The Effect of the Aerial Part of Lindera akoensis on Lipopolysaccharides (LPS)-Induced Nitric Oxide Production in RAW264.7 Cells

Four new secondary metabolites, 3α-((E)-Dodec-1-enyl)-4β-hydroxy-5β-methyldihydrofuran-2-one (1), linderinol (6), 4′-O-methylkaempferol 3-O-α-l-(4″-E-p-coumaroyl)rhamnoside (11) and kaempferol 3-O-α-l-(4″-Z-p-coumaroyl) rhamnoside (12) with eleven known compounds—3-epilistenolide D₁ (2), 3-epilistenolide D₂ (3), (3Z,4α,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (4), (3E,4β,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (5), matairesinol (7), syringaresinol (8), (+)-pinoresinol (9), salicifoliol (10), 4″-p-coumaroylafzelin (13), catechin (14) and epicatechin (15)—were first isolated from the aerial part of Lindera akoensis. Their structures were determined by detailed analysis of 1D- and 2D-NMR spectroscopic data. All of the compounds isolated from Lindera akoensis showed that in vitro anti-inflammatory activity decreases the LPS-stimulated production of nitric oxide (NO) in RAW 264.7 cell, with IC₅₀ values of 4.1–413.8 μM.     

New Labdane-Type Diterpenoids and Anti-Inflammatory Constituents from Hedychium coronarium

Four new labdane-type diterpenoids: hedychicoronarin (1), peroxycoronarin D (2), 7β-hydroxycalcaratarin A (3), and (E)-7β-hydroxy-6-oxo-labda-8(17),12-diene-15,16-dial (4), have been isolated from the rhizomes of Hedychium coronarium, together with 13 known compounds (5–17). The structures of these new compounds were determined through spectroscopic and MS analyses. Compounds 3, 5, 6, and 10 exhibited inhibition (IC₅₀ values ≤4.52 μg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 3–6, 10, and 11 inhibited fMLP/CB-induced elastase release with IC₅₀ values ≤6.17 μg/mL.     

Rapid Bioassay-Guided Isolation of Antibacterial Clerodane Type Diterpenoid from Dodonaea viscosa (L.) Jaeq.

Plant extracts are complex matrices and, although crude extracts are widely in use, purified compounds are pivotal in drug discovery. This study describes the application of automated preparative-HPLC combined with a rapid off-line bacterial bioassay, using reduction of a tetrazolium salt as an indicator of bacterial metabolism. This approach enabled the identification of fractions from Dodonaea viscosa that were active against Staphylococcus aureus and Escherichia coli, which, ultimately, resulted in the identification of a clerodane type diterpenoid, 6β-hydroxy-15,16-epoxy-5β, 8β, 9β, 10α-cleroda-3, 13(16), 14-trien-18-oic acid, showing bacteriostatic activity (minimum inhibitory concentration (MIC) = 64–128 µg/mL) against test bacteria. To the best of our knowledge, this is the first report on antibacterial activity of this metabolite from D. viscosa.     

In Vitro Antiprotozoal Activity of Abietane Diterpenoids Isolated from Plectranthus barbatus Andr.

Chromatographic separation of the n-hexane extract of the aerial part of Plectranthus barbatus led to the isolation of five abietane-type diterpenes: dehydroabietane (1); 5,6-didehydro-7-hydroxy-taxodone (2); taxodione (3); 20-deoxocarnosol (4) and 6α,11,12,-trihydroxy-7β,20-epoxy-8,11,13-abietatriene (5). The structures were determined using spectroscopic methods including one- and two-dimensional NMR methods. Compounds (1)–(3) and (5) are isolated here for the first time from the genus Plectranthus. The isolated abietane-type diterpenes tested in vitro for their antiprotozoal activity against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxicity was determined against fibroblast cell line MRC-5. Compound (2) 5,6-didehydro-7-hydroxy-taxodone showed remarkable activity with acceptable selectivity against P. falciparum (IC₅₀ 9.2 μM, SI 10.4) and T. brucei (IC₅₀ 1.9 μM, SI 50.5). Compounds (3)–(5) exhibited non-specific antiprotozoal activity due to high cytotoxicity. Compound (1) dehydroabietane showed no antiprotozoal potential.     

