exhibited moderate inhibitory effects on LPS‐induced NO production in RAW264.7 cells with IC₅₀ of 44.7 μM, and without cytotoxicity to RAW264.7 cells within 50 μM.     

An Unusual Tetrahydrofuran Lignan from the Roots of Zanthoxylum planispinum and the Potential Anti‐inflammatory Effects

An unusual tetrahydrofuran lignin, zanthplanispine ( 1 ), together with 14 known lignans ( 2 – 15 ) were isolated from the AcOEt‐soluble fraction from the MeOH extract of Z. planispinum roots. The structures of 1 was elucidated on the basis of 1D‐ and 2D‐NMR experiments as well as HR‐ESI‐MS analysis. The known compounds were identified by the comparison of their NMR data with previously reported in the literatures. Bioassay showed that compounds 1 – 4 could inhibit nitric oxide (NO) production in lipopolysaccharide (LPS) stimulated RAW 264.7 cells. In particular, compound 1 showed significant inhibitory activity with an IC₅₀ value of 36.8 μm .     

Toll‐like receptor signalling cross‐activates the autophagic pathway to restrict Salmonella Typhimurium growth in macrophages

It has been long recognised that activation of toll‐like receptors (TLRs) induces autophagy to restrict intracellular bacterial growth. However, the mechanisms of TLR‐induced autophagy are incompletely understood. Salmonella Typhimurium is an intracellular pathogen that causes food poisoning and gastroenteritis in humans. Whether TLR activation contributes to S. Typhimurium‐induced autophagy has not been investigated. Here, we report that S. Typhimurium and TLRs shared a common pathway to induce autophagy in macrophages. We first showed that S. Typhimurium‐induced autophagy in a RAW264.7 murine macrophage cell line was mediated by the AMP‐activated protein kinase (AMPK) through activation of the TGF‐β‐activated kinase (TAK1), a kinase activated by multiple TLRs. AMPK activation led to increased phosphorylation of Unc‐51‐like autophagy activating kinase (ULK1) at S317 and S555. ULK1 phosphorylation at these two sites in S. Typhimurium‐infected macrophages overrode the inhibitory effect of mTOR on ULK1 activity due to mTOR‐mediated ULK1 phosphorylation at S757. Lipopolysaccharide (LPS), flagellin, and CpG oligodeoxynucleotide, which activate TLR4, TLR5, and TLR9, respectively, increased TAK1 and AMPK phosphorylation and induced autophagy in RAW264.7 cells and in bone marrow‐derived macrophages. However, LPS was unable to induce TAK1 and AMPK phosphorylation and autophagy in TLR4‐deficient macrophages. TAK1 and AMPK‐specific inhibitors blocked S. Typhimurium‐induced autophagy and xenophagy and increased the bacterial growth in RAW264.7 cells. These observations collectively suggest that activation of the TAK1–AMPK axis through TLRs is essential for S. Typhimurium‐induced autophagy and that TLR signalling cross‐activates the autophagic pathway to clear intracellular bacteria.     

iTRAQ proteomic analysis of N‐acetylmuramic acid mediated anti‐inflammatory capacity in LPS‐induced RAW 264.7 cells

Lactobacillus acidophilus probiotic bacteria have lasting beneficial health effects in the gastrointestinal tract, including protecting against pathogens, improving immunomodulation, and producing beneficial bacteria‐derived molecules. In lipopolysaccharide (LPS) induced RAW 264.7 cells treated with peptidoglycan or N‐acetylmuramic acid (NAM) from L. acidophilus, 390 differentially expressed proteins (8.76%) were identified by iTRAQ analysis, 257 (5.77%) of which were upregulated and 133 (2.99%) were downregulated under LPS‐induced conditions. Most of these proteins were grouped into the following inflammation‐related cellular signaling: lysosome pathway, calcium signaling pathway, and Toll‐like receptor (TLR) signaling pathway. Among them, clathrin, SERCA, and interleukin 1 receptor antagonist were differentially expressed to a significant degree in peptidoglycan or NAM pretreated RAW 264.7 cells. Bioinformatics analysis indicated that NAM may mediate an anti‐inflammatory process via a Ca²⁺‐dependent NF‐κB pathway. These observations reveal new insights into the molecular mechanisms involved in the suppression of LPS‐induced macrophage inflammation by L. acidophilus.     

