Bursatella leachii Purple Ink Secretion Concentrate Exerts Cytotoxic Properties against Human Hepatocarcinoma Cell Line (HepG2): In Vitro and In Silico Studies.

Liver cancer is a leading cause of cancer death globally. Marine mollusc-derived drugs have gained attention as potential natural-based anti-cancer agents to overcome the side effects caused by conventional chemotherapeutic drugs during cancer therapy. Using liquid chromatography-mass spectrometry, the main biomolecules in the purple ink secretion released by the sea hare, named Bursatella leachii (B. leachii), were identified as hectochlorin, malyngamide X, malyngolide S, bursatellin and lyngbyatoxin A. The cytotoxic effects of B. leachii ink concentrate against human hepatocarcinoma (HepG2) cells were determined to be dose- and time-dependent, and further exploration of the underlying mechanisms causing the programmed cell death (apoptosis) were performed. The expression of cleaved-caspase-8 and cleaved-caspase-3, key cysteine-aspartic proteases involved in the initiation and completion of the apoptosis process, appeared after HepG2 cell exposure to the B. leachii ink concentrate. The gene expression levels of pro-apoptotic BAX, TP53 and Cyclin D1 were increased after treatment with the B. leachii ink concentrate. Applying in silico approaches, the high scores predicted that bioactivities for the five compounds were protease and kinase inhibitors. The ADME and cytochrome profiles for the compounds were also predicted. Altogether, the B. leachii ink concentrate has high pro-apoptotic potentials, suggesting it as a promising safe natural product-based drug for the treatment of liver cancer.

Detection and longitudinal distribution of GW1516 and its metabolites in equine hair for doping control using liquid chromatography/high-resolution mass spectrometry.

RATIONALE: GW1516 is a peroxisome proliferator-activated receptor-δ (PPAR-δ) agonist that is banned in horseracing and equestrian sports. Long-term detection and longitudinal distribution of GW1516 in the mane of a horse are reported for the first time and this hair analysis could prolong the detection window of GW1516 for doping control. METHODS: Mane hairs were divided into three segments (0-7, 7-15, and >15 cm from the cut end) and completely pulverized and homogenized for analysis. The pulverized hair samples were extracted with methanol followed by further purification and the extracts were analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using a Q-Exactive instrument. This method was successfully validated and applied to post-administration samples to confirm the presence of GW1516 and its metabolites and estimate the uptake amounts of GW1516. RESULTS: After administration of 150 mg of GW1516 to a thoroughbred mare, GW1516 was detected in one of two segments of all mane hairs, and four metabolites, namely GW1516 sulfoxide, GW1516 sulfone, 5-(hydroxymethyl)-4-methyl-2-(4-trifluoromethylphenyl)thiazole (HMTT), and 4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazole-5-carboxylic acid (MTTC), were also identified. The longitudinal distribution analysis results showed that the maximum uptake of GW1516 into hair (approximately 0.05 pg/mg) was observed at around 13 weeks post-administration and GW1516 could be detected and confirmed up to 6 months post-administration. CONCLUSIONS: The parent drug GW1516 was identified as the most appropriate monitoring target in equine hair for controlling its misuse in horses. The use of hair analysis could extend the detection time of GW1516 to at least 6 months after the administration of 150 mg of GW1516 to a thoroughbred mare.

Screening for Small Molecule Inhibitors of BMP-Induced Osteoblastic Differentiation from Indonesian Marine Invertebrates.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder with heterotopic ossification (HO) in soft tissues. The abnormal activation of bone morphogenetic protein (BMP) signaling by a mutant activin receptor-like kinase-2 (ALK2) leads to the development of HO in FOP patients, and, thus, BMP signaling inhibitors are promising therapeutic applications for FOP. In the present study, we screened extracts of 188 Indonesian marine invertebrates for small molecular inhibitors of BMP-induced alkaline phosphatase (ALP) activity, a marker of osteoblastic differentiation in a C2C12 cell line stably expressing ALK2(R206H) (C2C12(R206H) cells), and identified five marine sponges with potent ALP inhibitory activities. The activity-guided purification of an EtOH extract of marine sponge Dysidea sp. (No. 256) resulted in the isolation of dysidenin (1), herbasterol (2), and stellettasterol (3) as active components. Compounds 1-3 inhibited ALP activity in C2C12(R206H) cells with IC50 values of 2.3, 4.3, and 4.2 µM, respectively, without any cytotoxicity, even at 18.4-21.4 µM. The direct effects of BMP signaling examined using the Id1WT4F-luciferase reporter assay showed that compounds 1-3 did not decrease the reporter activity, suggesting that they inhibit the downstream of the Smad transcriptional step in BMP signaling.

