Chemical profiling and in-vitro anti-inflammatory activity of bioactive fraction(s) from Trichodesma indicum (L.) R.Br. against LPS induced inflammation in RAW 264.7 murine macrophage cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Trichodesma indicum (L.) R. Br. (family: Boraginaceae) is a medicinal herb largely used to treat arthralgia, rheumatoid arthritis, wound healing, dysentery, etc. It's mechanism of anti-inflammatory activity has not been systematically analyzed yet. AIM OF THE STUDY: The present study was undertaken to examine the anti-inflammatory effects of successive solvent extracts (n-hexane extract (HE), ethyl acetate extract (EA), ethanol extract (EE), aqueous extract (AE) and fractions of HE) from the aerial parts of Trichodesma indicum (TI) against lipopolysaccharide (LPS) stimulated inflammatory reaction using mouse macrophage RAW 264.7 cells. MATERIALS AND METHODS: Cytotoxic effects of the extracts and fractions of TI were assessed by MTT assay. The effect of extracts and fractions on the production of nitric oxide (NO) in RAW 264.7 macrophages were measured using the Griess reagent method. IL - 6, IL - 1ß, TNF-α, iNOS and COX-2 gene expressions were examined by a qRT-PCR method. RESULTS: RAW 264.7 macrophages pretreated with HE, EA, EE and AE of TI showed a significant decrease in the production of proinflammatory cytokines and NO without exhibiting cytotoxicity. The potent HE was fractionated using flash chromatography into FA, FB, FC, FD and FE. Among the five fractions, FE displayed a stronger ability to reduce IL - 1ß, TNF-α, iNOS, COX2 and NO importantly no cytotoxicity was observed. The phytochemical compounds present in FE were further screened by Gas chromatography - Mass spectroscopy (GC-MS). GC-MS analysis revealed that 1,2-benzenedicarboxylic acid diisooctyl ester is the major compound in FE. Molecular docking analysis showed good inhibition of 1,2-benzenedicarboxylic acid diisooctyl ester against TLR-4, NIK and TACE. CONCLUSION: Our results suggested that 1,2-benzenedicarboxylic acid diisooctyl ester could be a potential candidate in alleviating inflammatory reactions in TI.

Eremophilane Sesquiterpenes and Benzene Derivatives from the Endophyte Microdiplodia sp. WGHS5.

Three new eremophilane sesquiterpenes phomadecalins G-I (1-3) and two new benzene derivatives microdiplzenes A and B (12 and 13), together with nine known eremophilane sesquiterpenes (4-11 and 14) were isolated from an endophytic fungus, Microdiplodia sp. WGHS5. Their structures were elucidated by the interpretation of HR-ESI-MS and NMR data; meanwhile, the absolute configurations of new compounds were determined on the base of ECD calculations. All compounds were evaluated for the antimicrobial activities and antiproliferative effect on human gastric cancer cell lines (BGC-823).

7-HYB, a Phenolic Compound Isolated from Myristica fragrans Houtt Increases Cell Migration, Osteoblast Differentiation, and Mineralization through BMP2 and ß-catenin Signaling.

The seeds (nutmegs) of Myristica fragrans Houtt have been used as popular spices and traditional medicine to treat a variety of diseases. A phenolic compound, ((7S)-8'-(benzo[3',4']dioxol-1'-yl)-7-hydroxypropyl)benzene-2,4-diol (7-HYB) was isolated from the seeds of M. fragrans. This study aimed to investigate the anabolic effects of 7-HYB in osteogenesis and bone mineralization. In the present study, 7-HYB promotes the early and late differentiation of MC3T3-E1 preosteoblasts. 7-HYB also elevated cell migration rate during differentiation of the preosteoblasts with the increased phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2, p38, and JNK. In addition, 7-HYB induced the protein level of BMP2, the phosphorylation of Smad1/5/8, and the expression of RUNX2. 7-HYB also inhibited GSK3ß and subsequently increased the level of ß-catenin. However, in bone marrow macrophages (BMMs), 7-HYB has no biological effects in cell viability, TRAP-positive multinuclear osteoclasts, and gene expression (c-Fos and NF-ATc1) in receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. Our findings suggest that 7-HYB plays an important role in osteoblast differentiation through the BMP2 and ß-catenin signaling pathway. It also indicates that 7-HYB might have a therapeutic effect for the treatment of bone diseases such as osteoporosis and periodontitis.

Benwamycins A-G, Trialkyl-Substituted Benzene Derivatives from a Soil-Derived Streptomyces.

