Dissertations / Theses on the topic 'Luteolina'

Magister Pharmaceuticae - MPharm<br>Artemisia afra (A. afra) is traditionally used for a variety of ailments and contain flavonoids e.g. luteolin which may contribute to some of its activity. It is generally administered as a tea or decoction, and such liquid dosage forms present challenges as far as long term storage and stability are concerned, as well as sub-optimal oral bioavailability of actives they contain. Freeze dried aqueous extracts (FDAE) can alleviate such problems but may be hygroscopic and unstable. The use of modified forms of FDAE can counter the problem of hygroscopicity (e.g. use of alginate) and alleviate the issue of sub-optimal bioavailability of plant actives (e.g. polymethylmethacrylate). The objectives of this study, were to: (1) prepare the freeze dried aqueous extract (FDAE) and modified forms, which include alginate-extract beads (alginate-FDAE) and polymethylmethacrylate coated alginate matrix beads of herbal extract (PMMA-alginate-FDAE) of the FDAE of A. afra, (2) determine and compare the pharmaceutical characteristics of the above mentioned preparations of A. afra,(3) quantify and compare the total flavonoid and specifically luteolin levels of the different forms of A. afra,(4) evaluate and compare the release characteristics of FDAE of A. afra from the alginate-FDAE and PMMA-alginate-FDAE beads in gastrointestinal fluids and (5) determine the intestinal permeability of luteolin contained in selected modified Artemisia afra extract preparations. It was hypothesized that making the alginate beads and the polymethylmethacrylate coated alginate beads would make the FDAE less hygroscopic with a lower moisture content, that the rate of release of luteolin from A. afra FDAE into gastrointestinal fluids would be faster than from the modified forms, and that the effective gastrointestinal permeability of luteolin in the alginate-FDAE and PMMA-alginate-FDAE beads of A. afra is equal to that in FDAE. To realize these objectives, the FDAE was prepared by freeze drying the aqueous extract of the A. afra dried leaves, alginate-FDAE prepared by dispersing FDAE into 4% sodium alginate solution, then adding the resulting stock solution into a 2% calcium chloride solution and drying resulting beads and PMMA-alginate-FDAE prepared by a modified water-in-oil-in-water emulsion solvent evaporation method using water as an internal aqueous phase. Using pharmacopoeial methods and methods adapted from other workers the organoleptic and pharmaceutical characteristics were determined to compare the pharmaceutical quality of these preparations of A. afra. To identify and determine the levels of luteolin in the plant preparations, a validated HPLC assay was developed. Finally, the in situ perfused rat intestine model was used to determine the in vitro bioavailability, i.e. gastrointestinal permeability, of luteolin from solutions containing luteolin in pure form, FDAE, alginate-FDAE and PMMA-alginate-FDAE. The A. afra forms were obtained in moderate to good yields and FDAE was brown and hygroscopic in nature, the alginate beads dark brown free flowing and spherical in shape and the PMMA-alginate beads light brown in colour with rough edges. The A. afra plant forms on average contained 0.185 ± 0.24, 0.067 ± 0.014, 0.012 ± 0.071 μg/mg of free luteolin (n=3) in FDAE, alginate-FDAE and PMMA-alginate-FDAE respectively and 0.235 ± 0.026, 0.079 ± 0.093, 0.058 ± 0.082 μg/mg of total luteolin (n=3) in FDAE, alginate-FDAE and PMMAalginate- FDAE respectively. The Plumen values for intestinal uptake of luteolin were significantly higher from solutions of A. afra forms than the pure luteolin solution (i.e. Plumen values in the range of 0.02 - 0.035 cm/s for all plant forms vs Plumen values in the range of 0.010 - 0.014 cm/s for pure luteolin, t-test p = 0.0252). The permeability of luteolin in FDAE appeared to be slighter greater than that of the modified forms (Plumen values >0.03 cm/s for FDAE and Plumen values <0.03 cm/s for both modified forms). In summary, the results showed that, the modified A. afra forms; alginate-FDAE and PMMAalginate- FDAE were of acceptable pharmaceutical quality with luteolin better taken up in the plant forms than in its pure form. The A. afra forms prepared had similar rates of uptake (permeability) of free and total luteolin with the rates being highest for the FDAE. Collectively, these results indicate that alginate-FDAE and PMMA-alginate-FDAE bead forms should be suitable for use in a solid dosage form (e.g. tablet or capsule) of A. afra.

Colorectal cancer is one of the most common malignancies and a leading cause of cancer death in the world. More powerful and safer therapeutic approaches are urgently needed to reduce mortality and garner better curative effects. In this regard, dietary supplements capable of preventing carcinogenesis and inhibiting the growth of colon carcinoma cells have generated intense interest. Luteolin (LU), a common dietary flavonoids, has emerged as a powerful anticancer agents able to sensitize different cancer cells, including colon cancer ones, to therapeutic-induced cytotoxicity. However, the molecular mechanisms underlying LU effects in colon cancer are largely unknown. Sphingolipids have critical functions as signaling molecules. In particular, Ceramide (Cer) and Sphingosine-1-phosphate (S1P) are involved as key antagonist mediators in regulating crucial cell responses such as proliferation and apoptosis. Cer can act as a second messenger, and, by activating a variety of signaling pathways, is able to promote growth arrest, apoptosis, or cell differentiation. To the opposite, the sphingoid mediator S1P can act as a potent mitogen and survival factor for a variety of cell types. These two lipids together form the “sphingolipid rheostat” regulating the balance between cell growth and cell death. The objective of our study was to investigate the effects and the molecular mechanisms of LU in colon cancer, focusing on the role of the bioactive sphingoid molecules Cer and S1P. To this purpose, we used the Caco-2 cell line, obtained from a human colon adenocarcinoma, as cell model of both colon cancer cells, and differentiated intestinal enterocytes. Indeed, in long-term culture, these cells undergo a spontaneous differentiation into polarized cells, representing so far the best available in vitro model of absorptive enterocytes. These two models might thus allow to distinguish the potential LU effects on colon cancer cells in comparison to their healthy counterparts. At first, we characterized the morphological and biochemical features of both models. We found that Caco-2 cell differentiation was accompanied by the formation of “domes” across the monolayer, known as characteristic structures of differentiated cells, and indicative of their property of absorptive epithelium. In addition, the activity of the alkaline phosphatase, a membrane-associated hydrolase was found significantly increased in differentiated Caco-2 cells compared to the tumoural cancer ones. Furthermore, we found that Caco-2 differentiation was associated with a reduction of the phospholipids/protein ratio, and whereas phosphatidylcholine was the most abundant phospholipid species of tumoural cells, phosphatidylethanolamine prevailed in the differentiated ones. Moreover, phosphatidylethanolamine plasmalogen, a quantitative relevant species in the cancer cells, exhibited a marked decrease with intestinal differentiation. As far as the sphingolipid composition concerns, we first demonstrated that Caco-2 differentiation was associated to an increase in the total amount of sphingolipids, including Sphingomyelin (SM), the major component and precursor of both Cer and S1P, and above all ceramide. Since the SM hydrolysis, triggered by Sphingomyelinases (SMases), has been implicated in colon tumourigenesis, we then evaluated whether changes in the activity of the known different SMases (the neutral (N-SMase) and alkaline (Alk-SMase) enzymes) occur with cell differentiation. We found that the Alk-SMase activity which was barely detectable in tumoural cancer cells, significantly increased in differentiated ones. The high level of this enzyme is consistent with its presence in the apical brush border, characteristic of intestinal cells. Interestingly a significant increase in the N-SMase was observed in the differentiated Caco-2 cells suggesting a role for this enzyme in intestinal cells. Prompted by these data indicating that the SM hydrolytic potential is enhanced in differentiated Caco-2 cells, we then investigated SM metabolism in both tumoural and differentiated cells. We found that the tumoural cells were much more rapid in the production of Cer from SM than the differentiated ones, and that Cer turnover was present and rapid in both cell types. Inhibition of N-SMase activity, the most abundant enzyme, resulted in no variation of Cer formation in both types of cells, suggesting that the lysosomal acid SMase (A-SMase) is involved in SM degradation. After this characterization, we then evaluated the effect of LU on the tumoural and intestinal cells. Interestingly, we found that the flavonoid exhibited a potent cytotoxic effect on tumoural cells, by inducing apoptosis, without affecting the viability of differentiated cells. Instead, we found that LU induces an increase in intracellular Cer level with a more pronounced trend in tumoural cells. Based on these results we examined whether Cer is involved in the mechanism of LU-induced apoptosis. Notably we obtained that conditions leading to enhance the Cer content in colon cancer cells, as treatment with short-chain Cer analogues and inhibitor of sphingomyelin synthase (SM-synthase) were succeeded by inducing apoptosis in tumoural cells, thus mimicking the LU effect. Furthermore, we evaluated the possible effect of LU on the formation of Cer and S1P in tumoural Caco-2 cells. Pulse experiments showed that treatment with LU induced a dose-dependent increase in Cer, paralleled by a concomitant decrease of both SM and glycosphingolipids synthesis. This result suggested that LU most probably acts by reducing the availability of Cer as a substrate for both SM-synthase and glucosylceramide synthase enzymes localized in the Golgi complex, possibly by inducing a defect in the common vesicular route responsible for complex SLs biosynthetic process. Fluorescent studies using a BODIPY-C5-CER, a ceramide analogue known to mimic the ER-Golgi traffic of natural Cer in living cells, supported the presence of an aberrant traffic of Cer during LU treatment. Furthermore, pulse experiments using treatment with Brefeldin A, known to induce a retrograde merging of Golgi membranes with the ER, demonstrated that LU was not more able to exert alterations of Cer metabolism, thus indicating that the ER-Golgi traffic of Cer is the site of LU action. In parallel, different protein kinases, including Akt have been shown to regulate ER to Golgi traffic. In addition, a study in our laboratory reported for the first time a putative role of PI3K /Akt pathway in the regulation of the vesicle-mediated movements of Cer in the ER-Golgi district. Prompted by these findings, pulse experiments using LY294002, a representative PI3K inhibitor, showed that, LY294002 and subsequently the inhibition of PI3K/Akt had the same effect on the Cer metabolism observed during LU treatment. Furthermore, we obtained that LU strongly inhibited the Akt phosphorylation as a downstream response to PI3K inhibition. Taken together, it emerges that blockade of PI3K by LU and subsequently the downregulation of PI3K/Akt pathway is considered at least one of the mechanisms responsible for the effect of this flavonoid on Cer trafficking observed in our tumoural cell model Further analyses revealed that LU was able to alter not only Cer content but also to decrease the endogenous S1P level inducing thereby a shift of the “sphingolipid balance” to the side of death. Based on this result, we demonstrated for the first time that LU was able to act as inhibitor of Sphingosine kinase (SPHK) activity in tumoural cells, especially SPHKII, the up-regulated isoform in our cancer cell model, exhibiting only a modest effect on SPHKI. Furthermore, in order to gain deeper insights into the role of the “Sphingolipid rheostat” in mediating the effect of LU, we attempted to push the balance towards S1P with addition of exogenous S1P. The results demonstrated that S1P conferred a significant resistance of colon cancer cells to the cytotoxic effect of LU. The mechanism by which S1P acts as LU-antagonist was proved to be mainly by the up-regulation of the PI3K/Akt pathway, which is able to rescue colon cancer cells from the apoptotic effect of the LU-induced increase of Cer. Taken together, our results demonstrate, for the first time, that the dietary natural flavonoid LU induces apoptosis in colon cancer cells by targeting the “sphingolipid rheostat”, and directing the balance in favor of Cer. As the balance between Cer and S1P appears to be an important target for development of new and effective therapeutic strategies against tumour progression, LU could be a novel antitumoural agent not only in colon cancer but possibly in the treatment of other solid tumours.

