Cytostatic compounds in sludge and sediment: extraction and determination by a combination of microwave-assisted extraction and UHPLC-MS/MS.

Cytostatic compounds are an important group of micro-pollutants since they are used to kill cells or stop cell division. For this reason, they are also considered mutagenic. Several cytostatic compounds have been detected in hospital effluents, in the influents and effluents of wastewater treatment plants and even in river water. However, their detection in solid matrices is very scarce. In this work, we have developed a new procedure based on microwave-assisted extraction (MAE) for the extraction of cytostatic compounds from sludge and sediment before determination by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). To develop this procedure, we have chosen a group of eight widely used cytostatic compounds and carried out a systematic experimental design to optimize the extraction conditions. Under these optimal conditions, the studied cytostatic compounds are extracted with good sensitivity, with recoveries ranging from 65 to 122% in sludge and recoveries varying between 49 and 109% in sediment, with the exception of etoposide, which has a lower recovery from these types of samples. The limits of detection were from 0.42 to 79.8 ng g-1 in sludge and from 0.10 to 87.5 ng g-1 in sediment. Intraday and interday relative standard deviations (RSDs) were below 15% and 18%, respectively, in both matrices at the tested concentrations. The total procedure was applied to samples of sludge taken from the main wastewater treatment plant (WWTP) of the island of Gran Canaria (Spain) and for sediment samples obtained close to the marine outfalls of different wastewater treatment plants for the same island. Graphical abstract.

Removal of anthracycline cytostatics from aquatic environment: Comparison of nanocrystalline titanium dioxide and decontamination agents.

Anthracyclines are a class of pharmaceuticals used in cancer treatment have the potential to negatively impact the environment. To study the possibilities of anthracyclines (represented by pirarubicin and valrubicin) removal, chemical inactivation using NaOH (0.01 M) and NaClO (5%) as decontamination agents and adsorption to powdered nanocrystalline titanium dioxide (TiO2) were compared. The titanium dioxide (TiO2) nanoparticles were prepared via homogeneous precipitation of an aqueous solution of titanium (IV) oxy-sulfate (TiOSO4) at different amount (5-120 g) with urea. The as-prepared TiO2 samples were characterized by XRD, HRSEM and nitrogen physisorption. The adsorption process of anthracycline cytostatics was determined followed by high-performance liquid chromatography coupled with mass spectrometry (LC-MS) and an in-situ Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) technique. It was found that NaClO decomposes anthracyclines to form various transformation products (TPs). No TPs were identified after the reaction of valrubicin with a NaOH solution as well as in the presence of TiO2 nanoparticles. The best degree of removal, 100% of pirarubicin and 85% of valrubicin, has been achieved in a sample with 120 grams of TiOSO4 (TIT120) and TiO2 with 60 grams (TIT60), respectively.

Cytotoxic triterpenes from Salvia buchananii roots.

A pentacyclic triterpene, named salvibuchanic acid (1), together with five known compounds, were isolated from the roots of Salvia buchananii Hedge (Lamiaceae). The structural characterisation of all compounds was performed by spectroscopic analyses, including 1D and 2D NMR and HRESIMS experiments. The lupane triterpene (1) and hyptadienic acid (2) were investigated for their potential cytotoxic activity on Jurkat, HeLa and MCF7 cell lines. Both compounds showed an interesting antiproliferative activity with similar potency in all cell lines. By means of flow cytometric studies, hyptadienic acid (2) induced in HeLa cells a S cell cycle block, while 1 elicited both cytostatic and cytotoxic responses.

Withapubesides A-D: natural inducible nitric oxide synthase (iNOS) inhibitors from Physalis pubescens.

Four new steroid glycosides, withapubesides A-D (1-4), were isolated from the stems of Physalis pubescens L. Their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2 were deduced by single-crystal X-ray diffraction and ECD data analysis, respectively. Compound 3 has shown significant inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages with an IC50 value of 12.8 µM and moderate cytostatic activity against human carcinoma cells (786-O, C4-2B, 22Rvl, A375 and A375S2) with IC50 values in the range of 3.05-9.47 µM. Molecular docking simulation demonstrated that 3 is bound in the inducible nitric oxide synthase (iNOS) active site heme pocket very well, which suggests that 3 might be a candidate for the development of iNOS inhibitors.

