Methodological Modifications for Crude Thymus tiflisiensis Leaf Extract Screening for In Vitro Anti-Inflammatory and Antibacterial Activities
DOI:
https://doi.org/10.56580/GEOMEDI80Keywords:
Thymus tiflisiensis, BSA denaturation assay modification, broth microdilution, disk diffusion comparison, methodological optimizationAbstract
Thymus tiflisiensis Klokov & Des.-Shost. (Lamiaceae) is a medicinal plant traditionally used in the Caucasus region for inflammatory conditions and infectious diseases. However, standard in vitro assays often require methodological modifications when applied to crude plant extracts due to interference from coloured phytochemicals, tannins, and other matrix components. This study aimed to: (1) optimize the bovine serum albumin (BSA) denaturation assay for crude plant extracts by determining the optimal buffer pH and denaturation temperature, and (2) compare the broth microdilution (liquid culture) method versus disk diffusion (agar) method for antibacterial screening of plant extracts, and to demonstrate the superiority of liquid culture over agar diffusion for MIC determination. Leaves of T. tiflisiensis were extracted by maceration using 80% methanol. For BSA denaturation optimization, buffer pH (6.0-7.4) and denaturation temperature (65 °C, 70 °C, 75 °C) were systematically evaluated using a fixed BSA concentration (1 %). Optimal conditions were identified as pH 6.4 and incubation at 37 °C for 20 minutes, followed by heating at 70 °C for an additional 20 minutes. For antibacterial screening, broth microdilution is unequivocally superior to disk diffusion for crude plant extracts, as it eliminates diffusion-related artifacts and provides quantitative MIC data required for meaningful comparison. These optimized protocols provide a validated framework for ethnopharmacological screening of Caucasian medicinal plants.
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