Vassiliki Exarchou

In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum.

Tris(2-butoxyethyl) phosphate (TBOEP) is a plasticizer present in indoor dust, reaching levels of several micrograms per gram. Such levels could lead to significant daily exposure of adults and children. Currently, no toxicokinetic data are available to estimate TBOEP clearance in humans after uptake and therefore, one objective of this study was to investigate intrinsic clearance of TBOEP by human liver microsome (HLM) and serum enzymes. Another objective was to generate information to identify and prioritize several metabolites of TBOEP for investigation of human exposure by biomonitoring. 1D and 2D-NMR methodologies were successfully applied on a mixture of the metabolites to confirm the structure of 3-HO-TBOEP (bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate) and to tentatively assign structures to 1-HO-TBOEP and 2-HO-TBOEP. HO-TBOEP isomers and bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were further monitored by liquid chromatography-tandem mass spectrometry. Rates of formation of BBOEHEP and HO-TBOEP metabolites by liver enzymes were best described by the Michaelis-Menten model. Apparent Km values for BBOEHEP, 3-HO-TBOEP, and sum of 1- and 2-HO-TBOEP isomer formation were 152, 197 and 148μM, respectively. Apparent Vmax values for the formation of BBOEHEP, 3-HO-TBOEP, and the sum of 1- and 2-HO-TBOEP isomers were 2560, 643, and 254pmol/min/mg protein, respectively. No detectable formation of BBOEP occurred with liver or serum enzymes. Our findings indicate that intrinsic clearance of TBOEP is mainly catalyzed by oxidative enzymes in the liver and that its major in vitro metabolite is BBOEHEP. These findings can be applied in human biomonitoring studies and risk assessment.

Edible coating composed of chitosan and Salvia fruticosa Mill. extract for the control of grey mould of table grapes

BACKGROUNDnConsumer concerns regarding high-quality produce, free of pesticide residues, direct research towards disease management strategies that minimise or even exclude the use of synthetic chemistries in crop production. The efficacy of a chitosan-based edible coating combined with the acetonic extract of Salvia fruticosa Mill. (ASF) was assessed against the grey mould of table grapes.nnnRESULTSnHPLC-SPE-NMR and q-NMR analyses defined major constituents of ASF to be the flavonoids hispidulin, salvigenin and cirsimaritin and the diterpenes carnosic acid, carnosol and the 12-methoxycarnosic acid. The extract was found to be efficacious in reducing spore germination and mycelial growth of Botrytis cinerea in vitro at 10 and 25 °C. However, the combination of the ASF with chitosan 1% (w/v; CHIT) significantly improved fungal inhibition. Similarly, in fruit inoculation trials at 10 °C, the efficacy of the combined application of the ASF at 500 mg L-1 with CHIT against grey mould was statistically equal to the synthetic fungicide thiabendazole, ranging from 98.4% to 92.7% at 12 and 21 days post-inoculation, respectively. Furthermore, chitosan coating alone and in combination with ASF decreased the rate of fruit weight loss during cold storage, while preserved soluble solids content and titratable acidity. Chitosan-based coatings did not affect quality attributes and the bioactive compounds in table grapes.nnnCONCLUSIONnThe combined application of the ASF in the form of an edible coating with chitosan could effectively control B. cinerea without deteriorating quality and physico-chemical properties of grapes.

Phytochemical and Pharmacological Investigations on Nymphoides indica Leaf Extracts