Bioactive Chemical Constituents from the Brown Alga Homoeostrichus formosana

A new chromene derivative, 2-(4'',8''-dimethylnona-3''E,7''-dienyl)-8-hydroxy-2,6-dimethyl-2H-chromene (1) together with four known natural products, methylfarnesylquinone (2), isololiolide (3), pheophytin a (4), and β-carotene (5) were isolated from the brown alga Homoeostrichus formosana. The structure of 1 was determined by extensive 1D and 2D spectroscopic analyses. Acetylation of 1 yielded the monoacetylated derivative 2-(4'',8''-dimethylnona-3''E,7''-dienyl)-8-acetyl-2,6-dimethyl-2H-chromene (6). Compounds 1–6 exhibited various levels of cytotoxic, antibacterial, and anti-inflammatory activities. Compound 2 was found to display potent in vitro anti-inflammatory activity by inhibiting the generation of superoxide anion (IC₅₀ 0.22 ± 0.03 μg/mL) and elastase release (IC₅₀ 0.48 ± 0.11 μg/mL) in FMLP/CB-induced human neutrophils.     

Synthesis and Characterization of Some New Bis-Pyrazolyl-Thiazoles Incorporating the Thiophene Moiety as Potent Anti-Tumor Agents

A new series of 1,4-bis(1-(5-(aryldiazenyl)thiazol-2-yl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)benzenes 3a–i were synthesized via reaction of 5,5′-(1,4-phenylene)bis(3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide) (1) with hydrazonoyl halides 2a–i. In addition, reaction of 1 with ethyl chloroacetate afforded bis-thiazolone derivative 8 as the end product. Reaction of compound 8 with methyl glyoxalate gave bis-thiazolone derivative 10. The structures of the newly synthesized compounds were established on the basis of spectroscopic evidences and their alternative syntheses. All the synthesized compounds were evaluated for their anti-tumor activities against hepatocellular carcinoma (HepG2) cell lines, and the results revealed promising activities of compounds 3g, 5e, 3e, 10, 5f, 3i, and 3f with IC₅₀ equal 1.37 ± 0.15, 1.41 ± 0.17, 1.62 ± 0.20, 1.86 ± 0.20, 1.93 ± 0.08, 2.03 ± 0.25, and 2.09 ± 0.19 μM, respectively.     

Anti-Inflammatory Components from the Root of Solanum erianthum

Two new norsesquiterpenoids, solanerianones A and B (1–2), together with nine known compounds, including four sesquiterpenoids, (−)-solavetivone (3), (+)-anhydro-β-rotunol (4), solafuranone (5), lycifuranone A (6); one alkaloid, N-trans-feruloyltyramine (7); one fatty acid, palmitic acid (8); one phenylalkanoid, acetovanillone (9), and two steroids, β-sitosterol (10) and stigmasterol (11) were isolated from the n-hexane-soluble part of the roots of Solanum erianthum. Their structures were elucidated on the basis of physical and spectroscopic data analyses. The anti-inflammatory activity of these isolates was monitored by nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells. The cytotoxicity towards human lung squamous carcinoma (CH27), human hepatocellular carcinoma (Hep 3B), human oral squamous carcinoma (HSC-3) and human melanoma (M21) cell lines was also screened by using an MTT assay. Of the compounds tested, 3 exhibited the strongest NO inhibition with the average maximum inhibition (E_max) at 100 μM and median inhibitory concentration (IC₅₀) values of 98.23% ± 0.08% and 65.54 ± 0.18 μM, respectively. None of compounds (1–9) was found to possess cytotoxic activity against human cancer cell lines at concentrations up to 30 μM.     

