Discovery of Novel Derivatives of Catechin Gallate with Antimycobacterial Activity from Kirkia wilmsii Engl. Extracts

<i>Background/Objectives:</i> The increase in incidences of multidrug resistance exacerbates tuberculosis-related global health challenges and underscores a call for more efforts for development of new antitubercular drugs, including the use of medicinal plants, especially those that have been used for generations by traditional healers. Despite reports of antimicrobial activity and chemical profiling of <i>Kirkia wilmsii</i> (<i>K. wilmsii</i>) extracts, chemical structures of the bioactive agents have not been elucidated. Here, we used a combination of bioactivity-guided fractionation, mass spectrometry, and nuclear magnetic resonance to purify and elucidate the chemical structure of antimycobacterial agents contained in leaf and twig extracts for <i>K. wilmsii</i>. <i>Results:</i> After overnight extraction with acetone and 90 g of dry twigs and leaves produced 5.38 g (6%) and 4.56 g (5%) of product, which displayed moderate antimycobacterial activity of 0.5 and 1 mg/mL, respectively. The antimycobacterial activity was increased six- and three-fold, respectively, after the crude extracts were subjected to solvent-solvent partitioning. Due to many bioactive fractions being obtained after silica gel chromatography purification, fraction 5 of twig extract was prioritized for further purification due to its low minimum inhibitory concentration (MIC) (0.25 mg/mL) and cytotoxicity (20%, in THP-1 cells). Sequential purification of the fraction 5 (twig extract) extracts through the C18 cartridge and high-performance liquid chromatography (HPLC) produced four fractions, which were subjected to structural elucidation. The high-resolution mass spectrometric analyses revealed that the first two eluting peaks had the same mass ion of 441.0822 <i>m</i>/<i>z</i> (M - H^-), which corresponded to catechin monogallate, and so were the last two eluting peaks, which had a mass ion of 539.0932 <i>m</i>/<i>z</i> (M - H^-), corresponding to catechin digallate. Further analyses by ¹H, ¹³C, and 2D NMR confirmed the chemical structures of compounds eluting in the first two peaks on HPLC as structural isomers of catechin 3'-monogallate and catechin 4'-monogallate (MIC not determined). Similarly, compounds eluting in the last two peaks were identified as structural isomers catechin 3'-digallate and catechin 4'-digallate, with an MIC of 250 µg/mL against <i>Mycobacterium smegmatis</i> and <i>Mycobacterium tuberculosis</i> H37Rv and an MBC of 500 μg/mL against <i>M. smegmatis</i>. <i>Conclusions:</i> To the best of our knowledge, this study is the first to report the structure of catechin 3'- and 4'-digallate, their antimycobacterial activity, and the existence of acyl migration involving galloyl 3' and 4'-hydroxyl groups of catechin ring B.