Nymphoides indica (L.) Kuntze (Menyanthaceae) is traditionally used in the Indian subcontinent. However, scientific data reporting its constituents are poor. This study aimed at evaluating its phytochemical constituents and various biological activities. Phytochemical investigations of the extracts and fractions resulted in the isolation of 5 lipophilic compounds, i.e. azelaic (nonanedioic) acid (1) and 4‐methyl‐heptanedioic acid (3), hexadecanoic (2) and stearic acid (5) and the fatty alcohol hexadecanol (4); 3 seco‐iridoids, i.e. 7‐epiexaltoside (6), 6″,7″‐dihydro‐7‐epiexaltoside (7) and menthiafolin (8); 3 flavonoids, i.e. 3,7‐di‐O‐methylquercetin‐4′‐O‐β‐glucoside (9), 3‐O‐methylquercetin‐7‐O‐β‐glucoside (10) and 3,7‐di‐O‐methylquercetin (11); scopoletin (12) and ferulic acid (13); and the monoterpenoids foliamenthoic acid (14) and 6,7‐dihydrofoliamenthoic acid methyl ester (15). Compounds 1–5 showed moderate antimicrobial activities, whereas compound 9 presented mild antiprotozoal activities against Trypanosoma brucei (IC50 8u2009μM), Leishmania infantum (IC50 32u2009μM) and Trypanosoma cruzi (IC50 30u2009μM). Antiglycation activity was shown by compounds 7 (IC50 0.36u2009mM), 10 (IC50 0.42u2009mM) and 15 (IC50 0.61u2009mM). Finally α‐glucosidase inhibition was shown by compounds 7, 9, 11 and 13–15. It could be concluded that N. indica leaf extracts possess mild to moderate antimicrobial, antiprotozoal, antioxidant and antidiabetic activities. Copyright

Automated analytical standard production with supercritical fluid chromatography for the quantification of bioactive C17-polyacetylenes: A case study on food processing waste

Food processing enterprises produce enormous amounts of organic waste that contains valuable phytochemicals (e.g. C17-polyacetylenes). Knowledge on the phytochemicals content is a first step towards valorisation. Quantification of C17-polyacetylenes is however often hampered by the lack of commercially available standards or by tedious multistep in-house standard production procedures. In the current study, a new and straightforward supercritical fluid chromatography purification procedure is described for the simultaneous production of 2 analytical C17-polyacetylene standards. Respectively, 5 and 6 mg of falcarinol and falcarindiol were purified in 17 h on analytical scale. After confirming the identity and quality (97% purity) by Nuclear Magnetic Resonance, accurate mass-Mass Spectrometry (am-MS) and Photo Diode Array (PDA) detection the C17-polyacetylene standards were used for the analysis of industrial vegetable waste with Liquid Chromatography coupled to PDA and am-MS detection. Measurements showed varying concentrations of C17-polyacetylenes in the organic waste depending on its nature and origin.

Treatment with Rhus tripartita extract curtails isoproterenol-elicited cardiotoxicity and oxidative stress in rats

BackgroundConsumption of plant-derived nutraceuticals and crude drugs in traditional medicine is widely believed to confer beneficial effects in thwarting the progression of cardiovascular diseases. Rhus tripartita (family Anacardiaceae) has been traditionally used to treat a wide range of ailments.MethodsIn the present study we investigated the protective effects of an alcoholic extract of the stem part of Rhus tripartita male genotype (RTSM) on experimentally induced myocardial injury in rats. To this end, cardiac injury was induced by administration of isoproterenol (ISO) and serum enzyme markers, lipid profiles and cardiac tissue redox status were determined following RTSM treatment (250 and 500xa0mg/kg).ResultsAs a result, RTSM treatment significantly mitigated ISO-triggered upregulation of cardiac-specific markers of injury creatine kinase and lactate dehydrogenase. RTSM treatment significantly attenuated ISO-induced increase in serum cholesterol and triglycerides as well alterations in serum lipoproteins. Determination of oxidative balance showed that RTSM treatment significantly blunted ISO-induced increase in malondialdehyde and decrease in nonprotein sulfhydryl in cardiac tissue. Six compounds were isolated and identified as gallocatechin 1, taxifolin 2, myricetin-3-O-β-glucoside 3, catechin 4, epicatechin 5, and 3′,8-binaringenin 6. Compound 6 was isolated for the first time from the stem part of Rhus tripartita. Furthermore, RTSM treatment enhanced the survival fraction of cardiac cells exposed to oxidative stress in vitro.ConclusionWe conclude that the antioxidant properties of RTSM treatment underpin its cardioprotective pharmacological effects, thus, providing biological evidence for the treatment of cardiovascular diseases using Rhus tripartita in indigenous medicine.

