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Five new xanthones, named coxanthones A-E (1-5), together with 21 known secondary metabolites (6-26) that include seven xanthones, five flavonoids, two steroids and seven triterpenoids were isolated from the chemically unexplored whole plant Codonopsis ovata. The structures of new metabolites were elucidated by HRMS, interpretation of NMR spectra and other spectroscopic techniques. The absolute configuration of the stereogenic centre of coxanthone B (2) was determined by electronic circular dichroism (ECD) spectroscopy. This is the first report of xanthones from the genus Codonopsis. All isolated metabolites were evaluated for cytotoxic activity by SRB assay against six human cancer cell lines A549 (lung), PC-3 (prostate), HCT-116 (colon), MCF-7 (breast), SF-295 (CNS), and MDAMB-435 (melanoma). Among the new compounds, coxanthone B (2) exhibited significant inhibitory activity against SF-295 and MDAMB-435 with IC 50 values of 7.0 and 15.0 μM, respectively. Coxanthone A (1) displayed cytotoxicity against A549 cell line at IC 50 value of 22.5 μM. Cytotoxic activity of 1-hydroxy-3,5-dimethoxyxanthone (7), swertiperenine (9) and 1,7,8-trihydroxy-3-methoxyxanthone (10) are reported here first time that exhibited the IC 50 values of 3.0, 5.0 and 21.0 μM against A549, MDAMB-435, and A549 cell lines, respectively. Kaempferol (13) showed most potent cytotoxic activity with an IC 50 values in the 1.0-2.3 μM range against all tested cancer cell lines., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Timosaponin B-II (TB-II) is a main active saponin isolated from the rhizome of Anemarrhena asphodeloides Bge., which is widely used in traditional Chinese medicine. In this study, the effect of TB-II on learning and memory was investigated in a scopolamine-induced mouse model of Alzheimer's disease. The results of behavioral tests indicated that TB-II significantly increased the spontaneous alternation in the Y-maze test, and reversed the shortening of step-through latency induced by scopolamine in the passive avoidance test, showing protective effects on short-term and working memory. In the Morris water maze test, TB-II reduced the escape latency time in the training trial, and increased the swimming time in the target quadrant in the probe trial. Biochemical data demonstrated that TB-II significantly inhibited acetylcholinesterase (AChE) activity in the cerebral cortex and hippocampus of mice. Moreover, TB-II markably attenuated the reduction in glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, and decreased malondialdehyde (MDA) levels, which are key biomarkers of brain oxidative stress. These results indicated that TB-II offers protection against scopolamine-induced deficits in learning and memory, possibly by inhibiting AChE and preventing oxidative stress damage. The findings suggested that TB-II has a potential therapeutic effect on cognitive and behavioral impairment.

Objective Taccaoside, a steroidal saponin, has been shown to be cytotoxic, although the mechanism of cytotoxicity remains unclear. This study examined the effect of taccaoside on the human hepatocellular carcinoma (HCC) cell lines SMMC-7721 and Bel-7404. Methods The antiproliferative effect of taccaoside were measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Cells were stained with Hoechst 33258 to observe morphology. Cell cycle and apoptosis were analysed by flow cytometry. Caspase activation was detected using specific assays, and PARP, Bax and Bcl-2 expression were analysed using western blotting. Results Taccaoside showed antiproliferative effect on HCC cell lines growth in a concentration- and time-dependent manner. Taccaoside arrested cell cycle in the G2/M phase and induced caspase-dependent apoptosis. Western blotting indicated that taccaoside upregulated Bax expression and downregulated Bcl-2 expression. PARP cleavage was observed following taccaoside treatment. Conclusions This study showed that taccaoside may inhibit HCC cell proliferation by inducing apoptosis.

Two new steroidal glycosides (1-2) were isolated from the roots of Reineckia carnea, together with three known compounds (3-5). Their structures were determined on the basis of chemical methods and spectral data. Compounds 1-2 were the unique steroidal glycosides possessing structural feature of 14α-hydroxy-5β-steroids, and compounds 4-5 were isolated from R. carnea for the first time. The isolated compounds (1-5) were tested in vitro for their cytotoxic activities against the A549, HepG 2 and Caski cancer cell lines. Among them, the compounds 2 and 3 showed cytotoxicity against Caski cancer cell line with IC 50 values of 34.4 and 3.7μM, respectively., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To develop aqueous microseparation columns for bioactive compounds, a thermoresponsive polymer grafted polymer monolith was prepared inside silica capillaries having an I.D. of 100μm by polymerization of styrene (St) with m/p-divinylbenzene (DVB) in the presence of polydimethylsiloxane as porogen, followed by surface-initiated atom transfer radical polymerization (SI-ATRP) of N-isopropylacrylamide (NIPAAm). SEM analysis indicated that the resulting poly(N-isopropylacrylamide) (PNIPAAm) grafted polystyrene monolith had a consecutive three-dimensionally interconnected structure and through-pores, similar to the base polystyrene (PSt) monolith. The elution behavior of steroids with different hydrophobicity was evaluated using micro-high-performance liquid chromatography in sole aqueous mobile phase. Temperature dependent interaction changes were observed between steroids and the PNIPAAm modified surfaces. Furthermore, the interaction between bioactive compounds and the PNIPAAm grafted PSt surfaces was controlled and eventually separate these molecules with different hydrophobicities by simple temperature modulation in aqueous environment. The PNIPAAm grafted PSt monolithic capillary showed improved separation properties of bioactive compounds, compared with a PNIPAAm grafted hollow capillary in aqueous environment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Timosaponin AIII (TAIII) exhibits extensive pharmacological activities and has been reported as a potent antitumour agent for various human cancers. In the present study, a potential industrial process for producing TAIII that involves biotransformation directly in the crude extract liquid of rhizoma anemarrhenae (RA) was developed. β-D-glycosidase was used to transform timosaponin BII (TBII) into TAIII, and monofactor experiments were conducted to optimise the enzymolysis conditions. In addition, AB-8 macroporous resin column chromatography, preparative liquid chromatography, and crystallisation technique were applied for yielding TAIII crystals with a purity > 97%. Approximately, 7 g of TAIII with a high purity of > 97% was obtained from 1 kg of RA through this five-step preparation method, which can be used to produce TAIII on a large scale.

