Rapid determination of furanocoumarins in creams and pomades using SPE and GC

A solid-phase extraction and chromatography-flame ionization detection (GC-FID) method has been developed for the routine analysis of psoralen, bergapten, isopimpinellin and pimpinellin in creams and pomades employed in Brazil for the treatment of vitiligo. The calibration curve for psoralen was linear in the range 10-100 mu g ml(-1), for bergapten 5-90 mu g ml(-1), for pimpinellin 10-90 mu g ml(-1) and for isopimpinellin 5-100 mu g ml(-1). The best recoveries of the furanocoumarins in the creams analysed were 94-97%, whereas in the pomades, recoveries were 94-96%. The R.S.D. of the quantitative analysis of the furanocoumarins in the products analyses were within 5%. (C) 2000 Elsevier B.V. B.V. All rights reserved.

Simultaneous determination of furanocoumarins in infusions and decoctions from Carapia (Dorstenia species) by high-performance liquid chromatography

The use of furanocoumarins, which are photosensitizing compounds, combined with exposure to UV-A radiation is a common treatment for vitiligo, psoriasis, and a number of other skin diseases. Although furanocoumarins plus UV-A treatment is highly effective, several studies have shown that exposure to high doses increases the risk to development of cutaneus carcinoma. Several Dorstenia species are used in folk medicine, mainly against skin diseases, because of the presence of biologically active compounds. We present here analysis of the chemical composition of furanocoumarins from infusion and decoction of Carapia (Dorstenia species), which is used in Brazil against several diseases. We have employed high-performance liquid chromatography (HPLC) procedures for the quantitative determination of psoralen, bergapten, and isopimpinellin. The contents of furanocoumarins revealed an insignificant difference between infusion and decoction. Dorstenia tubicina and D. asaroides contained psoralen and bergapten only in the rhizomes, whereas D. vitifolia shows solely isopimpinellin in both rhizomes and aerial parts.

FTIR-spektroskopia kasviuutteiden analysoinnissa

FTIR-spektroskopia (Fourier-muunnosinfrapunaspektroskopia) on nopea analyysimenetelmä. Fourier-laitteissa interferometrin käyttäminen mahdollistaa koko infrapunataajuusalueen mittaamisen muutamassa sekunnissa. ATR-liitännäisellä varustetun FTIR-spektrometrin käyttö ei edellytä juuri näytteen valmistusta ja siksi menetelmä on käytössä myös helppo. ATR-liitännäinen mahdollistaa myös monien erilaisten näytteiden analysoinnin. Infrapunaspektrin mittaaminen onnistuu myös sellaisista näytteistä, joille perinteisiä näytteenvalmistusmenetelmiä ei voida käyttää. FTIR-spektroskopian avulla saatu tieto yhdistetään usein tilastollisiin monimuuttuja-analyyseihin. Klusterianalyysin avulla voidaan spektreistä saatu tieto ryhmitellä samanlaisuuteen perustuen. Hierarkkisessa klusterianalyysissa objektien välinen samanlaisuus määritetään laskemalla niiden välinen etäisyys. Pääkomponenttianalyysin avulla vähennetään datan ulotteisuutta ja luodaan uusia korreloimattomia pääkomponentteja. Pääkomponenttien tulee säilyttää mahdollisimman suuri määrä alkuperäisen datan variaatiosta. FTIR-spektroskopian ja monimuuttujamenetelmien sovellusmahdollisuuksia on tutkittu paljon. Elintarviketeollisuudessa sen soveltuvuutta esimerkiksi laadun valvontaan on tutkittu. Menetelmää on käytetty myös haihtuvien öljyjen kemiallisten koostumusten tunnistukseen sekä öljykasvien kemotyyppien havaitsemiseen. Tässä tutkimuksessa arvioitiin menetelmän käyttöä suoputken uutenäytteiden luokittelussa. Tutkimuksessa suoputken eri kasvinosien uutenäytteiden FTIR-spektrejä vertailtiin valikoiduista puhdasaineista mitattuihin FTIR-spektreihin. Puhdasaineiden FTIR-spektreistä tunnistettiin niiden tyypilliset absorptiovyöhykkeet. Furanokumariinien spektrien intensiivisten vyöhykkeiden aaltolukualueet valittiin monimuuttuja-analyyseihin. Monimuuttuja-analyysit tehtiin myös IR-spektrin sormenjälkialueelta aaltolukualueelta 1785-725 cm-1. Uutenäytteitä pyrittiin luokittelemaan niiden keräyspaikan ja kumariinipitoisuuden mukaan. Keräyspaikan mukaan ryhmittymistä oli havaittavissa, mikä selittyi vyöhykkeiden aaltolukualueiden mukaan tehdyissä analyyseissa pääosin kumariinipitoisuuksilla. Näissä analyyseissa uutenäytteet pääosin ryhmittyivät ja erottuivat kokonaiskumariinipitoisuuksien mukaan. Myös aaltolukualueen 1785-725 cm-1 analyyseissa havaittiin keräyspaikan mukaan ryhmittymistä, mitä kumariinipitoisuudet eivät kuitenkaan selittäneet. Näihin ryhmittymisiin vaikuttivat mahdollisesti muiden yhdisteiden samanlaiset pitoisuudet näytteissä. Analyyseissa käytettiin myös muita aaltolukualueita, mutta tulokset eivät juuri poikenneet aiemmista. 2. kertaluvun derivaattaspektrien monimuuttuja-analyysit sormenjälkialueelta eivät myöskään muuttaneet tuloksia havaittavasti. Jatkotutkimuksissa nyt käytettyä menetelmää on mahdollista edelleen kehittää esimerkiksi tutkimalla monimuuttuja-analyyseissa 2. kertaluvun derivaattaspektreistä suppeampia, tarkkaan valittuja aaltolukualueita.