Molecular Cloning and Characterization of the Calcineurin Subunit A from Plutella xylostella

Calcineurin (or PP2B) has been reported to be involved in an array of physiological process in insects, and the calcineurin subunit A (CNA) plays a central role in calcineurin activity. We cloned the CNA gene from Plutella xylostella (PxCNA). This gene contains an ORF of 1488 bp that encodes a 495 amino acid protein, showing 98%, and 80% identities to the CNA of Bombyx mori, and humans respectively. The full-length of PxCNA and its catalytic domain (CNA_1–341, defined as PxCNα) were both expressed in Escherichia coli. Purified recombinant PxCNA displayed no phosphatase activity, whereas recombinant PxCNα showed high phosphatase activity with a Km of 4.6 mM and a kcat of 0.66 S⁻¹ against pNPP. It could be activated at different degrees by Mn²⁺, Ni²⁺, Mg²⁺, and Ca²⁺. The optimum reaction pH was about 7.5 and the optimum reaction temperature was around 45 °C. An in vitro inhibition assay showed that okadaic acid (OA) and cantharidin (CTD) competitively inhibited recombinant PxCNα activity with the IC₅₀ values of 8.95 μM and 77.64 μM, respectively. However, unlike previous reports, pyrethroid insecticides were unable to inhibit recombinant PxCNα, indicating that the P. xylostella calcineurin appears not to be sensitive to class II pyrethroid insecticides.     

New Triterpenoids with Cytotoxic Activity from Actinidia Valvata

Two new triterpenoids, 30-O-β-d-glucopyranosyloxy-2α,3α,24-trihydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (1) and 2α,3β,3,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (2) were isolated from roots of Actinidia valvata Dunn. Their structures were elucidated by means of extensive spectroscopic studies. Both these two new compounds showed moderate cytotoxic activity in vitro against BEL-7402 and SMMC-7721 tumor cell line.     

Enzymatic Properties of Populus α- and β-NAD-ME Recombinant Proteins

Plant mitochondrial NAD-malic enzyme (NAD-ME), which is composed of α- and β-subunits in many species, participates in many plant biosynthetic pathways and in plant respiratory metabolism. However, little is known about the properties of woody plant NAD-MEs. In this study, we analyzed four NAD-ME genes (PtNAD-ME1 through PtNAD-ME4) in the genome of Populus trichocarpa. PtNAD-ME1 and -2 encode putative α-subunits, while PtNAD-ME3 and -4 encode putative β-subunits. The Populus NAD-MEs were expressed in Escherichia coli cells as GST-tagged fusion proteins. Each recombinant GST-PtNAD-ME protein was purified to near homogeneity by glutathione-Sepharose 4B affinity chromatography. Milligram quantities of each native protein were obtained from 1 L bacterial cultures after cleavage of the GST tag. Analysis of the enzymatic properties of these proteins in vitro indicated that α-NAD-MEs are more active than β-NAD-MEs and that α- and β-NAD-MEs presented different kinetic properties (V_max, k_cat and k_cat/K_m). The effect of different amounts of metabolites on the activities of Populus α- and β-NAD-MEs was assessed in vitro. While none of the metabolites evaluated in our assays activated Populus NAD-ME, oxalacetate and citrate inhibited all α- and β-NAD-MEs and glucose-6-P and fructose inhibited only the α-NAD-MEs.     

Preparation and Characterization of Tripterygium wilfordii Multi-Glycoside Nanoparticle Using Supercritical Anti-Solvent Process

The aim of this study was to prepare nanosized Tripterygium wilfordii multi-glycoside (GTW) powders by the supercritical antisolvent precipitation process (SAS), and to evaluate the anti-inflammatory effects. Ethanol was used as solvent and carbon dioxide was used as an antisolvent. The effects of process parameters such as precipitation pressure (15–35 MPa), precipitation temperature (45–65 °C), drug solution flow rates (3–7 mL/min) and drug concentrations (10–30 mg/mL) were investigated. The nanospheres obtained with mean diameters ranged from 77.5 to 131.8 nm. The processed and unprocessed GTW were characterized by scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy and thermal gravimetric analysis. The present study was designed to investigate the beneficial effect of the GTW nanoparticles on adjuvant-induced arthritis in albino rats. The processed and unprocessed GTW were tested against Freund’s complete adjuvant-induced arthritis in rats. Blood samples were collected for the estimation of interleukins (IL-1α, IL-1β) and tumor necrosis factor-α (TNF-α). It was concluded that physicochemical properties and anti-inflammatory activity of GTW nanoparticles could be improved by physical modification, such as particle size reduction using supercritical antisolvent (SAS) process. Further, SAS process was a powerful methodology for improving the physicochemical properties and anti-inflammatory activity of GTW.     