A new 10-demethylated steroid, nephtheasteroid A (1), a new 19-oxygenated steroid, nephtheasteroid B (2) as well as five known steroids 3-7 were isolated from the organic extract of a Taiwanese soft coral Nephthea erecta. The structure was determined by means of IR, MS, and NMR techniques. Among these metabolites, 1 is rarely found in steroids possessing a 19-norergostane skeleton. In vitro cytotoxicity study using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that compounds 3 and 4 exhibited cytotoxicity against human chronic myelogenous leukemia (K562), human acute lymphoblastic leukemia (Molt-4), human T lymphoblastoid (Sup-T1), and human leukemic monocyte lymphoma (U937), with IC 50 of 6.5-14.0 µM.

Steroid analysis is being conquered by liquid chromatography tandem mass spectrometry (LC-MS/MS) benefiting from higher standardization, selectivity and diversity. Regarding high throughput in routine diagnostics rapid chromatography is mandatory. Introducing MS(3) (MS/MS/MS), specificity of mass spectrometric detection can be enhanced without sacrificing analysis time. 100mL of human plasma/serum, saliva, urine and 10-20mg of hair are used for the simultaneous quantification of 17α-hydroxyprogesterone, aldosterone, androstenedione, cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), estradiol, progesterone, and testosterone using online solid phase extraction (SPE) LC-MS/MS or LC-MS(3). Steroids can be analyzed in 4min after a single manual dilution and protein precipitation step. In complex sample matrices like hair MS(3) detection was found to be appropriate for quantitation. Lower limits of quantitation ranged from 37pmol/L (estradiol) up to 3.1nmol/L (DHEAS). General accuracy was 89-107% with between-run imprecision ≤10%. Comparison to immunoassays revealed significant differences in quantitation for urinary cortisol (-71% mean), aldosterone (-40% mean) and plasma aldosterone (-45% mean). The comparison of MS(2) and MS(3) quantitation of hair cortisol also revealed significant differences. In general, quantitation via MS(3) was not applicable for a long time. But with the current generation of mass spectrometers quantitation via MS(3) can be superior to MS(2) regarding specificity and accuracy when dealing with matrix issues. However, drawbacks regarding flexibility and precision have to be taken into account. Concludingly, simple protein precipitation combined with rapid online SPE LC-MS/MS/MS allows us to quantify over broad, essential concentration ranges in human serum, saliva, urine and hair., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Two polar steroid compounds, taurochenodeoxycholic acid sodium salt (1) and a rare cyclic steroid glycoside luzonicoside A (2), were isolated from the tropical starfish Leiaster sp. and identified by extensive NMR and ESIMS techniques. The isolation of primary bile acid 1 is the first report from a representative of the class Asteroidea and on the whole in invertebrates. Its presence confirms the hypothesis about the digestive role of some polar steroids in starfish and possibly demonstrates the parallel evolution of fat emulsifying agents in vertebrates and some starfish. Compound 2 was also obtained from starfish belonging to this genus for the first time. This finding indicates that cyclic steroid glycosides are more widely distributed in starfish than the two species of the genus Echinaster from which they were isolated earlier.

Six steroids (1-6), including the two new compounds 3β,4α-dihydroxyergosta-5,24(28)-diene (1) and 24(S),28-epoxyergost-5-ene-3β,4α-diol (2), were isolated from the methanol extract of the Vietnamese soft coral Sinularia nanolobata. Their structures were elucidated by spectroscopic methods including one and two dimensional (1D- and 2D)-NMR, Fourier transform ion cyclotron resonance (FT-ICR)-MS, and circular dichroism (CD). Compound 2 exhibited moderate cytotoxicity against the acute leukemia (HL-60) cell line with IC50 value of 33.53±4.25 µM and weak effect on the hepatoma cancer (HepG2) and colon adenocarcinoma (SW480) cell lines with IC50 values of 64.35±7.00 and 71.02±4.00 µM, respectively.

Four new steroidal glycosides, protolinckiosides A - D (1 - 4, resp.), were isolated along with four previously known glycosides, 5 - 8, from the MeOH/EtOH extract of the starfish Protoreaster lincki. The structures of 1 - 4 were elucidated by extensive NMR and ESI-MS techniques as (3β,4β,5α,6β,7α,15α,16β,25S)-4,6,7,8,15,16,26-heptahydroxycholestan-3-yl 2-O-methyl-β-d-xylopyranoside (1), (3β,5α,6β,15α,24S)-3,5,6,8,15-pentahydroxycholestan-24-yl α-l-arabinofuranoside (2), sodium (3β,6β,15α,16β,24R)-29-(β-d-galactofuranosyloxy)-6,8,16-trihydroxy-3-[(2-O-methyl-β-d-xylopyranosyl)oxy]stigmast-4-en-15-yl sulfate (3), and sodium (3β,6β,15α,16β,22E,24R)-28-(β-d-galactofuranosyloxy)-6,8,16-trihydroxy-3-[(2-O-methyl-β-d-xylopyranosyl)oxy]ergosta-4,22-dien-15-yl sulfate (4). The unsubstituted β-d-galactofuranose residue at C(28) or C(29) of the side chains was found in starfish steroidal glycosides for the first time. Compounds 1 - 4 significantly decreased the intracellular reactive oxygen species (ROS) content in RAW 264.7 murine macrophages at induction by proinflammatory endotoxic lipopolysaccharide (LPS) from E. coli., (© 2016 Wiley-VHCA AG, Zürich.)