<p>The aim of this study was to investigate the effect the plant matrix and the structure of the flavonoid (i.e. whether aglycone or glycoside) may have on the gastrointestinal uptake and metabolism of luteolin derivatives from Artemisia afra traditional plant medicine. Specifically, how these two factors influenced the intestinal uptake and disposition of luteolin derivatives in pure and in Artemisia afra plant extract forms were to be assessed by investigating the uptake and metabolism of the luteolin derivatives in human intestinal epithelial Caco-2 cells and the perfused rat intestinal loop. To realize this aim, the following were determined: (1) identification and characterization of major luteolin derivatives found in Artemisia afra, (2) the effect of the plant matrix on the uptake of luteolin derivatives in Artemisia afra aqueous-extract forms across the Caco-2 cell monolayer, (3) the effect of the plant matrix on the absorption and metabolism of luteolin derivatives in Artemisia afra aqueous-extract forms in the perfused rat small intestine, (4) the effect of gut contents on the uptake and metabolism of luteolin derivatives in intestinal loop and (5) the metabolic profiles of luteolin derivatives obtained for the pure solutions versus plant aqueous extract solutions in Caco-2 cells and the rat intestine.</p>

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias. Programa de Pós-Graduação em Agroecossistemas.<br>Made available in DSpace on 2012-10-20T01:10:22Z (GMT). No. of bitstreams: 1 204182.pdf: 1646322 bytes, checksum: 3ffafc98e51d5f30dbbbc5381986485e (MD5)<br>A presente dissertação foi delineada com o objetivo de obter informações sobre características agrostológicas de duas leguminosas sub-tropicais, Vigna luteola e Vigna longifolia, espontâneas nas planícies litorâneas de Santa Catarina para subsidiar programas pastoris sustentáveis. O trabalho foi desenvolvido em quatro etapas distintas: 1) Resgate da descrição botânica e da distribuição geográfica, dessas espécies junto ao Herbário Barbosa Rodrigues, Itajaí, Santa Catarina, a pesquisadores da Universidade Federal do Rio Grande do Sul e na literatura; 2) Levantamento de dados fenológicos obtidos de excursões científicas aos locais de ocorrência, descritos naquele Herbário; 3) Experimentação realizada na Fazenda Experimental da Ressacada, Universidade Federal de Santa Catarina, em Florianópolis, para obter dados de germinação e de sobrevivência de plântulas após sobre-semeadura em pastagem naturalizada e 4) testes de laboratório, para comparar métodos de quebra de dormência de sementes dessas duas espécies. Registros encontrados no Herbário Barbosa Rodrigues informam que o hábito de crescimento da Vigna luteola é volúvel e o da Vigna longifolia é estolonífero. Ambas são plantas perenes. O limite norte da ocorrência espontânea dessas espécies no estado de Santa Catarina é o paralelo 27 Sul. Uma revisão na literatura mostrou que a Vigna luteola expande-se ao sul até a Argentina, paralelo 45 Sul, assim como vegeta espontaneamente em várias regiões do mundo. Não foram encontradas referências sobre a dispersão de Vigna longifolia, com exceção do Uruguai e Argentina onde é tida como nativa. Em excursões realizadas nas regiões de ocorrência apontadas no Herbário, em Santa Catarina, registrou-se que ambas as espécies ocorrem consorciadas, em ambiente caracterizado por solo com alta salinidade e umidade e baixa fertilidade (pobres em Ca e P). Ambas as espécies, nessas condições, apresentavam abundante massa verde, elevada produção de flores e sementes, nodulação apreciável nas raízes, porém a sua presença era muito mais notável em áreas protegidas do pastejo. A grande aceitabilidade pelo gado foi registrada, e por isso especulou-se ser essa a razão da pouca freqüência dessas espécies na pastagem. Amostras de forragem foram coletadas e encaminhadas para o laboratório de Nutrição Animal da Epagri, Lages para avaliar o valor nutritivo da forragem, colhida no mês de novembro de 2001. Os resultados dessas análises revelaram que a digestibilidade da matéria orgânica, o NDT, a Proteína Bruta, o Cálcio e Fósforo eram: 68,2% e 59,2 %; 56,3% e 50,0%; 20,4% e 18,9%; Cálcio 1,51% e 1,41%; e 0,23% e 0,23% na matéria seca, respectivamente para Vigna luteola e Vigna longifolia. Essas observações levaram o autor a testar a possibilidade de ampliar a freqüência dessas espécies através da sobre-semeadura das mesmas em pastagem natural. Este estudo foi desenvolvido na Fazenda Experimental da Ressacada. Dezoito parcelas, cada com 10m 2, foram, ao acaso, demarcadas e sobre-semeadas com ambas as espécies. Trinta dias após, uma medida do número de plantas germinadas foi registrada. Observou-se que apenas a germinação da Vigna luteola tinha ocorrido, mas nenhuma planta de Vigna longifolia havia germinado. Três meses depois, uma avaliação da presença dessas espécies na pastagem foi avaliada através do método BOTANAL. Aproximadamente 20% da matéria seca da pastagem amostrada provinha da Vigna luteola . Novamente, nenhuma planta de Vigna longifolia foi encontrada. Testes em câmara de germinação, confirmaram os dados de campo, indicando necessidade de quebra de dormência das sementes. Por isso, decidiu-se testar essa hipótese através da escarificação das sementes. Lixas, água quente, álcool, soda cáustica ou ácido sulfúrico foram os processos testados. A escarificação resultou igualmente eficiente em promover a germinação da Vigna longifolia e em melhorar a da Vigna luteola, quando comparados com a testemunha. Estes resultados permitem concluir que Vigna luteola e Vigna longifolia são leguminosas promissoras como forrageiras para implantação em pastagens do litoral Sul do Brasil, pela facilidade de adaptação a esse ambiente, alta apetecibilidade, adequado valor nutritivo, e elevada produção de sementes. Embora a presença de sementes duras possa beneficiar a germinação ao longo do ano, a escarificação promove quebra de dormência das sementes e acelera o aparecimento dessas leguminosas na pastagem após sobre-semeadura e a escarificação mecânica aparece como melhor alternativa pelo menor risco à saúde do operador.

The purpose of this investigation was to determine and characterise the anti-oxidant activity of the methanol extract of the leaves of Halleria lucida utilizing the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. The methanol extract of the leaves of H. lucida displayed promising anti-oxidant activity with an IC50 value of 8.49±0.12 μg/ml and was subsequently subjected to activity-guided fractionation resulting in the isolation of a flavone-type flavonoid and phenylpropanoid glycoside, namely luteolin-5-O-β-D-glucoside and verbascoside (acteoside), respectively. Both compounds displayed promising anti-oxidant activities with IC50 values of 6.12±0.40 and 7.18±0.08 μg/ml for luteolin-5-O-β-D-glucoside and verbascoside, respectively. Furthermore, isobologram construction was undertaken to determine pharmacological interactions between the isolated molecules resulting in a concentration-dependent additive and antagonistic interaction being recognised.

Memoria para optar al Título Profesional de Ingeniero Agrónomo Mención Sanidad Vegetal<br>Durante la temporada 2008-2009, se evaluó en laboratorio la toxicidad de cuatro insecticidas de origen botánico; Biomilbe, Biobug, Bugitol y Garlic Barrier, sobre larvas y adultos de la vaquita del olmo, Xanthogaleruca luteola Müller (Coleoptera: Chrysomelidae), colectados desde árboles ubicados en el Parque O`Higgins, Región Metropolitana. Los insecticidas se aplicaron en cuatro dosis distintas cada uno, mediante una torre de Potter, y su acción se evaluó de tres formas, directa, para determinar la toxicidad por contacto, sobre hojas, para medir el efecto residual, y una prueba con una bandeja que contenía por un lado hojas asperjadas con la mezcla insecticida y por el otro hojas sólo asperjadas con agua, para observar cualquier efecto de repelencia. Para los ensayos se utilizó un diseño estadístico completamente aleatorio, con los cuatro insecticidas en el primer y segundo ensayos, más un testigo sin insecticida, y cuatro repeticiones por tratamiento. La unidad experimental consistió en una placa Petri con 20 individuos. La mortalidad se evaluó 24 y 48 horas después de la aspersión sobre hojas, y a los 30 y 60 minutos en el experimento por aplicación directa. Los porcentajes de mortalidad se sometieron a pruebas no paramétricas sin normalización de datos y pruebas de separación de promedios. Para el ensayo en bandejas se comparó el porcentaje de larvas ubicadas en cada sector, tratado y no tratado. Los porcentajes de mortalidad obtenidos fueron muy bajos. El mayor promedio de mortalidad se obtuvo con Bugitol al 4%, con 46,3% y 37,5% de mortalidad en larvas y adultos respectivamente. En la prueba preliminar de repelencia se encontraron efectos marcados en las hojas tratadas con Biomilbe y Biobug, con las larvas concentrándose en mayor porcentaje sobre las hojas asperjadas sólo con agua.<br>The toxicity of four botanical insecticides, Biomilbe, Biobug, Bugitol, and Garlic Barrier, was evaluated in the laboratory onto larvae and adults of the elm leaf beetle Xanthogaleruca luteola Müller (Coleoptera: Chrysomelidae), which were collected in Parque O`Higgins, Santiago, Metropolitan Region of Chile. The insecticides were applied at four concentrations with a Potter spray tower, and their action was evaluated in three ways, directly, to determine contact toxicity, onto leaves, to measure contact effect of residues, and a test using a tray with leaves sprayed with the insecticide on one side, and leaves sprayed only with water in the other, to observe any effect of repellence. For the first two experiments, a completely random statistical design was used, with four insecticide treatments at four concentrations each, and an untreated control, and four replicates per treatment. The experimental unit was a Petri dish with 20 individuals. Mortality was evaluated at 24 and 48 hours after spraying onto leaves, and after 30 and 60 minutes in the experiment of immersion of leaves. Percentages of mortality were subjected to non-parametric tests without standardization of data and range tests to separate means. The percentages of mortality obtained were very low. The greatest mortality was obtained with Bugitol at 4%, with 46,3% y 37,5% of mortality of larvae and adults respectively. The preliminary repellency test show marked effects on the leaves treated with Biomilbe y Biobug, and higher percentage of larvae concentrated on spray leaves only with water.

Memoria para optar al Título Profesional de Ingeniero Forestal<br>Se evaluaron extractos insecticidas de hojas de Schinus molle Rev L. (Anacardiaceae) con solvente agua y etanol para el control de la vaquita del olmo, Xanthogaleruca (=Pyrrhalta) luteola Müller (Coleoptera: Chrysomelidae), en concentraciones de 2,0; 2,5; 3,5; 4,3 y 4,7% p/v para los extractos etanólicos, y 2,5; 3,0; 4,3 y 5,6% p/v para los extractos acuosos, bajo condiciones de laboratorio. Los extractos se aplicaron sobre hojas de olmo (Ulmus sp., Ulmaceae) para la alimentación de individuos adultos de X. luteola, y se determinó posteriormente la efectividad de los extractos de S. molle y su CL50. Los resultados indicaron que ambos extractos insecticidas fueron eficaces sobre X. luteola, al causar mortalidades mayores a 97% con el solvente etanol y las concentraciones más altas (4,3 y 4,7% p/v), y cercanas a 27% con el solvente agua y las concentraciones 4,3 y 5,6% p/v. Mediante el análisis Probit se obtuvo que la CL50 del extracto etanólico fue de 1,88% al 2do d (día) y fue menor que la alcanzada por el extracto acuoso, con una CL50 de 8,52% al 4to d. Adicionalmente, se evaluó el efecto antialimentario de los extractos etanólicos y acuosos de S. molle sobre adultos de X. luteola. Los extractos acuosos inhibieron totalmente la alimentación de X. luteola, mientras que los extractos etanólicos no tuvieron efecto antialimentario.

Memoria para optar al Título Profesional de Ingeniero Forestal<br>Se evaluó la eficacia insecticida y actividad antialimentaria de extractos obtenidos desde frutos verdes de Melia azedarach L. (Meliaceae) sobre larvas de Xanthogaleruca luteola Müller (Coleoptera: Chrysomelidae) para contribuir en el Manejo Integrado de esta plaga. El estudio consistió en cuatro etapas: 1. Colecta de larvas al azar desde árboles ornamentales de Ulmus minor Mill. (Ulmaceae) en Santiago (comuna de Macul), Chile; 2. Muestreo de frutos al azar desde árboles ornamentales de M. azedarach en la Facultad de Ciencias Forestales y de la Conservación de la Naturaleza, Universidad de Chile, Santiago; 3. Preparación de extractos desde frutos verdes en etanol y agua; 4. Evaluación de la mortalidad y efecto antialimentario de las larvas (n = 5) mediante bioensayos de laboratorio, con frutos verdes, agua y etanol (solventes) y seis concentraciones de los extractos, cuatro de ellas las mismas para los dos solventes (1,2; 1,5; 2,4 y 3,0% p/v), 4,4; 5,7% p/v para agua y 4,7; 6,1% p/v para etanol. Para evaluar la eficacia insecticida se hizo un análisis de varianza de un factor y bifactorial, y pruebas de Tukey (ρ≤0,05) al surgir diferencias significativas.