Discovery, biological evaluation and docking studies of novel N-acyl-2-aminothiazoles fused (+)-nootkatone from Citrus paradisi Macf. as potential α-glucosidase inhibitors.

Nowadays, the discovery and development of α-glucosidase inhibitors from natural products or their derivatives represents an attractive approach. Here we reported studies on a series of novel N-acyl-2-aminothiazoles fused (+)-nootkatone and evaluation for their α-glucosidase inhibitory activities. Most of (+)-nootkatone derivatives exhibited more potent α-glucosidase inhibitory ability than the positive drug acarbose. In particular, compounds II7 and II14 showed the most promising α-glucosidase inhibitory ability with IC50 values of 13.2 and 13.8 µM. II7 and II14 also exhibited relatively low cytotoxicities towards normal LO2 cells. Kinetic study indicated that compounds II7 and II14 inhibited the α-glucosidase in a noncompetitive manner, and molecular docking results were in line with the noncompetitive characteristics that II7 and II14 did not bind to the known active sites (Asp214, Glu276 and Asp349). Based on our findings, these (+)-nootkatone derivatives could be used as antidiabetic candidates.

Gradient HPLC-DAD method for quantification of novel oral anticoagulant "Edoxaban" in plasma: Its selective determination in presence of sixteen co-administered drugs.

As an anticoagulant, Edoxaban (EDX) is a high risk drug that may cause a life-threatening bleeding. Also, it is prescribed as a chronic therapy for atrial fibrillation and venous thromboembolism patients. They are special population that needs appropriate care and optimum dosing of EDX. Hence, its monitoring in the patient plasma is fundamental, especially in emergency and special circumstances. However, such patient mostly receives many drugs of different pharmacological classes, side by side with EDX. This study represents the first attempt to quantify EDX in plasma without interference of the plasma matrix or concomitant medications. An accurate RP-HPLC-DAD method was developed for this purpose. It succeeded to monitor EDX level, selectively, without interference of plasma matrix or 16 of its frequently co-administered drugs. All drugs were extracted from plasma samples by protein precipitation followed by evaporation and concentration. EDX was well resolved from the co-administered drugs on C8 column using linear gradient elution of methanol and phosphate buffer (pH 4), at a flow rate of 1 mL/min. EDX appeared at retention time 9.6 min and was quantified at its λmax (290 nm). It exhibited a linear response over the concentration range of 0.15-2.2 µg/mL plasma which covers the reported therapeutic concentration. The suggested method fulfilled the US FDA guidelines for bioanalytical method validation. The developed method is fully discussed in comparison with the reported techniques. An in vivo study was performed to ensure applicability of the method on real plasma samples without interference from plasma matrix, co-administered drugs or the expected metabolites. It presented a unique selectivity of the method that guarantees accurate laboratory monitoring of EDX in plasma in almost all combined treatments including such novel oral anticoagulant drug.

Surface-enhanced Raman Spectroscopy Coupled with Dispersive Solid-phase Extraction for the Rapid Detection of Tricyclazole Residues in Rice and Brassica campestris L. ssp. chinensis var. utilis Tsen.