Slurry photocatalytic membrane reactor technology for removal of pharmaceutical compounds from wastewater: Towards cytostatic drug elimination.

The potential of photocatalytic membrane reactors (PMR) to degrade cytostatic drugs is presented in this work as an emerging technology for wastewater treatment. Cytostatic drugs are pharmaceutical compounds (PhCs) commonly used in cancer treatment. Such compounds and their metabolites, as well as their degraded by-products have genotoxic and mutagenic effects. A major challenge of cytostatic removal stands in the fact that most drugs are delivered to ambulant patients leading to diluted concentration in the municipal waste. Therefore safe strategies should be developed in order to collect and degrade the micro-pollutants using appropriate treatment technologies. Degradation of cytostatic compounds can be achieved with different conventional processes such as chemical oxidation, photolysis or photocatalysis but the treatment performances obtained are lower than the ones observed with slurry PMRs. Therefore the reasons why slurry PMRs may be considered as the next generation technology will be discussed in this work together with the limitations related to the mechanical abrasion of polymeric and ceramic membranes, catalyst suspension and interferences with the water matrix. Furthermore key recommendations are presented in order to develop a renewable energy powered water treatment based on long lifetime materials.

Antiproliferative Effects of New Dimeric Ellagitannin from Cornus alba in Prostate Cancer Cells Including Apoptosis-Related S-Phase Arrest.

Activity-guided isolation of 80% acetone extract of Cornus alba, which is traditionally used as an anti-inflammatory, hemostatic and diuretic in Korea, yielded one novel compound, tentatively designated cornusiin H (13), together with 12 known compounds. The known compounds included four flavonoids (catechin (1), quercetin-3-O-ß-D-glucuronide (2), quercetin-3-O-ß-D-glucopyranoside (3), kaempferol-3-O-ß-D-glucopyranoside (4)) and eight hydrolysable tannins (gallic acid (5), 2,6-di-O-galloyl-hamamelofuranoside (6), 2-galloyl-4-caffeoyl-L-threonic acid (7) 2,3-di-O-galloyl-4-caffeoyl-L-threonic acid (8), 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranoside (9), cornusiin B (10), cornusiin A (11) and camptothin B (12)). All compounds exhibited potent 1,1-diphenyl-2-picrylhydrazyl (DPPH)-free radical scavenging activity. Especially, the radical scavenging activities of 6 and 9-13 were higher than that of vitamin C. Compounds 9, 11, 12 and 13 inhibited the production of nitric oxide (NO) in lipopolysaccharide-stimulated RAW264.7 cells to the same degree as N(G)-Monomethyl-L-arginine (L-NMMA). When the antiproliferative effects of the isolated compounds were assessed in prostate cancer cells, the dimeric ellagitannins (11-13) selectively inhibited LNCaP hormone-dependent prostate cancer cells. Flow cytometry analysis indicated that the dimeric ellagitannins induced apoptosis and S-phase arrest. These results suggest that dimeric ellagitannins from Cornus alba can be developed as functional materials or herbal medicines for prostate tumors such as benign prostate hyperplasia and early-stage prostate cancer.

Higginsianins A and B, Two Diterpenoid α-Pyrones Produced by Colletotrichum higginsianum, with in Vitro Cytostatic Activity.

