New Verticillene Diterpenoids, Eudesmane Sesquiterpenoids, and Hydroperoxysteroids from the Further Chemical Investigation of a Taiwanese Soft Coral Cespitularia sp.

An investigation of the chemical composition of a Formosan soft coral Cespitularia sp. led to the discovery of one new verticillene-type diterpenoid, cespitulactam M (1); one new eudesmane sesquiterpenoid, cespilamide F (2); and three new hydroperoxysteroids (3-5) along with twelve known analogous metabolites (6-17). In addition, one new derivative, cespitulactam M-6,2'-diacetate (1a), was prepared from compound 1. The structures were determined by detailed spectroscopic analyses, particularly HRESIMS and NMR techniques. Moreover, the in vitro cytotoxicity, anti-inflammatory, and antibacterial activity of 1-17 and 1a were evaluated.

Rapid Characterization of Bacterial Lipids with Ambient Ionization Mass Spectrometry for Species Differentiation.

Ambient ionization mass spectrometry (AIMS) is both labor and time saving and has been proven to be useful for the rapid delineation of trace organic and biological compounds with minimal sample pretreatment. Herein, an analytical platform of probe sampling combined with a thermal desorption-electrospray ionization/mass spectrometry (TD-ESI/MS) and multivariate statistical analysis was developed to rapidly differentiate bacterial species based on the differences in their lipid profiles. For comparison, protein fingerprinting was also performed with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) to distinguish these bacterial species. Ten bacterial species, including five Gram-negative and five Gram-positive bacteria, were cultured, and the lipids in the colonies were characterized with TD-ESI/MS. As sample pretreatment was unnecessary, the analysis of the lipids in a bacterial colony growing on a Petri dish was completed within 1 min. The TD-ESI/MS results were further performed by principal component analysis (PCA) and hierarchical cluster analysis (HCA) to assist the classification of the bacteria, and a low relative standard deviation (5.2%) of the total ion current was obtained from repeated analyses of the lipids in a single bacterial colony. The PCA and HCA results indicated that different bacterial species were successfully distinguished by the differences in their lipid profiles as validated by the differences in their protein profiles recorded from the MALDI-TOF analysis. In addition, real-time monitoring of the changes in the specific lipids of a colony with growth time was also achieved with probe sampling and TD-ESI/MS. The developed analytical platform is promising as a useful diagnostic tool by which to rapidly distinguish bacterial species in clinical practice.

Seasonal changes in bacterial communities associated with healthy and diseased Porites coral in southern Taiwan.

We compared the bacterial communities associated with healthy scleractinian coral Porites sp. with those associated with coral infected with pink spot syndrome harvested during summer and winter from waters off the coast of southern Taiwan. Members of the bacterial community associated with the coral were characterized by means of denaturing gradient gel electrophoresis (DGGE) of a short region of the 16S rRNA gene and clone library analysis. Of 5 different areas of the 16S rRNA gene, we demonstrated that the V3 hypervariable region is most suited to represent the coral-associated bacterial community. The DNA sequences of 26 distinct bands extracted from DGGE gels and 269 sequences of the 16S rRNA gene from clone libraries were determined. We found that the communities present in diseased coral were more heterogeneous than the bacterial communities of uninfected coral. In addition, bacterial communities associated with coral harvested in the summer were more diverse than those associated with coral collected in winter, regardless of the health status of the coral. Our study suggested that the compositions of coral-associated bacteria communities are complex, and the population of bacteria varies greatly between seasons and in coral of differing health status.

Halogenated Sesquiterpenoids from the Red Alga Laurencia tristicha Collected in Taiwan.

Chemical investigation of the red alga Laurencia tristicha led to the discovery of eight new halogenated chamigrane-type sesquiterpenoids (1-8) and one new bromocuparane-type sesquiterpene (9), along with nine known related metabolites (10-18). Their structures were elucidated on the basis of extensive spectroscopic analyses, and the absolute configurations of 1-8 were proposed by comparison to the biosynthetically related known compound 12. Cytotoxicity, antibacterial, and anti-inflammatory activities of these isolates were also investigated. The results showed that compound 11 exhibited good antibacterial activity against Serratia marcescens compared to the positive control ampicillin at a dosage of 100 µg/disk. Compound 17 showed strong inhibition toward elastase release generation at 10 µM.

Sinugyrosanolide A, an unprecedented C-4 norcembranoid, from the Formosan soft coral Sinularia gyrosa.