An efficient methodology has been developed to determine the tricyclazole residue in matrix based on surface-enhanced Raman scattering (SERS) coupled with dispersible matrix solid-phase extraction. After pretreatment and test conditions optimization, peaks at 1373 and 1317 cm-1 in the SERS spectrum were respectively selected as quantitative peaks for rice and Brassica campestris L. ssp. chinensis var. utilis Tsen, respectively. The matrix standard curve-external standard method was used to quantitatively conduct a statistical analysis. The correlation between the quantitative peak response and tricyclazole concentration showed a significant linear relationship with a correlation coefficient of R2 > 0.99. The lowest spiked concentration was determined to be the quantitative limit that was below the maximum residue limits of tricyclazole. This study provides a sensitive, stable and rapid approach for the analysis of tricyclazole in above matrix via SERS, and it will be a useful complement to the quantitative analysis of tricyclazole in a complex matrix.

A Validated LC Method for the Enantiomeric Separation of EAI045 on Chiral Stationary Phase.

A simple and accurate chiral liquid chromatographic method was developed for enantiomeric resolution and determination of 2-(5-fluoro-2-hydroxyphenyl)-2-(1-oxo-2,3-dihydro-1H-isoindol-2-yl)-N-(1,3-thiazol-2-yl)acetamide (EAI045). The enantiomers of EAI045 were baseline resolved on a Chiralpak AD-H (250 mm × 4.6 mm, 5 µm) column using a mobile phase system containing n-hexane: 2-propanol (75: 25 v/v) at a flow rate of 1 mL min-1 at 30°C. The eluted analytes were subsequently detected with an ultraviolet detector at 254 nm. The effects of organic modifiers and temperature on the enantioselectivity and resolution of the enantiomers were evaluated. The calibration curves were plotted within the concentration range between 2 and 600 µg mL-1 (n = 11), and recoveries between 98.74% and 101.52% were obtained, with relative standard deviation < 1.4%. The limit of detection and limit of quantitation for R-enantiomer were 0.94 and 3.07 µg mL-1 and for S-enantiomer were 0.86 and 2.84 µg mL-1, respectively. The validated method was found to be suitable for enantiomeric separation and sufficiently accurate for the determination of enantiomeric purity of EAI045 in bulk drugs.

Phenolic Compounds, Antioxidant Activities, and Inhibitory Effects on Digestive Enzymes of Different Cultivars of Okra (Abelmoschus esculentus).

In this study, the phenolic profiles and bioactivities of five representative cultivars of okra collected in China were investigated. Noticeable variations of phenolic compounds and their bioactivities were observed among these different cultivars of okra. The contents of total flavonoids (TFC) in "Shuiguo", "Kalong 8", "Kalong 3", "Wufu", and "Royal red" ranged from 1.75 to 3.39 mg RE/g DW, of which "Shuiguo" showed the highest TFC. Moreover, five individual phenolic compounds were found in okra by high performance liquid chromatography analysis, including isoquercitrin, protocatechuic acid, quercetin-3-O-gentiobioside, quercetin, and rutin, while isoquercitrin and quercetin-3-O-gentiobioside were detected as the main phenolic compounds in okra. Moreover, all tested okra exhibited significant antioxidant activities (2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity, 2,2'-azino-bis (3-ethylenzthiazoline-6-sulphonic acid) radical scavenging capacity, and ferric reducing antioxidant power) and inhibitory effects on digestive enzymes (lipase, α-glucosidase, and α-amylase). Indeed, "Shuiguo" exhibited much better antioxidant activities and inhibitory activities on digestive enzymes, which might be attributed to its high TFC. Results suggested that okra, especially "Shuiguo", could be developed as natural antioxidants and inhibitors against hyperlipidemia and hyperglycemia in the fields of functional foods and pharmaceuticals, which could meet the increasing demand for high-quality okra with health-promoting properties in China.

A new thiodiketopiperzaine from the marine sponge Tedania sp.

A new thiodiketopiperzaine, tedanizaine A (1), together with six known ones, were isolated from the marine sponge Tedania sp. Their structures were determined by spectroscopic analyses. The absolute configuration of 1 was established by ECD calculation. Compound 1 was the second example of thiodiketopiperazine bearing a thiazolidine unit. Cytotoxic activities of 1 were also evaluated.