Two new diterpenoid α-pyrones, named higginsianins A (1) and B (2), were isolated from the mycelium of the fungus Colletotrichum higginsianum grown in liquid culture. They were characterized as 3-[5a,9b-dimethyl-7-methylene-2-(2-methylpropenyl)dodecahydronaphtho[2,1-b]furan-6-ylmethyl]-4-hydroxy-5,6-dimethylpyran-2-one and 4-hydroxy-3-[6-hydroxy-5,8a-dimethyl-2-methylene-5-(4-methylpent-3-enyl)decahydronaphthalen-1-ylmethyl]-5,6-dimethylpyran-2-one, respectively, by using NMR, HRESIMS, and chemical methods. The structure and relative configuration of higginsianin A (1) were confirmed by X-ray diffractometric analysis, while its absolute configuration was assigned by electronic circular dichroism (ECD) experiments and calculations using a solid-state ECD/TDDFT method. The relative and absolute configuration of higginsianin B (2), which did not afford crystals suitable for X-ray analysis, were determined by NMR analysis and by ECD in comparison with higginsianin A. 1 and 2 were the C-8 epimers of subglutinol A and diterpenoid BR-050, respectively. The evaluation of 1 and 2 for antiproliferative activity against a panel of six cancer cell lines revealed that the IC50 values, obtained with cells reported to be sensitive to pro-apoptotic stimuli, are by more than 1 order of magnitude lower than their apoptosis-resistant counterparts (1 vs >80 µM). Finally, three hemisynthetic derivatives of 1 were prepared and evaluated for antiproliferative activity. Two of these possessed IC50 values and differential sensitivity profiles similar to those of 1.

Guaianolides and a seco-Eudesmane from the Resinous Exudates of Cushion Bush (Leucophyta brownii) and Evaluation of Their Cytostatic and Anti-inflammatory Activity.

A detailed phytochemical investigation of a dichloromethane extract of the resinous exudates of the cushion bush plant (Leucophyta brownii) resulted in the isolation of the new 8,12-guaianolides leucophytalins A (5) and B (6), the new 1,10-seco-eudesmane leucophytalin C (10), six rare 8,12-guaianolides (1-4, 7, and 8), and the xanthanolide tomentosin (9). The structures of all isolated compounds were elucidated on the basis of spectroscopic and spectrometric analyses. The structures of compounds isolated in crystalline form, including leucophytalins A and C, were further confirmed by X-ray crystallography. The crude extract exhibited moderate cytostatic activity against a breast cancer (MCF-7) and human colon cancer (HT-29) cell line with IC50 values of 9.3 and 18 µg/mL, respectively, and anti-inflammatory activity against the macrophage-like cell line RAW 264.7 with IC50 values of 3.9 and 6.1 µg/mL for thromboxane B2 and prostaglandin E2 production, respectively. The isolated compounds were evaluated for their cytostatic activity against MCF-7 and HT-29 cells (1, 3-10) and their anti-inflammatory activity against RAW 264.7 cells (1-10). All isolated compounds are most likely derived from (+)-germacrene A, and a biosynthetic pathway is proposed for these sesquiterpenoids.

Sulfated modification, characterization and property of a water-insoluble polysaccharide from Ganoderma atrum.

Sulfated modification was carried out to modify a water-insoluble polysaccharide from Ganoderma atrum (AGAP). The effects of sulfation on structure, physicochemical and functional properties of AGAP were investigated. Three sulfated derivatives were prepared, designated as S-1, S-2 and S-3 with degree of substitution (DS) of 0.35, 0.74 and 1.14, respectively. AGAP was elucidated as an α-(1→3)-glucan with few branches terminated by single mannose or xylose residues. The molecular weight (Mw) and radius of gyration (Rg) were estimated to be 1665 kDa and 65.49 nm, respectively. After sulfated modification, non-selective sulfation occurred preferably at O-6, partially at O-2 and O-4 positions of the glucosyl residues. The water-solubility of the derivatives was significantly improved in a DS-dependent manner. Mw of the derivatives showed a sharp decrease, and the chain conformation was estimated to be expanded stiff in phosphate buffer. In vitro tests showed that sulfated modification improved its antioxidant activities and anti-proliferative ability against S-180 tumor cells. This study suggested that sulfated modification was an effective approach to improve the water-solubility and functional properties of insoluble polysaccharides.

Hepatoprotective saikosaponin homologs from Comastoma pedunculatum.