New Dihydro-β-agarofuran Sesquiterpenes from Parnassia wightiana Wall: Isolation,Identification and Cytotoxicity against Cancer Cells

Five new (4–8) and three known (1–3) dihydro-β-agarofuran sesquiterpene polyesters were isolated from the whole plants of Parnassia wightiana. The structures of all compounds were elucidated through spectroscopic analysis including 2D-NMR and HR-MS. The absolute configuration of these compounds was established by X-ray diffraction analysis, comparison of NOESY spectra and biogenetic means. The cytotoxities of compounds 2–8 were evaluated in vitro against HL-60, SMMC-7721, A549, MCF-7 and SW480 cell lines. Compounds 5–7 exhibited the highest activities with IC₅₀ values of 11.8–30.1 μM in most cases. The SAR revealed that the introduction of hydroxyl group was able to significantly improve the activities of the compounds for most of the cell lines.     

Preparation and Characterization of a Chloroperoxidase-like Catalytic Antibody

The small molecule, meso-tetra(α,α,α,α-o-phenylacetamidophenyl) porphyrin (Mr1147.0) was used as complete antigen to elicit MAb through the immunization and cell fusion techniques. The MAb 1F2 obtained was demonstrated to be very pure by MALDI/TOFMS. The subtype of MAb 1F2 is IgG2a, which has a relative molecular weight of 156,678.8 Da.No significant change in the intensity of absorption peaks in UV and CD spectra was observed over a pH range between 6 and 12. The high stability of the abzyme and the tight binding between Fe porphyrin and antibody were also demonstrated. V_max, K_m, κcat, κcat/K_m for abzyme are 5.18 × 10⁻⁸ Ms⁻¹, 1.50 × 10⁻⁸ M, 0.518 s⁻¹, 3.45 × 10⁷ M⁻¹s⁻¹, respectively. The data obtained indicate that catalytic antibody has high catalytic activity. The chloroperoxidase activity of MAb 1F2-Fe porphyrin complex is stable from 10 °C to 60 °C.     

New Benzo[c]phenanthridine and Benzenoid Derivatives,and Other Constituents from Zanthoxylum ailanthoides: Effects on Neutrophil Pro-Inflammatory Responses

A new benzo[c]phenanthridine, oxynorchelerythrine (1), and two new benzenoid derivatives, methyl 4-(2-hydroxy-4-methoxy-3-methyl-4-oxobutoxy)benzoate (2) and (E)-methyl 4-(4-((Z)-3-methoxy-3-oxoprop-1-enyl)phenoxy)-2-methylbut-2-enoate (3), have been isolated from the twigs of Zanthoxylum ailanthoides, together with 11 known compounds (4–14). The structures of these new compounds were determined through spectroscopic and MS analyses. Among the isolated compounds, decarine (4), (−)-syringaresinol (6), (+)-episesamin (8), glaberide I (9), (−)-dihydrocubebin (10), and xanthyletin (11) exhibited potent inhibition (IC₅₀ values ≤ 4.79 μg/mL) of superoxide anion generation by human nutrophils in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 4, 8, and 11 also inhibited fMLP/CB-induced elastase release with IC₅₀ values ≤ 5.48 μg/mL.     