L'intérêt suscité par les colorants naturels nous a conduit à étudier deux plantes tinctoriales, la gaude (Reseda luteola) et la garance (rubia tinctorum). Les méthodes d'extraction des colorants présents dans ces plantes ont été mises au point en optimisant en particulier le solvant, la durée et la température d'extraction. La CLHP a permis l'identification et la quantification de ces molécules dans les différents extraits obtenus. L'application de ces molécules colorantes pour la teinture de deux fibres naturelles, la soie et le coton, a été étudiée. L'influence de l'alun et du bitartrate de potassium (pour la soie) ou du carbonate de sodium (pour le coton) sur les teintes obtenues a été évaluée par la méthode des plans d'expérimentation. Cette étude a permis de déterminer les quantités minimales de ces composés permettant l'obtention d'échantillons textiles présentant une teinte optimale. L'évaluation de ces quantités permet notamment de minimiser les teneurs en sels métalliques incorporées lors de l'étape de mordançage, et à fortiori de réduire les risques de pollution environnementale. La localisation des colorants, déterminée par microscopies optique et électronique à balayage, est apparue différente selon la nature de la fibre sur laquelle ils sont appliqués. Sur les fibres de soie, les colorants sont principalement localisés à l'intérieur des fibres, comme pour la majorité des teintures à l'aide de colorants synthétiques. En revanche, les colorants sont présents sur la surface externe des fibres cellulosiques, adsorbés par l'intermédiaire de complexes d'aluminium. L'étude de la solidité lumière des colorations des fibres de soie et de coton teintes à partir de ces colorants a révélé une meilleure résistance à la lumière des fibres de soie, ainsi qu'une meilleure résistance pour les molécules colorantes de la garance. Un traitement post-teinture à l'aide de différents absorbeurs UV et/ou antioxydants des fibres teintées a permis d'augmenter la solidité lumière des échantillons teints à partir de la gaude. Dans l'optique d'un développement Agro-Industriel, la coloration de peintures et autres produits par les extraits tinctoriaux de gaude et de garance a également été abordée<br>An increasing interest in natural dyes lead us to sudy two dye plants, the weld (Reseda luteola) and the madder (Rubia tinctorum). Extraction optimisation of these vegetables dyes was achieved by comparing several solvents, extraction times and extraction temperatures. Qualitative and quantitative analysis of the natural dyes were performed by HPLC. Dyeing properties of these compounds were studied on two natural textile fibres, the silk and the cotton. The effect of alun and potassium bitartrate (for silk) or sodium carbonate (for cotton) on the fibres coloration was studied by experimental designs. This study allowed us to determine minimal necessary quantities of these additives in order to obtain the best coloration and to minimize environmental pollution by metallic salts. Dyes localisation, analysed by microscopy, appeared to be different depending on the kind of the fibre. On silk fibres, weld and madder dyes are mainly present inside fibres while in cotton dyes are adsorbed on the external "area" of fiber through aluminium complexes. Such an external localisation had never been described before even with synthetic dyes. The light fastness of dyed fabrics was better for silk fibres than for cotton fibres and for madder dyes than for weld dyes. Treatment with UV absorbers and/or antioxidants improved the light fastness of fibres dyed with weld dyes. In order to extend dyes applications, coloration of paints and other products with dyes extracts of weld and madder was studied too

Orientador: Claudia Regina Baptista Haddad<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-08-05T00:53:28Z (GMT). No. of bitstreams: 1 Ferreira_MariaCristinadaCosta_M.pdf: 561166 bytes, checksum: 57a597ae699902d2f7063c9a77a30325 (MD5) Previous issue date: 2005<br>Resumo: O excesso de sais no solo afetao metabolismo geral da planta, causando alterações fisiológicas e morfológicas. Visando avaliar os efeitos do sal no crescimento e metabolismo sob estresse salino, foram comparadas as respostas de Vigna luteola (halófita) e Vigna unguiculata (sensível). As plantas foram cultivadas na presença de NaCl nas concentrações de: 100mM, 250mM e 500mM. V. unguiculata apresentou reduções significativas de massas fresca e seca, área foliar e número de folhas sob salinidade. Estes parâmetros não foram afetados em V. luteola. Há dados na literatura que mostram que plantas adaptadas à salinidade apresentam a razão raiz/parte aérea maior que plantas sensíveis. Contudo, este aumento foi evidenciado na espécie mais sensível ao sal, sob salinidade, mantendo-se constante em V. luteola. O aumento de suculência, que é considerado uma adaptação ao estresse salino, não foi verificado em nenhuma das espécies estudadas. A resistência ao estresse salino tem sido relacionada com a atividade de enzimas antioxidantes, que removem espécies ativas de oxigênio. O aumento da atividade de peroxidases totais foi observado apenas em V. luteola, associado ao estresse salino. Sob estresse salino, ocorreu o aumento da atividade de siringaldazina oxidase somente em V. unguiculata. Não foi observada relação entre crescimento de raízes e atividade desta enzima, pois nesta espécie a salinidade provocou maior crescimento da raiz. A atividade de peroxidases totais em Vigna luteola está localizada na epiderme, córtex e cilindro central e, em V. unguiculata na epiderme e cilindro central. Sob estresse salino, a atividade foi mais intensa nas duas espécies. Em V. luteola foi observada em toda raiz, em V. unguiculata, além da epiderme e do cilindro central, foi possível observa-la em porções do córtex. A respeito da localização da enzima siringaldazina oxidase, em V. unguiculata restringiu-se à epiderme, expandindo - se para o córtex e cilindro central sob estresse salino. Já em V. luteola estava presente em todas as regiões da raiz, intensificando - se com a aplicação de NaCl no meio de cultivo. A alteração da permeabilidade das membranas é um dos resultados da salinidade, que está relacionado ao aumento do teor de espécies ativas de oxigênio. Foi observado aumento de liberação de eletrólitos em ambas as espécies de Vigna. Entretanto, em V. unguiculata, o acréscimo neste parâmetro foi altamente significativo na presença de NaCl e com resultados sempre maiores aos mesmos tratamentos de V. luteola. A manutenção da razão Na+/K+ nas células é um fator relevante para a tolerância à salinidade. O acúmulo de Na+ foi observado em raízes e folhas das duas espécies de Vigna. Em raízes, obteve-se valores maiores em V. luteola, em decorrência a alta concentração de Na+ neste órgão. Em folhas, a maior razão Na+/K+ foi obtida em V. unguiculata. As análises de parâmetros bioquímicos sob estresse salino, indicaram que não houve variação no teor de proteínas (folhas), sacarose (folhas) e açúcares solúveis totais (raiz e folhas) em Vigna luteola. Sob salinidade, o teor de proteínas foi reduzido em folhas de V. unguiculata. Já as concentrações de açúcares solúveis totais (raízes e folhas), sacarose (folhas) e malondialdeído (raiz) ficaram inalterados. Mediante salinidade, não ocorreram alterações significativas nos parâmetros bioquímicos em V. luteola, o que permitiu seu crescimento e desenvolvimento normal. As mudanças de indicadores bioquímicos e ausência de resposta antioxidante em V. unguiculata sob salinidade, podem estar relacionadas com o comprometimento do seu crescimento sob estresse salino<br>Abstract: The excess of salt in the soil affects all the general metabolism of the plant causing physiologic and morphologic alterations. With the objective of evaluating the effects of salt in the growth and metabolism under saline stress, this study compares the responses of two Vigna species, with distinct sensitivity to salt, cultivated with the presence of NaCl in the following concentrations: 100mM, 250mM e 500mM. V. unguiculata presented significant reductions in fresh and dry mass, leaf area and number of leaves when subjected to salinity. However, these parameters were not affected in V. luteola. There is data in the literature which shows that plants that have adapted to salinity present the root/shoot ratio higher than sensitive plants. On the other hand, this increase was evidenced in the species which is most sensitive to salt, under salinity, keeping itself constant in V. luteola. The increase in juiciness, which is considered an adaptation to saline stress, was not present in either of the species studied. The resistance to saline stress has been associated to the activity of antioxidant enzymes, which remove reactive oxygen species. The increase in activity of total peroxidases was observed only in V. luteola. Under saline stress, only V. unguiculata presented an increase in siringaldazine oxidase activity. There was no evidence of relation between the growth of roots and the presence of this enzyme, since this species presented larger root growth due to salinity. The histochemical location of total peroxidases activity indicated that the distribution of the activity of these enzymes is similar in both species of Vigna (epidermis, cortex and central cylinder), however, there is stronger activity in V. luteola, especially under stress. In relation to the location of the seringaldazine oxidase enzyme, in V. unguiculatat was restricted to the epidermis, expanding to the cortex and central cylinder under saline stress. In V. luteola it was present in all parts of the root, intensifying when NaCl was applied during cultivation. The change in the membrane permeability is one of the results of salinity which is connected to the increase of reactive oxygen species concentration. An increase in eletrolites release was noticed in both Vigna species. Meanwhile, in V. unguiculata, the rise in this parameter was highly significative in the presence of NaCl and with results constantly higher than the results of the same treatments in V. luteola. The maintenance of the Na+/K+ ratio in the cells is a relevant factor to the tolerance of salinity. Na+ accumulation was observed in both Vigna species¿ roots and leaves. In roots, higher values were obtained in V. luteola, due to the higher Na+ concentration in this organ. In leaves, the highest Na+/K+ ratio change was obtained in V. unguiculata. The analysis of biochemical parameters under saline stress, show that there was no change in protein content (leaves), sucrose (leaves) and total soluble sugars (root and leaves) in Vigna luteola. Protein content was reduced in V. unguiculata leaves, while concentrations of total soluble sugars (root and leaves), sucrose (leaves) e malondialdeid (root) remained the same. Under salinity, there were no significant changes in the biochemical parameters of V. luteola, which allowed for its normal growth and development. The changes in biochemical indicators and lack of antioxidant response in V. unguiculata under salinity may be connected to its growth and development compromise under saline stress<br>Mestrado<br>Mestre em Biologia Vegetal

Activation of the maternal immune system and resultant maternal cytokine expression due to prenatal infection has been implicated as a significant contributor to the pathology of neuropsychiatric and neurodevelopmental disorders such as schizophrenia and Autism Spectrum Disorder (ASD). Increased maternal interleukin-6 (IL-6) expression, observed clinically and in animal models of prenatal infection, and resultant activation of key signaling pathways, has been shown to be a biological indicator of pathology, and a central component of the pathological mechanism. In animal models of prenatal infection and clinically in pregnancy disorders hallmarked by immunological irregularities and increased IL-6 expression, inhibition of IL-6 has been shown to reduce pathological symptoms both maternally and in the exposed offspring. This study aims to demonstrate the ability of IL-6 expression, resulting from prenatal infection, to induce neuropathological and behavioral outcomes that mirror clinical observations seen in disorders such as ASD. More importantly, it shows how flavones luteolin and diosmin, a subclass of the flavonoid family, through inhibition of IL-6 mediated activation of Signal Transducer and Activator of Transcription-3 (Stat3) can reduce these pathologies both in vitro and in vivo. Evidence suggests that flavonoids, a polyphenolic class of naturally occurring plant secondary metabolites, are potent anti-inflammatory agents that can attenuate the expression of cytokines such as IL-6, possibly through the modulation of tyrosine kinase activity. They have been shown to have significant therapeutic potential in disorders hallmarked by increased inflammation or disruptions in immune regulation, such as neurodegenerative disorders and certain cancers. Members such as diosmin have also been shown to be safe during pregnancy, and are currently utilized in the treatment of certain vascular disorders associated with pregnancy. In vitro work undertaken in this study showed that co-administration of luteolin with IL-6 in neural stem cells (NSC) was able to attenuate pathological outcomes induced by IL-6 including aberrant proliferation, over expression of astroglial marker, glial fibrillary acidic protein (GFAP) and changes in cellular morphology. In vivo studies involving luteolin and diosmin further confirmed the therapeutic efficacy of these compounds as similar attenuation of IL-6 mediated maternal and fetal pro-inflammatory cytokine expression and abnormal behaviors in prenatally exposed offspring was observed. Mechanistically, these effects were mediated through inhibition of Stat3 activation although other pathways activated by IL-6 were modulated by flavone co-treatment. Flavonoid treatment during periods of prenatal infection may prove to be a therapeutic intervention for the resultant pathological outcomes seen in offspring through attenuation of the maternal and fetal immune response to infection as well as modulation of signaling pathways in the fetal brain. These compounds may prove therapeutically efficacious for the application in perinatal conditions hallmarked by increased inflammation during pregnancy.

