The continuous re-isolation of the known and non-applicable compounds that is time-consuming and wasting resources is still a critical problem in the discovery of bioactive entities from natural resources. To efficiently address the problem, high performance liquid chromatography-diode array detector-microfractionation (HPLC-DAD-microfractionation) guided by disk agar diffusion assay was developed, and the active compounds were further identified using the tandem mass spectrometry (MS/MS)-based molecular networking. Of 150 fungal strains screened, the methanolic extracts of Phoma herbarum PPM7487, Cryptosporiopsis ericae PPM7405, and Albifimbria verrucaria PPM945 exhibited potent antimicrobial activity against Candida albicans SC5314 and Cryptococcus neoformans H99 in the preliminary agar diffusion assay. The concept of OSMAC (one strain many compounds) was employed in the fungal cultures in order to enrich the diversity of the 2nd metabolites in this study. HPLC coupled with off-line bioactivity-directed profiling of the extracts enabled a precise localization of the compounds responsible for the conspicuous antimicrobial activity. The purified active compounds were identified based mainly on MS/MS database, and further supported by 13C nuclear magnetic resonance (NMR) spectral data compared to the literatures. In addition to nineteen known compounds, a new trichothecene derivative 1, namely trichoverrin D, was isolated and identified through this protocol. The antifungal activities of all the pure isolates were evaluated, and the structure activity relationships were also inferred. This report has demonstrated the combination of HPLC microfractination and MS/MS coupled by NMR spectral dereplication for speeding up the antimicrobial natural products discovery process.

中文翻译：

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通过串联 MS 和 NMR 数据库有效鉴定真菌抗菌原理

对已知和不适用的化合物进行连续重新分离既耗时又浪费资源，这仍然是从自然资源中发现生物活性实体的关键问题。为了有效解决这一问题，开发了以盘琼脂扩散法为指导的高效液相色谱-二极管阵列检测器-微分馏（HPLC-DAD-microfractionation），并使用串联质谱（MS/MS）进一步鉴定活性化合物-基于分子网络。在筛选的 150 株真菌菌株中，Phoma herbarum PPM7487、Cryptosporiopsis ericae PPM7405 和 Albifimbria verrucaria PPM945 在初步琼脂扩散试验中表现出对白色念珠菌 SC5314 和新型隐球菌 H99 的有效抗菌活性。在真菌培养中采用了 OSMAC（一种菌株多种化合物）的概念，以丰富本研究中第二种代谢物的多样性。HPLC 与提取物的离线生物活性定向分析相结合，能够精确定位负责显着抗菌活性的化合物。纯化的活性化合物主要基于 MS/MS 数据库进行鉴定，并通过 13C 核磁共振 (NMR) 光谱数据与文献进行比较进一步支持。除了 19 种已知化合物外，还通过该协议分离和鉴定了一种新的单端孢菌素衍生物 1，即 trichoverrin D。评价了所有纯菌株的抗真菌活性，并推断了构效关系。