Metabolomics Assay Identified a Novel Virulence-Associated Siderophore Encoded by the High-Pathogenicity Island in Uropathogenic Escherichia coli.

To date, yersiniabactin remains the only identified siderophore encoded by the high pathogenicity island (HPI) in uropathogenic Escherichia coli (UPEC). In the present study, we aim to discover and identify new siderophores in the HPI-dependent biosynthetic pathway using a combinational strategy of metabolomics and genetics. A global metabolome assay of wild-type UTI89, UTI89ΔybtS, and UTI89ΔybtS with the substrate addition of salicylic acid found numerous unknown metabolite features that were encoded by the HPI with an obvious substrate dependency on salicylic acid. One metabolite feature with m/ z 307.0206 was shown to have a similar phenotype as yersiniabactin. Furthermore, isotope mass spectrum calculations and MS/MS annotations were combined to identify this metabolite as HPTzTn-COOH. HPTzTn-COOH was verified as a new siderophore in this study, and it was observed to have a robust capacity to chelate different metals, including Al3+, Ni2+, and Ca2+, in addition to binding Fe3+. Our data revealed that HPTzTn-COOH has a stronger diagnostic ability over the more conventionally used yersiniabactin, as characterized by its high production throughout UPEC strains harboring HPI. Altogether, our discoveries revise the siderophore family, and HPTzTn-COOH can be classified as an additional key siderophore along with yersiniabactin.

Karamomycins A-C: 2-Naphthalen-2-yl-thiazoles from Nonomuraea endophytica.

Karamomycins A-C (2-4), the first natural 2-naphthalen-2-yl-thiazole derivatives, were isolated along with a plausible precursor molecule, 1-hydroxy-4-methoxy-2-naphthoic acid (1), uracil, 1-acetyl-ß-carboline, and actinomycin C2 from the culture broth of the terrestrial actinomycete strain GW58/450, identified as Nonomuraea endophytica. These compounds were characterized by analysis of their NMR and mass spectrometry (MS) data; the absolute configurations of 2 and 4 were determined by comparison of 13C NMR, NOESY, and circular dichroism (CD) spectra with density functional theory (DFT)-calculated data. In karamomycin C (4), the thiazole of 2 is connected to an unusual iminothiazolo[4,3- c][1,4]thiazepinone, for which we proposed a biosynthetic origin from two cysteine residues. It is closely related to ulbactin F; however, the heterocycle is enantiomeric to the latter and connected to phenol instead of 4-methoxy-1-naphthol. Karamomycins A (2) and C (4) were cytotoxic.

Development of magnetic carbon nanotubes for dispersive micro solid phase extraction of the cyanide metabolite, 2-aminothiazoline-4-carboxylic acid, in biological samples.

2-aminothiazoline-4-carboxylic acid (ATCA) is a minor metabolite of cyanide and is suggested to be a promising biomarker for cyanide exposure due to its specificity to cyanide metabolism and its excellent short- and long-term stability during storage. In this study, magnetic carbon nanotubes, including magnetic multi-walled carbon nanotubes (Mag-MWCNT) and magnetic single-walled carbon nanotubes (Mag-SWCNT) were synthesized as a novel sorbent for dispersive micro solid phase extraction (d-µSPE) to extract ATCA from biological matrices. ATCA spiked deionized water samples with the addition of the isotopic internal standard (ATCA - 13C, 15N) were subjected to Mag-CNT/d-µSPE to confirm extraction efficiency of this new technique. The extracted ATCA was derivatized and quantitated using gas chromatography/mass spectrometry (GC/MS) analysis. The extraction parameters were optimized and a detection limits of 15 and 25 ng/mL were obtained for synthetic urine and bovine blood respectively with a linear dynamic range of 30-1000 ng/mL. The optimized Mag-CNT/d-µSPE method facilitated efficient extraction of ATCA using 2 mg of Mag-MWCNT with a 10-minute extraction time. The current assay was also found to be effective for the extraction of ATCA with average recoveries of 97.7 ±â€¯4.0% (n = 9) and 96.5 ±â€¯12.1% (n = 9) from synthetic urine and bovine blood respectively. The approach of using Mag-CNT to facilitate d-µSPE offered a novel alternative to extract ATCA from complex biological matrices.