Four new steroids, namely klyflaccisteroids G-J (1-4) were isolated from the Formosan soft coral Klyxum flaccidum. The structures of compounds 1-4 were established by spectral data analysis (IR, MS, 1D and 2D NMR) and comparison of spectral data with those of the related known compounds. Cytotoxicity assay revealed that 4 exhibited inhibition activity against the growth of HT-29, P388 and K562 cancer cell lines, whereas 2 showed selective cytotoxicity toward P388 cells. Compound 4 was also found to display significant anti-inflammatory activity for suppressing superoxide anion generation (O2(-)) and elastase release., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cancer remains a major killer worldwide. To search for novel naturally occurring compounds that are cytotoxic to cancer cells to be used as lead structures for drug development, five new steroids (1-5) along with seven known ones (6-12) were isolated from the rhizome of Anemarrhena asphodeloides Bge. Their structures were established by detailed spectral studies, including 1D-NMR, 2D-NMR, HR-ESI-MS and by comparison with literature data. These compounds exhibited different levels of growth inhibition against A549, HepG2, Hep3B, Bcap37 and MCF7 cell lines in vitro. Compounds 9, 10 and 11 showed potent inhibitory against all the tested cell lines with IC50 values ranging from 0.35±0.15 to 25.53±0.31μM. The three compounds displayed stronger inhibitory activities against A549, HepG2 and Hep3B cell lines compared with the positive control 5-fluorouracil. The experimental data obtained permit us to identify the roles of the sugar moieties, hydroxyl group, double bond and F-ring with regard to their cytotoxic activities., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Timosaponin A-III (TSA-III), a saponin isolated from the rhizome of Anemarrhena asphodeloides, exhibits potent cytotoxicity and has the potential to be developed as an anticancer agent. However, the molecular mechanism underlying the anticancer activity of TSA-III has not been fully elucidated. In this study, the apoptotic effects of TSA-III were investigated in HepG2 cells. Treatment with TSA-III significantly inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis in HepG2 cells. This induction was associated with increased fluorescence intensity of Annexin V-FITC, activation of caspases, and altered expression of inhibitor of apoptosis protein (IAP) family members. In addition, TSA-III mediated mitochondrial dysfunction with the release of HtrA2/Omi, Smac/Diablo, and cytochrome c. These findings suggest that TSA-III induces mitochondria-mediated and caspase-dependent apoptosis in HepG2 cells by altering expression of the IAP family. Thus, TSA-III could possibly be used to treat other types of cancer with similar pathologic mechanisms., (Copyright © 2016. Published by Elsevier B.V.)

Nephthea are soft coral species rich in sesquiterpenoids and steroids. An organic extract of Nephthea sp. resulted in the isolation of a new steroid (1), as well as several previously reported metabolites (2-9). Structures were elucidated by employing NMR and HR-EI-MS analyses. The total extract, fractions and purified compounds exhibited differential cytotoxicity against the breast cancer MCF-7 cell line.