Seven new trialkyl-substituted benzene derivatives named benwamycins A-G (1-7), together with three known congeners, 8-10, were isolated from culture broth of the soil-derived Streptomyces sp. KIB-H1471. Their structures were elucidated by using 1D and 2D NMR analyses in combination with HRESIMS data. The absolute configurations of 1-9 were determined by chemical conversion and comparison of circular dichroism spectra and confirmed for 1 by single-crystal X-ray crystallography. Compounds 6 and 7 have a unique γ-pyrone-like ring on one side chain. Compounds 2 and 6 inhibited human T cell proliferation with IC50 values of 14.3 and 12.5 µM, respectively, without obvious cytotoxicity for naïve human T cells. Compounds 3 and 6 could weakly enhance insulin-stimulated glucose uptake.

A novel anti-platelet aggregation target of chinensinaphthol methyl ether and neojusticin B obtained from Rostellularia procumbens (L.) Nees.

This study explored the possible bioactive ingredients and target protein of Rostellularia procumbens (L.) Nees. The results of optical turbidimetry revealed that the ethyl acetate extraction obtained from R. procumbens (L.) Nees could inhibit platelet aggregation. Gene chip was used to investigate differentially expressed genes. According to the results of the gene chip, the targets of compounds isolated from the ethyl acetate extraction were predicted by network pharmacology. Computational studies revealed that chinensinaphthol methyl ether and neojusticin B may target the integrin αIIbß3 protein. The results of Prometheus NT.48 and microscale thermophoresis suggested that the molecular interactions between the two compounds with purified integrin αIIbß3 protein in the optimal test conditions were coherent with the docking results. To our best knowledge, this is the first report to state that chinensinaphthol methyl ether and neojusticin B target the integrin αIIbß3 protein.

Chemical Constituents from Chloranthus anhuiensis and Their Cytotoxic Activities.

Three hiherto unknown phenylpropanoid compounds, namely (7S,8R)-1-(1-ethoxy-2-hydroxypropyl)-2-methoxy-3,4-(methylenedioxy)benzene (1), (7S,8S)-1-(1-ethoxy-2-hydroxypropyl)-2-methoxy-3,4-(methylenedioxy)benzene (2), and (7S,8R)-1-(1-methoxy-2-hydroxypropyl)-2-methoxy-3,4-(methylenedioxy)benzene (3), along with 12 known compounds (4 - 15) were obtained from the extract of whole plant of Chloranthus anhuiensis. Among them, 7 and 13 were obtained from nature for the first time. The structures of these natural compounds were characterized by extensive spectroscopic analysis and calculated electronic circular dichroism (ECD) data. Furthermore, their cytotoxic and neuroprotective activities were evaluated using MDA-MB-231, 4T1, HepG2, and PC12 cell lines. Compounds 8 and 13 exhibited moderate cytotoxic activities against MDA-MB-231 cell line with the IC50 values of 39.7 and 25.8 µm, respectively. And all the isolated compounds have no neuroprotective activities.

Profile of Polyphenolic and Essential Oil Composition of Polish Propolis, Black Poplar and Aspens Buds.

In this work, we studied similarities and differences between 70% ethanol in water extract (70EE) and essential oils (EOs) obtained from propolis, black poplars (Populus nigra L.) and aspens (P. tremula L.) to ascertain which of these is a better indicator of the plant species used by bees to collect propolis precursors. Composition of 70EE was analyzed by UPLC-PDA-MS, while GC-MS was used to research the EOs. Principal component analyses (PCA) and calculations of Spearman's coefficient rank were used for statistical analysis. Statistical analysis exhibited correlation between chemical compositions of propolis and Populus buds' 70EE. In the case of EOs, results were less clear. Compositions of black poplars, aspens EOs and propolises have shown more variability than 70EE. Different factors such as higher instability of EOs compared to 70EE, different degradation pattern of benzyl esters to benzoic acid, differences in plant metabolism and bees' preferences may be responsible for these phenomena. Our research has therefore shown that 70EE of propolis reflected the composition of P. nigra or complex aspen⁻black poplar origin.

Lab-In-Syringe automation of stirring-assisted room-temperature headspace extraction coupled online to gas chromatography with flame ionization detection for determination of benzene, toluene, ethylbenzene, and xylenes in surface waters.