In Vitro and In Silico Antidiabetic and Antimicrobial Evaluation of Constituents from Kickxia ramosissima (Nanorrhinum ramosissimum)

Background and Aims: Kickxia ramosissima (Wall.) Janch (or Nanorrhinum ramosissimum (Wall.) Betsche is a well-known medicinal plant in Pakistan that is traditionally used in diabetic and inflammatory conditions. Because little information is available on its phytochemical composition, a range of constituents were isolated and evaluated in vitro in assays related to the traditional use. Methods: Dried whole plant material was extracted and chromatographically fractionated. Isolated constituents were evaluated in silico and in vitro in assays related to the traditional use against diabetes (inhibition of α-glucosidase activity; inhibition of advanced glycation endproducts) and in inflammatory conditions (inhibition of AAPH induced linoleic acid peroxidation, inhibition of 15-LOX, antimicrobial activity). Results: Phytochemical analysis of the extracts and fractions led to isolation of 7 compounds, including the iridoids kickxiasine (being a new compound), mussaenosidic acid, mussaenoside and linarioside; the flavonoids pectolinarigenin and pectolinarin; and 4-hydroxy-benzoic acid methyl ester. The iridoids showed weak antiglycation activity. The flavonoids, however, showed interesting results as pectolinarigenin was highly active compared to pectolinarin. In the α-glucosidase inhibition assay, only weak activity was observed for the iridoids. However, the flavonoid pectolinarigenin showed good activity, followed by pectolinarin. In the 15-LOX experiment, moderate inhibition was recorded for most compounds, the iridoids mussaenosidic acid and mussaenoside being the most active. In the AAPH assay, weak or no inhibition was recorded for all compounds. The in silico assays for the α-glucosidase and 15-LOX assays confirmed the results of respective in vitro assays. Pectolinarigenin showed moderate antimicrobial activity against Staphylococcus aureus, Plasmodium falciparum K1, and Trypanosoma cruzi, but it was not cytotoxic on a human MRC-5 cell line. Conclusion: Our findings may in part contribute to explain the traditional use of K. ramosissima.

A new cyclopeptide alkaloid from Hymenocardia acida

Hymenocardia acida Tul. (Euphorbiaceae) is a shrub or a small tree of about 6 m high, which grows in the savannah regions of Africa. The extracts are widely used in traditional African medicine, for example for their antiplasmodial and antidiabetic potential. Previous phytochemical studies on this plant have established the presence of alkaloids, anthocyanins, anthraquinones, cardiac glycosides, flavonoids, phenols, saponins, steroids, stilbenoids, tannins and triterpenoids [1, 2], but not much is known yet about the active compounds. In this study the isolation of alkaloids from H. acida is described. The dried root bark of H. acida (2.7 kg) was extracted by percolation with 80% methanol and fractionated by liquid-liquid separation and flash chromatography. To indicate the presence of alkaloids the Dragendorff and iodoplatinate reagent, two alkaloid specific TLC visualization reagents, were used and fractions which showed a positive reaction with both were selected for further analysis. The main compounds in these fractions were then isolated by semi-preparative HPLC-DAD-MS. In order to elucidate the structures of these compounds, 1H-, 13C- and a variety of 2D-NMR spectra were recorded. Compound 1, hymenocardine, has been isolated from this plant before [3] and the structure was confirmed by comparison with previously reported 1 H- and 13C-NMR spectra [3, 4]. Compound 2 was almost identical to compound 1, but the spectroscopic data showed that it contained a hydroxyl group instead of a carbonyl group and thus it was named hymenocardinol. To our knowledge it is the first time hymenocardinol was isolated from H. acida or any other natural source, although it has been prepared by reduction of hymenocardine. Thus, hymenocardinol is reported here as a new cyclopeptide alkaloid. Keywords: Hymenocardia acida, Euphorbiaceae, cyclopeptide alkaloids, hymenocardine, hymenocardinol References: [1] Starks C., Williams R. et al. (2014) Phytochemistry 98: 216 – 222 [2] Manga F., Khattabi C. et al (2013)J Ethnoparmacol 146: 623 – 631 [3] Pais M., Marchand J. et al. (1968) Bull Soc Chim Fr 7: 2979 – 2984 [4] Pais M., Jarreau F. et al. (1979) Phytochemistry 18: 1869 – 1872