Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Jalan UMS, 88400, Kota Kinabalu, Sabah, Malaysia.
Department of Food and Nutrition, Institute of Food and Nutrition Science, Eulji University, Seongnam, 13135, Republic of Korea.
J Microbiol. 2021 Feb;59(2):164-174. doi: 10.1007/s12275-021-0551-8. Epub 2020 Dec 23.
There have been relatively few studies which support a link between Ganoderma boninense, a phytopathogenic fungus that is particularly cytotoxic and pathogenic to plant tissues and roots, and antimicrobial compounds. We previously observed that liquid-liquid extraction (LLE) using chloroformmethanol-water at a ratio (1:1:1) was superior at detecting antibacterial activities and significant quantities of antibacterial compounds. Herein, we demonstrate that antibacterial secondary metabolites are produced from G. boninense mycelia. Antibacterial compounds were monitored in concurrent biochemical and biophysical experiments. The combined methods included high performance thin-layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopy. The antibacterial compounds derived from mycelia with chloroform-methanol extraction through LLE were isolated via a gradient solvent elution system using HPTLC. The antibacterial activity of the isolated compounds was observed to be the most potent against Staphylococcus aureus ATCC 25923 and multidrug-resistant S. aureus NCTC 11939. GC-MS, HPLC, and FTIR analysis confirmed two antibacterial compounds, which were identified as 4,4,14α-trimethylcholestane (m/z = 414.75; lanostane, CH) and ergosta-5,7,22-trien-3β-ol (m/z = 396.65; ergosterol, CHO). With the aid of spectroscopic evaluations, ganoboninketal (m/z = 498.66, CHO), which belongs to the 3,4-seco-27-norlanostane triterpene family, was additionally characterized by 2D-NMR analysis. Despite the lack of antibacterial potential exhibited by lanostane; both ergosterol and ganoboninketal displayed significant antibacterial activities against bacterial pathogens. Results provide evidence for the existence of bioactive compounds in the mycelia of the relatively unexplored phytopathogenic G. boninense, together with a robust method for estimating the corresponding potent antibacterial secondary metabolites.
目前，仅有少量研究表明，博宁栓菌（Boninense），一种对植物组织和根部具有较强细胞毒性和致病性的植物病原菌，与抗菌化合物之间存在关联。我们之前观察到，使用氯仿-甲醇-水（1:1:1）进行液-液萃取（LLE）在检测抗菌活性和大量抗菌化合物方面具有优势。在此，我们证明了博宁栓菌菌丝体可产生抗菌次生代谢产物。在同步的生化和生物物理实验中监测了抗菌化合物。这些联合方法包括高效薄层层析（HPTLC）、气相色谱-质谱（GC-MS）、高效液相色谱（HPLC）、傅里叶变换红外（FTIR）和核磁共振（NMR）光谱。通过 LLE 用氯仿-甲醇提取的菌丝体产生的抗菌化合物，通过 HPTLC 采用梯度溶剂洗脱系统进行分离。分离得到的化合物对金黄色葡萄球菌 ATCC 25923 和多药耐药金黄色葡萄球菌 NCTC 11939 的抗菌活性最强。GC-MS、HPLC 和 FTIR 分析证实了两种抗菌化合物，它们被鉴定为 4,4,14α-三甲基胆甾烷（m/z = 414.75；麦角甾烷，CH）和麦角甾-5,7,22-三烯-3β-醇（m/z = 396.65；麦角固醇，CHO）。在光谱评估的帮助下，还通过 2D-NMR 分析对属于 3,4-裂环-27-降胆甾烷三萜类的 ganoboninketal（m/z = 498.66，CHO）进行了表征。尽管麦角甾烷没有表现出抗菌潜力；但麦角固醇和 ganoboninketal 均对细菌病原体表现出显著的抗菌活性。结果为相对未探索的植物病原菌博宁栓菌菌丝体中存在生物活性化合物提供了证据，并提供了一种估计相应有效抗菌次生代谢产物的强大方法。