Memoria para optar al Título Profesional de Ingeniero Forestal<br>Se evaluó el efecto insecticida de extractos elaborados con harina de hojas de boldo (Peumus boldus Molina, Monimiaceae) y dos solventes (agua y etanol), en varias concentraciones para el control de Xanthogaleruca luteola (Pyrrhalta) Müller (Coleoptera: Chrysomelidae) en laboratorio. Se compararon harinas obtenidas de follaje juvenil y maduro de boldo con ambos solventes. Se determinó el porcentaje de mortalidad de los insectos, la concentración letal 50% (CL50) y la duración de los estados de pupa y adulto. Además se caracterizaron física y químicamente las hojas juveniles y maduras mediante análisis proximal. El diseño experimental fue completamente al azar con análisis de efectos fijos para cada uno de los solventes; para la comparación de los estados de madurez se utilizó un diseño bifactorial. Los extractos fueron eficaces y causaron una mortalidad promedio en adultos de X. luteola superior al 75% con hojas juveniles y en las concentraciones mayores, con diferencias significativas en relación a las dosis más bajas. La CL50 menor fue de 1% y se obtuvo con etanol como solvente a los 2 días. Los parámetros del análisis proximal, rendimiento, humedad, cenizas y fibra cruda de las hojas arrojaron diferencias significativas entre estados de madurez, no así en el porcentaje de lípidos. El desarrollo de pupas y adultos duró 7 y 21 días, respectivamente.

Ces travaux portent principalement sur l’étude de trois plantes tinctoriales : la garance, le nerprun et la gaude. Ces végétaux ont fait l’objet de nombreuses cultures en Provence et constituaient la principale matière première en colorants rouges et jaunes pour les teinturiers et les artistes. Une optimisation des conditions d’extraction des colorants de la garance assistée par ultrasons a été effectuée en utilisant un modèle statistique. Ce procédé d’extraction simple, rapide et efficace, a été comparé à deux autres techniques utilisées conventionnellement. Une étude cytohistologique des racines de garance a permis d’examiner les effets apportés par les différents procédés d’extraction. Les cellules traduisent après extraction par ultrasons, de profondes déstructurations fournissant une explication au plus important rendement en colorant extraits en comparaison aux extractions classiques. Une étude fondamentale sur l’identification des colorants extraits à partir des fruits immatures d’espèces appartenant au genre Rhamnus a été effectuée. Une approche chromatographique utilisant la CLHP/UVVisible/ SM a permis d’identifier la partie flavonol. Elle présente principalement des composés glycosylés dont la partie rhamninoside est liée sur le flavonol en position 3 ou 4'. Des flavonols 3-O-acétyl-rhamninoside ont également été caractérisés et sont spécifiques de Rh. saxatilis. Les fruits matures renferment aussi des anthraquinones qui ont été séparées des flavonols et concentrées en utilisant l’Extraction sur Phase Solide (SPE). Après analyse par RMN, des dérivés rhamnoside et arabinoside acétylés de l’émodine, jamais décrits dans la littérature, ont été identifiés dont le 6-O-(3',4' diacétyl)-arabinopyranoside d’émodine et 6-O-(2',3',4'-triacétyl)- arabinopyranoside d’émodine présents seulement dans Rh. alaternus. Les colorants jaunes de la gaude (Reseda luteola) ont été analysés par électrophorèse capillaire. En comparaison avec la CLHP, un gain important de la durée d’analyse a été observé tout en conservant une séparation convenable. L’ensemble de ces résultats expérimentaux a pu être appliqué avec succès à l’étude de colorants extraits à partir d’objets et d’échantillons historiques provenant de collections muséales et comprenant notamment des indiennes du XIXème siècle. Enfin, des essais de teintures ont été réalisés, en collaboration avec la société Les Olivades dans le but de développer une gamme de tissus à base de colorants naturels<br>This work concerns the study of three tinctorial plants: madder, buckthorn and weld. These plant species produced many cultures in Provence and represented the principal raw material in red and yellow dyes for dyers and artists. An optimisation of extraction conditions for madder dyes, using ultrasounds, was carried out with a statistical model. This easy, fast and effective extraction process was compared with two other conventional techniques. A cytohistological study on madder roots permits to examine effects produced by the different extraction processes. Cells reveal, after ultrasonic extraction, profound structural alterations, explaining the high yield in extracted dyes in comparison with classical methods. A fundamental study on the dyes identification extracted from Rhamnus species green fruits was carried out. A chromatographic approach using HPLC-UV-MS permits to identify the flavonol fraction. It is mainly composed of glycosiled compounds where the rhamninosid part is linked in position 3 or 4’ on the flavonol nucleus. 3-O-acetyl-rhamninosid derivatives were also characterised and they are specific to Rh. saxatilis species. Ripe fruits contained anthraquinonic compounds that were separated from flavonols and concentrated using Solid Phase Extraction (SPE). After NMR analyse, acetyl rhamnosid and arabinosid derivatives of émodine, never described in the specialised literature, were identified as emodin-6-O-(3',4'-diacetyl)-arabinopyranosid and emodin-6-O-(2',3',4' triacetyl)-arabinopyranosid were only present in Rh. alaternus. Yellow dyes of weld (Reseda luteola) were analysed by capillary electrophoresis. In comparison with HPLC, a reduced run time was observed while preserving a suitable separation. These experimental results were successfully applied to the study of ancient samples belonging from museums and including “indiennes” of the XIXth century. Finally, dying tests were carried out, in collaboration with Les Olivades society, in the aim to develop textiles containing natural dyes

La bactérie psychrotrophe P. Fluorescens MF0 est plus sensible aux antibiotiques de type β-lactamines à une température de croissance de 8°C qu'à une température de 28°C. Cette différence est parfaitement corrélée avec les valeurs de conductance obtenues pour les porines majoritaires de la membrane externe OprF à 8°C et 28°C puisqu'elles sont respectivement de 80 pS et 250 pS dans du NaCl 1M. Pour expliquer ce phénomène, les OprFs 8°C et 28°C ont été purifiées et les études de spectrométrie de masse et de dichroïsme circulaire ne montrent pas de différence dans leur structure primaire. Des mesures de cinétiques de digestion à la pronase ont conduit à une différence des valeurs de constantes de vitesse de dégradation impliquant ainsi une modification de la conformation protéique en fonction de la température de croissance. Les fragments protéolytiques de 18 kDa, résistant à l'action de la pronase, ont été purifiés et identifiés comme la partie N-terminale de la protéine native (177 acides aminés). Les deux fragments forment des canaux ioniques avec des valeurs de conductances similaires (65 et 75 pS dans NaCl 1M) impliquant que la région C-terminale est seule responsable de la modulation des canaux formes par l'OprF en fonction de la température de culture. La caractérisation du LPS associé aux OprFs met en évidence une différence de phosphorylation des LPS obtenus à partir de culture à 28°C et 8°C. Ainsi, l'association LPS-porine serait différente selon les températures de culture. Nous avons ensuite voulu rechercher une éventuelle généralisation de ce phénomène et nous avons choisi la bactérie psychrotrophe Chryseomonas luteola MCF10. La protéine majoritaire de la membrane externe (31 kDa) a été purifiée à 8°C et 28°C et son étude fonctionnelle montre des valeurs de conductances unitaires similaires quelle que soit la température de croissance. Cependant, la détermination de la séquence N-terminale ainsi que la séquence déduite du gène la caractérise comme une OmpA. L'utilisation des anticorps montre que C. Luteola est plus proche des entérobactéries que des pseudomonas. Ce résultat montre que le mécanisme de résistance décrit chez P. Fluorescens n'est pas systématiquement applicable à toute bactérie psychrotrophe.