Online coupling of Lab-In-Syringe automated headspace extraction to gas chromatography has been studied. The developed methodology was successfully applied to surface water analysis using benzene, toluene, ethylbenzene, and xylenes as model analytes. The extraction system consisted of an automatic syringe pump with a 5 mL syringe into which all solutions and air for headspace formation were aspirated. The syringe piston featured a longitudinal channel, which allowed connecting the syringe void directly to a gas chromatograph with flame ionization detector via a transfer capillary. Gas injection was achieved via opening a computer-controlled pinch valve and compressing the headspace, upon which separation was initialized. Extractions were performed at room temperature; yet sensitivity comparable to previous work was obtained by high headspace to sample ratio VHS/VSample of 1.6:1 and injection of about 77% of the headspace. Assistance by in-syringe magnetic stirring yielded an about threefold increase in extraction efficiency. Interferences were compensated by using chlorobenzene as an internal standard. Syringe cleaning and extraction lasting over 10 min was carried out in parallel to the chromatographic run enabling a time of analysis of <19 min. Excellent peak area repeatabilities with RSD of <4% when omitting and <2% RSD when using internal standard corrections on 100 µg L-1 level were achieved. An average recovery of 97.7% and limit of detection of 1-2 µg L-1 were obtained in analyses of surface water.

Micro-solid phase extraction of benzene, toluene, ethylbenzene and xylenes from aqueous solutions using water-insoluble ß-cyclodextrin polymer as sorbent.

Water-insoluble ß-cyclodextrin polymer was synthesized by chemical cross-linking using epichlorohydrin (EPI) as a cross-linker agent. The produced water-insoluble polymer was used as a sorbent for the micro-solid phase extraction (µ-SPE) of benzene, toluene, ethylbenzene and xylenes (BTEX) from water samples. The µ-SPE device consisted of a sealed tea bag envelope containing 15mg of sorbent. For the evaluation of the extraction efficiency, parameters such as extraction and desorption time, desorption solvent and salt concentration were investigated. At an extraction time of 30min in the course of the extraction process, analytes were extracted from a 10mL aqueous sample solution. The analytes were desorbed by ultrasonication in 200µL of acetonitrile for 20min. Analysis of the analytes was done by a gas chromatography-flame ionization detector (GC-FID) system. The enrichment factor (EF) was found to be in the range 23.0-45.4 (EFmax=50.0). The method provided linearity ranges of between 0.5 and 500.0ng/mL (depending on the analytes), with good coefficients of determination (r2) ranging between 0.997 and 0.999 under optimized conditions. Detection limits for BTEX were in the range of between 0.15 and 0.60ng/mL, while corresponding recoveries were in the range of 46.0-90.0%. The relative standard deviation of the method for the analytes at 100.0ng/mL concentration level ranged from 5.5 to 11.2% (n=5). The proposed method was concluded to be a cost effective and environmentally-friendly extraction technique with ease of operation and minimal usage of organic solvent.

Lorneic Acid Analogues from an Endophytic Actinomycete.

Our natural products discovery program utilizes endophytic actinomycetes associated with plants and employs biological assays and HPLC-based metabolite profiles as the preliminary screen to identify strains of interest, followed by large-scale fermentation and isolation, leading to new and/or bioactive natural products. Six new trialkyl-substituted aromatic acids, namely, lorneic acids E-J (1-6), together with two known analogues (7 and 8), were isolated and identified from the culture extract of Streptomyces sp. KIB-H1289, an endophytic actinomycete obtained from the inner tissue of the bark of Betula mandshurica Nakai. The structures were characterized by interpretation of their spectroscopic data, mainly 1D and 2D NMR. Among them, compound 5 contains a unique disulfide bond that is presumably derived from N-acetylcysteine. All isolated metabolites were evaluated for their inhibitory activity on tyrosinase.

New amide and dioxopiperazine derivatives from leaves of Breynia nivosa.

The first chemical investigation of leaves of Breynia nivosa from Nigeria resulted in the isolation of two new amide derivatives breynivosamides A and B (1 and 2) and two new dioxopiperazine derivatives breynivosines A and B (4 and 5) together with seven known compounds (3, 6-11). The structures of the new compounds were elucidated by 1D, 2D NMR and HRESIMS data as well as by comparison with the literature. All isolated compounds were tested for the cytotoxic and antimicrobial activities. Only cristatin A (6) showed cytotoxicity against the L5178Y mouse lymphoma cell line with an IC50 value of 13.9µM while breynivosamide A (1) exhibited moderate antimicrobial activity against Mycobacterium tuberculosis with an MIC value of 25µM.

New Benzenoid Derivatives and Other Constituents from Lawsonia inermis with Inhibitory Activity against NO Production.