Theoretical and experimental approach to hydrophilic interaction dispersive solid-phase extraction of 2-aminothiazoline-4-carboxylic acid from human post-mortem blood.

In this paper, we proposed an innovative hydrophilic interaction dispersive solid-phase extraction (HI-d-SPE) protocol suitable for the isolation of the potential cyanide intoxication marker, 2-aminothiazoline-4-carboxylic acid (ATCA), from such complicated matrix as post-mortem blood. To create an optimal HI-d-SPE protocol, two sorbents were used: a molecularly imprinted polymer (MIP) and commercially available Oasis-MCX®. The latter sorbent was identified as more recovery-efficient with higher clean-up abilities in a carefully optimized process. Computational analysis was employed to provide insight into the adsorption mechanism of the two selected sorbents. The theoretical results were in agreement with the experiment regarding the efficiency of the sorbent. HI-d-SPE was successfully applied to the analysis of ATCA in 20 post-mortem blood samples using LC-MS/MS. The analytical performance of the method was finally compared to prior existing methods, in turn revealing its superiority.

Removal of Two Neonicotinoid Insecticides and Mineralization of 14C-Imidacloprid in Biomixtures.

Environmental contamination with neonicotinoid insecticides represents an issue of wide concern due to their negative effects on pollinators. The goal of this work was to evaluate the potential use of biomixtures employed in biopurification systems (BPS) to remove two neonicotinoid pesticides, imidacloprid and thiamethoxam, from wastewater of agricultural origin. The removal was assayed by quantification of the parent compounds and the detection of putative transformation products of imidacloprid by means of LC-MS/MS, and mineralization of radiolabeled imidacloprid. Two biomixtures (B1, B2) were prepared using coconut fiber, compost and two soils pre-exposed to imidacloprid (volumetric composition 50:25:25). After spiking of neonicotinoids and 228 days of treatment, the removal ranged from 22.3%-30.3% and 38.6%-43.7% for imidacloprid and thiamethoxam, respectively. Transformation products imidacloprid-urea, desnitro-imidacloprid and desnitro-olefin-imidacloprid were detected in both biomixtures. The mineralization of 14C-imidacloprid revealed DT50 (mineralization half-lives) values of 3466 and 7702 days in the biomixtures B1 and B2, respectively, markedly lower than those in the soil used in their preparation (8667 and 9902 days, respectively). As demonstrated by these findings, the high persistence of these compounds in the BPS suggests that additional biological (or physicochemical) approaches should be explored in order to decrease the impact of neonicotinoid-containing wastewater of agricultural origin.

Bioaugmentation potential of a newly isolated strain Sphingomonas sp. NJUST37 for the treatment of wastewater containing highly toxic and recalcitrant tricyclazole.

In order to develop an effective bioaugmentation strategy for the removal of highly toxic and recalcitrant tricyclazole from wastewater, a tricyclazole degrading strain was firstly successfully isolated and identified as Sphingomonas sp. NJUST37. In batch reactors, 100 mg L-1 tricyclazole could be completely removed within 102 h, which was accompanied by significant biomass increase, TOC and COD removal, as well as toxicity reduction. Chromatography analysis and density functional theory simulation indicated that monooxygenation occurred firstly, followed by triazole ring cleavage, decyanation reaction, hydration reaction, deamination, dihydroxylation and final mineralization reaction. Tricyclazole biodegradation condition by NJUST37 was optimized in terms of temperature, pH, tricyclazole concentration and additional carbon and nitrogen sources. After the inoculation of NJUST37 into a pilot-scale powdered activated carbon treatment tank treating real fungicide wastewater, tricyclazole removal efficiency increased to higher than 90%, demonstrating the great potential of NJUST37 for bioaugmentation particularly on tricyclazole biodegradation in practice.

