Discovery of antimicrobial compounds from Hakka Traditional Medicine using high resolution mass spectrometric approaches

Bacteria and fungi continue to cause serious infections world-wide, whose treatment by existing antibiotics is increasingly hampered by the emergence of drug-resistant strains. The development of new antimicrobial drugs must therefore be a focus of new drug research and development. This requires the discovery of new active compounds (preferably with a novel mechanism of antimicrobial action) and elucidation of the molecular mechanism of drug resistance. As a kind of folk medicine, Hakka traditional medicine (HTM) originated from the migration of Hakka people, who belongs to a branch of the Han nationality, and are not an ethnic minority. The Hakkas moved South from the Central Plains, and settled down in South China. After this southward migration, they could not easily access many resources of the Traditional Chinese Medicine (TCM) used in the North for disease prevention and treatment, such as Glycyrrhizae Radix et Rhizoma, Rhei Radix et Rhizoma, Astragali Radix, Angelicae sinensis Radix, Ginseng Radix et Rhizoma, etc. Therefore, the Hakkas turned to developing and utilizing local medicine resources, and gradually formed a Hakka medical model with regional characteristics. Therefore, HTM is considered as a characteristic Chinese folk medicine, developed by the Hakka people during the long migration process under the guidance of the theory of TCM. However, the systematic research and development of HTM resources is still largely non-existent. Hence, the study of HTM is of great significance and can provide a valuable resource for the discovery of new pharmacological substances. This proposal is intended to systematically research and rationally develop the Chinese medicine resources in the Hakka area, and to provide a scientific basis for local medical economic development. The lab of Prof. Walter Luyten uses ethnopharmacological information to select plants with demonstrated effectiveness for the treatment of certain diseases of interest. So far, with the help of research colleagues at Tsinghua University in Beijing (Prof. Guoan Luo, etc.) and Gannan Medical University in Ganzhou (Prof. Hao Huang etc.), and also in collaboration with other KU Leuven research groups, they have studied infectious diseases as well as epilepsy, and are expanding into parasitic diseases and ageing. Presently available antiviral and antibacterial bioassays include: hepatitis B virus, influenza virus, herpesvirus, Coxsackie virus, and respiratory syncytial virus (in collaboration with the Rega Institute), Bacillus cereus, Bacillus subtilis, Lactococcus lactis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Salmonella typhimurium, Shigella flexneri, and Yersinia enterocolitica, etc., as well as molecular, cell and animal models in drug evaluation, with additional assays under development. The previous collaboration with Prof. Hao Huang focused on antibacterial activities of TCM plant extracts. The extracts of 60 medicinal plants were tested on 11 species of bacteria. Many extracts showed activity on one or more bacterial strains, and one extract was selected for further study. Some extracts, such as Saxifraga stolonifera, Taraxacum mongolicum, Solanum nigrum, Syzygium aromaticum, etc., prepared with different solvents, showed reproducible strong inhibitory activity in liquid culture on Staphylococcus aureus for all wild type and clinical drug-resistant strains tested. This suggests that the active compound(s) may have different target(s) and mechanism(s) compared with currently used antibiotics. This proposal will focus on compounds from HTM showing activity on bacteria, and the mechanism by which resistance to these compounds can be generated.  Firstly, we selected around 100 plants (see below) used in Hakka regions as having some anti-bacterial effects. Small-scale extracts will be prepared from 1 g of botanical material in four solvents: water, ethanol, acetone and hexane. These extracts will be tested in anti-bacterial assays on the above-mentioned bacteria. Then the activity-based separation, purification and identification will be carried out by using various chromatographic methods, followed by a combination of mass spectrometry and NMR analysis of purified compounds. Secondly, the mechanism of action of purified active compounds will be studied by selecting resistant bacteria and sequencing their genome. Specific drug-resistant mutants will be selected by repeatedly sub-culturing bacteria grown in the presence of increasing concentrations of an active compound. The genomic DNA of resistant strains and the wild type strain will be sequenced and compared. Thus, we will find the key genes relevant for drug resistance, and reveal the mechanism of antimicrobial action by using genomic methods. This research will not only reveal the anti-bacterial compound(s) of HTM, but also find new target(s) and mechanism for curing diseases caused by these bacteria.