Entwicklung einer biosensorischen Durchfluss-Messmethode für die Qualitätskontrolle von pflanzlichen Arzneimitteln und gesundheitsrelevanten Nahrungsbestandteilensowie Untersuchungen zum Einfluss von ionisierender Strahlung auf Cysteinsulfoxide und Proteine

Im Sinne der präventiven Medizin und angesichts des hohen Bedarfs an präparierten Naturprodukten stehen Heilpflanzen zunehmend höher im Kurs. In der vorliegenden Arbeit wurde deshalb eine spezifische Bestimmungsmethode (FIA-Biosensor) für die heilsam wirkenden S-haltigen Aminosäuren wie die Cystein...

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Bibliographic Details
Main Author: Tannous, Fadi
Contributors: Keusgen, Michael (Professor Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2008
Pharmazeutische Chemie
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Table of Contents: For the purposes of the preventive medicine and in view of the high need of prepared natural products, medicinal plants are becoming increasingly popular. Therefore, in the present work, a specific method for determination (biosensor FIA) was developed for the wholesome S-containing amino acids like the Cysteine sulfoxides of the genus Allium. With the watery extract of the tests it was possible to execute measurements at minute intervals and make a statement in the ppm-range about the whole Cysteine sulfoxides in the test. Garlic as a carrier of valuable natural substances and egg white as a healthy donor of proteins, both in pure form or as an ingredient of a preparation, with their high quality components are in the focus of this work. To gain access to the relevant substances in recent years, a competition was started in the context of instrumental analysis. The fastest and most environmentally friendly method has been called “biosensor”. Accordingly, in this study, a biosensor was developed, which - for the first time – operated on a sensitive, fluorescence-specific analysing and measuring technique (FIA Biosensor), with a sufficient sensitivity to a detection limit of 0.9 μ M NH3 ± 2%. Hereby a possibility of scanning was found tracking the entire preliminary stage of S-Alk (en) yl-L-Cysteine sulfoxides as an evidence of the value of medical plants containing Cysteine sulfoxides. These precursors have recently been demonstrated in mushrooms as well. For the development of the FIA –Biosensor, the biochemical occurrences released by the squashing of garlic were simulated and implemented in a technical apparatus. Thus the enzymatic conversion (C-S- Lyase) of the non-proteinogene amino acids takes place (Cysteine sulfoxides as precursors, represented by Alliin, Isoalliin, Propiin, Methiin) in the strongly smelling and irritant substance of Cystein as well as in other products like Pyruvates and Ammonia. Ammonia is released under the effect the Alliinase [EC 4.4.1.4] at a ratio of 1:1 to the amount of the occuring Cysteine sulfoxides in the investigated plant. Because of that Ammonia was used as a key for hunting up the Cysteine sulfoxides in the FIA-biosensor development. This enzymatic reaction was made possible for the Biosensor due to a buffer and Carrier system, which optimized the alkaline medium (pH 10.5) and transferred the released Ammonia into a detectable fluorescent derivative. The enzyme reactor had been developed particularly for this purpose, was created by the immobilization and optimization of the enzyme (diluted at the ratio of 1:10) on the substrate (Concanavalin Agarose/Con A). The arrangement took place in a conveying technique (FIA). Within the 3 - to 4-minute measurement, it was possible to determine the entire content of Cysteine sulfoxides in aqueous (40 µl as sample volumes) plant extract. Lengthy steps such as pre-derivatization processes and incubations, which are usually used with the HPLC or comparable analysing systems, had become superfluous. After the further optimization and after connecting the system to an autosampler, the entire content of Cysteine sulfoxides (CS) of dozens or even hundreds of samples could be analyzed reliably in unbeatable time and arranged in a chemo-taxonomical data base. the change of CS through ionising radiation (beta radiation 5-10 MeV) could be proved with the FIA sensor for the first time due to the work on hand. Sulfurous proteins such as CS are considered as very sensitive to radiation. To that extent it was possible, in particular in the unprocessed variants of garlic (i.e. normal fresh garlic cloves), to obtain a significant result with FIA sensor starting from 1 kGy, whereby with 5 kGy and 3.5 kGy 50% and 70% of CS-loss could be noted respectively. The other subject was egg white as a healthy protein donor. For radiation-induced changes, the varieties of egg white were examined with two developed techniques with the aim to distinguish the irradiated samples. developed: 1) capillary electrophoresis (CE). 2) MIR-ATR spectroscopy. With CZE, it was possible to track significantly under UV detection some changes in liquid egg white caused by radiation. In particular the sensibility of Lysozyme (MW: 14,3) was detected, whereby a reduction of 70% of the absorption was noted with 3 kGy. Likewise other undefined proteins, which have longer migration times than Lysozyme, namely Ovalbumin and Ovomocoid (over 76 k Dalton), showed a similarly important decrease in their UV absorption. In addition, a relatively proportional dependence of the reduction of the UV absorption with rising doses was noted. A significant distinction of the irradiated liquid, fresh egg white could be reached through CZE-LIF. Around 70% of fluorescence reduction with 3 kGy of the initial value was registered, which can be considered as a decisive new significant knowledge and indication. On the contrary, it was not possible to recognize any change in the dry varieties, not even with 10 kGy. This is an indication for the fact that desired effects of irradiation such as the reduction of the microbial load and the resulting improvement in durability of dry varieties can be applied softly with less changes in proteins. With the aid of CGE separation, a new peak within the range of 15 to 23 kDalt could be discovered after the irradiation with 3 kGy. for fresh liquid egg white. The new peak increased with rising dose, in particular with 3; 4 kGy up to 100% of his value with 1 kGy, and was detected with SDS more clearly than with native CGE. MIR-ATR method. The different pre-treatments of the samples lead to clearly different MIR-spectra: The spectra of the pasteurized samples differed from those of the untreated ones and those irradiated with 10 kGy. In particular within the range of the NH-oscillations with 3600 to 3200 cm-1 and within the finger print range at 1390 as well as at 1278 and 1252 cm-1, differences are recognizable.