Development and evaluation of a fluorescent method for measuring phospholipase A2 activity and inhibition and its application in inhibitory effect of Chinese herbs

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Development and evaluation of a fluorescent method for measuring phospholipase A2 activity and inhibition and its application in inhibitory effect of Chinese herbs

 

Author: Yuen, Yiu-kwan
Title: Development and evaluation of a fluorescent method for measuring phospholipase A2 activity and inhibition and its application in inhibitory effect of Chinese herbs
Degree: M.Sc.
Year: 2008
Subject: Hong Kong Polytechnic University -- Dissertations.
Phospholipase A2.
Phospholipase A2 -- Inhibitors.
Herbs -- Therapeutic use -- China.
Medicine, Chinese.
Department: Dept. of Health Technology and Informatics
Pages: xiv, 179 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2198998
URI: http://theses.lib.polyu.edu.hk/handle/200/605
Abstract: Phospholipase A2 (PLA2) catalyzes the hydrolysis of cellular phospholipids at the sn-2 position of the triglyceride moiety, releasing arachidonic acid and lysophospholipids, and generating a family of pro-inflammatory escosanoids and platelet activating factor. Experimental and clinical evidence suggests that PLA2 may have a primary regulatory role in the development of inflammatory disorders. Studies of specific inhibitors of sPLA2 activation could play an important role in development of anti-inflammatory drugs. Therefore, development and evaluation of a sensitive and specific measurement of PLA2 activity and the effect of putative inhibitors are areas of interest in anti-inflammatory research. The primary aim of this study was to develop and evaluate a fluorimetric assay for the study of the inhibition effect of herbs as potential PLA2 inhibitors. By comprehensively reviews the existing technologies for PLA2 activity measurement, a suitable fluorescence substrate was selected. Modification of published methods was required in order to make the assay feasible, practical and able to be performed with acceptable sensitivity with the resources available within the laboratory of the Department of Health Technology & Informatics at The Hong Kong Polytechnic University. The secondary aim was to study the effect of various PLA2 inhibitors including those with known potent PLA2 inhibiting effects and extracts of eight herbs, selected for their possible anti-inflammatory (PLA2 inhibition) effects based on their reputed health effects. Finally, the fluorimetric assay developed was compared against other methods of measuring PLA2 activity, including an established isotope method, an established pH based method and a commercial spectrophotometric based method. Results have indicated that the fluorimetric assay developed was able to produce results comparable to a well established E.coli assay and a commercial assay using human enzymes with acceptable sensitivity. Some of the eight herbs tested in this study, notably produced significantly inhibitory effect on sPLA2. Others had little or no inhibitory effect, suggesting that their reported anti-inflammatory activity may act via other key enzymes relating to inflammation. In conclusion, the fluorimetric method developed has acceptable sensitivity and, in comparison with other test methods available, has the advantages of being technically simple, inexpensive, with reasonable sensitivity, reagents are stable, and the test uses an easily obtained substrate. These features make it an attractive option for testing potential anti-inflammatory effects of herbs and drugs.

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