[EN] The main goal of this Thesis was to determine the influence of the main processing stages involved in obtaining natural extracts with high antioxidant potential from byproducts originating in the olive oil industry. Firstly, the effect of freezing and/or the drying methods applied to olive oil byproducts on the polyphenol content and antioxidant capacity of the extracts subsequently obtained was addressed. For this purpose, two byproducts were considered: olive leaves and olive pomace. Secondly, the feasibility of intensifying the extraction of olive leaf polyphenols by means of a new technology, such as power ultrasound, was approached taking both compositional and kinetic issues into account. Thirdly, how the processing conditions (drying and extraction) influence the extract's stability was evaluated. Thus, on the one hand, extracts obtained from olive leaves were subjected to in vitro digestion or dehydrated and stored at different conditions. Finally, the possibility of obtaining a dried vegetable matrix (apple) rich in olive leaf phenolic compounds was explored by addressing the influence of apple pretreatments (blanching and freezing) and drying on the final retention of infused phenolics. The antioxidant potential of extracts and the retention of infused polyphenols in apple were evaluated by means of the total phenolic content and antioxidant capacity analysis, as well as the identification and quantification of the main olive leaf polyphenols by HPLC-DAD/MS-MS. Moreover, in apple samples, the polyphenol oxidase and peroxidase activity and microstructure were also analyzed. The experimental results highlighted that both drying and freezing methods significantly (p<0.05) influenced the concentration of the main polyphenols identified in the olive leaf extracts. Thus, drying at the highest temperature tested was the best processing condition in which to obtain extracts with high antioxidant capacity and phenolic content. Ultrasound application was found to be a relevant, non-thermal way of speeding-up the antioxidant extraction from olive leaves. Thus, by appropriately tuning-up the process variables, the ultrasonic assisted extraction shortened the extraction time from the 24 h needed in conventional extraction to 15 min, without modifying either the extract composition or the antioxidant potential. As far as extract stability is concerned, the processing conditions used for obtaining the olive leaf extracts did not have a meaningful influence on bioaccessibility. Regardless of the method used, stabilizing the extracts by means of dehydration only reduced both the antioxidant capacity and the total phenolic content by around 10 %. Moreover, storage conditions did not show a significant (p<0.05) effect on the antioxidant potential of the extracts for 28 days of storage. A stable dried product (apple), rich in natural phenolic compounds (from olive leaves or tea extracts), was obtained by combining drying-impregnation-drying steps. However, it should be considered that the role of fresh apple drying on the retention of infused olive leaf polyphenols was more important than the further drying of the impregnated apple. In overall terms, olive leaves can be considered a potential source of natural phenolic compounds. Notwithstanding this, the previous drying and freezing steps applied in the raw material processing are decisive factors in the obtaining of natural extracts with high antioxidant potential. Moreover, enhancing the extraction by applying power ultrasound was stated as a non-thermal way of shortening processing times. The stability of olive polyphenols during storage and in vitro digestion was closely related to the individual component considered. Finally, the exploitation of olive leaf extracts as a means of enriching solid foodstuffs requires the use of porous solid matrices free of oxidative enzymes.<br>[ES] El objetivo principal de esta Tesis fue determinar la influencia de las principales etapas de procesado implicadas en la obtención de extractos naturales con alto potencial antioxidante a partir de los subproductos originados en la industria del aceite de oliva. En primer lugar, se evaluó el efecto de los métodos de congelación y/o secado de la materia prima (hojas y orujo), sobre el contenido polifénolico y la capacidad antioxidante de los extractos. En segundo lugar, se abordó la intensificación de la extracción de polifenoles de hoja de olivo con ultrasonidos de potencia, teniendo en cuenta: composición y la cinética del proceso. A continuación, se estudió cómo las condiciones de procesado (secado y extracción) podían influir en la estabilidad de los extractos. Así, extractos de hojas de olivo fueron sometidos a digestión in vitro o deshidratados y almacenados a distintas condiciones. Por último, se exploró la posibilidad de obtener una matriz vegetal deshidratada (manzana) y rica en compuestos fenólicos de hoja de olivo. Para ello, se evaluó la influencia de los pretratamientos de la manzana (escaldado y congelación) y del secado en la retención final de los polifenoles impregnados. El potencial antioxidante se determinó a través del contenido total en compuestos fenólicos y la capacidad antioxidante y la identificación y cuantificación (HPLC-DAD/MS-MS) de los principales polifenoles. Además, en manzana, se midió la actividad enzimática de la polifenol oxidasa y peroxidasa y se analizó la microestructura. Los resultados manifestaron que el método de secado y el de congelación influyeron significativamente (p<0.05) en la concentración de los principales polifenoles en los extractos. Así, el secado a mayor temperatura resultó ser el mejor tratamiento para obtener extractos con alta capacidad antioxidante y alto contenido fenólico. La aplicación de ultrasonidos resultó ser una alternativa no térmica muy interesante para acelerar la extracción de antioxidantes de hojas de olivo. Con la combinación adecuada de las variables del proceso, la aplicación de ultrasonidos redujo el tiempo de extracción de 24 h necesarias en extracción convencional a 15 min, sin modificar la composición de los extractos y su potencial antioxidante. En cuanto a la estabilidad del extracto, las condiciones de procesado no tuvieron una influencia significativa en la bioaccesibilidad de los extractos. Independientemente del método utilizado, la estabilización de extractos por deshidratación sólo redujo la capacidad antioxidante y el contenido total en compuestos fenólicos en torno a un 10 %. Además, las condiciones de almacenamiento no mostraron ningún efecto significativo (p<0.05) sobre el potencial antioxidante durante los 28 días de almacenamiento. Combinando secado-impregnación-secado, fue posible desarrollar un producto deshidratado (manzana), estable y rico en compuestos fenólicos naturales (de hojas de olivo o extractos de té). No obstante, cabe destacar que el secado de la manzana fresca jugó un papel más importante en la retención de los polifenoles de hoja de olivo infundidos que el secado final de la manzana impregnada. En términos generales, las hojas de olivo pueden considerarse como una fuente potencial de compuestos fenólicos naturales. No obstante, el secado y la congelación durante el procesado de la materia prima son factores decisivos para la obtención de extractos naturales con alto potencial antioxidante. Además, la aplicación de ultrasonidos de potencia durante la extracción puede resultar una alternativa no térmica muy interesante de cara a acortar el tiempo de procesado. La estabilidad de los polifenoles de la hoja de olivo, durante el almacenamiento y la digestión in vitro, dependió claramente del compuesto individual considerado. Finalmente, el empleo del extracto de hoja de olivo como medio para enriquecer alimentos sólidos requiere del uso de matrices s<br>[CAT] L'objectiu principal d'aquesta tesi va ser determinar la influència de les principals etapes de processament implicades en l'obtenció d'extractes naturals amb alt potencial antioxidant procedents de subproductes de la indústria de l'oli d'oliva. En primer lloc, es va estudiar l'efecte de la congelació i/o els mètodes d'assecatge aplicats a fulles d'olivera i pinyolada sobre el contingut fenòlic i la capacitat antioxidant dels extractes. En segon lloc, es va avaluar, tenint en compte la composició i la cinètica del procés, la intensificació de l'extracció de polifenols de fulla d'olivera amb ultrasons de potència. En tercer lloc, es va avaluar com les condicions de processament (assecatge i extracció) poden influir en l'estabilitat dels extractes. Així, extractes de fulles d'olivera van ser sotmesos a una digestió in vitro o deshidratats i emmagatzemats a distintes condicions. Finalment, es va explorar la obtenció d'una matriu vegetal deshidratada (poma) i rica en compostos fenòlics de fulla d'olivera considerant la influència del pretractament de la poma (escaldament i congelació) i de l'assecatge sobre la retenció final dels fenòlics introduïts en la poma. El potencial antioxidant es va avaluar determinant el contingut fenòlic total i la capacitat antioxidant, així com identificant i quantificant els principals polifenols (HPLC-DAD/MS-MS). A més, en poma l'activitat enzimàtica de la polifenoloxidasa i la peroxidasa i la microestructura. Els resultats experimentals van destacar que el mètode d'assecatge i el de congelació van influir significativament (p<0,05) en la concentració dels principals polifenols identificats en els extractes. L'assecatge a la temperatura més alta que es va provar va resultar la millor condició de processament per a obtenir extractes amb una alta capacitat antioxidant i un alt contingut fenòlic. L'aplicació d'ultrasons va ser una manera rellevant i no tèrmica d'accelerar l'extracció d'antioxidants de les fulles d'olivera. Així, amb la combinació adequada de les variables del procés, l'extracció assistida per ultrasons va escurçar el temps d'extracció, de les 24 h requerides en l'extracció convencional a 15 min, sense modificar la composició de l'extracte ni el potencial antioxidant. Quant a l'estabilitat de l'extracte, les condicions de processament utilitzades per a l'obtenció dels extractes de fulla d'olivera no van tenir una influència significativa en la bioaccessibilitat. Independentment del mètode utilitzat, l'estabilització dels extractes per mitjà de la deshidratació només va reduir la capacitat antioxidant i el contingut fenòlic total al voltant d'un 10 %. A més, les condicions d'emmagatzematge (temperatura i forma de l'extracte: líquid o pols) no van mostrar cap efecte significatiu (p<0,05) en el potencial antioxidant dels extractes durant els 28 dies d'emmagatzematge. Combinant etapes d'assecatge-impregnació-assecatge fou possible obtenir un producte assecat estable (poma) i ric en compostos fenòlics naturals (de fulles d'olivera o te). No obstant això, cal destacar que l'assecatge de la poma fresca va ser més important i determinant en la retenció dels polifenols de fulla d'olivera que no l'assecatge de la poma impregnada. En termes generals, les fulles d'olivera es poden considerar com una font potencial de compostos fenòlics naturals. No obstant això, l'aplicació d'assecatge i congelació durant el processament de la matèria primera són factors decisius per a l'obtenció d'extractes naturals amb un alt potencial antioxidant. A més, l'aplicació d'ultrasons de potència durant l'extracció resultà ser una forma no tèrmica de millorar el procés, tot reduint-ne el temps d'extracció. L'estabilitat dels polifenols d'olivera durant l'emmagatzematge i la digestió in vitro va dependre del compost individual considerat. Finalment, la utilització d'extractes de fulla d'olivera per a desenvolupar aliments sòlids enriquits requ<br>Ahmad-Qasem Mateo, MH. (2015). ASSESSMENT OF THE INFLUENCE OF PROCESSING CONDITIONS ON THE ANTIOXIDANT POTENTIAL OF EXTRACTS OBTAINED FROM OLIVE OIL INDUSTRY BYPRODUCTS [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/53452<br>TESIS<br>Premiado

Ces travaux portent principalement sur l'étude de trois plantes tinctoriales : la garance, le nerprun et la gaude. Ces végétaux ont fait l'objet de nombreuses cultures en Provence et constituaient la principale matière première en colorants rouges et jaunes pour les teinturiers et les artistes. Une optimisation des conditions d'extraction des colorants de la garance assistée par ultrasons a été effectuée en utilisant un modèle statistique. Ce procédé d'extraction simple, rapide et efficace, a été comparé à deux autres techniques utilisées conventionnellement. Une étude cytohistologique des racines de garance a permis d'examiner les effets apportés par les différents procédés d'extraction. Les cellules traduisent après extraction par ultrasons, de profondes déstructurations fournissant une explication au plus important rendement en colorant extraits en comparaison aux extractions classiques. Une étude fondamentale sur l'identification des colorants extraits à partir des fruits immatures d'espèces appartenant au genre Rhamnus a été effectuée. Une approche chromatographique utilisant la CLHP/UVVisible/ SM a permis d'identifier la partie flavonol. Elle présente principalement des composés glycosylés dont la partie rhamninoside est liée sur le flavonol en position 3 ou 4'. Des flavonols 3-O-acétyl-rhamninoside ont également été caractérisés et sont spécifiques de Rh. saxatilis. Les fruits matures renferment aussi des anthraquinones qui ont été séparées des flavonols et concentrées en utilisant l'Extraction sur Phase Solide (SPE). Après analyse par RMN, des dérivés rhamnoside et arabinoside acétylés de l'émodine, jamais décrits dans la littérature, ont été identifiés dont le 6-O-(3',4' diacétyl)-arabinopyranoside d'émodine et 6-O-(2',3',4'-triacétyl)- arabinopyranoside d'émodine présents seulement dans Rh. alaternus. Les colorants jaunes de la gaude (Reseda luteola) ont été analysés par électrophorèse capillaire. En comparaison avec la CLHP, un gain important de la durée d'analyse a été observé tout en conservant une séparation convenable. L'ensemble de ces résultats expérimentaux a pu être appliqué avec succès à l'étude de colorants extraits à partir d'objets et d'échantillons historiques provenant de collections muséales et comprenant notamment des indiennes du XIXème siècle. Enfin, des essais de teintures ont été réalisés, en collaboration avec la société Les Olivades dans le but de développer une gamme de tissus à base de colorants naturels

Ces travaux portent principalement sur l’étude de trois plantes tinctoriales : la garance, le nerprun et la gaude. Ces végétaux ont fait l’objet de nombreuses cultures en Provence et constituaient la principale matière première en colorants rouges et jaunes pour les teinturiers et les artistes. Une optimisation des conditions d’extraction des colorants de la garance assistée par ultrasons a été effectuée en utilisant un modèle statistique. Ce procédé d’extraction simple, rapide et efficace, a été comparé à deux autres techniques utilisées conventionnellement. Une étude cytohistologique des racines de garance a permis d’examiner les effets apportés par les différents procédés d’extraction. Les cellules traduisent après extraction par ultrasons, de profondes déstructurations fournissant une explication au plus important rendement en colorant extraits en comparaison aux extractions classiques. Une étude fondamentale sur l’identification des colorants extraits à partir des fruits immatures d’espèces appartenant au genre Rhamnus a été effectuée. Une approche chromatographique utilisant la CLHP/UVVisible/ SM a permis d’identifier la partie flavonol. Elle présente principalement des composés glycosylés dont la partie rhamninoside est liée sur le flavonol en position 3 ou 4'. Des flavonols 3-O-acétyl-rhamninoside ont également été caractérisés et sont spécifiques de Rh. saxatilis. Les fruits matures renferment aussi des anthraquinones qui ont été séparées des flavonols et concentrées en utilisant l’Extraction sur Phase Solide (SPE). Après analyse par RMN, des dérivés rhamnoside et arabinoside acétylés de l’émodine, jamais décrits dans la littérature, ont été identifiés dont le 6-O-(3',4' diacétyl)-arabinopyranoside d’émodine et 6-O-(2',3',4'-triacétyl)- arabinopyranoside d’émodine présents seulement dans Rh. alaternus. Les colorants jaunes de la gaude (Reseda luteola) ont été analysés par électrophorèse capillaire. En comparaison avec la CLHP, un gain important de la durée d’analyse a été observé tout en conservant une séparation convenable. L’ensemble de ces résultats expérimentaux a pu être appliqué avec succès à l’étude de colorants extraits à partir d’objets et d’échantillons historiques provenant de collections muséales et comprenant notamment des indiennes du XIXème siècle. Enfin, des essais de teintures ont été réalisés, en collaboration avec la société Les Olivades dans le but de développer une gamme de tissus à base de colorants naturels<br>This work concerns the study of three tinctorial plants: madder, buckthorn and weld. These plant species produced many cultures in Provence and represented the principal raw material in red and yellow dyes for dyers and artists. An optimisation of extraction conditions for madder dyes, using ultrasounds, was carried out with a statistical model. This easy, fast and effective extraction process was compared with two other conventional techniques. A cytohistological study on madder roots permits to examine effects produced by the different extraction processes. Cells reveal, after ultrasonic extraction, profound structural alterations, explaining the high yield in extracted dyes in comparison with classical methods. A fundamental study on the dyes identification extracted from Rhamnus species green fruits was carried out. A chromatographic approach using HPLC-UV-MS permits to identify the flavonol fraction. It is mainly composed of glycosiled compounds where the rhamninosid part is linked in position 3 or 4’ on the flavonol nucleus. 3-O-acetyl-rhamninosid derivatives were also characterised and they are specific to Rh. saxatilis species. Ripe fruits contained anthraquinonic compounds that were separated from flavonols and concentrated using Solid Phase Extraction (SPE). After NMR analyse, acetyl rhamnosid and arabinosid derivatives of émodine, never described in the specialised literature, were identified as emodin-6-O-(3',4'-diacetyl)-arabinopyranosid and emodin-6-O-(2',3',4' triacetyl)-arabinopyranosid were only present in Rh. alaternus. Yellow dyes of weld (Reseda luteola) were analysed by capillary electrophoresis. In comparison with HPLC, a reduced run time was observed while preserving a suitable separation. These experimental results were successfully applied to the study of ancient samples belonging from museums and including “indiennes” of the XIXth century. Finally, dying tests were carried out, in collaboration with Les Olivades society, in the aim to develop textiles containing natural dyes