Aminothiazoles inhibit RANKL‐ and LPS‐mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells

Periodontitis is characterized by chronic inflammation and osteoclast‐mediated bone loss regulated by the receptor activator of nuclear factor‐κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase‐1 (mPGES‐1) on RANKL‐ and lipopolysaccharide (LPS)‐mediated osteoclastogenesis and prostaglandin E₂ (PGE₂) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) or 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644). Aminothiazoles significantly decreased the number of multinucleated tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells in cultures of RANKL‐ and LPS‐stimulated RAW 264.7 cells, as well as reduced the production of PGE₂ in culture supernatants. LPS‐treatment induced mPGES‐1 mRNA expression at 16 hrs and the subsequent PGE₂ production at 72 hrs. Conversely, RANKL did not affect PGE₂ secretion but markedly reduced mPGES‐1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL‐activated RAW 264.7 cells, TH‐848 and TH‐644 down‐regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE₂ production was also confirmed in LPS‐stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS‐ and RANKL‐mediated osteoclastogenesis and PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.     

Homochiral bifunctional L‐prolinamide‐ and L‐bis‐prolinamide‐catalyzed asymmetric aldol reactions performed in wet solvent‐free conditions

In this study, the novel bifunctional homochiral thiourea‐L‐prolinamides 1–4 , tertiary amino‐L‐prolinamide 5 , and bis‐L‐prolinamides 6 and 7 were prepared from enantiomerically pure (11R,12R)‐11,12‐diamino‐9,10‐dihydro‐9,10‐ethanoanthracene 8 and (11S,12S)‐11,12‐diamino‐9,10‐dihydro‐9,10‐ethanoanthracene ent‐8 . Highly enantioselective and diastereoselective aldolic intermolecular reactions (up to 95% enantiomeric excess, 93:7 anti/syn) between aliphatic ketones (20 equiv) and a range of aromatic aldehydes (1 equiv) were successfully carried out in the presence of water (10 equiv) and monochloroacetic acid (10 mol%), solvent‐free conditions, at room temperature over 24 h using organocatalysts 1–7 (5 mol%). Stereoselective induction using density functional theory–based methods was consistent with the experimental data.     

Chemical Components from Phaeanthus vietnamensis and Their Inhibitory NO Production in BV2 Cells

Phaeanthus vietnamensis Bân is a well‐known medicinal plant which has been used for the treatment of various inflammatory diseases in traditional medicine. Using various chromatographic methods, three new compounds, (7S,8R,8′R)‐9,9′‐epoxy‐3,5,3′,5′‐tetramethoxylignan‐4,4′,7‐triol ( 1 ), 8α‐hydroxyoplop‐11(12)‐en‐14‐one ( 5 ), and (1R,2S,4S)‐4‐acetyl‐2‐[(E)‐(cinnamoyloxy)]‐1‐methylcyclohexan‐1‐ol ( 12 ) along with twelve known compounds were isolated from the leaves of P. vietnamensis. Their chemical structures were elucidated by physical and chemical methods. All compounds were evaluated for the inhibitory activities of nitric oxide production in LPS‐stimulated BV2 cells. As the results, compound 6 showed the most potent inhibitory activity on LPS‐stimulated NO production in BV2 cells with the IC₅₀ values of 15.7 ± 1.2 μm . Compounds 2 , 7 , and 8 significantly inhibited inflammatory NO production with IC₅₀ values ranging from 22.6 to 25.3 μm .     

Structure–cytotoxic activity relationship of sesquilignan,morinol A

The cytotoxic activities of sesquilignans, (7S,8S,7′R,8′R)- and (7R,8R,7′S,8′S)-morinol A and (7S,8S,7′S,8′S)- and (7R,8R,7′R,8′R)-morinol B were compared, showing no significant difference between stereoisomers (IC₅₀ = 24–35 μM). As a next stage, the effect of substituents at 7, 7′, and 7″-aromatic ring on the activity was evaluated to find out the higher activity of (7S,8S,7′R,8′R)-7,7′,7″-phenyl derivative 18 (IC₅₀ = 6–7 μM). In the research on the structure–activity relationship of 7″-position of (7S,8S,7′R,8′R)-7,7′,7″-phenyl derivative 18, the most potent compounds were 7,7′,7″-phenyl derivative 18 (IC₅₀ = 6 μM) against HeLa cells. Against HL-60 cells, 7″-(4-nitrophenyl)-7,7′-phenyl derivative 33 and 7″-hexyl-7,7′-phenyl derivative 37 (IC₅₀ = 5 μM) showed highest activity. We discovered the compounds showed four to sevenfold potent activity than that of natural (7S,8S,7′R,8′R)-morinol A. It was also confirmed that the 7′-benzylic hydroxy group have an important role for exhibiting activity, on the other hand, the resonance system of cinnamyl structure is not crucial for the potent activity.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Sesquiterpenoids from Chloranthus henryi and Their Anti‐neuroinflammatory Activities