Selective adsorbents for solid-phase extraction are needed to meet the low concentration requirements of new environmental quality standard directives, especially for the analysis of estrogens in wastewater. In this work, bulk polymerization procedures were first optimized for the synthesis of non-imprinted polymers (NIP) with low non-specific adsorption of nonpolar compounds in aqueous environments. Water-compatible molecularly imprinted polymers (MIP) were then synthetized by increasing the selectivity of the polymer towards steroids with a testosterone template (average imprinting factor > 10). In addition, the affinity of synthetized entrapped β-cyclodextrin-epichlorohydrin polymers (ECD) towards steroids was clarified. The polymers were applied to the extraction of spiked wastewater effluent samples and their performance compared to commercially available adsorbents. The selectivity of the studied adsorbents was evaluated utilizing liquid chromatography ‒ mass spectrometry as well as comprehensive two-dimensional gas chromatography ‒ time-of-flight mass spectrometry. Affinity between adsorbents and steroids as well as matrix removal potential were measured with targeted methodologies, and two novel non-targeted methodologies were proposed to quantitatively measure adsorbent selectivity by utilizing chemometrics. Semi-quantitative selectivity was measured from the ratio of peak areas between steroidal and other compounds. Semi-qualitative selectivity was calculated from the ratio between the number of tentatively identified steroidal and other compounds. The synthetized polymers provided good matrix removal potential (ion suppression 15-30%) and semi-qualitative selectivity (∼4 units) compared to the commercial adsorbents (ion suppression 45-80%, selectivity < 3 units). Simple non-targeted approaches provided a novel method of quantifying the selectivity of extraction., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Introduction: Several taccalonolides with various bioactivities have been isolated from Tacca species but no studies to isolate taccalonolides with anti-trypanosomal activity from Tacca leontopetaloides have been reported., Objectives: To analyse extracts of the roots of Tacca leontopetaloides, purify the extracts by column chromatography and identify isolated compounds by spectroscopic methods. The compounds and fractions will be tested for antitrypanosomal activity in vitro against Trypanosoma brucei brucei., Material and Methods: Dried roots or tubers of Tacca leontopetaloides, chromatographic separation and spectroscopic identification., Results: A novel taccalonolide A propanoate and some known taccalonolides were isolated and their structures were determined by NMR and mass spectrometry, Conclusion: Several taccalonolides were isolated from Tacca leontopetaloides and were found to have in vitro antitrypanosomal activity against Trypanosoma brucei brucei and EC50 values for the isolated compounds were from 0.79 µg/mL. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The antifungal activities of eleven C21-steroidal compounds isolated from Cynanchum wilfordii, together with thirty-six derivatives of caudatin and qingyangshengenin were evaluated on Sclerotinia sclerotiorum and other five fungal strains by the mycelium growth rate method. Four derivatives 1k, 1y, 10d, and 10j exhibited much stronger inhibitions on growth of S. sclerotiorum with IC50 values of 0.0084, 0.0049, 0.0053, and 0.0034 μM, respectively., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this study, we describe the synthesis of graphene oxide functionalized with the ionic liquid 1-butyl-3-aminopropyl imidazolium chloride and its use as an adsorbent for the dispersive solid-phase microextraction (micro SPE) of four anabolic steroids and six β-blockers from aqueous samples of environmental importance, prior to their HPLC-diode array detector analysis. As the ionic liquid is covalently attached to graphene oxide sheets, it is made possible for it to participate in the dispersive micro SPE procedure. The limits of detection and limits of quantification of the proposed method were found to be in the range of 7-23ng/L and between 20 and 70ng/L, respectively. The linearity was satisfactory, with the determination coefficients to range from 0.9940 to 0.9998 while the within- and between-day relative standard deviation of the method ranged between 3.1 and 7.6% and from 4.0 to 8.5%, respectively. In order to test the applicability of the proposed method in real-life samples, the effluent from a municipal wastewater treatment plant as well as natural water samples from two rivers and a lake were collected and analyzed. After the analysis of samples, the effluent from municipal wastewater treatment plant was fortified with the analytes, at concentrations equal to 2 and 10 times the LOQs. Recoveries were calculated after subtracting the native (no-spike) concentrations of analytes, when needed. All the recoveries were in the range of 87-98%. A comparison study attests to the superiority of the developed nanomaterial over graphene oxide and graphene for the dispersive micro SPE of steroids and β-blockers., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Impatiens glandulifera has been imported from Himalaya in Europe and is considered as an invasive alien plant whose spreading arouses increasing interest among scientific literature. Via anti-cancer bioguiding, two new glucosylated steroids, named glanduliferins A and B, were isolated from the dried stem of I. glandulifera plants, together with the well-known α-spinasterol and 2-methoxy-1,4-naphthoquinone, which are also isolated from roots and leaves. They were characterized as 17-(2-hydroxy-2-pentamethylcyclopropyl-ethyl)-10,13-dimethyl-2,3,4,5,6,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopents[a]phenathren-3-O-(4-O-acetyl)-α-D-glucopyranoside and 17-(4-ethyl-1,5-dimethyl-hex-2-enyl)-10,13-dimethyl-2,3,4,5,6,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopents[a]phenathren-3-O-(6-O-acetyl)-β-D-glucopyranoside using various NMR and HRESIMS techniques and chemical methods. In vitro determination of the growth inhibitory activity of the four isolated compounds using the MTT colorimetric assay revealed mean IC50 growth inhibitory value of ~30 μM for glanduliferin A while glanduliferin B and α-spinasterol were poorly active till 100 μM. 2-methoxy-1,4-naphthoquinone revealed to be active in the single micromolar digit range as previously described. Quantitative videomicroscopy analyses of the effects of glanduliferins A and B suggested cytostatic rather than cytotoxic activity in U373 glioblastoma (GBM) cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The bulb of Fritillaria thunbergii has been utilised as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk medicine., Hypothesis/purpose: We investigated whether verticine, ebeiedine and suchengbeisine isolated from the bulbs of Fritillaria thunbergii inhibit the gene expression and production of MUC5AC mucin from human airway epithelial cells., Study Design: Confluent NCI-H292 cells were pretreated with verticine, ebeiedine or suchengbeisine for 30 min and then stimulated with EGF, PMA or TNF-α for 24h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA., Results: (1) Verticine, ebeiedine or suchengbeisine inhibited the expression of MUC5AC mucin gene induced by EGF, PMA or TNF-α; (2) The production of MUC5AC mucin protein induced by EGF, PMA or TNF-α were also inhibited by treatment of verticine, ebeiedine or suchengbeisine., Conclusion: These results suggest that verticine, ebeiedine and suchengbeisine isolated from the bulbs of Fritillaria thunbergii inhibit the gene expression and production of MUC5AC mucin, by directly acting on airway epithelial cells, and the results are consistent with the traditional use of Fritillaria thunbergii as remedy for diverse inflammatory pulmonary diseases., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Bulbus of Fritillaria pallidiflora, the dried bulb of F. pallidiflora, is widely used in Chinese folk medicine due to its powerful biological activities. The aim of this study was to investigate in vitro and in vivo antitumor activity of different fractions and isosteroidal alkaloids from bulbus of F. pallidiflora and clarify its putative mechanism of antitumor activity. Firstly, we assayed in vitro antitumor effects of different fractions from bulbus of F. pallidiflora and found that chloroform extracts and purified total alkaloids of bulbus of F. pallidiflora showed higher cytotoxic activity than other tested extracts. We further isolated four main alkaloids, chuanbeinone, imperialine-β-N-oxide, isoverticine and isoverticine-β-N-oxide, from the total alkaloids of bulbus of F. pallidiflora and found that they display significant cytotoxicity, whereby chuanbeinone showed the highest activity against Lewis lung carcinoma cells. Moreover, we found that chuanbeinone induced S phase arrest and further increased apoptosis of Lewis lung carcinoma cells. The results of Western blotting experiments showed that the expression of the antiapoptotic Bcl-2 was reduced by chuanbeinone treatment, while the proapoptotic protein Bax and caspase-3 were increased. Moreover, we investigated the in vivo antitumor activity of chuanbeinone and characterized its putative antitumor mechanism of action by the TUNEL assay and by histological and immunohistochemical analyses. Our results showed that chuanbeinone exhibited significant antitumor activity in vivo, while notably inhibiting tumor angiogenesis and inducing apoptosis characterized by an increased expression of caspase-3. Our findings show that chuanbeinone exhibits significant antitumor activity in vitro and in vivo, and points to possible therapeutic potential for this compound as well as for its natural source, bulbus of F. pallidiflora., (Georg Thieme Verlag KG Stuttgart · New York.)

