Universiẗat, Diss., 2004--Kiel.

Entsprechend den Vorarbeiten von C. Heid-Jehn1 wurde aus den Wurzelknöllchen der Pflanze Cyperus esculentus durch wässrige Extraktion ein Faktor isoliert, der als Cyperus-esculentus-Faktor (CEF) bezeichnet wurde. Die vorliegende Arbeit hatte das Ziel, die Isolierung und Reinigung zu optimieren, die Reinheit zu prüfen und die chemische Zusammensetzung näher zu charakterisieren. Des Weiteren sollte die biologische Aktivität gegen die Mykotoxine-bildenden Fusariosen im Hinblick auf eine mögliche physiologische Funktion als Bestandteil des pflanzlichen Abwehrsystems gegen Pflanzenpathogene, untersucht werden. Inbesonders sollte die Funktion des früher gefundenen Silikatanteils in die Untersuchungen aufgenommen werden. Für die Isolierung des agglutinierenden Faktors aus Cyperus esculentus erwies sich eine zweimalige Alkalisierung des Extraktes pH 6.4 mit anschließender Gelfiltration an Sephadex G-25 als vorteilhaft. Diese Methode lieferte eine Ausbeute von 81 % und einen Reinigungsfaktor von 143. Erst nach Erhöhung der Ionenstärke konnte der CEF durch hydrophobe Interaktionschromatographie weiter gereinigt werden. Diese Methode hatte nach Vereinigung der mittels eines Stufengradienten von Verunreinigungen abgetrennten relevanten Fraktionen, Gelfiltration an Sephadex G-10, Dialyse und abschließender Lyophilisation eine Ausbeute von 81 % mit einem Reinigunsfaktor von 25 zur Folge. Somit ergab sich ein Gesamtreinigungsfaktor von 3600 nach zweimaliger Alkalisierung, HIC an Phenyl Sepharose HP, Gelfiltration an Sephadex G-10 und Dialyse. Die Charakterisierung ergab, daß Humanerythrozyten unabhängig von der Blutgruppe gebunden werden, mit einer leichten Präferenz für die Blutgruppe 0. Die Agglutination wird von den Zukkern D-Glucosamin, D-Mannosamin und D-Galactosamin (Grenzkonzentration 25 mmol/ l), nicht aber von deren acetylierten Formen, sowie von den Glycoproteinen Ovomucoid und Asialoovomucoid (0.125 - 0.25 mg/ ml) inhibiert. Weitere Oligosaccharid-strukturen werden nicht von der agglutinierenden Aktivität aus Cyperus esculentus gebunden. Der Proteinanteil wurde mit den zur Verfügung stehenden Methoden zu 4.25 % bestimmt. Zu den durch Aminosäureanalyse erfaßten Aminosäuren zählen Arginin, Alanin und Phenylalanin. Der Cyperus-esculentus-Faktor enthält kaum Neutralzucker und nahezu keine Uronsäuren, aber Aminozucker. Der mittlere Aschegehalt von 43.33 % bekräftigt zunächst das Ergebnis meiner Vorgängerin, die einen organischen Anteil von 53.25 % und einen Glührückstand von 48.7 % ermittelte. Der sehr hohe Anteil an Siliciumdioxid (22 %1) konnte allerdings nicht bestätigt werden. Nach direkter Veraschung und anschließender gravimetrischer Silicatbestimmung wurde ein Silicatanteil von nur 5.3 % ermittelt. Das für die strukturelle Aufklärung durchgeführte 29Si CP MAS NMR-Spektrum läßt für das Silicium die Koordinationszahl vier mit benachbarten Sauerstoff-Atomen annehmen. Allerdings wurde dieses Resultat nur in einer von drei unabhängigen Messungen erhalten, so daß der Siliciumgehalt eher chargenabhängig von der Beschaffenheit des Bodens und weiteren Wachstumsbedingungen zu sein scheint. Das Gesamtmolekulargewicht wurde mittels Gelfiltration auf 11376 ± 1000 Da bestimmt. In der Aminosäureanalyse fehlen die chromogenen Aminosäuren Tyrosin und Tryptophan. In Übereinstimmung dazu wurde ein ungewöhnlich niedriger Absorptionskoeffizient von e1%(280 nm) = 0.148 gefunden. Die Untersuchung des CEF auf seine biologische Aktivität als Abwehrsubstanz gegen Pflanzenpathogene zeigte bei den getesteten Fusariosen Fusarium solani f. pisi, Fusarium moniliformis und Fusarium culmorum eine Hemmwirkung auf das Pilzwachstum, die nicht auf eine Chitinase-Aktivität zurückzuführen ist. Die Hemmwirkung korrelierte mit der Zunahme des Reinheitsgrades des CEF. Die agglutinierende Aktivität nach HIC, Gelfiltration und Dialyse war am wirksamsten. Die Effektivität blieb bis 42 Stunden Inkubation erhalten. Allerdings beschränkte sich die fungistatische Wirkung auf Pflanzenpathogene. Gegenüber Candida albicans trat keine Hemmung auf. Welche Struktur bzw. strukturelle Komponente für die antifungale Wirkung des Cyperus-esculentus-Faktors verantwortlich ist, bleibt allerdings weiter ungeklärt. 