Dissertação de mestrado em Química Farmacêutica Industrial, apresentada à Faculdade de Farmácia da Universidade de Coimbra.<br>A eficácia terapêutica de activos farmacêuticos, APIs, para administração oral, a estabilidade físico-química, a processabilidade, são, com frequência, condicionadas pelas características estruturais da forma sólida. A investigação de ocorrência de polimorfos e, recentemente, a pesquisa de co-cristais são de elevada relevância para o avanço da ciência e em pré-formulação farmacêutica. O trabalho desenvolvido nesta tese incide sobre um flavonóide, a luteolina. Trata-se um composto de origem natural, de uma família que tem vindo a ganhar destaque pelas suas potencais aplicações farmacológicas. Foi efectuada a pesquisa de polimorfos/solvatos por cristalização em solução e a investigação de formação de co-cristais com co-formadores que têm capacidade de formar diferentes sintões supramoleculares com a luteolina. Para a análise dos sólidos obtidos recorreu-se a espectroscopia de infravermelho, a vários métodos de análise térmica, calorimetria diferencial de varrimento, termogravimetria e termomicroscopia, e ainda a difracção de raios-X de pó. O estudo preliminar dos sólidos obtidos por cristalização em solventes permitiu identificar quatro novas formas sólidas, duas das quais se propõe serem solvatos. Foi identificado e caracterizado um outro polimorfo de luteolina, forma 2, obtido por aquecimento do hemi-hidrato comercial e dos sólidos obtidos por cristalização em solução. Obtiveram-se novas entidades cristalinas, co-cristais (1:1) com os co-formadores isonicotinamida, teofilina e cafeína, utilizando métodos de síntese com recurso a quantidades diminutas de solvente ou memso na ausência deste. Com os dois anti-inflamatórios nãoesteróides estudados, ácidos carboxílicos, e com a pirazinamida não ocorreu formação de novas estruturas cristalinas, nas condições utilizadas. Palavras-chave:<br>The therapeutic efficacy of active pharmaceutical ingredients, APIs, for oral administration, their physicochemical stability and processability are often constrained by the structural characteristics of the solid form. The investigation of polymorphs and, recently, the research of co-crystals are of high relevance to science and also in the context of applications in pharmaceutical pre-formulation. The work presented in this thesis focuses on a flavonoid, luteolin. It is a compound of natural origin, from a family that has been gaining prominence for its potencial pharmacological applications. In this work, research on polymorphs/solvates of luteolin by crystallization from solution, and on co-crystal formation with co-formers which are able of giving rise to different supramolecular heterosynthons, was performed. The solids obtained were analysed by infrared spectroscopy, thermal analysis methods, differential scanning calorimetry, thermogravimetry and thermomicroscopy, and also by X-ray powder diffraction. The preliminary study of the solids obtained by crystallization from solvents allowed the identification of four new solid forms, two of which are proposed to be solvates. Another luteolin polymorph, Form 2, was identified and characterized. It is obtained by heating the commercial hemihydrate or the solid formss obtained by crystallization from solution. Novel crystal entities, (1:1) co-crystals, were obtained with the co-formers, isonicotinamide, theophylline and caffeine. In the methods used for their synthesis only small quantities of solvent were used or no solvent at all. With both anti-inflammatory nonsteroidal drugs used, carboxylic acids, and with pyrazinamide there was no formation of new crystalline structures, in the experimental conditions used.

Praca przedstawia wyniki badań spektroskopowych alizaryny, indygo i luteoliny. Poprzez połączenie różnych technik spektroskopii oscylacyjnej i elektronowej absorpcyjnej oraz obliczeń kwantowo-chemicznych i chemometrii możliwa była identyfikacja in situ, analiza strukturalna, badanie dystrybucji i oddziaływań wybranych barwników naturalnych zarówno w materiale roślinnym jak i w miejscu ich zastosowania - tkaninie. Ze względu na różnorodne właściwości prezentowanych związków i w konsekwencji zróżnicowaną problematykę badawczą, układ rozprawy nie jest standardowy. Rozdział pierwszy stanowi wprowadzenie, ukazuje pracę w kontekście ogólnym i przedstawia motywację podjęcia badań. Kolejne trzy rozdziały (II. Alizaryna, III. Indygo, IV. Luteolina) przedstawiają szczegółowo przeprowadzone badania, a każdy z nich poprzedzony jest przeglądem literaturowym, gdzie przedstawiony jest i krótko scharakteryzowany badany związek. Każdorazowo została omówiona metodologia badań, a następnie zaprezentowano otrzymane wyniki i ich dyskusję. Rozdział V. podsumowuje osiągnięcia pracy w kontekście metodologicznym i poznawczym oraz prezentuje możliwości wykorzystania zdobytej wiedzy i perspektywy dalszych badań. Na końcu załączono zestawienie cytowanej literatury (VI) oraz spis publikacji własnych (VII).

碩士<br>國立陽明大學<br>傳統醫藥研究所<br>98<br>The flowers of Dendranthema morifolium (Ramat.) Tzvel. (Compositae) are used in traditional medicine for the treatment of headache, vertigo, and sore throat. Luteolin and luteolin-7-O-glucoside are important ingredients of D. morifolium. The aims of this study were to isolate the herbal ingredients, luteolin and luteolin-7-O-glucoside, from D. mori-folium, and access their pharmacokinetics in rats. We prepared the luteolin-7-O-glucoside and luteolin from the ethanolic extract of D. morifolium. Utilized solvent partition processes to enrich the contents of target compounds. After column purification, luteolin-7-O-glucoside was purified from the n-butanol layer and luteolin from ethyl acetate layer. Their structures were confirmed by spectral means. We used different solvent extraction to compare the contents of luteolin-7-O-glucoside and luteolin. The content of former was 4.19 % in ethanolic extract and 6.56 % in water ex-tracts. However, the content of latter was 0.19 % in ethanolic extract and not detected in water extract. The pharmacokinetic studies of luteolin-7-O-glucoside and luteolin were explored in a free-moving rat model. Four sets of experimental were designed, two for luteo-lin-7-O-glucoside and the other two for luteolin. The intravenous (i.v.) administration groups of luteolin-7-O-glucoside and luteolin had similar pharmacokinetic parameters. The oral bioavailability of luteolin-7-O-glucoside and luteolin were 10 % and 26 %, respec-tively. In the oral administration group of luteolin-7-O-glucoside, a biotransformed product luteolin was detected, but it did not detected in i.v. administration group of luteo-lin-7-O-glucoside. The transforming ratio of luteolin-7-O-glucoside to luteolin was about 76 %. The mean residence time (MRT) of biotransformed product luteolin increased about 70 % in comparison of oral administration group of luteolin. These results mean that the oral administration of luteolin-7-O-glucoside could be fast transformed to luteolin and this transformed product luteolin has a longer residence time, which may offer the bioactivity benefit of luteolin in organism.

碩士<br>萬能科技大學<br>化妝品應用與管理研究所<br>106<br>Luteolin is a natural flavonoid which is present in a variety of plants and has antioxidant, anti-inflammatory and antibacterial properties. However, trehalose is a non-reducing disaccharide molecule that exists in nature and is used to maintain cell viability. In the preparation of cosmetics and pharmaceuticals, it is an important component of moisturizing cosmetics. In this experiment, the first stage product was synthesized by using luteolin and propylene glycol and a catalyst, and then the polyethylene glycol (PEG400, 600, 1000, 2000) with good dispersibility was synthesized as the hydrophilic segment and succinic anhydride. The second stage product is then added to the second stage product for synthesis, and finally trehalose is added to the final product to obtain a series of water soluble polyphenol/glucose surfactants. The synthesized product was analyzed by infrared spectroscopy (FT-IR), and the surface tension of the water-soluble luteolin/trehalose surfactant was measured by a surface tension meter. The particle size analysis and the interfacial potentiometer were used to test the emulsification performance of the synthesized product. The dyeing test is carried out, and the designed new surfactant has good interfacial activity, can increase the wettability of the wool, and exerts a good leveling effect in the low temperature dyeing of wool.

碩士<br>國立嘉義大學<br>微生物免疫與生物藥學系研究所<br>107<br>Dendritic cells (DCs) are professional antigen-presenting cells. They are the key to initiate immune responses and induce immune tolerance. The generated tolerogenic DCs might be a useful tool for the induction of specific unresponsiveness. Luteolin is a kind of flavonoid that exists in many types of plants including vegetables, fruits, and medicinal herbs. It has multiple biological activities like anti-cancer and anti-oxidation. Besides, luteolin has been evidenced its anti-inflammatory effect through blockade of NF-κB activation in macrophages. Whether luteolin treated DCs can differentiate to suppressive status is still unknown. In this study, we investigated the influence of luteolin on the physiological characteristics and functions of DCs, and evaluate their ability to modulate T cell responses. Mouse DCs were matured with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α). Allogeneic T cells, isolated from spleens of BALB/c mice, were co-cultured with luteolin-treated DCs, and then the proliferation of T cells was assessed. After treatment with luteolin, the expression of surface molecules (MHC-II, CD80, CD86, CD83, CD40, CD54 and CD274) of DCs was determined by FACS analysis. Besides, the level of mRNA in luteolin-treated DCs was also measured. The results showed that luteolin-treated DCs could strongly inhibit allogenic T-cell proliferation in a dose-dependent manner. Compared with non-treated DCs, the treatment of luteolin clearly decreased the expression of CD80 and CD86 molecules on DCs. Furthermore, the cytokine mRNA of IL-12p40, IL-10 and STAT-3 in DCs were inhibited by luteolin, and the cytokine mRNA of TGF-β was increased. These results suggested that luteolin changed the DCs to suppressive properties. It might be a tool to use in transplantation or DC-associated disease.

碩士<br>高雄醫學大學<br>天然藥物研究所<br>90<br>In order to develop therapeutical drugs for liver cancer, we evaluated the cytotoxic effect of luteolin, 2-(3,4-Dihydro xyphenyl)-5,7-dihydroxy-4H-1- benzopyran-4-one, on human hepatoma cell lines. Five different human hepatoma cell lines, namely HepG2, SK-HEP-1, PLC/PRF/5, Hep 3B and HA22T/VGH were used as target cells. The colorimetric assay that involved reduction of XTT tetrazolium to soluble orange formazan was used to evaluate the cytotoxic activity of luteolin. The results indicated that luteolin exhibited potent anti-hepatoma activity, and also found that luteolin expressed different levels of cytotoxicity on five human hepatoma cell lines. According to the results, luteolin displayed predominant cytotoxicity against HBV-DNA(+) hepatoma cell lines of PLC/PRF/5 and Hep 3B with IC50 values of 8.40, 15.04μg/ml, respectively. Besides, this study showed that luteolin-treated PLC/PRF/5 cells exhibited typical changes of apoptosis. Flow cytometric analysis exhibited a prominent apoptotic marker (sub-G1 peak) after luteolin treatment. After gel electrophoresis, the fragmented DNA showed a characteristic ladder pattern. Cell cycle analysis revealed that luteolin induced PLC/PRF/5 cell cycle arrested at G0/G1 phase.

碩士<br>中山醫學大學<br>生化暨生物科技研究所<br>97<br>Luteolin, 3&apos;&apos;,4&apos;&apos;,5,7-tetrahydroxyflavone, is a common flavonoid that exists in many types of plants including fruits, vegetables, and medicinal herbs. Plants rich in luteolin have been used in Chinese traditional medicine for treating various diseases such as hypertension, inflammatory disorders, and cancer. Histone acetylation can modulate gene expression. Many cancers are involved in the chromatin condensation caused by histone deacetylaton which makes transcriptional repression of several tumor suppressor genes. However, the relationship between histone modification mediating gene regulation and the mechanism of the luteolin-induced apoptosis have not yet been clarified. Here, we found that luteolin was able to induce HL-60 cells apoptosis. For further understanding of this molecular mechanism we applied western blot, DNA electrophoresis, PCR, RT-PCR and ChIP assay. As a result we discovered that luteolin activated JNK MAPK pathway facilitating c-jun transcription factor translocation from cytosol to nuclear which concomitant with integration of histone acetyltransferase, p300 to FasL promoter region. This caused hyperacetylation of FasL promoter and change chromatin structure which increased transcriptional activity of FasL. This de novo FasL induced caspase 8 and caspase 3 activation and PARP cleavage in regulating the luteolin-induced apoptosis. On the other hand, we also discovered, interestingly, that histone decetylase 3 (HDAC 3) was cleaved follow caspase 7 activation. This could be the main reason of the hyperacetylated Fas promoter which induced apoptotsis through extrinsic pathway. Taken together, luteolin induced HL-60 cells apoptosis through JNK/c-Jun signaling pathway and histone modification. Besides cystic tumor, we also evaluated the anti-tumor activity of luteolin on solid tumor. It showed that while 35 μM luteolin led to cell cycle arrest and 75μM luteolin induced apoptosis of MDA-MB-231 cells. No matter low or high dose of luteolin both can cause histone H3 acetylation. Overall, luteolim presents antitumor activity involving mediating histone H3 acetylation.

