A Simple and Efficient Two-Dimensional High-Speed Counter-Current Chromatography Linear Gradient and Isocratic Elution Modes for the Preparative Separation of Coumarins from Roots of Toddalia asiatica (Linn.) Lam.

Toddalia asiatica (L.) Lam. (Rutaceae) has shown a broad spectrum of biological properties, such as anti-inflammatory, antioxidant, antimicrobial, anti-HIV, and anticancer properties. The present study is concerned with the separation of the main components with broad partition coefficients (K_D values) from T. asiatica, using linear gradient high-speed counter-current chromatography (LGCCC) combined with an off-line two-dimensional (2D) mode. Similar to the binary gradient HPLC, the LGCCC mode is operated by the adjustment of the proportion between the mobile phase of 5:5:1:9 (v/v) (pump A) and 5:5:4.5:5.5 (v/v) (pump B) in an n-hexane/ethyl acetate/methanol/water solvent system. The off-line 2D-CCC mode was used in this study for the secondary separation of two similar K_D value compounds with n-hexane/ethyl acetate/methanol/water (5:5:4:6, v/v). Notably, six coumarins, namely, tomentin (1), toddalolactone (2), 5,7,8-trimethoxycoumarin (3), mexoticin (4), isopimpinellin (5), and toddanone (6), were efficiently separated. The structures of the pure compounds were elucidated by spectral techniques and compared with the literature.     

Isolation and antiproliferative activity of triterpenoids and fatty acids from the leaves and stem of Turraea vogelii Hook. f. ex benth

Chloroform extract from the leaves of Turraea vogelii Hook f. ex Benth demonstrated cytotoxic activity against a chronic myelogenous leukemia cell, K-562 with IC₅₀ of 14.27 μg/mL, while chloroform, ethyl acetate and methanol extracts from the stem of the plant inhibited K-562 cells growth with IC₅₀ of 19.50, 24.10 and 85.40 μg/mL respectively. Bioactive chloroform extract of Turraea vogelii leaves affords two triterpenoids: oleana-12,15,20-trien-3β-ol (1), and oleana-11,13-dien-3β,16α,28-triol (2), with six fatty esters, ethyl hexaeicos-5-enoate (3), 3-hydroxy-1,2,3-propanetriyltris(tetadecanoate) (4), 1,2,3-propanetriyl(7Z,7′Z,7′′Z)tris(-7-hexadecenoate) (5), 1,2,3-propanetriyl(5Z,5′Z,5′′Z)tris(-5-hexadecenoate) (6), 1,2,3-propanetriyltris(octadecanoate) (7), and 2β-hydroxymethyl tetraeicosanoate (8). Tetradecane (9), four fatty acids: hexadecanoic acid (10), tetradecanoic acid (11), (Z)-9-eicosenoic acid (12), and ethyl tetradec-7-enoate (13) were isolated from chloroform extract of Turraea vogelii stem. 1,2,3-propanetriyltris(heptadecanoate) (14), (Z)-9-octadecenoic acid (15) and (Z)-7-tetradecenoic acid (16) were isolated from ethyl acetate extract while (Z)-5-pentadecenoic acid (17) was obtained from methanol extract of the plant stem. Compounds 1, 2, 5, 6, 11, 12, 15, 16 and 17 exhibited pronounced antiproliferative activity against K-562 cell lines.     

Annurca peel extract: from the chemical composition,through the functional activity,to the formulation and characterisation of a topical oil-in-water emulsion

The aim of this study was to produce a hydro-alcoholic safe antioxidant Malus pumila Miller cv Annurca peel extract (APE) useful as functional ingredient in an oil-in-water emulsion. Results showed that APE contains a hydroxycinnamic acid (chlorogenic acid), flavonol glycosides (quercetin derivatives) and a dihydrochalcone, phloridzin (phloretin-2-O-glucoside). The isoquercitrin (quercetin-3-O-glucoside) content was quantified in 0.3% w/w of extract. APE showed a significant and concentration-dependent free-radical scavenging activity correlated to its polyphenols content. No cytotoxic effect was observed in primary human epidermal keratinocyte adults and dermal fibroblast cell lines. The formulative approach led to produce a stable emulsion able to load a high amount of APE, up to 6.0% w/w. The homogenous distribution of APE in the emulsion was clearly demonstrated by fluorescence microscopy analysis. The emulsion resulted able to enhance the in vitro release rate of APE through synthetic membranes with respect to the raw material.     

Development and validation of a LC‐MS/MS method for the in vitro analysis of 1‐hydroxymidazolam in human liver microsomes: application for determining CYP3A4 inhibition in complex matrix mixtures

Complementary and alternative medicines (CAM) can affect the pharmacokinetics of anticancer drugs by interacting with the metabolizing enzyme cytochrome P450 (CYP) 3A4. To evaluate changes in the activity of CYP3A4 in patients, levels of 1‐hydroxymidazolam in plasma are often determined with liquid chromatography–quadrupole mass spectrometry (LC‐MS/MS). However, validated LC‐MS/MS methods to determine in vitro CYP3A4 inhibition in human liver microsomes are scarce and not optimized for evaluating CYP3A4 inhibition by CAM. The latter is necessary because CAM are often complex mixtures of numerous compounds that can interfere with the selective measurement of 1‐hydroxymidazolam. Therefore, the aim was to validate and optimize an LC‐MS/MS method for the adequate determination of CYP3A4 inhibition by CAM in human liver microsomes. After incubation of human liver microsomes with midazolam, liquid–liquid extraction with tert‐butyl methyl ether was applied and dried samples were reconstituted in 50% methanol. These samples were injected onto a reversed‐phase chromatography consisting of a Zorbax Extend‐C₁₈ column (2.1 × 150 mm, 5.0 µm particle size), connected to a triple quadrupole mass spectrometer with electrospray ionization. The described LC‐MS/MS method was validated over linear range of 1.0–500 nm for 1‐hydroxymidazolam. The results revealed good inter‐assay accuracy (≥85% and ≤115%) and within‐day and between‐day precisions (coefficient of variation ≤ 4.43%). Furthermore, the applicability of this assay for the determination of CYP3A4 inhibition in complex matrix mixtures was successfully demonstrated in an in vitro experiment in which CYP3A4 inhibition by known CAM (β‐carotene, green tea, milk thistle and St. John's wort) was determined. Copyright © 2013 John Wiley & Sons, Ltd.     

Room temperature phosphorescence in the liquid state as a tool in analytical chemistry

A wide-ranging overview of room temperature phosphorescence in the liquid state (RTPL¹) is presented, with a focus on recent developments. RTPL techniques like micelle-stabilized (MS)-RTP, cyclodextrin-induced (CD)-RTP, and heavy atom-induced (HAI)-RTP are discussed. These techniques are mainly applied in the stand-alone format, but coupling with some separation techniques appears to be feasible. Applications of direct, sensitized and quenched phosphorescence are also discussed. As regards sensitized and quenched RTP, emphasis is on the coupling with liquid chromatography (LC) and capillary electrophoresis (CE), but stand-alone applications are also reported. Further, the application of RTPL in immunoassays and in RTP optosensing—the optical sensing of analytes based on RTP—is reviewed. Next to the application of RTPL in quantitative analysis, its use for the structural probing of protein conformations and for time-resolved microscopy of labelled biomolecules is discussed. Finally, an overview is presented of the various analytical techniques which are based on the closely related phenomenon of long-lived lanthanide luminescence. The paper closes with a short evaluation of the state-of-the-art in RTP and a discussion on future perspectives.