1. Heid-Jehn, C. (1997) Isolierung und Charakterisierung des agglutinierenden Faktors aus Cyperus esculentus und dessen antifungale Wirkung, Dissertation, Würzburg. / According to the preceding work of C. Heid-Jehn1, we isolated from the tubers of Cyperus esculentus by aqueous extraction a factor called Cyperus-esculentus-Factor (CEF). Aim of the present work was to optimize the isolation and purification, to control the purification degree and to characterize the chemical composition. Furthermore the biological activity against mycotoxin-forming fusarioses with regard to potential physiological function as constituent of the plant-defence-system against plant pathogens should be analyzed. In particular the function of the previously found silica portion should be part of the study. The isolation of the agglutinating factor from Cyperus esculentus, a once-repeated alkaline precipitation of the pH 6.4 extract, followed by gel filtration on Sephadex G-25, proved to be advantageous. This method led to a yield of 81 % and to a purification factor of 143. Attempts to purify the factor by ion-exchange-chromatography on carboxymethylcellulose did not succeed as well. First after increasing the ionic strength stepwise the CEF could be further purified by hydrophobic interaction chromatography. After pooling the relevant fractions and subsequent gel filtration on Sephadex G-10, dialysis and final freeze-drying, this method resulted in a yield of 81 % with a purification factor of 25. Thus the total purification factor was 3600 starting from the crude extract after once-repeated alkaline precipitation, HIC on Phenyl Sepharose HP, gel filtration on Sephadex G-10 and dialysis. The characterization resulted in a blood group independent agglutination of human erythrocytes with a slight preference for blood group 0. The agglutination is inhibited by D-glucosamine, D-mannosamine and D-galactosamine (concentration limit 25 mmol/ l), but neither by their acetylated forms, nor by the glycoproteins ovomucoid and asialoovomucoid (0.125 - 0.25 mg/ ml). Other oligosaccharides as far as tested are not bound by the agglutinating activity of Cyperus esculentus. The protein part was determined by the available methods to 4.25 %. The amino acids determined by amino acid analysis are arginine, alanine and phenylalanine. The Cyperus-esculentus-Factor contains hardly neutral sugars and almost no uronic acids, but amino sugars. At first the average content of 43.33 % did underline the result of my antecessor, who calculated for the organic fraction 53.25 % and an ash content of 48.7 %. The very high silica content (22 %1) however, could not be confirmed. After direct combustion and subsequent gravimetric silica determination, only 5.3 % were detected. The 29Si CP MAS NMR-spectra for the structural elucidation suggests a coordination number of four for silicon with neighbouring oxygen atoms. But this result was only obtained in one of three independent measurements, so that the amount of silicon means to be rather charge-dependent on the soil consistency and futher on the growing conditions. The total molcular weight was determined by gel filtration to be 11376 ± 1000 Da. Amino acid analysis revealed the absence of chromogenic amino acids tyrosine and tryptophan. In accordance to this, we found a exceptionally low absorption coefficient of e1%(280 nm) = 0.148. Investigations of CEF concerning its biological activity as defence substance against plant pathogens resulted in an inhibition of fungal growth for the tested fusarioses Fusarium solani f. pisi, Fusarium moniliformis and Fusarium culmorum which is not attributed to a chitinase activity. The inhibition correlated to the increase of the purification factor of CEF. The agglutinating activity after HIC, gel filtration and dialysis was the most active one. The efficiency persisted until 42 hours of incubation. The fungistatic effect, however, was limited to plant pathogens. Against Candida albicans there was no inhibition. Which structure or structural component, respectively, is responsable for the antifungal activity of the Cyperus-esculentus-Factor remains unsettled. 1. Heid-Jehn, C. (1997) Isolierung und Charakterisierung des agglutinierenden Faktors aus Cyperus esculentus und dessen antifungale Wirkung, Dissertation, Würzburg.