Three new benzenoid derivatives, lawsoinermone (1), inermidioic acid (2), and inermic acid (3) have been isolated from the aerial part of Lawsonia inermis, together with 11 known compounds (4-14). The structures of three new compounds were determined through spectroscopic and MS analyses. Compounds 1, 4-6, 13 and 14 were evaluated for inhibition of nitric oxide production in LPS-stimulated product of nitrite in RAW 264.7 cells with IC50 values of 6.12, 16.43, 18.98, 9.30, 9.30 and 14.90 µg/mL, respectively.

Graphitic carbon nitride nanofibers in seaweed-like architecture for gas chromatographic separations.

Seaweed-like graphitic carbon nitride (g-C3N4) has a unique porous architecture composed of interlocking g-C3N4 nanofibers (NF-C3N4) with much higher surface area than bulk g-C3N4 and shows good potential in separation science. This work investigated the separation performance of NF-C3N4 as stationary phase for capillary gas chromatographic (GC) separations. The NF-C3N4 column exhibits weak polarity and high column efficiency of 4728 plates/m for n-dodecane. Importantly, it displays good separation performance for a wide range of analytes and shows different retention behaviors from the bulk g-C3N4 column and commercial HP-5MS column with 5% phenylpolysiloxane. Particularly, it shows high resolving capability for both aliphatic and aromatic isomers. In addition, NF-C3N4 column has high thermal stability up to 280°C and good separation repeatability with relative standard deviation (RSD) values in the range of 0.29-0.61% for intra-day, 0.56-1.1% for inter-day and 2.0-4.9% for between-column, respectively. Moreover, it was applied for the determination of isomer impurities in real samples, showing good potential in GC applications.

Zeolite/iron oxide composite as sorbent for magnetic solid-phase extraction of benzene, toluene, ethylbenzene and xylenes from water samples prior to gas chromatography⬜mass spectrometry.

This study reports a new composite based on ZSM-5 zeolite decorated with iron oxide magnetic nanoparticles as a valuable sorbent for magnetic solid-phase extraction (MSPE). A proposal is made to determine benzene, toluene, ethylbenzene and xylenes (BTEX) as model analytes in water samples using gas chromatography-mass spectrometry. A two-step multivariate optimization strategy, using Plackett⬜Burman and circumscribed central composite designs, was employed to optimize experimental parameters affecting MSPE. The method was evaluated under optimized extraction conditions (i.e., amount of sorbent, 138mg; extraction time, 11min; sample pH, pH of water (i.e., 5.5⬜6.5); eluent solvent volume, 0.5mL; and elution time, 5min), obtaining a linear response from 1 to 100µgL(↙1) for benzene; from 10 to 100µgL(↙1) for toluene, ethylbenzene and o-xylene; and from 10 to 75µgL(↙1) for m,p-xylene. The repeatability of the proposed method was evaluated at a 40µgL(↙1) spiking level and coefficients of variation ranged between 8 and 11% (n=5). Limits of detection were found to be 0.3µgL(↙1) for benzene and 3µgL(↙1) for the other analytes. These values satisfy the current normative of the Environmental Protection Agency and European Union for BTEX content in waters for human consumption. Finally, drinking water, wastewater and river water were selected as real water samples to assess the applicability of the method. Relative recoveries varied between 85% and 114% showing negligible matrix effects.

Sulfotanone, a new alkyl sulfonic acid derivative from Streptomyces sp. IFM 11694 with TRAIL resistance-overcoming activity.

One new alkyl sulfonic acid derivative, sulfotanone (1), and the known panosialin wA (2) were isolated from the methanolic extract of mycelium of Streptomyces sp. 11694. The structure of the new compound (1) was established by a combination of spectroscopic techniques, including HRESIMS, IR, 1D and 2D NMR measurements. Compound 1 (40 µM) in combination with TRAIL showed synergistic activity in sensitizing TRAIL-resistance in human gastric adenocarcinoma cell lines.

Suncheonosides A-D, Benzothioate Glycosides from a Marine-Derived Streptomyces sp.

A marine-derived Streptomyces strain, SSC21, was isolated from the sediment of Suncheon Bay, Republic of Korea. Chemical analysis of the bacterial strain resulted in the isolation of four new metabolites, suncheonosides A-D (1-4, respectively), each bearing a sulfur atom. The planar structures of the suncheonosides were identified as hexasubstituted benzothioate glycosides by combined spectroscopic analyses. Analysis of the configuration of the sugar moieties based on ROESY nuclear magnetic resonance correlations, one-bond (1)H-(13)C coupling constant analysis, and chemical derivatizations indicated that the suncheonosides incorporate only l-rhamnose. Suncheonosides A, B, and D promoted adiponectin production in a concentration-dependent manner during adipogenesis in human mesenchymal stem cells, suggesting antidiabetic potential.