Chemical investigation of the soft coral Lobophytum lobophytum collected from the Red Sea led to the isolation of a new compound gorgostan-5,25-dien-3β-ol (1), and two known compounds gorgosterol (2), and alismol (3). Structures were elucidated by employing extensive NMR and HR-ESI-MS experiments.

Eight highly hydroxylated steroids (1-8), including three new compounds as sodium salts of (24S)-5α-cholestane-3β,4β,5,6α,7β,8,14,15α,24-nonaol 6-sulfate (1), (24E)-5α-cholest-24-ene-26-yde-3β,6α,8,14,15α-pentaol 15-sulfate (2), and 5α-cholest-3β,6α,8,14,15α,24,25,26-octaol 15-sulfate (3), were isolated and elucidated from the methanol extract of the Vietnamese starfish Archaster typicus. The structure elucidation was done by spectroscopic methods including one and two dimensional (1D-, 2D-)NMR and Fourier transform ion cyclotron resonance (FT-ICR)-MS. The isolated compounds can be used as chemical markers for taxonomic identification of the starfish A. typicus.

The structures of two novel steroidal derivatives (1 and 4) were elucidated. Steroidal derivatives namely fologenin (1) and hoyagenin (4) have been isolated from chloroform-soluble extract of the whole plant of Hoya longifolia (family: Asclepiadaceae), and their structures were determined by using (1)H NMR, (13)C NMR, (1)H-(1)H COSY and FAB-MS techniques as well as chemical degradation and derivatisation.

Anaerobic treatment of municipal wastewater offers the prospect of a new paradigm by reducing aeration costs and minimizing sludge production. It has been successfully applied in warm climates, but does not always achieve the desired outcomes in temperate climates at the biochemical oxygen demand (BOD) values of municipal crude wastewater. Recently the concept of 'fortification' has been proposed to increase organic strength and has been demonstrated at the laboratory and pilot scale treating municipal wastewater at temperatures of 10-17°C. The process treats a proportion of the flow anaerobically by combining it with primary sludge from the residual flow and then polishing it to a high effluent standard aerobically. Energy consumption is reduced as is sludge production. However, no new treatment process is viable if it only addresses the problems of traditional pollutants (suspended solids - SS, BOD, nitrogen - N and phosphorus - P); it must also treat hazardous substances. This study compared three potential municipal anaerobic treatment regimes, crude wastewater in an expanded granular sludge blanket (EGSB) reactor, fortified crude wastewater in an EGSB and crude wastewater in an anaerobic membrane bioreactor. The benefits of fortification were demonstrated for the removal of SS, BOD, N and P. These three systems were further challenged with the removal of steroid estrogens at environmental concentrations from natural indigenous sources. All three systems removed these compounds to a significant degree, confirming that estrogen removal is not restricted to highly aerobic autotrophs, or aerobic heterotrophs, but is also a faculty of anaerobic bacteria.

Sensitive and specific biomarkers are required for the diagnosis and treatment of depression because the existing diagnostic criteria are subjective and could produce false positives or negatives. Some endogenous neuroactive steroids that have shown either antidepressant effects or concentration changes in individuals with depression could provide potential biomarkers. In this study, a simple and specific method was developed to simultaneously determine seven endogenous neuroactive steroids in biological samples: cortisone, cortisol, dehydroepiandrosterone, estradiol, progesterone, pregnenolone, and testosterone. After liquid-liquid extraction, chromatographic separation was achieved on a C18 column with gradient elution using water-methanol at a flow rate of 300 μL min(-1). Detection and quantitation were performed by tandem mass spectrometry with atmospheric pressure chemical ionization and selected reaction monitoring. Plasma and brain neuroactive steroid levels were then determined in control rats and rats exposed to forced swimming, a classical rodent model of depression. The results showed that the plasma concentrations of testosterone, pregnenolone, and progesterone significantly increased in rats exposed to the forced swimming test. In contrast, brain homogenate levels of cortisol, estradiol, and progesterone decreased, while pregnenolone levels were elevated in this model of depression. In conclusion, a new method to quantify neuroactive steroids was successfully developed and applied to their investigation in rat plasma and brain. The findings of this study indicated that plasma testosterone, pregnenolone, and progesterone levels could provide potential biomarkers for the diagnosis and treatment of depression.

Phytochemical investigation of the stems and leaves of Paris polyphylla var. yunnanensis led to isolation of 12 steroidal saponins, chonglouosides SL-9-SL-20, which had not been described previously, along with 13 known compounds. Their structures were established on the basis of extensive spectroscopic analysis and chemical methods. Four of the twelve steroidal saponins possessed three steroidal aglycones which have not been reported in nature. Steroidal saponins were also evaluated for their cytotoxicities against two human cancer cell lines (HepG2 and HEK293) and anti-HCV effects. One known steroidal saponin was the most cytotoxic compound overall with IC50 values of 2.9 ± 0.5 μM and 5.0 ± 0.6 μM against HepG2 and HEK293 cell lines, respectively, while none showed anti-HCV activity at a concentration of 20 μM., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Four new polyoxygenated sterol derivatives (1-4) along with the compounds (5-7) previously known from other biological sources were isolated from the gorgonian Menella woodin, collected from the Vietnamese waters. Structures of 1-4 were elucidated by the detailed NMR spectroscopic and mass-spectrometric analyses as well as comparison with those reported in literature data. Compounds 1, 4, and 6 decrease the production of reactive oxygen species (ROS) by the murine macrophages of RAW 264.7 line at induction by endotoxic lipopolysaccharide (LPS) from Escherichia coli., (Copyright © 2015 Elsevier Inc. All rights reserved.)