Prédiction des impacts pharmacocinétiques des interactions médicamenteuses impliquant des CYP3A et les glycoprotéines-P : développement de modèles physiologiques et analyse de sensibilité

Les propriétés pharmacocinétiques d’un nouveau médicament et les risques d’interactions médicamenteuses doivent être investigués très tôt dans le processus de recherche et développement. L’objectif principal de cette thèse était de concevoir des approches prédictives de modélisation du devenir du médicament dans l’organisme en présence et en absence de modulation d’activité métabolique et de transport. Le premier volet de recherche consistait à intégrer dans un modèle pharmacocinétique à base physiologique (PBPK), le transport d’efflux membranaire gouverné par les glycoprotéines-P (P-gp) dans le cœur et le cerveau. Cette approche, basée sur des extrapolations in vitro-in vivo, a permis de prédire la distribution tissulaire de la dompéridone chez des souris normales et des souris déficientes pour les gènes codant pour la P-gp. Le modèle a confirmé le rôle protecteur des P-gp au niveau cérébral, et a suggéré un rôle négligeable des P-gp dans la distribution tissulaire cardiaque pour la dompéridone. Le deuxième volet de cette recherche était de procéder à l’analyse de sensibilité globale (ASG) du modèle PBPK précédemment développé, afin d’identifier les paramètres importants impliqués dans la variabilité des prédictions, tout en tenant compte des corrélations entre les paramètres physiologiques. Les paramètres importants ont été identifiés et étaient principalement les paramètres limitants des mécanismes de transport à travers la membrane capillaire. Le dernier volet du projet doctoral consistait à développer un modèle PBPK apte à prédire les profils plasmatiques et paramètres pharmacocinétiques de substrats de CYP3A administrés par voie orale à des volontaires sains, et de quantifier l’impact d’interactions médicamenteuses métaboliques (IMM) sur la pharmacocinétique de ces substrats. Les prédictions des profils plasmatiques et des paramètres pharmacocinétiques des substrats des CYP3A ont été très comparables à ceux mesurés lors d’études cliniques. Quelques écarts ont été observés entre les prédictions et les profils plasmatiques cliniques mesurés lors d’IMM. Cependant, l’impact de ces inhibitions sur les paramètres pharmacocinétiques des substrats étudiés et l’effet inhibiteur des furanocoumarins contenus dans le jus de pamplemousse ont été prédits dans un intervalle d’erreur très acceptable. Ces travaux ont contribué à démontrer la capacité des modèles PBPK à prédire les impacts pharmacocinétiques des interactions médicamenteuses avec une précision acceptable et prometteuse.

Growth inhibition of mammalian cells by synthetic and natural photosensitising agents

A mammalian cell line, J774, was susceptible to both synthetic and natural photosensitising agents after irradiation with long-wave ultraviolet light. Both UV-A light and psoralen did not affect cell growth individually; a reduction in visual confluency was achieved only when psoralen and UV-A light were used in combination. The maximum visual confluency decreased by 55% when 50 ppm psoralen was added to a growing culture and irradiated with UV light for 3 min. Decreasing the UV-A exposure times from 3 min to 3 s did not greatly affect the maximum total visual confluence reached using different synthetic psoralen concentrations, but did affect the rate at which cell death occurred. The 3 min exposure time resulted in a rapid decrease in cell numbers in comparison to 3 s exposure time. Synthetic psoralen was found to have an increasing photosensitising activity with increasing concentration using a logarithmic shift between 0.5 ppm and 50 ppm. A visual confluency of 45% was achieved using concentrations of 50 ppm psoralen, and 70% visual confluency using 0.5 ppm. Natural mixtures of furanocoumarins containing psoralens, obtained from two separate parsley sources, were found to have greater efficacy at inhibiting the growth cycle of the cells when compared to the synthetic psoralen.