Five new and seven known mono‐sesquiterpenoids ( 1 – 5 and 6 – 12 , resp.) together with five known lindenane‐type disesquiterpenoids, 13 – 17 , were isolated from the whole plant of Chloranthus henryi. Based on spectroscopic methods, the new structures were established to be (5S,6R,8S,10R)‐6‐hydroxyeudesma‐4(15),7(11)‐diene‐12,8‐olide ( 1 ), 6α‐hydroxyeudesma‐4(15),7(11),8(9)‐triene‐12,8‐olide ( 2 ), 8,12‐epoxy‐1β‐hydroxyeudesma‐4(15),7,11‐trien‐6‐one ( 3 ), 12‐oxochloraniolide A ( 4 ), and (4α)‐8‐hydroxy‐12‐norcardina‐6,8,10‐trien‐11‐one ( 5 ), respectively. Among the isolates, compound 2 , zederone epoxide ( 8 ), spicachlorantin G ( 13 ), chloramultilide A ( 14 ), shizukaol B ( 15 ), and spicachlorantin B ( 17 ) showed significant anti‐neuroinflammatory effects by inhibiting nitric‐oxide (NO) production in lipopolysaccharide (LPS)‐stimulated murine BV‐2 microglial cells with relatively low cytotoxicity.     

Antimicrobial activity,bactericidal mechanism and LPS‐neutralizing activity of the cell‐penetrating peptide pVEC and its analogs

pVEC is a cell‐penetrating peptide derived from the murine vascular endothelial‐cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)‐neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4–16 μM) against Gram‐positive and Gram‐negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 μM. These peptides induced a near‐complete membrane depolarization (more than 80%) at 4 μM against Staphylococcus aureus and a significant dye leakage (35–70%) from bacterial membrane‐mimicking liposome at a concentration as low as 1 μM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog‐derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor‐α release in LPS‐stimulated mouse macrophage RAW264.7 cells at 10 to 50 μM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS‐neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

CME‐1, a novel polysaccharide,suppresses iNOS expression in lipopolysaccharide‐stimulated macrophages through ceramide‐initiated protein phosphatase 2A activation

CME‐1, a novel water‐soluble polysaccharide purified from Ophiocordyceps sinensis mycelia, has anti‐oxidative, antithrombotic and antitumour properties. In this study, other major attributes of CME‐1, namely anti‐inflammatory and immunomodulatory properties, were investigated. Treating lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells with CME‐1 concentration‐dependently suppressed nitric oxide formation and inducible nitric oxide synthase (iNOS) expression. In the CME‐1‐treated RAW 264.7 cells, LPS‐induced IκBα degradation and the phosphorylation of p65, Akt and mitogen‐activated protein kinases (MAPKs), including extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38, were reduced. Treatment with a protein phosphatase 2A (PP2A)‐specific inhibitor, significantly reversed the CME‐1‐suppressed iNOS expression; IκBα degradation; and p65, Akt and MAPK phosphorylation. PP2A activity up‐regulation and PP2A demethylation reduction were also observed in the cells. Moreover, CME‐1‐induced PP2A activation and its subsequent suppression of LPS‐activated RAW 264.7 cells were diminished by the inhibition of ceramide signals. LPS‐induced reactive oxygen species (ROS) and hydroxyl radical formation were eliminated by treating RAW 264.7 cells with CME‐1. Furthermore, the role of ceramide signalling pathway and anti‐oxidative property were also demonstrated in CME‐1‐mediated inhibition of LPS‐activated primary peritoneal macrophages. In conclusion, CME‐1 suppressed iNOS expression by up‐regulating ceramide‐induced PP2A activation and reducing ROS production in LPS‐stimulated macrophages. CME‐1 is a potential therapeutic agent for treating inflammatory diseases.     