An efficient microwave-assisted extraction (MAE) technique was applied to isolate total steroid saponins from Dioscorea zingiberensis C.H. Wright (DZW). The optimal extracting conditions were established as 75% ethanol as solvent, ratio of solid/liquid 1:20 (g/ml), temperature 75 °C, irradiation power 600 W and three extraction cycles of 6 min each. Scanning electron microscopy (SEM) images of DZW processed by four different extractions provided visual evidence of the disruption effect on DZW. Diosgenin was quantified by HPLC and examined further by LC-ESI/MS after acid hydrolysis. Total steroid saponins were calculated using diosgenin from total steroid saponins. The MAE procedure was optimized, validated and compared with other conventional extraction processes. This report provides a convenient technology for the extraction and quantification of total saponins of DZW combining MAE with HPLC and LC-ESI/MS for the first time., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Many synthesized drugs with clinical severe side effects have been used for diabetic nephropathy (DN) treatment. Therefore, it is urgent and necessary to identify natural and safe agents to remedy DN. Timosaponin B-II (TB-II), a major steroidal saponin constituent in Anemarrhena asphodeloides Bunge, exhibits various activities, including anti-inflammatory and hypoglycemic functions. However, the anti-DN effects and potential mechanism(s) of TB-II have not been previously reported., Purpose: To investigate the effect of TB-II on DN in alloxan-induced diabetic mice., Methods: TB-II was isolated and purified from A. asphodeloides Bunge using macroporous adsorption resin and preparative high-performance liquid chromatography. The effect of TB-II on DN was evaluated in alloxan-induced diabetic mice using an assay kit and immunohistochemical determination in vivo. The expression of mammalian target of rapamycin (mTOR), thioredoxin-interacting protein (TXNIP), and nuclear transcription factor-κB (NF-κB) signaling pathways was also measured using Western blot analysis., Results: TB-II significantly decreased the blood glucose levels and ameliorated renal histopathological injury in alloxan-induced diabetic mice. In addition, TB-II remarkably decreased the levels of renal function biochemical factors, such as kidney index, blood urea nitrogen, serum creatinine, urinary uric acid, urine creatinine, and urine protein, and it reduced lipid metabolism levels of total cholesterol and triglycerides and the levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-α in alloxan-induced mice. Furthermore, TB-II inhibited the expression of mTOR, TXNIP, and NF-κB., Conclusion: The results revealed that TB-II plays an important role in DN via TXNIP, mTOR, and NF-κB signaling pathways. Overall, TB-II exhibited a prominently ameliorative effect on alloxan-induced DN.

Eight steroidal saponins (1-8), including one previously unreported derivative (1), have been isolated from the mesocarp of Raphia farinifera fruits by combined column chromatography and RP-HPLC methods. They were characterized by spectroscopic (1D and 2D NMR experiments) and spectrometric (ESIMS) methods, as well as by comparison of their spectral data with those of reported compounds in the literature. All the isolated compounds were tested for cytotoxicity against urinary bladder carcinoma cells (ECV-304). Our results revealed that, for a high cytotoxicity, a sugar chain of at least three sugar moieties attached to C-3 of the steroidal saponin is necessary.

Understanding the genetic basis of phenotypic diversity is a challenge in contemporary biology. Domestication provides a model for unravelling aspects of the genetic basis of stress sensitivity. The ancestral Red Junglefowl (RJF) exhibits greater fear-related behaviour and a more pronounced HPA-axis reactivity than its domesticated counterpart, the White Leghorn (WL). By comparing hormones (plasmatic) and adrenal global gene transcription profiles between WL and RJF in response to an acute stress event, we investigated the molecular basis for the altered physiological stress responsiveness in domesticated chickens. Basal levels of pregnenolone and dehydroepiandrosterone as well as corticosterone response were lower in WL. Microarray analysis of gene expression in adrenal glands showed a significant breed effect in a large number of transcripts with over-representation of genes in the channel activity pathway. The expression of the best-known steroidogenesis genes were similar across the breeds used. Transcription levels of acute stress response genes such as StAR, CH25 and POMC were upregulated in response to acute stress. Dampened HPA reactivity in domesticated chickens was associated with changes in the expression of several genes that presents potentially minor regulatory effects rather than by means of change in expression of critical steroidogenic genes in the adrenal.

The fungus Nodulisporium sp. (No. 65-12-7-1), which can produce a rare class of steroids (4-methyl-progesteroids) in rice solid medium, was subjected to a one strain-many compounds (OSMAC) approach. It was found to produce ten new 4-methyl-progesteroid derivatives named nodulisporisteroids C-L (1-10) in potato-dextrose-broth (PDB) medium, which showed that the fungus was a prolific producer of 4-methyl-progesteroids. The structures of 1-10 were elucidated by spectroscopic and X-ray crystallographic analysis. Their cytotoxic activities against five human cancer cell lines had been evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Derivatization is one of the most important steps during sample preparation in doping control analysis. Its main purpose is the enhancement of chromatographic separation and mass spectrometric detection of analytes in the full range of laboratory doping control activities. Its application is shown to broaden the detectable range of compounds, even in LC-MS analysis, where derivatization is not a prerequisite. The impact of derivatization initiates from the stage of the metabolic studies of doping agents up to the discovery of doping markers, by inclusion of the screening and confirmation procedures of prohibited substances in athlete's urine samples. Derivatization renders an unlimited number of opportunities to advanced analyte detection.