Chemical investigations on the acetone extract of the Formosan soft coral Sinularia gyrosa have obtained a novel C-4 norcembranoid possessing an unprecedented tricyclo[9.3.0.0(3,8)]tetradecane skeleton, namely sinugyrosanolide A. The NMR spectroscopic data of the novel norcembranoid were completely assigned by using a combination of 2D NMR experiments including (1)H-(1)H COSY, HSQC, HMBC, and NOESY. The cytotoxicities, anti-HCMV (human cytomegalovirus) endonuclease activities and antibacterial activities were evaluated in vitro. It showed moderate cytotoxicity against P-388 (mouse lymphocytic leukemia) cancer cell line with an EC50 of 11.8µM.

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 ß-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.

Analysis of the coral associated bacterial community structures in healthy and diseased corals from off-shore of southern Taiwan.

The methods of denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to analyze the ribotypes of microbial communities associated with corals. Both healthy and diseased coral of different species were collected at three locations off the southern coast of Taiwan. Ribotyping results suggested that the microbial communities were diverse. The microbial community profiles, even among the same species of corals from different geographical locations, differ significantly. The coral-associated bacterial communities contain many bacteria common to the habitants of various invertebrates. However, some bacteria were unexpected. The presence of some unusual species, such as Staphylococcus, Clostridium and Legionella, associated with corals that were likely the results of human activities. Human activities, such as thermal pollution from the nearby nuclear plant, active fishing and tourism industries in the region might have all contributed to the change in bacterial communities and the death of coral colonies around the region.

Antiviral and anti-inflammatory diterpenoids from the soft coral Sinularia gyrosa.

Chemical investigation of the soft coral Sinularia gyrosa led to the purification of three new diterpenoids, designated as gyrosanols A-C (1-3). The structures of 1-3 were elucidated through extensive spectroscopic analyses. Compounds 1 and 2 exhibited antiviral activity against HCMV with IC(50)'s of 2.6 and 3.7 microM, respectively. In addition, compounds 1 and 2 showed significant anti-inflammatory activity by reducing the levels of the COX-2 protein (19.6 + or - 3.9% and 29.1 + or - 9.6%, respectively) in RAW 264.7 macrophages.

Bioactive norditerpenoids from the soft coral Sinularia gyrosa.

Chemical investigations of the soft coral Sinularia gyrosa resulted in the isolation of six new norcembranolides, gyrosanolides A-F (1-6), a new norcembrane, gyrosanin A (7), and 11 known norditerpenoids 8-18. The structures of the isolated compounds were elucidated through extensive spectroscopic data and by comparison with reported data in the literature. Compounds 1-3, 7-9, 12, and 13 at concentration of 10microM did not inhibit the COX-2 protein expression, but significantly reduced the levels of the iNOS protein (55.2+/-14.6%, 18.6+/-6.7%, 10.6+/-4.6%, 66.9+/-5.2%, 10.2+/-5.1%, 17.4+/-7.2%, 47.2+/-11.9%, and 56.3+/-5.1%, respectively) by LPS stimulation. Compound 8 showed significant antiviral activity against HCMV (human cytomegalovirus) cells with an IC(50) of 1.9microg/mL.

Cembranoids from the octocoral Sarcophyton ehrenbergi.

Chemical investigation of the octocoral Sarcophyton ehrenbergi led to the isolation of six new cembranoids, (+)-12-carboxy-11Z-sarcophytoxide (1), (+)-12-methoxycarbonyl-11Z-sarcophine (3), ehrenberoxides A-C (4-6), and lobophynin C (2), along with two known compounds, (+)-sarcophytoxide (7) and (+)-sarcophine (8). The structures of these isolated metabolites were elucidated through extensive spectroscopic analyses, while the relative configuration of 1 was confirmed by X-ray diffraction analyses. The chemical evidence combined with spectroscopic and physical data suggested that the locations of the epoxide and the methyl carboxylate for lobophynin C should be exchanged. Moreover, metabolites 1-6 were evaluated in vitro for their cytotoxicity against selected cancer and normal cells lines, antiviral activity against human cytomegalovirus, and antibacterial activity against Salmonella enteritidis.

Nanoparticles incorporated in pH-sensitive hydrogels as amoxicillin delivery for eradication of Helicobacter pylori.

A variety of approaches have been studied to overcome the problems encountered with using antibiotics, which are ineffective in treating Helicobacter pylori infections. In our study, chitosan/poly-gamma-glutamic acid nanoparticles incorporated into pH-sensitive hydrogels were developed as an efficient carrier for amoxicillin delivery. Our results indicate that hydrogels are pH-sensitive, leading to protecting nanoparticles from being destructed by gastric acid. The results of drug releasing in vitro study clearly indicate that the amount of amoxicillin released from nanoparticles incorporated in hydrogels at pH 1.2 was relatively low (14%), compared to that from only nanoparticles (50%). Confocal laser scanning microscopy revealed that nanoparticles could infiltrate cell-cell junctions and interact with H. pylori infection sites in the intercellular spaces. Additionally, the incorporation of amoxicillin-loaded nanoparticles in a hydrogel protected the drug from the actions of the gastric juice and facilitated amoxicillin interaction specifically with intercellular spaces, the site of H. pylori infection.

