Através de Testes de Diluição em Sistema de Tubos Múltiplos determinou-se in vitro a Intensidade de Atividade de Inibição Bacteriana (IINIB/bacteriostasia) e a Intensidade de Atividade de Inativação Bacteriana (IINAB/bactericidia) de soluções contendo extratos etanólicos a 50% de diferentes acessos in natura de rizomas (alcoolaturas), bem como diferentes amostras comerciais de pó de rizomas (hidroalcoolaturas) de açafrão-da-terra (Curcuma longa L.), sobre inóculos bacterianos padrões de Staphylococcus aureus (ATCC 25923), Salmonella Enteritidis (ATCC 11076), Enterococcus faecalis (ATCC 19433) e Escherichia coli (ATCC 11229). Os extratos hidroalcoólicos apresentaram baixa atividade de inibição/inativação entre as quatro bactérias testadas, enquanto que a forma de extração alcoólica sem reidratação (extrato bruto) apresentou atividade antibacteriana seletiva e significativamente mais intensa. Salmonella Enteritidis e Enterococcus faecalis foram às bactérias mais sensíveis, frente a ambos os extratos (hidroalcoólico e alcoólico), enquanto que Escherichia coli e Stahphylococcus aureus apresentaram a menor sensibilidade. Houve diferença positiva significativa para os extratos não reconstituídos (bruto) quando comparados aos reconstituídos. / Through Test Dilution System in Multiple Tubes were determined in vitro the intensity bacterial inhibition activity (IINIB/bacteriostasys) and the Intensity of bacterial inactivation activity (IINAB/bactericidie) solutions containing 50% ethanol extracts of different accessions fresh root (alcoholic preparations) as well as commercial samples of powdered root (hidroalcoolaturas) from ground-saffron (Curcuma longa L.). patterns of bacterial inocula of Staphylococcus aureus (ATCC 25923), Salmonella Enteritidis (ATCC 11076), Enterococcus faecalis (ATCC 19433) and Escherichia coli (ATCC 11229). The hydroalcoholic extracts showed low activity inhibition/inactivation among the four tested bacteria, while the form of alcohol extraction without rehydration (crude extract) showed selective antibacterial activity and significantly more intense. Salmonella enteritidis and Enterococcus faecalis bacteria were more sensitive, compared to both extracts (hydroalcoholic and alcohol), while Escherichia coli and Staphylococcus aureus Stahphylococcus showed the least sensitivity. There was a significant positive difference to the extracts not rehydrated (raw) when compared to rehydrated.

Einleitung: Verschiedene Erkrankungen gehen bei Pferden mit einer erhöhten Entzündungsbereitschaft einher. Das entzündliche Geschehen kann dabei in engem Zusammenhang mit dem Auftreten von Stress des endoplasmatischen Retikulums (ER) stehen. Im Zuge der Immunantwort entsteht eine Signalkaskade, die über Entzündungsmarker quantifiziert werden kann. Bei der Expression verschiedener Entzündungsmediatoren rückt insbesondere die Leber in den Fokus. Die anti-inflammatorische und ER-Stress-modifizierende Wirkung von oralen Polyphenolen aus einem Grüntee-Kurkuma-Extrakt (GKE) wurde bereits in anderen Tierspezies, wie z.B. Wiederkäuern oder Schweinen nachgewiesen. Ziel der Studie: Das Ziel der vorliegenden Studie war die Überprüfung der Effekte einer GKE-Supplementierung auf eine Lipopolysaccharid (LPS)-induzierte Entzündung bei Pferden und Ponys. Die Hypothese war, dass die Supplementierung des GKE zu einer Reduktion der entzündlichen Reaktion und des Zellstresses bei Pferden und Ponys führt. Tiere, Material und Methoden: In einer randomisierten, placebo-kontrollierten Studie wurden fünf gesunde adulte Warmblutpferde (Angaben in Mittelwert ± Standardabweichung, Alter 19 ± 5 Jahre; Körpermasse (KM) 589 ± 81 kg) und sechs gesunde adulte Shetlandponys (Angaben in Mittelwert ± Standardabweichung, Alter 9 ± 3 Jahre; KM 126 ± 8 kg) einbezogen. Die tägliche Basisration bestand aus Heu (2 kg/100 kg KM). Im Cross-over erhielten die Tiere für 21 Tage 10 g eines GKE (20% Polyphenolgehalt) nach Angaben des Herstellers, oder alternativ ein Placebo (Kalziumcarbonat) zusammen mit 1 kg (Pferde) oder 0,2 kg (Ponys) eines kommerziellen Ergänzungsfuttermittels für Pferde. Nach der 21-tägigen Supplementierungsphase wurde bei allen Tieren zur Induktion eines moderaten, generalisierten Entzündungsgeschehens LPS (10 ng/kg KM) intravenös appliziert. 