Eight new triterpenoid saponins, the saikosaponin homologs comastomasaponins A-H (1-8), as well as a known triterpenoid (9) and eight known saponins (10-17) were isolated from the aerial portions of Comastoma pedunculatum. The structures of these compounds were elucidated spectroscopically, and their hepatoprotective activity and cytotoxic activity were evaluated against five human tumor cell lines in vitro. Compounds 1, 5-12, 14, 15, and 17 exhibited potent hepatoprotective activity, and compound 11 displayed cytotoxic activity against HCT-8, Bel-7402, BGC-825, A549, and A2780 human tumor cell lines.

Isolation and cytotoxic effect of anthraquinones from Morinda umbellata.

Four new anthraquinones, 1,6-dihydroxy-2-methoxymethylanthraquinone (1), 6-hydroxy-7-methoxy-2-methoxymethylanthraquinone (3), 3,6-dihydroxy-7-methoxy-2-methylanthraquinone (4), and 6-hydroxy-2-methoxymethylanthraquinone (8), together with 12 known anthraquinones and 6 other known compounds, were isolated from the EtOAc extract of Morinda umbellata. Among the isolated compounds, 1, rubiadin (14), and, 3-hydroxy-2-hydroxymethylanthraquinone (16) exhibited significant cytotoxicities against HepG2 cells, with GI50 values of 4.4, 3.6, and 4.8 µM, respectively.

Occurrence of cytostatic compounds in hospital effluents and wastewaters, determined by liquid chromatography coupled to high-resolution mass spectrometry.

The occurrence of 26 commonly used cytostatic compounds in wastewaters was evaluated using an automated solid-phase extraction (SPE) method with liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Detection was optimized using Oasis HLB SPE cartridges at pH 2. Two hospital effluents and their two receiving wastewater treatment plants were sampled over five days. In hospital effluents, eight cytostatics were detected at levels up to 86.2 µg L(-1) for ifosfamide, 4.72 µg L(-1) for cyclophosphamide, and 0.73 µg L(-1) for irinotecan, the three most relevant compounds identified. Cyclophosphamide and megestrol acetate were found in wastewaters at concentrations up to 0.22 µg L(-1) for the latter. The predicted environmental concentrations (PEC) in sewage effluents of ifosfamide (2.4-4.3 ng L(-1)), capecitabine (11.5-14.2 ng L(-1)), and irinotecan (0.4-0.6 ng L(-1)), calculated from consumption data in each hospital, published excretion values for the target compounds, and wastewater elimination rates, were in agreement with experimental values.

Chemical properties and biological activities of cyclopentenediones: a review.

Cyclopentenediones (CPDs) are secondary metabolites of higher plants, fungi, algae, cyanobacteria and bacteria. A common denominator of CPDs is the cyclopent-4-ene-1,3-dione skeleton (1), which is modified by several functional groups. The heterogeneity of these substitutions is reflected in around one hundred CPDs reported to date. Most of the derivatives were isolated primarily from plant sources. Synthetic analogues were then prepared with new biological activities and more interesting pharmacological potential. Antifungal substances called coruscanones (2, 3) are the most studied of the CPDs. Other intensely investigated CPDs include lucidone (4), linderone (5), asterredione (6), involutone (7), nostotrebin 6 (8), TX-1123 (9), G2201-C (10), madindolines (11, 12) and many others. In addition to antibacterial and antifungal effects, a broad spectrum of biological activities for CPDs has been reported in the past two decades, especially anti-inflammatory, cytostatic and specific enzyme inhibitory activities. The CPD skeleton has been identified in a number of substances isolated from the plant kingdom; hence, CPDs can be referred to as a new group of natural bioactive substances. The main goal of this review is to define CPDs with respect to basic chemistry, isolation, synthetic approaches and description of their biological effects. Special attention is given to a detailed view into biological activities of CPDs in vitro and their phamacological potential.

Glioblastoma-specific anticancer activity of pheophorbide a from the edible red seaweed Grateloupia elliptica.