Cyclic AMP signaling enhances lipopolysaccharide sensitivity and interleukin‐33 production in RAW264.7 macrophages

While it has been suggested that IL‐33 plays pathogenic roles in various disorders, the factors that stimulate IL‐33 production are poorly characterized. In the present study, the effect of cyclic adenosine monophosphate (cAMP) signaling on IL‐33 production in RAW264.7 macrophages in response to various doses of LPS was examined. High‐dose LPS treatment induced IL‐33 and TNF protein production in RAW264.7 macrophages. In contrast, low‐dose LPS failed to induce IL‐33 production while significantly inducing TNF production. In the presence of the membrane‐permeable cAMP analog 8‐Br‐cAMP, low‐dose LPS induced vigorous IL‐33 production. This phenomenon was consistent with amounts of mRNA. Similarly, the cAMP‐increasing agent adrenaline also enhanced the sensitivity of RAW264.7 macrophages to LPS as demonstrated by IL‐33 production. The protein kinase A (PKA) inhibitor H89 blocked the effects of 8‐Br‐cAMP and adrenaline on IL‐33 production, suggesting that PKA is involved in IL‐33 induction. Taken together, cAMP‐mediated signaling pathway appears to enhance the sensitivity of RAW264.7 macrophages to LPS with respect to IL‐33 production. Our findings suggest that stress events and the subsequent secretion of adrenaline enhance macrophage production via IL‐33; this process may be associated with the pathogenesis of various disorders involving IL‐33.     

Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti‐inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)‐induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti‐inflammatory and anti‐endotoxin activities of Os and Os‐C, peptides derived from the carboxy‐terminal of a tick defensin, were investigated. Both Os and Os‐C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin‐binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os‐C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os‐C showed no scavenging activity. Os and Os‐C inhibited LPS/IFN‐γ induced NO and TNF‐α production in RAW 264.7 cells in a concentration‐dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF‐α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os‐C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os‐C, both peptides have in addition anti‐inflammatory and anti‐endotoxin properties. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Enantiomeric 9‐mer peptide analogs of protaetiamycine with bacterial cell selectivities and anti‐inflammatory activities

Protaetiamycine is an insect defensin, derived from the larvae of the beetle Protaetia brevitarsis. In our previous work, we designed 9‐mer peptide analogs of protaetiamycine, including 9Pbw2 (RLWLAIKRR‐NH₂), 9Pbw3 (RLWLAIWRR‐NH₂), and 9Pbw4 (RLWLAWKRR‐NH₂). 9Pbw2 and 9Pbw4 showed high antimicrobial activity without cytotoxicity, while 9Pbw3 with higher hydrophobicity compared to 9Pbw2 and 9Pbw4 showed high cytotoxicity as well as high antimicrobial activity (Shin et al., J. Pept. Sci. 2009; 15: 559–568). In this study, we investigated the anti‐inflammatory activities of 9Pbw2, 9Pbw3, and 9Pbw4 by quantitation of NO production in LPS‐stimulated RAW264.7 cells. The results showed that only 9Pbw3 has strong inhibition of NO production, implying that Trp⁷ as well as optimum level of hydrophobicity may play key roles in the anti‐inflammatory activity of 9Pbw3. In order to design potent anti‐inflammatory peptide with lower cytotoxicity as well as high stability from cleavage by protease compared to 9Pbw3, we synthesized 9Pbw3‐D , the all‐D ‐amino acid analog of 9Pbw3. 9Pbw3‐D showed less cytotoxicity against RAW264.7 cells as well as considerably stronger inhibition of NO production and inflammation‐induced cytokine production in LPS‐stimulated RAW264.7 cells than 9Pbw3. 9Pbw3‐D inhibited the gene expression of inflammatory‐induced cytokine significantly more than 9Pbw3 and showed high resistance to proteolytic digestion. Binding of 9Pbw3‐D with LPS caused higher enhancement of the FITC fluorescence as a result of its stronger interaction with LPS compared to that of 9Pbw3 and this result is in good agreement with their anti‐inflammatory activities. 9Pbw3‐D with higher anti‐inflammatory activity as well as lower cytotoxicity against mammalian cell compared to 9Pbw3 can be a potent noncytotoxic antibiotic candidates. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Bioactivity‐Guided Isolation of Anti‐Inflammatory Constituents of the Rare Mushroom Calvatia nipponica in LPS‐Stimulated RAW264.7 Macrophages

Calvatia species, generally known as puffball mushrooms, are used both as sources of food and as traditional medicine. Among the Calvatia genus, Calvatia nipponica (Agaricaceae) is one of the rarest species. Using bioassay‐guided fractionation based on anti‐inflammatory effects, five alkaloids ( 1 – 5 ), two phenolics ( 6 and 7 ), and a fatty acid methyl ester ( 8 ) were isolated from the fruiting bodies of C. nipponica. Compound 8 was identified from C. nipponica for the first time, and all isolates ( 1 – 8 ) were tested for inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophages. Compound 7 showed mild inhibition while compound 8 significantly inhibited NO production with an IC₅₀ value of 27.50 ± 0.08 μm . The mechanism of NO inhibition of compound 7 was simulated by molecular docking analysis against nitric oxide synthase (iNOS), which revealed the interactions of 7 with the key amino acid residue and the heme in the active site. With the most potent inhibition against LPS‐induced inflammation, compound 8 was further investigated with respect to its mechanism of action, and the activity was found to be mediated through the inhibition of iNOS and COX‐2 expression.     

