On-line identification of minor flavones from sugarcane juice by LC/UV/MS and post-column derivatization

This work describes the on-line characterization of minor flavones from sugarcane (Saccharum officinarum) juice by high-performance liquid chromatography coupled to diode array UV detection and mass spectrometry (LC/UV/MS) using atmospheric pressure chemical ionization-collision-induced dissociation (APCI-CID-MS/MS) and post-column derivatization using UV shift reagents. HPLC-UV analysis with shift reagents provided information about the substitution pattern in the flavonoid skeleton and, combined with MS data, these techniques allowed for the on-line identification of five "garapa" flavones: luteolin-8-C-glucosyl-7-O-glucuronide; tricin-7-O-neohesperoside-4'-O-rhamnoside; tricin-7-O-methylglucuronate-4'-O-rhamnoside; tricin-7-O-methylglucuronide; swertisin, while four other compounds were partially identified as glycosylflavones. Only swertisin (7-O-methylapigenin-6-C-glucoside) was reported previously in sugarcane molasses.Este trabalho apresenta a identificação "on line" de flavonas minoritárias do suco da cana-de-açúcar (Saccharum officinarum), por cromatografia líquida de alta eficiência com detector UV acoplada à espectrometria de massas (CL/UV/EM) com ionização química à pressão atmosférica, dissociação induzida por colisão (IQPA-DIC-EM/EM) e derivatização pós-coluna utilizando reagentes de deslocamento de UV. As análises CLAE-UV com reagentes de deslocamento forneceram informações sobre a posição da substituição no esqueleto dos flavonóides e, em combinação com dados de EM, estas técnicas permitiram a identificação "on-line" de cinco flavonas da garapa: luteolina-8-C-glucosil-7-O-glucuronídeo; tricina-7-O-neoesperosideo-4'-O-ramnosídeo; tricina-7-O-metilglucuronato-4'-O-ramnosídeo; tricina-7-O-metilglucuronídeo; swertisina; e outras quatro substâncias foram parcialmente identificadas como flavonas glicosiladas. Somente a swertisina (7-O-metilapigenina-6-C-glicosídeo) foi anteriormente descrita no bagaço da cana-de-açúcar.FAPESPCNPqSwiss NSF National Science FoundationUniversity of Lausanne - Fondation Herbett

Kinetic Properties of α-Galactosidase and the Localization of Total Proteins in Erwinia chrysanthemi

Erwinia chrysanthemi is an enterobacterium that causes soft-rot in plants in general, resulting in enormous economic losses annually. For the pathogen to survive in the host plant, it has to use the readily assimilable compounds from the host fluids and degrade the host tissue. To accomplish this, E. chrysanthemi produces several extracellular and intracellular enzymes. Among the intracellular enzymes there is a special digestive class, the galactosidases, which can be either periplasmic or cytoplasmic. α-Galactosidase is known to degrade melibiose and raffinose into glucose and galactose, and into galactose and sucrose respectively. The aim of the present study was to investigate the kinetic properties of α-galactosidase in E. chrysanthemi, and the localization of total proteins, after culturing it in the presence of raffinose and melibiose. The α-galactosidase that degrades melibiose seems to be the same enzyme that is also responsible for the breakdown of raffinose in E. chrysanthemi. It is localized mainly in the cytoplasm with a fraction of between 2.4 and 5.4 % localized in the periplasm. The majority of E. chrysanthemi proteins have cytoplasmic localization