The chlorophyll-related compound pheophorbide a (Pa) was successively purified from an edible red seaweed, Grateloupia elliptica, using silica, octadecyl silica column chromatography and reversed phase-high-performance liquid chromatography, as well as the cell cycle inhibitory and apoptotic effects of Pa being investigated in U87MG glioblastoma cells. The Pa exhibited strong anticancer effects in the absence of direct photo-irradiation against various cancer cell lines, including U87MG, SK-OV-3, and HeLa cells. Among the cancer cells, the strongest anticancer activity of Pa exhibited on U87MG cells with IC50 values of 2.8 µg/ml. In addition, Pa specifically had cytostatic activity on glioblastoma cells rather than human umbilical vein endothelial cells. Analysis of the cell cycle distribution showed that Pa induced G0/G1 arrest of U87 MG cells. In addition, arrested cells induced late apoptosis and DNA degradation under dark condition. These results suggest that Pa isolated from G. elliptica is a potential glioblastoma-specific anticancer agent without side effects on normal cells.

Multianalyte determination of 24 cytostatics and metabolites by liquid chromatography-electrospray-tandem mass spectrometry and study of their stability and optimum storage conditions in aqueous solution.

A multianalyte liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) method for determination of 19 cytostatics and 5 metabolites, from 6 different therapeutic families, has been developed, and the structures of the main characteristic fragment ions have been proposed. Instrumental limits of detection and quantification are in the range 0.1-10.3 and 1.0-34.3 ng mL(-1), respectively. Moreover, the stability of the compounds in aqueous solution was investigated in order to establish the best conditions for preparation and storage of both calibration standards and water samples. Dimethylsulphoxide (DMSO) was selected as solvent for preparation of the stock solutions. At room temperature (25 °C), 11 of the 24 target compounds were shown to be unstable in water (percentage of organic solvent 4%), with concentration losses greater than 20% in less than 24 h. At 4 °C (typical storage temperature for water samples) all compounds, except MTIC and chlorambucil, were stable for 24h, but the number of stable compounds decreased to 10 after 9 days. Freezing of the aqueous solutions improved considerably the stability of various compounds: after 3 months of storage at -20 °C, 10 compounds, namely, 5-fluorouracil, carboplatin, gemcitabine, temozolomide, vincristine, vinorelbine, ifosfamide, cyclophosphamide, etoposide, and capecitabine, remained stable (in contrast to only carboplatin and capecitabine at 4 °C). The addition of acid improved the stability of methotrexate and its metabolite hydroxy-methotrexate but not that of the rest of compounds. The addition of organic solvent (50% methanol or DMSO) prevented the degradation at 4 °C of the otherwise unstable compounds oxaliplatin, methotrexate, erlotinib, doxorubicin, tamoxifen, and paclitaxel. To the authors' knowledge, five of the analytes investigated have never been searched for in the aquatic environment (imatinib, 6α-hydroxypaclitaxel, endoxifen, (Z)4-hydroxytamoxifen, and temozolomide), and for many of them the stability data provided, and even the analytical LC-MS/MS conditions, are the first ever published.

Antiproliferative activity of Rhinella marina and Rhaebo guttatus venom extracts from Southern Amazon.

The venom of amphibians is a fascinating source of active substances. In view of their medical importance and aiming to explore the amazing Brazilian biodiversity, we conducted bioprospecting of antiproliferative activity in extracts of Rhinella marina and Rhaebo guttatus toads occurring in the Southern Amazon of Mato Grosso, Brazil. LC-MS and HPLC analysis of the venom extracts of R. marina revealed four bufadienolides (telocinobufagin, marinobufagin, bufalin and resibufogenin. R. guttatus venom extracts contained only marinobufagin. First, R. marina and R. guttatus venom extracts were evaluated for cytotoxicity against tumor cell lines by the MTT assay. All extracts revealed cytotoxicity, where R. marina extracts were comparable to doxorubicin (IC50 values ranging from 0.01 to 0.23 µg/mL). Only extracts of R. guttatus toad venom caused membrane disruption of human erythrocytes. The extracts were investigated for selective activity by determining their effect on stimulated human peripheral blood mononuclear cells (PBMC) with the Alamar Blue™ assay. The extracts were up to 80-fold more selective against leukemia cells when compared to dividing leukocytes. Aiming to confirm these antiproliferative effects, BrdU incorporation into DNA was measured in HL-60 treated cells with R. marina venom extracts. These extracts decreased BrdU incorporation at both concentrations tested. In summary, nine extracts of R. marina and R. guttatus venom showed pronounced lethal and discriminating effects on tumor lines, especially those from R. marina, highlighting toad parotoid gland secretions as a promising source for novel lead anticancer chemicals.