Triterpenoids and Flavonoids from the Leaves of Astragalus membranaceus and Their Inhibitory Effects on Nitric Oxide Production

Four new cycloartane triterpenes, named huangqiyegenins V and VI and huangqiyenins K and L ( 1 – 4 , resp.), together with nine known triterpenoids, 5 – 13 , and eight flavonoids, 14 – 21 , were isolated from a 70%‐EtOH extract of Astragalus membranaceus leaves. The structures of the new compounds were elucidated by detailed spectroscopic analyses, and the compounds were identified as (9β,11α,16β,20R,24S)‐11,16,25‐trihydroxy‐20,24‐epoxy‐9,19‐cyclolanostane‐3,6‐dione ( 1 ), (9β,16β,24S)‐16,24,25‐trihydroxy‐9,19‐cyclolanostane‐3,6‐dione ( 2 ), (3β,6α,9β,16β,20R,24R)‐16,25‐dihydroxy‐3‐(β‐D ‐xylopyranosyloxy)‐20,24‐epoxy‐9,19‐cyclolanostan‐6‐yl acetate ( 3 ), and (3β,6α,9β,16β,24E)‐26‐(β‐D ‐glucopyranosyloxy)‐16‐hydroxy‐3‐(β‐D ‐xylopyranosyloxy)‐9,19‐cyclolanost‐24‐en‐6‐yl acetate ( 4 ). All isolated compounds were evaluated for their inhibitory activities against LPS‐induced NO production in RAW264.7 macrophage cells. Compounds 1 – 3, 14, 15 , and 18 exhibited strong inhibition on LPS‐induced NO release by macrophages with IC₅₀ values of 14.4–27.1 μM .     

Iridoids from Pedicularis verticillata and Their Anti‐Complementary Activity

Three new iridoids named as pediverticilatasin A – C ( 1 – 3 , resp.), together with five known iridoids ( 4 – 8 , resp.) were isolated from the whole plants of Pedicularis verticillata. The structures of three new compounds were identified as (1S,7R)‐1‐ethoxy‐1,5,6,7‐tetrahydro‐7‐hydroxy‐7‐methylcyclopenta[c]pyran‐4(3H)‐one ( 1 ), (1S,4aS,7R,7aS)‐1‐ethoxy‐1,4a,5,6,7,7a‐hexahydro‐7‐hydroxy‐7‐methylcyclopenta[c]pyran‐4‐carboxylic acid ( 2 ), (1S,4aS,7R,7aS)‐1‐ethoxy‐1,4a,5,6,7,7a‐hexahydro‐7‐hydroxy‐7‐methylcyclopenta[c]pyran‐4‐carbaldehyde ( 3 ). Their structures were elucidated on the basis of spectroscopic methods and compared with the NMR spectra data in the literature. All compounds were evaluated for their anti‐complementary activity on the classical pathway of the complement system in vitro. Among which, compounds 1 , 3 , and 6 exhibited anti‐complementary effects with CH₅₀ values ranging from 0.43 to 1.72 mm , which are plausible candidates for developing potent anti‐complementary agents.     

A study on the interaction between 3‐spiro‐piperidones and bovine serum albumin using spectroscopic approaches

The interaction between 3‐spiro‐2′‐pyrrolidine‐3′‐spiro‐3″‐piperidine‐2,3″‐dione (PPD) and bovine serum albumin (BSA) in aqueous solution was studied using fluorescence and UV–vis spectroscopy. Fluorescence emission data revealed that BSA (1.00 × 10^‐5 mol/L) fluorescence was statically quenched by PPD at various concentrations, which implies that a PPD–BSA complex was formed. The binding constant (K_A), the number of binding sites (n) and the specific binding site of the PPD with BSA were determined. Energy‐transfer efficiency parameters were determined and the mechanism of the interaction discussed. The thermodynamic parameters, ΔG, ΔH and ΔS, were obtained according to van't Hoff's equation, showing the involvement of hydrophobic forces in these interactions. The effect of PPD acting on the BSA conformation was detected by synchronous fluorescence. Copyright © 2012 John Wiley & Sons, Ltd.