24 Stunden vor und 12 Stunden nach der LPS-Challenge wurden Blutproben und Lebergewebe entnommen. Die Entnahme des Lebergewebes erfolgte minimalinvasiv unter Ultraschallkontrolle. Während der anschließenden 3-monatigen Wash-out Phase erhielten alle Tiere ausschließlich Heu zur freien Aufnahme. Hiernach fand im Cross-over Verfahren ein Wechsel der Fütterungsgruppen statt. Eine erneute LPS-Challenge und die anschließende Probennahme wurden nach den oben genannten Kriterien durchgeführt. In allen Blutproben wurden die Parameter Serum Amyloid A (SAA), Haptoglobin (Hp) und Retinol-bindendes Protein 4 (RBP4) gemessen. Mittels RT-qPCR wurden in allen Leberbioptaten die mRNA-Level ausgewählter Entzündungsmarker bestimmt (Hp, TNF-α, IL-1β, IL-6, CD68, FGF21, NF-kB, ATF4). Zusätzlich erfolgte eine histologische Untersuchung auf entzündliche Reaktionen im Gewebe. Die Daten, mit Ausnahme der immunhistochemischen Auswertungen, wurden unter Verwendung der kommerziellen Software Statistica® statistisch ausgewertet. Die Daten wurden mittels des Shapiro-Wilks-Testes auf Normalverteilung überprüft. Da die Daten nicht normalverteilt waren, wurde der Wilcoxon-Test für nicht-parametrische Daten angewendet. Das Signifikanzniveau lag bei p < 0,05. Die Darstellung der Daten erfolgt als Median und dem 25./75. Perzentil. Ergebnisse: Vor der LPS-Challenge lagen alle Werte für SAA ≤ 2,6 mg/l. Nach der LPS-Challenge stiegen die Werte signifikant an (Angabe Median und in Klammern 25./75. Perzentil, Placebo-Gruppe: 98,4 (70,2/118) μg/ml, Zeit p = 0,008; GKE-Gruppe: 70,7 (46,8/111) μg/ml, Zeit p = 0,003). Innerhalb der GKE-Gruppe stieg der Wert für Hp im Serum nach der LPS-Challenge signifikant an (Zeit p = 0,005). Es konnten keine weiteren signifikanten Unterschiede in den Blutparametern im Vergleich der GKE-Gruppe zur Placebogruppe nach LPS Stimulation gefunden werden. Im Lebergewebe zeigte sich nach der LPS-Challenge für die GKE-Gruppe eine 2,6-fach geringere mRNA-Expression für das pro-inflammatorische Zytokin IL-1β im Vergleich zur Placebogruppe (Supplementierung p = 0,04). Die mRNA-Level von CD68 (Zeit p = 0,04) und Hp (Zeit p = 0,03) zeigten nach der LPS-Challenge im Lebergewebe einen signifikanten Anstieg innerhalb der Placebogruppe. Innerhalb der GKE-Gruppe zeigten sich keine Veränderungen durch die LPS-Challenge. Ein signifikanter Unterschied zwischen der Placebo- und der GKE Gruppe nach LPS wurde nicht gefunden. Andere Parameter im Lebergewebe, wie z.B. Transkriptionsfaktor NF-κB und die Zytokine IL-6 und TNF-α, zeigten keine signifikanten Veränderungen nach der LPS-Challenge in beiden Fütterungsgruppen. Für die Ergebnisse der Immunhistochemie konnten keine signifikanten Unterschiede in Abhängigkeit zur LPS-Challenge oder der Fütterung gezeigt werden. Schlussfolgerung: Die LPS-Challenge induzierte eine generalisierte Entzündung bei Pferden und Ponys. Die Supplementierung eines GKE für 21 Tage zeigte über die Reduktion des Entzündungsmarkers IL-1β in der Leber ein anti-inflammatorisches Potential. Andere Blut- und Leberparameter zeigten keinen Supplementierungseffekt. Somit konnte kein umfassender anti-inflammatorischer Effekt der GKE Supplementierung festgestellt werden.:1 Introduction 2 Literature review 2.1 Inflammation 2.1.1 Acute phase reaction 2.1.1.1 Serum amyloid A 2.1.1.2 Haptoglobin 2.1.1.3 Retinol-binding protein 4 2.1.2 Transcription factor NF-κB 2.1.2.1 Pro-inflammatory cytokines (Il-1β, IL-6, TNF-α) 2.1.3 Macrophages 2.1.3.1 Cluster of differentiation 68 2.2 Stress of the endoplasmic reticulum 2.2.1 Fibroblast growth factor 21 2.2.2 Activating transcription factor 4 2.3 Oxidative stress 2.4 Polyphenols 2.4.1 Green tea 2.4.2 Curcuma 2.4.3 Bioavailability 2.4.4 Anti-inflammatory effects 2.4.5 Antioxidative effects 3 Publication 3.1 Effects of a blend of green tea and curcuma extract supplementation on lipopolysaccharide-induced inflammation in horses and ponies 4 Discussion 4.1 Effects of the LPS challenge 4.1.1 Induction of inflammation by LPS 4.1.2 Induction of ER stress and oxidative stress by LPS 4.2 Effects of the GCE supplementation 4.2.1 Anti-inflammatory effects 4.2.2 Reduction of ER stress and oxidative stress 4.2.3 Comparison of in vivo effects in equines and farm animals 4.3 Daily dosages of polyphenols 4.4 Conclusion 5 Zusammenfassung 6 Summary 7 References 8 