Atividade de extratos vegetais e seus derivados sobre o crescimento do fungo simbionte de Atta sexdens L. e outros microrganismos

Pós-graduação em Ciências Biológicas (Microbiologia Aplicada) - IBRC

Estudo comparativo dos constituintes químicos de Brosimum gaudichaudii Trécul e do medicamento V

Pós-graduação em Química - IQ

Inhibiting effect of Dorstenia asaroides extracts on cariogenic properties of Streptococcus mutans

The aim of this study was to examine the effects of Dorstenia asaroides extracts on cariogenic properties of the most cariogenic bacteria, Streptococcus mutans. Hexane (HFr), ethyl-acetate (EFr) and chloroform (CFr) extracts obtained from D. asaroides rhizomes were submitted to chemical analyses, Minimal Inhibitory Concentrations (MIC), glycolysis assay and S. mutans 12-h-old initial biofilms. Chemical characterization showed that all the extracts present furanocoumarins. The MIC values were 80 (HFr and CFr) and 50 mu g/mL (EFr). Acid production by S. mutans cells was significantly disrupted by HFr (12.5 mg/mL), EFr (at 2.5; 6.25 and 12.5 mg/mL) and CFr (at 2.5, 6.25 and 12.5 mg/mL) (p < 0.01). Topical applications of HFr, EFr and CFr significantly reduced the colony forming units of S. mutans biofilms compared with those treated with control group in order to 20,30 and 25% respectively (p < 0.01). The results of the present study suggest that rhizomes of D. asaroides had inhibitory effects on cariogenic properties of S. mutans. (C) 2012 Elsevier Ltd. All rights reserved.

Chemical phenotype matching between a plant and its insect herbivore

Two potential outcomes of a coevolutionary interaction are an escalating arms race and stable cycling. The general expectation has been that arms races predominate in cases of polygenic inheritance of resistance traits and permanent cycling predominates in cases in which resistance is controlled by major genes. In the interaction between Depressaria pastinacella, the parsnip webworm, and Pastinaca sativa, the wild parsnip, traits for plant resistance to insect herbivory (production of defensive furanocoumarins) as well as traits for herbivore “virulence” (ability to metabolize furanocoumarins) are characterized by continuous heritable variation. Furanocoumarin production in plants and rates of metabolism in insects were compared among four midwestern populations; these traits then were classified into four clusters describing multitrait phenotypes occurring in all or most of the populations. When the frequency of plant phenotypes belonging to each of the clusters is compared with the frequency of the insect phenotypes in each of the clusters across populations, a remarkable degree of frequency matching is revealed in three of the populations. That frequencies of phenotypes vary among populations is consistent with the fact that spatial variation occurs in the temporal cycling of phenotypes; such processes contribute in generating a geographic mosaic in this coevolutionary interaction on the landscape scale. Comparisons of contemporary plant phenotype distributions with phenotypes of herbarium specimens collected 9–125 years ago from across a similar latitudinal gradient, however, suggest that for at least one resistance trait—sphondin concentration—interactions with webworms have led to escalatory change.

Conserved promoter elements in the CYP6B gene family suggest common ancestry for cytochrome P450 monooxygenases mediating furanocoumarin detoxification.

Despite the fact that Papilio glaucus and Papilio polyxenes share no single hostplant species, both species feed to varying extents on hostplants that contain furanocoumarins. P. glaucus contains two nearly identical genes, CYP6B4v2 and CYP6B5v1, and P. polyxenes contains two related genes, CYP6B1v3 and CYP6B3v2. Except for CYP6B3v2, the substrate specificity of which has not yet been defined, each of the encoded cytochrome P450 monooxygenases (P450s) metabolizes an array of linear furanocoumarins. All four genes are transcriptionally induced in larvae by exposure to the furanocoumarin xanthotoxin; several are also induced by other furanocoumarins. Comparisons of the organizational structures of these genes indicate that all have the same intron/exon arrangement. Sequences in the promoter regions of the P. glaucus CYP6B4v2/CYP6B5v1 genes and the P. polyxenes CYP6B3v2 gene are similar but not identical to the -146 to -97 region of CYP6B1v3 gene, which contains a xanthotoxin-responsive element (XRE-xan) important for basal and xanthotoxin-inducible transcription of CYP6B1v3. Complements of the xenobiotic-responsive element (XRE-AhR) in the dioxin-inducible human and rat CYP1A1 genes also exist in all four promoters, suggesting that these genes may be regulated by dioxin. Antioxidant-responsive elements (AREs) in mouse and rat glutathione S-transferase genes and the Barbie box element (Bar) in the bacterial CYP102 gene exist in the CYP6B1v3, CYP6B4v2, and CYP6B5v1 promoters. Similarities in the protein sequences, intron positions, and xanthotoxin- and xenobiotic-responsive promoter elements indicate that these insect CYP6B genes are derived from a common ancestral gene. Evolutionary comparisons between these P450 genes are the first available for a group of insect genes transcriptionally regulated by hostplant allelochemicals and provide insights into the process by which insects evolve specialized feeding habits.