Six steroid derivatives, 1-6, and five butyrolactone derivatives, 7-11, were isolated from the fermentation broth of a gorgonian-derived Aspergillus sp. fungus. Their structures were elucidated on the basis of NMR and MS spectral data. Compound 1 is a new, highly conjugated steroid. The NMR and MS data of 7 and 8 are reported for the first time, as their structures were listed in SciFinder Scholar with no associated reference. Compounds 1, 4, 5, and 8-11 inhibited the larval settlement of barnacle Balanus amphitrite with EC50 values ranging from 0.63 to 18.4 μg ml(-1) . Butyrolactone derivatives 7 and 8 showed pronounced antibacterial activities against Staphylococcus aureus with the same MIC values as the positive control ciprofloxacin (MIC 1.56 μM for all three compounds)., (Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.)

Three new steroidal glycosides (1-3) and a novel natural product 4 firstly obtained from a plant source, together with two known steroidal glycosides (5-6) have been isolated from the whole plant of Reineckia carnea. Their structures were determined by physicochemical properties and spectroscopic methods, and their cytotoxic activities against human 1299 tumor cells were evaluated by the MTT method. Compounds 4, 5 and 6 exhibited cytotoxicity with IC50 values of 50.3 μmol·L(-1), 67.2 μmol·L(-1) and 61.8 μmol·L(-1), while compounds 1, 2, and 3 showed no cytotoxicity with the cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Six new C21 steroidal glycosides (1-6) and one dideoxysaccharide (7), named atratcynosides A-F and atratcynose A, were isolated from the 80% ethanol extract of the root of Cynanchum atratum, together with three known compounds (8-10). The structures of the new compounds were determined on the basis of extensive spectral analyses and qualitative chemical methods. All compounds were subjected to detect the immunosuppressive activities by an in vitro model of concanavalin A-induced proliferation of T-lymphocytes from mice. Compounds 1-3 showed significant immunosuppressive activities in dose-dependent manners with the IC50 values from 3.3 to 7.0 μM. Moreover, the structure-activity relationship of the steroidal glycosides on the immunosuppression was analyzed., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Evolution in preparation of chromatographic columns has created the need for studying and evaluating them with the use of smart software. This research is an attempt to compare the retention mechanism between two stationary phases (butyl and phenyl) with the use of multivariate analysis for a large number of probes. Partial least squares has the ability to spot either major or minor differences in the chromatographic behavior of probes, with regard to changes in the stationary or mobile phases. The models developed refer to a total of 108 miscellaneous chemical compounds, described by 63 X variables (physicochemical properties and structural features) and one Y variable (retention time). The results showed that in both columns and mobile phases (40% methanol or 40% acetonitrile) the retention of an analyte is mainly affected by its lipophilicity, molar volume, and refractivity, which tend to cause delayed elution. On the contrary, solubility in water, polar surface area, and hydrogen bond donor or acceptor properties promote faster elution. The most important difference proved to be the effect of the presence of the carboxylic group and the solubility that affected the retention in a similar way in both columns but not with both mobile phases.

A set of 71 triterpenoid and steroid compounds from Ganoderma were periodically classified using a procedure based on information entropy with artificial intelligence. Six features were used in hierarchical order to classify the triterpenoids and steroids structurally. The phytochemicals belonging to the same group in the periodic table present similar antioxidant activity, and those compounds belonging to the same period exhibit maximum resemblance. The periodic classification is related to the experimental bioactivity and antioxidant potency data that are available in the literature: a steroid with a three-ketone group conjugated with two carbon-carbon double bonds in the right side of the periodic table exhibits the greatest antioxidant activity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A new C23 steroid, (3β,5β,14β)-methyl (3-hydroxy-14,15-epoxy-20-oxo-21-norcholan-24-oate) (1), together with four known ones (2-5), were isolated from the venom of Bufo bufo gargarizans. Their structures were elucidated on the basis of extensive spectroscopic analysis. The cytotoxicity of these compounds was also evaluated against human hepatocarcinoma HepG2 cells. Compound 3 showed significant cytotoxicity with an IC50 value of 16.8 +/- 0.7 μM.

The marine ecosystem has been a key resource for secondary metabolites with promising biological roles. In the current study, bioassay-guided phytochemical investigations were carried out to assess the presence of enzyme inhibitory chemical constituents from the methanolic extract of marine green alga-Codium dwarkense. The bioactive fractions were further subjected to chromatographic separations, which resulted in the isolation of a new triterpenic acid; dwarkenoic acid (1) and the known sterols; androst-5-en-3β-ol (2), stigmasta-5,25-dien-3β,7α-diol (3), ergosta-5,25-dien-3β-ol (4), 7-hydroxystigmasta-4,25-dien-3-one-7-O-β-d-fucopyranoside (5), 7-hydroxystigmasta-4,25-dien-3-one (6), and stigmasta-5,25-dien-3β-ol (7). The structure elucidation of the new compound was carried out by combined mass spectrometry and 1D (1H and 13C) and 2D (HSQC, HMBC, COSY, and NOESY) NMR spectroscopic data. The sub-fractions and pure constituents were assayed for enzymatic inhibition of alpha-glucosidase. Compound 1 showed significant inhibition at all concentrations. Compounds 2, 3, 5, and 7 exhibited a dose-dependent response, whereas compounds 4-6 showed moderate inhibition. Utilizing such marine-derived biological resources could lead to drug discoveries related to anti-diabetics.