Appendix 8.1 List of presentations as part of this thesis 8.2 Further publications 8.2.1 Published articles 8.2.2 Published conference contributions 9 Acknowledgement / Introduction: Different medical conditions are linked with an inflammatory status in horses and ponies. Inflammation can be induced by endoplasmic reticulum (ER) stress, leading to an immune response which can be measured by several markers of the signalling cascade. The liver plays an important role as production site of these inflammatory markers. The influence of feeding a polyphenol-rich supplement consisting of green tea and curcumin extract (GCE) on ER stress and inflammation has been already described in several species such as cattle and pigs. Aim of the study: The aim of the study was to investigate the effect of a GCE supplementation on lipopolysaccharide (LPS)-induced inflammation in horses and ponies. It was hypothesized that the GCE supplementation will reduce inflammatory reactions and cell stress compared to the placebo group. Animals, material and methods: In a randomized, placebo controlled study, five healthy adult warmblood horses (mean (±SD) age 19 ± 5 years, mean (±SD) body weight (BW) 589 ± 81 kg) and six healthy adult Shetland ponies (mean (±SD) age 9 ± 3 years and mean (±SD) BW 126 ± 8 kg) were included. Animals were fed daily with meadow hay (2 kg/ 100 kg BW). In a cross-over design, animals were supplemented for 21 days with 10 g of a GCE (20% total polyphenol content) according to manufacturer’s instructions or a placebo (calcium carbonate) in a mixture with 1 kg (horses) or 0.2 kg (ponies) of a commercial feed for horses. After supplementation for 21 days, all animals underwent an intravenous LPS (10 ng/kg BW) challenge to induce a moderate systemic inflammation. 24 hours before and 12 hours after the LPS challenge, blood and liver samples were collected. Liver tissue was taken transcutaneously under ultrasound control. Subsequently, a 3-months-lasting wash-out period was conducted where the animals were fed with meadow hay ad libitum. A change of feeding groups according to the cross-over design was performed. A second LPS challenge and sample collection were performed according to the above-mentioned protocol. All blood samples were analysed for serum amyloid A (SAA), haptoglobin (Hp) and Retinol-binding protein 4 (RBP4). RT-qPCR was used to analyse liver mRNA levels of selected markers of inflammation (Hp, TNF-α, IL-1β, IL-6, CD68, FGF21, NF-kB, ATF4). In addition, liver tissue was histologically examined for inflammatory responses. All data except for immunohistochemical results were statistically assessed with a commercial software package (Statistica). Data were checked for normal distribution using the Shapiro-Wilks test. As data set was not normally distributed the Wilcoxon test for non-parametric data was used for analyses. Level of significance was set at P < 0.05. Data are presented as median and 25./75. percentile. Results: All SAA values before LPS were ≤ 2.6 mg/L. After LPS challenge, all SAA values were significantly higher compared to baseline (data expressed as medians and 25./75. Percentile in brackets, placebo group: 98.4 (70.2/118) μg/mL, time P = 0.008; GCE group: 70.7 (46.8/111) μg/mL, time P = 0.003). GCE group showed a significant increase for serum Hp after LPS challenge (time P = 0.005). Other significant differences in blood parameters comparing placebo and GCE group after LPS could not be found. In the liver, the pro-inflammatory cytokine IL-1β showed a 2.6-fold lower mRNA level after LPS challenge in the GCE group compared to placebo group (supplementation P = 0.04). Hepatic mRNA levels of CD68 (time P = 0.04) and Hp (time P = 0.03) increased significantly in the placebo group, but not in the GCE supplementation group. A significant difference between the two feedings groups after LPS could not be found. Other liver parameters, such as transcription factor NF-κB and the cytokines IL-6 and TNF-α, were not different after LPS challenge in both feeding groups. Immunohistochemical results showed no difference between GCE and placebo group after LPS challenge. Conclusion: The LPS challenge induced inflammation in the horses and ponies. Feeding a GCE for 21 days showed some potential to alter LPS-induced systemic inflammatory