Flavonoid profiling among wild type and related GM wheat varieties

Pleiotropic effects are one of the main concerns regarding genetically modified organisms (GMOs). This includes unintended side effects of the transgene or its genome insertion site on the regulation of other endogenous genes, which could potentially cause the accumulation of different secondary metabolites that may have not only an impact on diet as repeatedly worried by the public but also on the environment. Regarding amount and possible environmental effects, flavonoids represent the most prominent group of secondary metabolites in wheat. Many flavonoids function as signalling or defence molecules. We used a robust and reproducible analytical method to compare the flavonoid content of genetically modified (GM) wheat (Triticum aestivum L., Gramineae) expressing genes that confer increased fungal resistance with their non-GM siblings. The transgenes provide either a broad-spectrum fungal defence (chitinase/glucanase from barley) or bunt-specific resistance by a viral gene (KP4). Significant differences in flavonoid composition were found between different wheat varieties whereas different lines of GM wheat with increased antifungal resistance showed only minor differences in their flavonoid composition relative to their non-GM siblings. In a field test, no significant differences were detectable between infected and non-infected wheat of the same variety regardless of the presence of the transgene. Our results are in agreement with the hypothesis that the transgenes we used to increase wheat defence to fungal pathogens do not interfere with the flavonoid biosynthesis pathway. More significantly, the genetic background resulting from conventional breeding has a direct impact on the biological composition of flavonoids, and thus possibly on the environmen

Absolute Configuration of a Tetrahydrophenanthrene from Heliotropium ovalifolium by LC-NMR of Its Mosher Esters

A new tetrahydrophenanthrene (1, (1R,2R)-1-hydroxy-2-methoxy-6,9-dimethyl-2,3-dihydrophenanthren-4(1H)-one (heliophenanthrone)) has been isolated from the aerial parts of Heliotropium ovalifolium. Its structure was elucidated on the basis of spectroscopic data, and the absolute configuration of the asymmetric centers was determined from LC-NMR data of the Mosher ester derivatives

Phytochemistry and in vitro Anti-sickling activity of Senna Occidentalis L. (Fabaceae)

Introduction Sickle cell disease is an inherited genetic disorder characterized by the presence of abnormal hemoglobin, leading to the deformation of red blood cells and serious complications. It is a major public health problem in many countries of inter-tropical Africa. In the Democratic Republic of the Congo, over a million people (2%) are affected by this hemoglobinopathy. Purpose This study aimed to scientifically validate the anti-sickle cell activity of aqueous extracts of S. occindentalis seeds and to identify the chemical constituents responsible for this activity. Methods In this study, we used S. occidentalis seeds harvested at Ilebo in Central Kasai Province, while the blood samples used were taken from sickle-cell patients. The phytochemical composition was determined according to the standard method described previously by Iteku et al. and Nkasa et al. The Emmel test was carried out according to the standard protocol described previously by Bongo et al. Results The results obtained in this study showed that the seeds of this plant are rich in secondary metabolites such as total polyphenols (flavonoids, anthocyanins, leuco-athocyanins, tannins, and saponins), di-terpenes, alkaloids, and bound quinones. However, these seeds do not contain triterpenoids and steroids. Total seed extracts from this plant showed significant anti-sickle cell activity. Conclusion This study identified a medicinal plant used by the sickle cell disease community

Determination of trace amounts of ginkgolic acids in Ginkgo biloba L. leaf extracts and phytopharmaceuticals by liquid chromatography-electrospray mass spectrometry.

Ginkgolic acids (GAs) are toxic phenolic compounds present in the fruits and leaves of Ginkgo biloba L. (Ginkgoacae). Their maximum level in phytopharmaceuticals containing ginkgo extracts has been recently restricted to 5 microg/g by the Commission E of the former Federal German Health Authority. In order to detect ginkgolic acids at these low levels, a sensitive and selective analytical method, based on liquid chromatography-electrospray mass spectrometry (LC-ES-MS) has been developed. The three main phenolic acids (1-3) of the chloroform fruit extract were isolated and used as standards for quantification. In the LC-ES-MS negative ion mode, calibration curves with good linearities (r=0.9973, n=6) were obtained in the range of 0.5-10 microg/g for compounds 1, 2 and between 0.1 and 7.5 microg/g (r=0.9949, n=6) for ginkgolic acid 3. The detection limits at a SIN ratio of 3 were 0.1 (3) and 0.25 microg/g (1, 2). Recoveries were around 101% at 5 microg/g for the substances detected in the leaf extracts. Good precision was achieved with relative standard deviations of less than 4% (n=6). The optimised method was applied to verify whether the amount of gingkolic acids was below 5 microg/g in a standardised leaf extract which is a constituent of a phytopreparation