For the purpose of advancing knowledge of the structural variations available in the natural cephalostatins contained in the marine worm Cephalodiscus gilchristi, the isolation and structure of the 20th member (1) has been accomplished (10(-7) % yield). In turn cephalostatin 20 (1) proved to be enough for an initial SAR study comprising six important human cancer cell lines. A parallel objective was aimed at the possible discovery of a natural cephalostatin with a more accessible structure for total synthesis and/or synthetic modifications, but with powerful cancer cell growth inhibition.

Five new steroid glycosides, luzonicosides B-E (2-5), belonging to a rare structure group of marine glycosides, containing carbohydrate moieties incorporated into a macrocycle, and a related open carbohydrate chain steroid glycoside, luzonicoside F (6), were isolated from the starfish Echinaster luzonicus along with the previously known cyclic steroid glycoside luzonicoside A (1). The structures of compounds 2-6 were established by extensive NMR and ESIMS techniques as well as chemical transformations. Luzonicoside A (1) at concentrations of 0.01-0.1 μM was shown to be potent in lysosomal activity stimulation, intracellular ROS level elevation, and NO synthesis up-regulation in RAW 264.7 murine macrophages. Luzonicoside D (4) was less active in these biotests.

Steroids have important roles in the progress of pregnancy, and their study in maternal urine is a non-invasive method to monitor the steroid metabolome and its possible abnormalities. However, the current screening techniques of choice, namely immunoassays and gas and liquid chromatography-mass spectrometry, do not offer means for the rapid and non-targeted multi-analyte studies of large sample sets. In this study, we explore the feasibility of two ambient mass spectrometry methods in steroid fingerprinting. Urine samples from pregnant women were screened by desorption electrospray ionization (DESI) and desorption atmospheric pressure photoionization (DAPPI) Orbitrap high resolution mass spectrometry (HRMS). The urine samples were processed by solid phase extraction for the DESI measurements and by enzymatic hydrolysis and liquid-liquid-extraction for DAPPI. Consequently, steroid glucuronides and sulfates were detected by negative ion mode DESI-HRMS, and free steroids by positive ion mode DAPPI-HRMS. In DESI, signals of eleven steroid metabolite ions were found to increase as the pregnancy proceeded, and in DAPPI ten steroid ions showed at least an order of magnitude increase during pregnancy. In DESI, the increase was seen for ions corresponding to C18 and C21 steroid glucuronides, while DAPPI detected increased excretion of C19 and C21 steroids. Thus both techniques show promise for the steroid marker screening in pregnancy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ten new ergosteroids, gloeophyllins A-J (1-10), have been isolated from the solid cultures of Gloeophyllum abietinum. The absolute configurations of 1, 2, and 9 were determined by X-ray crystallographic analysis. Compound 1 has a rare C-nor-D-homosteroid skeleton. Compound 9 possesses an unusual ergostane skeleton having a 10-oxabicyclo [4.3.1] decane moiety replacing 6/5 fused C/D rings. Compound 10 represents the first ergosteroid featuring the cleavage of a C8-C14 bond. The cytotoxicity of 1-10 was tested against the human cancer cell lines K562 and HCT116. The biosynthetic pathway for 1-10 is postulated.

Solanioic acid (1), a degraded and rearranged steroid that exhibits in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), has been isolated from laboratory cultures of the fungus Rhizoctonia solani obtained from tubers of the plant Cyperus rotundus collected in Sri Lanka. The structure of solanioic acid (1) was elucidated by detailed analysis of NMR data, a single crystal X-ray diffraction analysis of a reduction product 2, and Mosher ester analysis on a derivative of the natural product. Solanioic acid (1) has an unprecedented carbon skeleton.

Veratrum nigrum is recognized as a medicinal plant used for the treatment of hypertension, stroke, and excessive phlegm. Chemical investigation of the roots and rhizomes led to the isolation of five new steroidal alkaloids, jervine-3-yl formate (1), veramarine-3-yl formate (2), jerv-5,11-diene-3β,13β-diol (3), (1β,3β,5β)-1,3-dihydroxyjervanin-12(13)-en-11-one (4), and veratramine-3-yl acetate (5). Compounds 1 and 5 exhibited potent inhibitory activity (11.3 and 4.7 μM, respectively) against protein tyrosine phosphatase 1B (PTP1B), which has emerged as a viable target for treatment of type 2 diabetes mellitus. On the basis of their PTP1B inhibitory activity, the compounds were evaluated for their potential to enhance glucose uptake in C2C12 skeletal muscle cells. The insulin-stimulated glucose uptake was enhanced upon treatment with compounds 1 and 5 (10 μM) by 49.9 ± 6.5% and 56.0 ± 9.7%, respectively, in a more potent manner than that with the positive control rosiglitazone (47.3 ± 3.4% at 30 μM). These results suggest that steroidal alkaloids serve as practical antidiabetes mellitus leads capable of enhancing glucose uptake.

Chemical investigation of a Korean marine sponge, Monanchora sp., led to the isolation of three new steroids (1-3). Compounds 1 and 2, designated as monanchosterols A and B, respectively, represent the first examples of steroids possessing the bicyclo[4.3.1] A/B ring system from a natural source. Compounds 1-3 were investigated for their anti-inflammatory activity by evaluating their inhibitory effects on the mRNA expression of IL-6, TNF-α, and COX-2 in the LPS-stimulated murine RAW264.7 macrophage cells. Compounds 2 and 3 exhibited significant inhibitory effects on the mRNA expression of IL-6 without notable cytotoxicity to the cells in a dose-dependent manner.