Dokument

Summary:
Inflammation is the bodies response to infection or tissue injury in order to restore and maintain homeostasis. Prostaglandin E2 (PGE-2) derived from arachidonic acid (AA), via up-regulation of cyclooxygenase-2 (COX-2) is a key mediator of inflammation and can also be induced by several other factors including stress, chromosomal aberration, or environmental factors. Targeting prostaglandin production by inhibiting COX-2 is hence relevant for the successful resolution of inflammation. Waltheria indica is a traditional medicinal plant with reported anti-inflammatory properties whose extracts have demonstrated COX-2 inhibitory activity. However, the compounds responsible for the activity were merely assumed and, in most cases, unknown or assigned to molecules, such as tiliroside, whose content in the tested extracts during the investigations was unknown. For the preparation of extracts with effective anti-inflammatory properties, characterization of these substances is vital. In this thesis, this challenge was addresses by characterizing of substances responsible for the COX-2 inhibitory activity in the extracts and the generation of a prediction models to quantify the COX-2 inhibitory activity without the biological testing. The inhibitory potential of the probes against the COX-2 enzyme was assessed with a fluorometric COX-2 inhibition assay. The results obtained demonstrated that Waltheria indica extracts inhibited the inflammatory key mediator COX-2 with the activity related to the extraction parameters governing the composition of the extract. The examinations on tiliroside activity and content in the extracts revealed that although tiliroside demonstrated COX-2 inhibitory activity in a concentration-dependent manner, the content of tiliroside in the extracts was not sufficient to contribute to the observed activity with the extracts. For the identification of contributors to the observed COX-2 inhibitory activity, an extract was separated into fractions by means of centrifugal partition chromatography (CPC) and their COX-inhibitory activity evaluated. The characterization of compounds in fractions with highest COX-2 inhibitory activity were done by high resolution mass spectrometry (HPLC-MS/MS). It was found that these fractions contained alpha-linolenic acid, linoleic acid and oleic acid, identified and reported for the first time in Waltheria indica leaf extracts. After analyzing their content in extracts obtained varying extraction parameters, it could be demonstrated that these fatty acids contribute up to 41% to COX-2 inhibition observed with Waltheria indica extract. Additional quantification of phytochemicals in the extract fractions established that substances from the group of steroidal-saponins and triterpenoid-saponins also contribute to the COX-2 inhibitory activity. Based on the content of compounds contributing to COX-2 inhibition two mathematical models were successfully developed both with a root mean square error (RMSE) = 1.6% COX-2 inhibitory activity, demonstrating a high correspondence between predicted versus observed values. The results of the predictive models further suggest that the compounds contribute to COX-2 inhibition in the order linoleic acid > alpha linolenic acid > steroidal-saponins > triterpenoid-saponins. Based on the developed mathematical models in this project, a more targeted development of extraction procedures is possible in order to obtain Waltheria indica extracts with improved anti-inflammatory properties related to the inhibition of COX-2. Furthermore, the transfer of the approach presented in this work to the prediction of other biological endpoints would be of great interest for future studies.

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