碩士<br>東海大學<br>食品科學系<br>97<br>Microglia, resident immune cells in the central nervous system (CNS), respond to extracellular insults by releasing diversity of pro-inflammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF-alpha), interleukin 1beta(IL-1beta), and IL-6, leading to the initiation and promotion of inflammation. Evidence indicates that over-activated microglia and the associated pro-inflammatory mediators cause neural cell damage and contribute to several neurological disorders such as Alzheimer’s disease. Luteolin, a flavonoid compound, exhibits anti-oxidative, anti-neoplastic, anti-allerigc, and anti-inflammatory effects. Study suggests the neuroprotective effect of luteolin and proposes an anti-inflammatory mechanism. While the anti-inflammatory action is well demonstrated in the peripheral system, the role and potential action mechanisms of luteolin against inflammation in the CNS are largely unclear. The study was aimed to elicit the potential anti-inflammatory effect and mechanisms of luteolin against inflammatory responses in BV2 microglia cell lines after lipopolysaccharide (LPS)/interferon (IFN-gamma) stimulation. LPS/IFN-gamma stimulation caused BV2 cells elevating several pro-inflammatory mediators biosynthesis and releasing such as TNF-, iNOS, IL-6, and cyclooxygenase-2. Non-toxic level of luteolin reduced LPS/IFN-gamma-induced pro-inflammatory mediator production at the level of mRNA and protein. That is, the anti-inflammatory effect of luteolin is mediated by transcriptional regulation. Generally, intracellular signaling molecules such as ERK, JNK, p38, Akt, Jak family members, and Src could transduce extracellular signals to induce gene expression particularly pro-inflammatory genes through the modulation of transcription factors. Transcription factors such as NF-kappaB, IRF, and STAT are commonly downstream effectors of these signaling pathways. The inhibition of ERK, JNK, p38, Akt, and Jak activity by pharmacological inhibitors attenuated LPS/IFN-gamma-induced pro-inflammatory mediator production. LPS/IFN-gamma stimulation increased ERK, JNK, p38, Akt, Jak1, Jak2, Tyk2, and Src activity in BV2 cells, as evidenced by the elevated protein phosphorylation. Luteolin decreased ERK, JNK, p38, Akt, and Src but not Jak1, Jak2, and Tyk2 activity in LPS/IFN-gamma-treated cells. LPS/IFN-gamma stimulation caused IkappaB-alpha phosphorylation and triggered p50, p65, and RelB NF-kappaB subunit nuclear accumulation, indicating an activation of NF-kappaB. Luteolin attenuated LPS/IFN-gamma-induced IkappaB-alpha phosphorylation and p65 and RelB nuclear accumulation leading to a resolution of NF-kappaB activation. LPS/IFN-gamma increased IRF-1 protein expression resulting in activation and the increased IRF-1 expression was attenuated by luteolin. LPS/IFN-gamma stimulated STAT-1 and STAT-3 activity through the increased protein phosphorylation. Luteolin reduced LPS/IFN-gamma-induced STAT-1 and STAT-3 phosphorylation. Although luteolin had little effect on Jak family protein activity, another STAT upstream activator Src was inactivated in LPS/IFN-gamma stimulated cells by luteolin. Besides, luteolin increased SOCS3 protein expression, a negative regulator of STAT activation. Taken together, our experimental findings indicate that the anti-inflammatory effects of luteolin in microglia cells could be mediated by the down-regulation of ERK, JNK, p38, Akt, Src signaling molecules and NF-kappaB, IRF-1, STAT-1, and STAT-3 transcription factors.

碩士<br>中山醫學大學<br>應用化學系碩士班<br>101<br>Flavonoids, a large group of natural polyphenols existing in a wide range of daily fruits and vegetables, have been evidenced to progress various prominent bio-activities. Recently, members of flavonoids have found a variety of anticancer effect such as cell growth, apoptosis induction, suppression of the secretion of matrix metalloproteinases and tumor invasive behavior in various studies. First, our experiments chose two high malignant breast cancer cells (MDA-MB-231 and 4T1), and we used boyden chamber assay to investigate that the inhibitory effects of four flavonoids in two breast cancer cells. It showed that luteolin exhibited the highest anti-invasive capacity. However, the molecular mechanism regulation of inhibition of metastatic in breast cancer is not well clarified. We assayed the cell viability, cell invasion/motility, matrix metalloproteinases (MMPs) activity of 4T1 cells with luteolin treatment by MTT assay, trypan blue dye exclusion assay, wound healing, invasion assay, adhesion assay and gelatin zymography. The result showed that luteolin can inhibit 4T1 cell migration, movement, and adhesion. In addition, luteolin inhibited matrix metalloproteinases activity and expression. Luteolin also inhibits tumor metastasis in front of epithelial-mesenchymal transition process, so that increased cell-cell-marker protein :E-cadhetin and reduced the mesenchymal marker: vimentin and N-cadherin.Some research also demonstrated that OPN have high expression in malignant breast cancer cells. Therefore, further to explore that luteolin inhibited OPN, whereas in the past that literature mentioned OPN is regulated by ERK, Akt, stat3 and NF-κB signaling pathway. Our results showed that luteolin inhibited ERK, Akt, Stat3, NF-κB phosphorylation inhibition. Then using four inhibitor PD98059 (ERK inhibitor), Wortmannin (PI3K/Akt inhibitor), S3I-201 (stat3 inhibitor), PDTC (NF-κB inhibitor), respectively, discovered that adding ERK, Akt, NF-κB, Stat3 inhibitors will inhibit OPN, vimentin, MMP-2 and promote of E-cadherin expression. Finally explore further transfected with si-OPN and epithelial - mesenchymal conversion between correlation results confirmed inhibition of OPN may inhibit MMP-2, vimentin and promotion of E-cadherin protein expression. Above of all, these results demonstrated anti-invasion effect of luteolin likely through inhibition of ERK, Akt, NF-κB, Stat3 signaling pathway resulting in inhibition of the activity of transcription factors of OPN then reversing EMT''s ability to inhibit cancer metastasis.

碩士<br>臺北醫學大學<br>藥學系<br>91<br>Part I: Flavonoids are polyphenolic compounds occurring in nature, commonly presents in plants. They have anti-inflammatory and immuno-regulatory effects. Cyclic nucleotides play an important role in inflammatory cells. Their concentration is related to the relaxation of airway smooth muscle. Therefore we are interested in investigating the relationships between structure and inhibitory effect of flavonoids on various PDE isozymes separated from guinea-pig lungs and hearts. Isolated guinea-pig lungs and hearts were separately homogenized and centrifuged. The supernatant was chromatographed over a column of Q-sepharose, and eluted with various concentrations of NaCl. In the following order, PDE subtype 1, 5, 2, 4 from lungs, and 3 from hearts were separated. Acccording to the method described by Thompson and Appleman, the activities of PDE isozymes were determined in the presence of cAMP and [3H]-cAMP or cGMP and [3H]-cGMP as substrate. The results revealed that luteolin had effectively inhibitory actions on PDE1~5. Diosmetin, which OCH3 group substitutes the C-4 OH group of luteolin, enhanced inhibition on PDE2, but lost inhibition on PDE3 activity. Apigenin, lacking of C-3'' OH group from luteolin, lost inhibition both on PDE4 and 5. Chrysin lacking of both C-3'' and C-4'' OH groups from luteolin lost all inhibition on PDE1~5. Luteolin-7-glucoside glycosylated from luteolin attenuated inhibition on PDE2 and 4, lost inhibition on PDE1, 3, and 5. Quercetin hydroxylated from luteolin at position of C-3 enhanced inhibition on PDE4, but lost inhibition on PDE5. Myricetin, hydroxylated from luteolin at both position of C-3 and C-5'', enhanced inhibition on PDE4, but lost inhibition on PDE5. Eriodictyol saturated luteolin between C-2 and 3 attenuated inhibition on PDE3, but lost inhibition on other PDE isozymes. Hesperetin saturated from diosmetin between C-2 and 3 attenuated inhibition on PDE4, but lost inhibition on other PDE isozymes. Part II: Luteolin effectively but non-selectively inhibited PDE1~5. The aim of this present study is to investigate whether it has anti-asthmatic action. The present results revealed that luteolin inhibited ovalbumin (OVA)-induced airway hyperresponsiveness (AHR). Luteolin (3-30 mmol/kg, i.p.) dose-dependently attenuated the enhanced pause (Penh) value induced by aerosolized methacholine (MCh, 25~50 mg/ml) in sensitized mice after secondary allergen challenge. Luteolin at 30 mmol/kg even significantly inhibited MCh (12.5 mg/ml)-induced increase of Penh value. Mice administered luteolin (3-30 mmol/kg) did not significantly differ from those sensitized but not challenged with aerosolized OVA (non-treatment). Luteolin (3-30 mmol/kg, i.p.) also suppressed total inflammatory cells, neutrophils and eosinophils, but not lymphocytes. Luteolin at a dose of 30 mmol/kg even significantly reduced marcrophages. Luteolin (3-30 mmol/kg) significantly reduced the IL-2, IL-4, IFN-g and TNF-a production in bronchoalveolar lavage fluid (BALF). At a dose of 30 mmol/kg, luteolin even inhibited release of IL-5. Luteolin (3~30 mM) significantly attenuated OVA (10 mg/ml)-induced tracheal contractions in vitro. From Lineweaver-Burk analysis, luteolin (3~30 mM) competitively inhibited various PDE isozymes. In conclusion, luteolin non-selectively but competitively inhibited PDE1~5. At low doses of 3~30 mmol/kg (0.858~8.58 mg/kg), luteolin has both anti-inflammatory and bronchodilatory effects, and has a potential in the treatment of asthma.

博士<br>中國醫藥大學<br>中國藥學研究所<br>96<br>The present study investigated the antifibrotic effect and its molecular mechanism of luteolin on lung fibrosis in both in vivo and in vitro models. C57BL/6J mice were administered a single intratracheal injection with bleomycin and then treated orally with luteolin. Lung inflammation was examined by direct counting inflammatory cell population and cytokine levels in the bronchoalveolar lavage fluid at day 7 and day 14. Antifibrotic effect of luteolin was determined by investigating histological changes, collagen contents and induction of TGF-β1 mRNA expression at day 21. To elucidate the antifibrotic mechanism of luteolin, the expression of α-SMA, collagen 1 and phosphorylated Smad3 protein were analysized by immunofluorescence staining and Western blotting in TGF-β1 stimulated primary mouse lung fibroblasts. The morphological changes as well as epithelial and mesenchymal marker were examined in TGF-β1-stimulated A549 cells. In vitro study showed that luteolin attenuated TGF-β1-induced α-SMA and collagen1 upregulation and Smad3 phosphorylation in mouse lung fibroblasts. Furthermore, TGF-β1 mediated E-cadherin downregulation as well as fibronectin and collagen 1 upregulation was significantly inhibited by luteolin in A549 cells. Our data suggest that luteolin may be useful as a therapy for pulmonary fibrosis and its antifibrotic effect at least partly through blockade of TGF-β1 signaling pathway. Luteolin dose-dependently inhibited the expression and production of these inflammatory genes and mediators in macrophages stimulated with LPS. Semi-quantitative reverse-transcription polymerase chain elongation reaction assay further confirmed the suppression of LPS-induced TNF-α,IL-6, iNOS and COX-2 gene expression by luteolin in a transcriptional level. Luteolin also reduced the DNA-binding activity of NF-kB in LPS-activated macrophages. Moreover, luteolin blocked the degradation of IkB-α and nuclear translocation of NF-kB p65 subunit. In addition, luteolin significantly inhibited the LPS-induced DNA binding activity of AP-1. We also found that luteolin attenuated the LPS-mediated Akt and IKK phosphorylation, as well as reactive oxygen species production. Our observations suggest a possible therapeutic application of this agent for treating inflammatory disorders in lung.