responses by reducing mRNA levels of the inflammatory marker IL-1β in liver tissue. Nevertheless, an overall anti-inflammatory effect by GCE was missing, since other blood and liver parameters were not affected by the GCE supplementation.:1 Introduction 2 Literature review 2.1 Inflammation 2.1.1 Acute phase reaction 2.1.1.1 Serum amyloid A 2.1.1.2 Haptoglobin 2.1.1.3 Retinol-binding protein 4 2.1.2 Transcription factor NF-κB 2.1.2.1 Pro-inflammatory cytokines (Il-1β, IL-6, TNF-α) 2.1.3 Macrophages 2.1.3.1 Cluster of differentiation 68 2.2 Stress of the endoplasmic reticulum 2.2.1 Fibroblast growth factor 21 2.2.2 Activating transcription factor 4 2.3 Oxidative stress 2.4 Polyphenols 2.4.1 Green tea 2.4.2 Curcuma 2.4.3 Bioavailability 2.4.4 Anti-inflammatory effects 2.4.5 Antioxidative effects 3 Publication 3.1 Effects of a blend of green tea and curcuma extract supplementation on lipopolysaccharide-induced inflammation in horses and ponies 4 Discussion 4.1 Effects of the LPS challenge 4.1.1 Induction of inflammation by LPS 4.1.2 Induction of ER stress and oxidative stress by LPS 4.2 Effects of the GCE supplementation 4.2.1 Anti-inflammatory effects 4.2.2 Reduction of ER stress and oxidative stress 4.2.3 Comparison of in vivo effects in equines and farm animals 4.3 Daily dosages of polyphenols 4.4 Conclusion 5 Zusammenfassung 6 Summary 7 References 8 Appendix 8.1 List of presentations as part of this thesis 8.2 Further publications 8.2.1 Published articles 8.2.2 Published conference contributions 9 Acknowledgement

Made available in DSpace on 2017-07-10T17:40:42Z (GMT). No. of bitstreams: 1 Heloisa_Maria_Formentini_tese.pdf: 1264963 bytes, checksum: d416be804a86c6ba7273d6aacedf8878 (MD5) Previous issue date: 2012-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In Brazil, the tomato is one of the vegetable species of great importance both economically and socially, however several factors are limiting its production as an example the diseases caused by nematodes of the genus Meloidogyne that limit the production in infested areas. Seeking new measures of protection and control of plant disease induced resistance is na alternative considerering that little attention has been directed to the possibility of induced resistance to root pathogens like nematodes. Therefore, this study aimed to verify the effectiveness of the chemical inducer acibenzolar-S-methyl, the biotic inductor Bacillus cereus and plant extracts of rosemary (Rosmarinus officinalis) and turmeric (Curcuma longa) in the induced resistance in susceptible and resistant tomato in plants infected with Meloidogyne incognita race 3. Two experiments were conducted simultaneously both followed the 2 x 6 factorial design with two tomato genotypes, one susceptible to M. incognita (Santa Clara) and a resistant (Ivety) and six treatments: ASM (125 mg i.a L -1 ), Bacillus cereus (6.10 7 CFU mL -1 ), rosemary 10%, turmeric 10%, water and a control (no inoculum and no spraying in the aerial part), with five replicates. Each vase with a capacity of 2 L, were filled with a mixture of soil, sand and compost previously autoclaved and homogenized in the ratio 2:2:1 and were transplanted to each one three tomato seedlings susceptible and resistant. The treatments were sprayed in the aerial part in tomato plants in all vases except the absolute control. At 72 h after the first application of treatments was carried out the inoculation of 407/100 cm 3 of J2 and eggs per vase. In the first experiment, using destructive samples of tomato treated and inoculated with M. incognita were determined the response of both genotypes to the treatments applied to the enzymatic activity of peroxidase, polyphenol oxidase, chitinase, β-1,3 glucanase and phenylalanine ammonia-lyase from roots of tomato plants that were macerated and homogenized to withdrawals in time 0 h, 24 h, 48 h, 96 h and 120 h after the first application of the treatments. In the second experiment, the variables analyzed to determine the effect of treatments on nematode population were the number of root-knots, juveniles and eggs in the soil accomplished at 56 days after the first application of the treatments that were reapplied every seven days during this period. From the results obtained it was concluded that there was a reduction in the