Quantification of benzene, toluene, ethylbenzene and o-xylene in internal combustion engine exhaust with time-weighted average solid phase microextraction and gas chromatography mass spectrometry.

A new and simple method for benzene, toluene, ethylbenzene and o-xylene (BTEX) quantification in vehicle exhaust was developed based on diffusion-controlled extraction onto a retracted solid-phase microextraction (SPME) fiber coating. The rationale was to develop a method based on existing and proven SPME technology that is feasible for field adaptation in developing countries. Passive sampling with SPME fiber retracted into the needle extracted nearly two orders of magnitude less mass (n) compared with exposed fiber (outside of needle) and sampling was in a time weighted-averaging (TWA) mode. Both the sampling time (t) and fiber retraction depth (Z) were adjusted to quantify a wider range of Cgas. Extraction and quantification is conducted in a non-equilibrium mode. Effects of Cgas, t, Z and T were tested. In addition, contribution of n extracted by metallic surfaces of needle assembly without SPME coating was studied. Effects of sample storage time on n loss was studied. Retracted TWA-SPME extractions followed the theoretical model. Extracted n of BTEX was proportional to Cgas, t, Dg, T and inversely proportional to Z. Method detection limits were 1.8, 2.7, 2.1 and 5.2 mg m(-3) (0.51, 0.83, 0.66 and 1.62 ppm) for BTEX, respectively. The contribution of extraction onto metallic surfaces was reproducible and influenced by Cgas and t and less so by T and by the Z. The new method was applied to measure BTEX in the exhaust gas of a Ford Crown Victoria 1995 and compared with a whole gas and direct injection method.

The effect of the potential fuel additive isobutanol on benzene, toluene, ethylbenzene, and p-xylene degradation in aerobic soil microcosms.

Isobutanol is being considered as a fuel additive; however, the effect of this chemical on gasoline degradation (following a spill) has yet to be fully explored. To address this, the current study investigated the effect of isobutanol on benzene, toluene, ethylbenzene and p-xylene (BTEX) degradation in 14 sets of experiments in saturated soils. This involved four hydrocarbons for three soils (12 experiments) and two extra experiments with a lower level of isobutanol (for toluene only). Each soil and hydrocarbon combination involved four abiotic control microcosms and 12 sample microcosms (six with and six without isobutanol). The time for complete degradation of each hydrocarbon varied between treatments. Both toluene and ethylbenzene were rapidly degraded (5-13 days for toluene and 3-13 days for ethylbenzene). In contrast, the time for complete degradation for benzene ranged from 5 to 47 days. The hydrocarbon p-xylene was the most recalcitrant chemical (time for removal ranged from 14 to 86 days) and, in several microcosms, no p-xylene degradation was observed. The effect of isobutanol on hydrocarbon degradation was determined by comparing degradation lag times with and without isobutanol addition. From the 14 treatments, isobutanol only affected degradation lag times in three cases. In two cases (benzene and p-xylene), an enhancement of degradation (reduced lag times) was observed in the presence of isobutanol. In contrast, toluene degradation in one soil was inhibited (increased lag time). These results indicate that co-contamination with isobutanol should not inhibit aerobic BTEX degradation rates.

Anodic alumina coating for extraction of volatile organic compounds in human exhaled breath vapor.

The objective of the study is to develop a facile and highly sensitive solid phase microextraction-gas chromatography/mass spectrometry method for the analysis of volatile organic compounds in human exhaled breath vapor. For the purpose, a highly ordered nanoporous anodic alumina coating was prepared by a two-step anodic oxidization method based on aluminum substrate. To have a good knowledge of the fiber, some features were characterized and the results indicate that the coating has several advantages, including excellent chemical and thermal stability, high mechanical strength, large surface area and good extraction performance. In addition, some parameters related to extraction efficiency were also studied. Under the optimal conditions, the coating was used to quantitatively extract volatile organic compounds. Good linearity and wide linear range were obtained with correlation coefficients (R(2)) ranging from 0.9933 to 0.9999. The detection limits of benzene homologues, aldehydes and ketones were between 0.7 and 3.4 ng L(-1). Relative standard deviations (n=5) ranged from 1.8 to 15.0%. Satisfied recovery (89-115%) was obtained at two spiked concentration levels. Finally, the developed method was successfully applied for the analysis of volatile organic compounds in human exhaled vapor samples of lung cancer patients and the controls, and the results were statistically analyzed with Independent-Sample T Test. The proposed method exhibits some outstanding merits, including convenience, non-invasion, low cost and sensitivity. It provides a potential tool for rapid detection of volatile organic compounds in human exhaled breath.