Antibacterial activity and mechanisms of action of the semi-purified fractions from melaleuca cajuputi leaves against selected bacterial strains

• Main Author: Musa, Isah
• Format: Thesis
• Language: English
• Published: 2024
• Subjects: R Medicine; RA440-440.87 Study and teaching. Research; RC109-216 Infectious and parasitic diseases
• Online Access: http://eprints.usm.my/61007/1/ISAH%20MUSA-FINAL%20THESIS%20P-SKD002321%28R%29-E.pdf; http://eprints.usm.my/61007/
• Institution: Universiti Sains Malaysia

The increasing incidence of bacterial infections and the rapid spread of antimicrobial resistance underscores the need to find novel alternative medications from natural sources, especially medicinal plants. Thus, this study aimed to investigate the antibacterial activities of Melaleuca cajuputi leaf extract and unveil the possible antibacterial mechanisms of the most potent semi-purified fractions against selected bacterial strains. The mineral content in M. cajuputi leaf was analyzed using inductively coupled plasma-mass-spectrometry (ICP-MS). Methanolic, ethanolic, and aqueous extracts were obtained by cold maceration. Subsequently, the most potent crude extract was fractionated to obtain semi-purified fractions by bioassay-guided fractionation technique. The antibacterial activity of the crude extracts and semi-purified Melaleuca fractions (MFs) was evaluated using a broth microdilution assay. Ultraviolet-visible (UV-Vis), Fourier transform infrared (FTIR) spectroscopies, and gas chromatography-mass spectrometry (GC-MS) were employed to identify the chemical compositions of the most potent extract and semi-purified MFs. Mechanisms of action of the most potent MFs were investigated using time-kill assay, cell morphology examination, and in-silico molecular docking studies. The toxicity profile was evaluated using the brine shrimp lethality test (BSLT). Macronutrients such as potassium (7182.042 mg/kg), sodium (3895.795 mg/kg), calcium (3730.259 mg/kg), and trace elements including iron (89.394 mg/kg), manganese (57.070 mg/kg), and zinc (51.626 mg/kg) were detected in M. cajuputi leaf extract. The antibacterial assays demonstrated that MF2c and MF2d were the most potent, with minimum inhibitory concentration (MIC) values ranging from 0.13 mg/mL to 0.25 mg/mL and 0.063 mg/mL to 0.25 mg/mL, respectively. The bioactive compounds identified in MF2c were β-eudesmol (71.96%), α-eudesmol (18.83%), and γ-eudesmol (9.21%). Meanwhile, 2-isopropyl-10-methylphenanthrene (83.09%), 10-methylanthracene-9-carboxaldehyde (10.95%), trimethyl gallic acid (2.60%), methyl-lathodoratin (2.10%), and methoxyamine (0.28%) were identified in MF2d. Time-kill assay revealed that MF2c and MF2d exhibited concentration-dependent bactericidal effects against the tested bacterial strains. The scanning electron micrographs of the treated bacteria showed apparent cell membrane damage characterized by abnormal cell elongation, shrinkage, and organic debris on the cell surfaces. Furthermore, the in-silico molecular docking analysis revealed that 2-isopropyl-10-methylphenanthrene had the highest binding propensity against DNA-dependent RNA polymerase, D-alanyl transferase, DNA gyrase, and dihydropteroate synthase, with docking energy scores of -8.4, -6.9, -6.5, and -6.1 kcal/mol respectively. Based on the toxicity results, M. cajuputi methanolic extract (LC50 781 μg/mL showed mild toxicity, whereas MF2c (LC50 6621 μg/mL) and MF2d (LC50 1165 μg/mL) were non-toxic. In conclusion, the semi-purified MFs showed remarkable antibacterial effects and were non-toxic. The findings hold promise for developing alternative therapeutic strategies to combat bacterial infection