Neem cake is a product of the cold pressing from the neem kernels to obtain neem oil. Bio-active substances from neem cake extracted solutions were evaluated for their potential to control the root knot nematodes and other pests of plants. In this study different concentrations of the solution extracted from neem cake was tested against the second stage juveniles of the plant parasitic nematode Meloidogyne spp. and four phytopathogenic fungi: Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytopthora capsici. Toxicity of neem cake extractions is represented by the EC50 value for the second-stage juvenile (J2) of Meloidogyne spp. and the four phytopathogenic fungi via Probit analysis. A 5% dilution of the solvent extracting from neem cake already caused 100% larval mortality after 24 hours exposure. Undiluted neem cake extraction effectively inhibited the growth of the four phytopathogenic fungi. The EC50 value of neem cake on J2-larvae of Meloidogyne nematode and on the fungi Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytophthora capsici was 0.51, 0.74, 0.30, 0.51 and 4.33%, respectively. / Bánh dầu neem là sản phẩm của quá trình ép nhân hạt neem để lấy dầu. Các hoạt chất sinh học từ dịch chiết bánh dầu neem đã được đánh giá có tiềm năng lớn trong phòng trừ tuyến trùng nốt sưng và các loài dịch hại khác của nhiều loại cây trồng. Trong nghiên cứu này các nồng độ dịch chiết khác nhau của bánh dầu neem đã được thử nghiệm khả năng diệt tuyến trùng (ấu trùng tuổi 2 thuộc giống Meloidogyne spp.) và ức chế 4 loài nấm bệnh như: Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. và Phytopthora capsici. Độc tính của dịch chiết bánh dầu neem được biểu diễn bởi giá trị EC50 đối với ấu trùng tuổi 2 của tuyến trùng Meloidogyne spp. và các loài nấm bệnh thông qua phân tích Probit. Dịch chiết bánh dầu neem ở nồng độ 5% đã làm chết 100% cá thể IJ2 của Meloidogyne spp sau 24 giờ phơi nhiễm. Dịch nguyên chất bánh dầu neem ức chế cả 4 loài nấm bệnh. Giá trị EC50 của bánh dầu neem lên ấu trùng tuổi 2 của Meloidogyne spp và các loài nấm bệnh Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytophthora capsici tương ứng là 0.51, 0.74, 0.30, 0.51 và 4.33%.

Neem cake is a product of the cold pressing from the neem kernels to obtain neem oil. Bio-active substances from neem cake extracted solutions were evaluated for their potential to control the root knot nematodes and other pests of plants. In this study different concentrations of the solution extracted from neem cake was tested against the second stage juveniles of the plant parasitic nematode Meloidogyne spp. and four phytopathogenic fungi: Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytopthora capsici. Toxicity of neem cake extractions is represented by the EC50 value for the second-stage juvenile (J2) of Meloidogyne spp. and the four phytopathogenic fungi via Probit analysis. A 5% dilution of the solvent extracting from neem cake already caused 100% larval mortality after 24 hours exposure. Undiluted neem cake extraction effectively inhibited the growth of the four phytopathogenic fungi. The EC50 value of neem cake on J2-larvae of Meloidogyne nematode and on the fungi Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytophthora capsici was 0.51, 0.74, 0.30, 0.51 and 4.33%, respectively. / Bánh dầu neem là sản phẩm của quá trình ép nhân hạt neem để lấy dầu. Các hoạt chất sinh học từ dịch chiết bánh dầu neem đã được đánh giá có tiềm năng lớn trong phòng trừ tuyến trùng nốt sưng và các loài dịch hại khác của nhiều loại cây trồng. Trong nghiên cứu này các nồng độ dịch chiết khác nhau của bánh dầu neem đã được thử nghiệm khả năng diệt tuyến trùng (ấu trùng tuổi 2 thuộc giống Meloidogyne spp.) và ức chế 4 loài nấm bệnh như: Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. và Phytopthora capsici. Độc tính của dịch chiết bánh dầu neem được biểu diễn bởi giá trị EC50 đối với ấu trùng tuổi 2 của tuyến trùng Meloidogyne spp. và các loài nấm bệnh thông qua phân tích Probit. Dịch chiết bánh dầu neem ở nồng độ 5% đã làm chết 100% cá thể IJ2 của Meloidogyne spp sau 24 giờ phơi nhiễm. Dịch nguyên chất bánh dầu neem ức chế cả 4 loài nấm bệnh. Giá trị EC50 của bánh dầu neem lên ấu trùng tuổi 2 của Meloidogyne spp và các loài nấm bệnh Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytophthora capsici tương ứng là 0.51, 0.74, 0.30, 0.51 và 4.33%.