Application of magnetic molecularly imprinted polymers for extraction of imidacloprid from eggplant and honey.

A magnetic molecularly imprinted polymer (MMIP) adsorbent for imidacloprid was prepared using non-covalent approach with functionalized nano Fe3O4 particles (magnetic cores), imidacloprid (template), acrylic acid (functional monomer), ethylene glycol dimethacrylate (cross linker) and azobisisobutyronitrile (initiator) and used for selective separation of imidacloprid from honey and vegetable samples. The polymers were characterized using FT-IR spectroscopy, SEM and TEM images. For analysis of imidacloprid LC-MS/MS equipment was used. Adsorption kinetics was best explained by pseudo-second-order kinetic model. Adsorption data fitted well into linearized Freundlich equation (R2 > 0.98). Scatchard plot analysis indicates the presence of two classes of binding sites in the MMIPs with the Cmax of 1889.6 µg g-1 and 65448.9 µg g-1, respectively. MMIPs demonstrated much higher affinity for imidacloprid over structurally similar analogues acetamiprid (α = 23.59) and thiamethoxam (α = 17.15). About 87.1 ±â€¯5.0% and 90.6 ±â€¯5.6% of the added imidacloprid was recovered from MMIPs in case of fortified eggplant and honey samples, respectively.

Secondary metabolites from the Colletotrichum gloeosporioides A12, an endophytic fungus derived from Aquilaria sinensis.

Two new cyclohexene derivatives colletotricones A and B (1 and 2) and a new thiazole derivative colletotricole A (5), along with six known natural metabolites were isolated from the extract of Colletotrichum gloeosporioides A12, an endophytic fungus derived from Aquilaria sinensis. Among them, the colletotricones A and B possess a cyclohexenone skeleton, whereas the colletotricole A is a thiazole derivative. Their structures were fully assigned with the aid of extensive spectroscopic analysis and data from the literature. Moreover, cytotoxic activity in vitro of compounds 1 and 3-9 were evaluated against MCF-7, NCI-H460, HepG-2 and SF-268 tumour cell lines. The new compound 1 exhibited growth inhibitory activity against all the four tumour cell lines with IC50 values ranging from 15.7 to 46.8 µM.

Novel Bioactive Paulomycin Derivatives Produced by Streptomyces albus J1074.

Four novel paulomycin derivatives have been isolated from S. albus J1074 grown in MFE culture medium. These compounds are structural analogs of antibiotics 273a2α and 273a2ß containing a thiazole moiety, probably originated through an intramolecular Michael addition. The novel, thiazole, moiety-containing paulomycins show a lower antibiotic activity than paulomycins A and B against Gram-positive bacteria. However, two of them show an improved activity against Gram-negative bacteria. In addition, the four novel compounds are more stable in culture than paulomycins A and B. Thus, the presence of an N-acetyl-l-cysteine moiety linked to the carbon atom of the paulic acid isothiocyanate moiety, via a thioester bond, and the subsequent intramolecular cyclization of the paulic acid to generate a thiazole heterocycle confer to paulomycins a higher structural stability that otherwise will conduce to paulomycin degradation and into inactive paulomenols.

Lipid Peroxidation and Cyclooxygenase Enzyme Inhibitory Compounds from Prangos haussknechtii.

Purification of extracts from Prangos haussknechtii Bioss afforded prenylated coumarins 1 and 2, monoterpenoid 3, amino acid derivative 4, and seven known compounds. Spectroscopic methods permitted establishment of the structures and relative configuration of these compounds. The pure isolates were tested for antioxidant and anti-inflammatory activities using lipid peroxidation (LPO), 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and cyclooxygenase (COX-1 and -2) enzyme inhibitory assays. Compounds 1-4 inhibited LPO with IC50 values between 43 and 114 µM and reduced MTT to formazan blue between 48 and 128 µM. In anti-inflammatory assays using cyclooxygenase enzymes, COX-1 and -2, these compounds showed inhibition, with IC50 values ranging from 34 to 56 µM.