On-line solid phase extraction-liquid chromatography-tandem mass spectrometry for the determination of 17 cytostatics and metabolites in waste, surface and ground water samples.

A fully automated on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method has been developed for the determination of 13 cytostatics and 4 metabolites in aqueous matrices, including groundwater, surface water, and raw and treated wastewater. On-line SPE is performed by loading 5 mL of water sample at pH 2 through a PLRP-s cartridge. MS/MS is performed with an electrospray (ESI) interface operating in the positive ion mode and registering two selected reaction monitoring (SRM) transitions per compound. Quantification is carried out by the isotope dilution method using 15 different isotope-labelled compounds, specific for the target analytes, as internal standards (IS). The main advantages of the method are high sensitivity, with limits of determination in groundwater, surface water, and raw and treated wastewater below 5 ng L(-1) for all compounds except for gemcitabine (6.9-9.3 ng L(-1)), temozolomide (26-50 ng L(-1)), imatinib (80-180 ng L(-1)) and etoposide (38-65 ng L(-1)), repeatability, with relative standard deviations (RSDs) in most cases below 15%, and selectivity and reliability of results. The method is also fairly simple and fast, with an analysis time per sample (excluding the manual steps, i.e., sample filtration, pH adjustment, and addition of IS) of 40 min. Application of the method to influent wastewater samples collected daily during eight consecutive days from a wastewater treatment plant (WWTP) from Catalonia showed the presence of methotrexate, ifosfamide, capecitabine, tamoxifen and 6(α)-hydroxypaclitaxel but at fairly low concentrations (up to 43 ng L(-1)).

Inhibition of leukemic U937 cell growth by induction of apoptosis, cell cycle arrest and suppression of VEGF, MMP-2 and MMP-9 activities by cytotoxin protein NN-32 purified from Indian spectacled cobra (Naja naja) venom.

A cytotoxin NN-32 (6.7 kDa) from Indian cobra (Naja naja) venom inhibited human leukemic U937 cell growth as observed by Trypan blue dye exclusion method and cytotoxicity was confirmed by MTT assay. NN-32 induced apoptosis of U937 cell and cell cycle arrest of sub-G1 phase were revealed by FACS analysis. Increased Bax/Bcl-2 ratio, increased caspase 3 and 9 activities, cleaved PARP, decreased VEGF, MMP-2 and MMP-9 activities were observed after NN-32 treatment of U937 cell. Antileukemic activity of NN-32 on U937 cell may be due to activation of apoptosis, arresting cell cycle and antiangiogenesis activities.

Secondary metabolites from a culture of the fungus Neosartorya pseudofischeri and their in vitro cytostatic activity in human cancer cells.

Four known (1, 2, 3, and 6) and three new compounds including a 1,4-diacetyl-2,5-dibenzylpiperazine derivative (4), a quinazolinone-containing indole derivative (5), and a new ester of 2,4-dihydroxy-6-methylbenzoic acid (7) were isolated from the fungus Neosartorya pseudofischeri S. W. Peterson. Compound 2 displayed in vitro growth inhibitory activity that ranged between the activities of etoposide and carboplatin, chosen as reference compounds, in six distinct cancer cell lines. Compound 1 displayed less activity than 2. Computer-assisted phase-contrast microscopy-related analysis revealed that 2 displayed cytostatic, not cytotoxic, effects in human U373 glioblastoma and A549 non-small cell lung cancer apoptosis-resistant cells with marked inhibition of mitotic rates. Cancer cells in the remaining phases of the cell cycle were unchanged. Flow cytometry analysis further confirmed that 2 does not induce apoptotic features in U373 or A549 cancer cells. Thus, 2 represents a novel chemical scaffold from which derivatives for anticancer cytostatic compounds can be derived.