Bioassay-Guided Interpretation of Antimicrobial Compounds in Kumu, a TCM Preparation From Picrasma quassioides' Stem via UHPLC-Orbitrap-Ion Trap Mass Spectrometry Combined With Fragmentation and Retention Time Calculation.

Haibo Hu,Dan Sun,Alokesh Kumar Ghosh,Ajmal Khan,Walter Luyten,Jinnian Peng,Changling Hu

doi:10.3389/fphar.2021.761751

Abstract

The stem of Picrasma quassioides (PQ) was recorded as a prominent traditional Chinese medicine, Kumu, which was effective for microbial infection, inflammation, fever, and dysentery, etc. At present, Kumu is widely used in China to develop different medicines, even as injection (Kumu zhusheye), for combating infections. However, the chemical basis of its antimicrobial activity has still not been elucidated. To examine the active chemicals, its stem was extracted to perform bioassay-guided purification against Staphylococcus aureus and Escherichia coli. In this study, two types of columns (normal and reverse-phase) were used for speedy bioassay-guided isolation from Kumu, and the active peaks were collected and identified via an UHPLC-Orbitrap-Ion Trap Mass Spectrometer, combined with MS Fragmenter and ChromGenius. For identification, the COCONUT Database (largest database of natural products) and a manually built PQ database were used, in combination with prediction and calculation of mass fragmentation and retention time to better infer their structures, especially for isomers. Moreover, three standards were analyzed under different conditions for developing and validating the MS method. A total of 25 active compounds were identified, including 24 alkaloids and 1 triterpenoid against S. aureus, whereas only β-carboline-1-carboxylic acid and picrasidine S were active against E. coli. Here, the good antimicrobial activity of 18 chemicals was reported for the first time. Furthermore, the spectrum of three abundant β-carbolines was assessed via their IC50 and MBC against various human pathogens. All of them exhibited strong antimicrobial activities with good potential to be developed as antibiotics. This study clearly showed the antimicrobial chemical basis of Kumu, and the results demonstrated that HRMS coupled with MS Fragmenter and ChromGenius was a powerful tool for compound analysis, which can be used for other complex samples. Beta-carbolines reported here are important lead compounds in antibiotic discovery.

Highlights

A previous study showed that the extract of total alkaloids exhibited antimicrobial activities against Streptococcus hemolytic-β, Staphylococcus aureus, Shigella castellani, Bacillus subtilis subsp, and Sporosarcina (Meng, 2007)
The results show that Kumu extracts can inhibit five G+, four G– bacteria, and one fungus (Figure 1), in which Kumu exhibited strongest activity against Staphylococcus aureus (IC50 275 μg/ml)
The results show that methanol is the best solvent to extract Kumu for its antimicrobial activity, and it was used to perform the large-scale extracting for bioassay guided purification work

Summary

Introduction

Don) Benn, a prominent medicinal herb from southern and eastern Asia, named “Kumu,” “Kudanmu,” “Shanxiongdan,” and “Kupizi” in China due to its extremely bitter and lasting taste, is a deciduous tree of the Simaroubaceae family. It was described with a scientific name in 1884 (Flora of China Editorial Committee, 1997; Flora of China Editorial Committee, 2008), but as a medicine, its stem in slices or powder was first recorded as “Shanxiongdan” in Xin Yi Xue (AD 1972) for the treatment of dysentery, infections of the biliary tract, and burns and wounds (Medical Team of, 1972; Chinese Materia Medica Editoral Committee of National Administration of TCM, 1999). In Korea and Japan, the heartwood of PQ, named ‘Picrasma wood’, is used as a herbal drug, consisting of chips, slices, or short pieces of wood (Ministry of Food and Drug Safety, 2012; The ministry of health and labour and welfare, 2016)

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Conclusion