Downregulation of de Novo Fatty Acid Synthesis in Subcutaneous Adipose Tissue of Moderately Obese Women

The purpose of this work was to evaluate the expression of fatty acid metabolism-related genes in human adipose tissue from moderately obese women. We used qRT-PCR and Western Blot to analyze visceral (VAT) and subcutaneous (SAT) adipose tissue mRNA expression involved in de novo fatty acid synthesis (ACC1, FAS), fatty acid oxidation (PPARα, PPARδ) and inflammation (IL6, TNFα), in normal weight control women (BMI < 25 kg/m², n = 35) and moderately obese women (BMI 30–38 kg/m², n = 55). In SAT, ACC1, FAS and PPARα mRNA expression were significantly decreased in moderately obese women compared to controls. The downregulation reported in SAT was more pronounced when BMI increased. In VAT, lipogenic-related genes and PPARα were similar in both groups. Only PPARδ gene expression was significantly increased in moderately obese women. As far as inflammation is concerned, TNFα and IL6 were significantly increased in moderate obesity in both tissues. Our results indicate that there is a progressive downregulation in lipogenesis in SAT as BMI increases, which suggests that SAT decreases the synthesis of fatty acid de novo during the development of obesity, whereas in VAT lipogenesis remains active regardless of the degree of obesity.     

Terpenoids from the Octocoral Sinularia gaweli

Two eudesmane sesquiterpenoids, verticillatol (1) and 5α-acetoxy-4(14)-eudesmene-1β-ol (2) and two cembrane diterpenoids, (–)-leptodiol acetate (3) and sinulacembranolide A (4) were isolated from the octocoral Sinularia gaweli and compounds 2–4 are new isolates. The structures of new terpenoids 2–4 were elucidated by spectroscopic methods and by comparison the spectral data with those of known analogues. Terpenoid 4 was found to inhibit the accumulation of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein of the lipopolysaccharide (LPS)-stimulated RAW264.7 marcophage cells.     

Cloning,Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris

A thermostable esterase gene (hydS14) was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG) and catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases), has three conserved regions, and contains the novel motif (GY(F)SLG), which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14) was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The K_m and V_max values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6), displaying optimal activity with p-nitrophenyl-C4 (K_cat/K_m = 11.74 mM⁻¹·S⁻¹). In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.     

Deletion of Specific Conserved Motifs from the N-Terminal Domain of αB-Crystallin Results in the Activation of Chaperone Functions

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54–61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54–61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21–28 residues from the activated αBΔ54–61 (to get αBΔ21–28, Δ54–61) on the structure–function of recombinant αBΔ21–28, Δ54–61. The αBΔ21–28, Δ54–61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21–28, ∆54–61 was found to prevent β-amyloid (Aβ_1–42) fibril formation in vitro and suppressed Aβ_1–42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54–61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54–61 in oxidatively stressed cells. Our study shows that the residues 21–28 and 54–61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21–28 residues further potentiates the activated αBΔ54–61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21–28, Δ54–61.     

β-Defensin 2 Ameliorates Lung Injury Caused by Pseudomonas Infection and Regulates Proinflammatory and Anti-Inflammatory Cytokines in Rat

An important member of the defensin family, β-defensin 2, is believed to play an important role in defense against foreign pathogens. In the present study, we constructed lentiviral vectors to express and knockdown β-defensin 2 in rat lungs. The results showed that the infection of β-defensin 2 overexpression lentivirus and β-defensin 2 shRNA effectively increased and suppressed the expression of β-defensin 2 in rat lung, respectively. The overexpression of β-defensin 2 mediated by the lentiviral vector protected lung from infection of Pseudomonas aeruginosa, but shRNA targeting β-defensin 2 aggregated the damage of lung. In addition, we also found that β-defensin 2 overexpression increased basal expression of anti-inflammatory cytokine such as IL-4, IL-10 and IL-13 and decreased levels of proinflammatory cytokines which include IL-1α, IL-1β, IL-5, IL-6, IL-8, IL-18, and TNF-α. Moreover, in the process of cytokine regulation, NF-κB pathway may be involved. Taken together, these data suggest that β-defensin 2 has protective effects against infection of Pseudomonas aeruginosa in rat and plays a role in inflammatory regulation by adjusting cytokine levels.