Anti-inflammatory cembranolides from the soft coral Lobophytum durum.

Chemical investigation of the soft coral Lobophytum durum resulted in the isolation of seven new cembranolides, durumolides F-L (1-7), as well as one previously characterized cembranolides, sinularolide D (8). The molecular structures of these isolated metabolites were determined mainly through NMR techniques and HRESIMS analysis. Moreover, the absolute configurations of 1 and 5 were established by application of modified Mosher's method. The antibacterial activities, anti-inflammatory effects, and anti-HCMV (Human cytomegalovirus) endonuclease activity of metabolites 1-8 were also evaluated in vitro. Anti-inflammatory activity of metabolites 1 and 6 (10 microM) significantly reduced the levels of the iNOS protein to 0.8+/-0.6% and 5.7+/-2.2%, respectively, and COX-2 protein to 47.8+/-9.0% and 71.6+/-5.8%, respectively. Metabolites 1-8 (100 microg/disk) exhibited weak antibacterial activity against Salmonella enteritidis.

Development of pH-responsive chitosan/heparin nanoparticles for stomach-specific anti-Helicobacter pylori therapy.

The microorganism now known as Helicobacter pylori is considered to be an important factor in the etiology of peptic ulcers. It can secrete urease enzyme and buffer gastric acids to survive in the stomach. H. pylori can colonize the gastric mucosa and preferentially adheres near the cell-cell junctions of the gastric mucous cells. In this study, pH-responsive nanoparticles were produced instantaneously upon the addition of heparin solution to a chitosan solution with magnetic stirring at room temperature. The nanoparticles appeared to have a particle size of 130-300 nm, with a positive surface charge, and were stable at pH 1.2-2.5, allowing them to protect an incorporated drug from destructive gastric acids. We also demonstrated that the prepared nanoparticles can adhere to and infiltrate cell-cell junctions and interact locally with H. pylori infection sites in intercellular spaces.

Unprecedented hemiketal cembranolides with anti-inflammatory activity from the soft coral Lobophytum durum.

Chemical investigations of the soft coral Lobophytum durum have led to the isolation of three unprecedented hemiketal cembranolides, durumhemiketalolides A-C (1-3). Full NMR spectroscopic assignments are provided for all isolated metabolites. The anti-inflammatory activities of 1-3 were evaluated in vitro.

Ceramide and cerebrosides from the octocoral Sarcophyton ehrenbergi.

Chemical investigation of the octocoral Sarcophyton ehrenbergi, collected at the Dongsha Islands, Taiwan, has led to the isolation of a known ceramide (1) and two new cerebrosides, sarcoehrenosides A (2) and B (4), along with three known cerebrosides (3, 5, and 6). The structures of the new compounds were established by spectroscopic and chemical methods. Sarcoehrenoside A (2) differs from previously known marine cerebrosides in that it possesses a rare alpha-glucose moiety. Compounds 1-6 were evaluated for antimicrobial activity against a small panel of bacteria and for anti-inflammatory activity using RAW 264.7 macrophages.

Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2.

The structural gene encoding the extracellular lipase of Aeromonas hydrophila MCC-2 was cloned and found to be expressed in Escherichia coli using its own promoter. When the cloned gene (lip) was expressed in E. coli minicells, an 80 kDa protein was identified. Subcellular fractionation of E. coli carrying the lip gene indicated that the Lip protein was mainly associated with the membrane fraction. Nucleotide sequence analysis revealed that the gene is 2253 bp long, coding for a 79-9 kDa protein with an estimated pl of 10.36. The deduced protein contains two putative signal peptide cleavage sites: one is a typical signal peptidase cleavage site and the other bears a strong resemblance to known lipoprotein leader sequences. Radioactivity from [3H]palmitate was incorporated into the Lip protein when expressed in E. coli. The deduced protein contains a sequence of VHFLGHSLGA which is very well conserved among lipases. It shows 67% and 65% overall identity to the amino acid sequences of lipase from A. hydrophila strains H3 and JMP636, respectively, but shows little homology to those of other lipases. The Lip protein was purified to homogeneity from both A. hydrophila and recombinant E. coli. In hydrolysis of p-nitrophenyl esters and triacylglycerols, using purified enzyme, the optimum chain lengths for the acyl moiety on the substrate were C10 to C12 for ester hydrolysis and C8 to C10 for triacylglycerol hydrolysis.