博士<br>中山醫學大學<br>生化暨生物科技研究所<br>95<br>PART I The dried fruits of Crataegus pinnatifida, a local soft drink material and medical herb, demonstrated antioxidant effect in our previous study. In the present study, we investigate the anti-inflammatory potential of flavonoid contents from dried fruit of Crataegus pinnatifida (CF-Fs). The preliminary investigation showed that CF-Fs (0.10 - 0.75 mg/ml) decreased the release of PGE2 and nitric oxide as induced by lipopolysaccharide (LPS, an endotoxin) in macrophage RAW 264.7 cells. The in vivo assay showed that pretreatment of rats with CF-Fs (50 mg/kg-200 mg/kg dosed by gavage) for 5 days significantly decreased the serum levels of the hepatic enzyme markers alanine- and aspartate aminotransferase (ALT and AST) induced by the 6-hour treatment with LPS (ip; 5 mg/kg). Histopathological evaluation of the rat livers revealed that CF-Fs reduced the incidence of liver lesions such as neutrophil infiltration and necrosis induced by LPS. Furthermore, we found that pretreatment with CF-Fs decreased the hepatic expression of iNOS and COX-2 induced by LPS in rats. These results demonstrate that CF-Fs present anti-inflammatory potential in vitro and in vivo, and that they may play a role in hepatoprotection. PART II The dried fruits of Crataegus pinnatifida have been used traditionally as oriental medicine and local soft drink material recently. Previously, we demonstrated that C. pinnatifida exhibited anti-oxidation and anti-inflammatory potential. To clarify the active components in anti-transformation and anti-tumor promotion, we collected the polyphenol fraction (CF-TP) of hot-water extracts from dried fruits of C. pinnatifida for the following study. By anchorage-independent transformation assay, CF-TP significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation in JB6 P+ cells. Moreover, we found that CF-TP inhibited the expression of osteopontin (OPN), a transformational marker, and the activation of NF-κB and AP-1 induced by TPA in JB6 P+ cells. In addition, we evaluated the effect of CF-TP on TPA application to ICR mouse skin with measurement of H2O2 production, myeloperoxidase (MPO) activity, edema formation, epidermal thickness and leukocyte infiltration. As a result, CF-TP significantly inhibited the generation of reactive oxygen species (ROS) and the phenomena of inflammation induced by TPA. It also suppressed the expression of COX-2 and iNOS, and the activation of ornithine decarboxylase (ODC). Furthermore, CF-TP inhibited benzo[a]pyrene (B[a]P)/ TPA-induced skin tumor formation and decreased the incidence of tumor. These results indicate that CF-TP possesses potential as a cancer chemopreventive agent against tumor promotion. PART III Dietary fatty acid intake is associated with the risk of development and progression of prostate, colon and breast cancer. Arachidonic acid (AA), an essential polyunsaturated fatty acid, is the major precursor of biological active eicosanoids, which include prostaglandin E2 (PGE2), thromboxanes and leukotrienes. In human prostate, AA was catalyzed to PGE2, the only significant eicosanoid product, by cyclooxygenase (COX) and has demonstrated that PGE2 stimulates prostate cell proliferation and progress to cancer. In previous study, arachidonic acid, an ω-6 fatty acid possess the potential to stimulate the proliferation of human prostate cancer cells. In this study, we demonstrated that arachidonic acid also induced cell proliferation of human benign prostate hyperplasia cells (BPH-1), and this phenomena may involved phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Moreover the expression of GSTP1, the biomarker in diagnosis of prostate cancer also decreased in arachidonic acid treated BPH-1 cells. The effect of some flavonoids, which are components of fruits, vegetables, and peas, on the cell proliferation of human benign prostate hyperplasia cells (BPH-1) was also investigated in this study. Luteolin inhibited the BPH-1 cell growth and phosphorylation of ERK and Akt. In addition, luteolin induced GST-P1 protein expression. Together, these results suggest that arachidonic acid induced BPH-1 cell proliferation and the ability of anti-proliferation of flavonoids may via inhibited the ERK and Akt signal pathway and Bcl-2 protein expression. The flavonoids could be an improved strategy for prevention and/or treatment of benign prostatic hyperplasia.

碩士<br>亞洲大學<br>生物科技學系碩士班<br>99<br>Oxidative stress has recently been implicated in the pathogenesis of atherosclerotic cardiovascular diseases. Dietary supplement of antioxidants has been reported to have beneficial effects on prevention of atherogenic diseases. Reactive oxygen species (ROS) such as superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (•OH) are continuously produced in cells as products of cellular oxidation–reduction processes and as the mechanisms of biophylaxis. Luteolin, 3,4,5,7-tetrahydroxyflavone, a polyphenolic compound, usually occurs as glycosylated forms in celery, green pepper, perilla leaf and camomile tea. It has been found to possess antimutagenic, antitumorigenic antioxidant and anti-inflammatory properties. However, the effects of luteolin on cardiovascular systems are still largely unknown. Therefore, the aim of the present study is to test whether luteolin could protect against oxidative stress-induced endothelial cell injury and explore the possible mechanisms. Primary human umbilical vein endothelial cells (HUVECs) were pretreated with luteolin (2.5-20 uM) for 2 hours followed by hydrogen peroxide (600 uM) for indicated time period. Cell viability was determined by MTT and LDH release. The generation of reactive oxygen species (ROS) and superoxide were determined by using the fluorescent probe 2’, 7’-dichloro-fluorescein acetoxymethyl ester (DCF-AM), dihydroethidium (DHE), respectively. The protein levels of ROS-mediated signaling pathways were measured by Western blot. In addition, AMPK knockdown were employed to explore whether the AMPK/PKC/NADPH oxidase involved in the process. Furthermore, several apoptotic features which showed NF-kB activation, alteration of mitochondria membrane potential, cytochrom c release and subsequent activation of caspase 3 were also inverstigated. Our results showed that luteolin protected hydrogen peroxide-induced cell death in a dose-dependent manner. Luteolin ameliorated ROS production and impaired antioxidant enzyme SOD-I caused by hydrogen peroxide. Luteolin suppressed hydrogen peroxide-induced superoxide anion radical generation and membrane assembly of NADPH oxidase subunits via modulating AMPK-suppressed PKC activation. Application of AMPK1-specific siRNA resulted in increased activation of PKC and p47phox. In addition, hydrogen peroxide also decreased AMPK-mediated Akt/eNOS/NO signaling and induced the phosphorylation of MAPK which in turn activated NF-B-mediated inflammatory responses such as the iNOS expression leading to a overproduction of NO and protein nitrosylation. We also found that hydrogen peroxide increased phosphorylation levels of transcription factor P53 which in turn disturbed the balance of Bcl-2 family proteins, destabilized mitochondrial membrane potential, and triggered subsequent cytochrome c release into the cytosol and activation of caspase 3. Pretreatment with luteolin, however, exerted significant cytoprotective effects in a dose-dependent manner. Results from this study may provide insight into a possible molecular mechanism underlying luteolin suppression of the oxidative stress-induced endothelial dysfunction.

碩士<br>輔仁大學<br>基礎醫學研究所碩士班<br>102<br>Doxorubicin is an antineoplastic agent used for more than 40 years. Its use was limited by the effect of dose dependent cardiomyopathy that can induce heart failure. The mechanism of doxorubicin affecting cardiomyocytes was still not fully clear, but most studies revealed that oxidative stress and inhibition of topoisomerase II play the main roles. Connexin 43 was the major constitution protein of gap junction in rat cardiomyocyte. Our study revealed that the low dose doxorubicin didn’t affect survival rate of cells but significantly decreased the expression of connexin 43. Luteolin is a member of flavonone which was constituted by a C6-C3-C6 backbone structure with 2 benzene rings connected with 3 carbons. Previous studies revealed that luteolin has anti-proliferative, pro-apoptotic properties as well as protective effect during ischemia stress in cardiomyocytes. Therefore, we postulated that luteolin might exert a protective effect to prevent the down-regulation of connexin 43 induced by doxorubicin, a widely used chemotherapeutic drug, in H9c2 cells. We used reverse transcription polymerase reaction and Western blot to evaluate the effect of doxorubicin on expression of connexin 43. We also used the fluorescence microscope to visualize the density and location of the connexin 43 with treatment of doxorubicin and co-treatment with luteolin. Finally, we used scrape loading method to check the protective effect of luteolin on doxorubicin-induced gap junction remodeling in H9c2 cells. The mechanism that doxorubicin decreased the expression of connexin 43 was still unclear, we tried to seek the possible intracellular pathways that response for the effect of doxorubicin and found that JNK and STAT3 seemed to play important roles in the process and luteolin ameliorated doxorubicin-induced gap junction remodeling in H9c2 cells via inhibiting the JNK and STAT3 signaling pathways.

博士<br>國立中興大學<br>分子生物學研究所<br>102<br>Luteolin (3’,4’,5,7-tetrahydroxyflavone) is a common flavonoid in many types of plants and has several beneficial biological effects, including anti-inflammation, anti-oxidant, and anti-cancer properties. However, the detail mechanisms of luteolin in suppressing tumor invasion and metastasis are poorly understood. Here, we investigated the effects of luteolin on suppressing glioblastoma tumor cell invasion and migration activity. Under the non-cytotoxic doses (15 and 30 μM), luteolin exhibited an inhibitory effect on migration and invasion in U87 and T98G glioblastoma cells. Additionally, filopodia assembly in U87 cells was markedly suppressed after luteolin treatment. The treatment of luteolin also showed a decrease of Cdc42 (cell division cycle 42) protein levels and reduced PI3K/AKT activation, whereas there was no association between this decrease and phosphorylated ERK or altered transcription levels of Cdc42. Over expression of constitutive Cdc42 (Q61L) using transient transfection in U87 cells induced a partial cell migration, but did not affected the degradation of the protein levels of Cdc42 after luteolin treatment. Moreover, inhibition of the proteaosome pathway by MG132 caused a significant recovery in the migration ability of U-87 cells and augmented the Cdc42 protein levels after luteolin treatment, suggesting that pharmacological inhibition of migration via luteolin treatment is likely to preferentially facilitate the protein degradation of Cdc42. Taken together, the study demonstrated that flavonoids of luteolin prevent the migration of glioblastoma cells by affecting PI3K/AKT activation, modulating the protein expression of Cdc42 and facilitating their degradation via the proteaosome pathway. In secondary section, dynamin-related protein 1 (DRP1) is an 80-kDa GTPase, which is involved in mitochondrial fission, mitochondrial protein imports and cisplatin cytotoxicity, suggesting an association with disease progression of cancer. This study investigated the predictive value of DRP1 in glioblastoma multiforme (GBM) to radiotherapy. Using immunohistochemistry, DRP1 expression was measured in 47 GBM patients. Expression of DRP1 was confirmed by immunoblotting. Correlation between DRP1 expression and clinicopathological parameters was analyzed by statistical analysis. Difference of patient’s survival was compared by a log-rank test. The Results showed that DRP1 expression was detected in 41 GBM patients. Among these, nuclear DRP1 (DRP1nuc) was detected in 33 (80.5%) patients. A significant difference was found in cumulative survival between patients with high DRP1 levels and those with low DRP1 levels (p = 0.0398). In vitro, DRP1 was identified in both T98G and U87 cell lines. DRP1 expression in T98G was higher than U87 cells. Silencing of DRP1 reduced cell proliferation, metastatic potential, and radiation resistance in both T98G and GBM stem cells. Shikonin, a compound extracted from Chinese medicinal herb Lithospermum erythrorhizon, inhibited DRP1 expression and induced autophagy. Although SAHA did not evidently decrease DRP1 levels, it increased the frequency of apoptotic cells. Moreover, both shikonin and SAHA reduced nuclear DRP1 and increased radiosensitivity, suggesting that both drugs radiosensitized GBM cells. In conclusion, our results suggest that DRP1 overexpression is a prospective radioresistant phenotype in GBM. DRP1 could be a potential target for improving radiotherapy.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Title, Name, Surname of candidate: Barbora Šalovská Title, Name, Surname of tutor: Doc. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Modulation effects of apigenin and luteolin on activity and expression of cytochrome P4501A2 in intestinal cells Flavonoids are a large group of polyphenolic compounds that occur naturally in plant kingdom. They have been shown to posses a variety of biological activities including anti-inflammatory, anti-oxidant and anti-mutagenic effects and play a prominent role in cancer prevention. The aim of this work was research of the effect of flavones apigenin and luteolin on an activity and expression of subfamily 1A of cytochromes P450 in human colon cancer cell line HCT-8. The CYP1A isozymes enzymatic activity was measured by EROD/MROD activity assay; the quantity of CYP1A's protein was set by Western blotting. We found out that apigenin and luteolin significantly decreased CYP1A activity in HCT-8 cells. During synchronous application of model inductor CYP1A β-naftoflavon (β-NF) with apigenin or luteolin (at the concentrations of 5µM and higher), the induction effect of β-NF on CYP1A was reduced. Apigenin and luteolin significantly decreased the CYP1A activity...