number of root-knots in the roots of tomato plants showing no difference between the two genotypes for plants that received the treatments with acibenzolar-S-methyl, turmeric, rosemary and B. cereus. There was a reduction in the formation of root-knots in susceptible cultivar, confirming their potential in protecting the genotypes used against the attack of M. incognita. For the enzimatic activity peroxidase was the enzyme strongly associated with resistance with the highest activity in resistant genotype if compared to susceptible regardless of inducer treatment. In susceptible tomato B. cereus stood out in the induction of chitinase and peroxidase whereas for the resistant tomato rosemary induced peroxidase and polyphenol oxidase and rosemary extracts and turmeric induced chitinase enzyme to the susceptible genotype / No Brasil o tomate é uma das espécies de hortaliças de grande importância tanto no ponto de vista econômico quanto social, no entanto vários fatores são limitantes para sua produção como exemplo as doenças causadas por fitonematoides principalmente as espécies do gênero Meloidogyne que inviabilizam a produção nas áreas infestadas. Buscando novas medidas de proteção e controle de doenças de plantas a indução de resistência é uma alternativa haja vista que pouca atenção tem sido direcionada a possibilidade de indução de resistência à patógenos do sistema radicular como os fitonematoides. Assim este trabalho teve como objetivo verificar a eficácia do indutor químico acibenzolar-S-metil, do indutor biótico Bacillus cereus e de extratos vegetais de alecrim (Rosmarinus officinalis) e cúrcuma (Curcuma longa) na indução de resistência em tomateiros suscetível e resistente infectados com Meloidogyne incógnita raça 3. Foram conduzidos dois experimentos simultaneamente ambos seguiram o esquema fatorial 2 x 6 com dois genótipos de tomateiro um suscetível à M. incognita (Santa Clara) e um resistente (Ivety) e seis tratamentos: ASM (125 mg i.a L -1 ), Bacillus cereus (6.10 7 UFC mL -1 ), alecrim 10%, cúrcuma 10%, água e uma testemunha absoluta (sem inóculo e sem pulverização na parte aérea), com cinco repetições. Cada vaso, com capacidade para 2 L, foram preenchidos com a mistura de solo, areia e composto orgânico previamente autoclavados e homogeneizados na proporção 2:2:1 e para cada vaso foram transplantados três mudas de tomateiro suscetível e resistente. Os tratamentos foram pulverizados na parte aérea dos tomateiros em todos os vasos com exceção da testemunha absoluta. Às 72 h após a primeira aplicação dos tratamentos foi realizada a inoculação de 407/100 cm 3 de J2 e ovos por vaso. No primeiro experimento, utilizando amostras destrutivas de tomateiros tratados e inoculados com M. incognita foram determinadas a resposta dos dois genótipos aos tratamentos aplicados para a atividade enzimática das enzimas peroxidase, polifenoloxidase, quitinase, β-1,3 glucanase e fenilalanina amônia-liase a partir do macerado homogeneizado das raízes dos tomateiros para o tempo de coleta 0 h, 24 h, 48 h, 96 h e 120 h após a aplicação dos tratamentos. No segundo experimento, as variáveis analisadas para determinar o efeito dos tratamentos sobre a população do nematoide foram o número de galhas, juvenis e ovos presentes no solo realizado aos 56 dias após a primeira aplicação dos tratamentos que foram reaplicados a cada sete dias durante este período. A partir dos resultados obtidos concluiu-se que houve uma redução no número de galhas no sistema radicular dos tomateiros não apresentando diferença entre os dois genótipos para as plantas que receberam os tratamentos com acibenzolar-S-metil, cúrcuma, alecrim e Bacillus cereus. Houve uma redução na formação de galhas na cultivar suscetível, confirmando seu potencial na proteção dos genótipos utilizados contra o ataque do M. incognita. Para a atividade enzimática a peroxidase foi a enzima que esteve fortemente associada à resistência com a atividade superior no genótipo resistente em relação ao suscetível independentemente do tratamento indutor. No tomateiro suscetível o B. cereus destacou-se na indução de peroxidase e quitinase enquanto que para o tomateiro resistente o alecrim induziu peroxidase e polifenoloxidase e os extratos de alecrim e cúrcuma induziram a enzima quitinase para o genótipo suscetível