High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

Summary

We describe a general protocol that integrates high-resolution mass spectrometry analysis and molecular docking in traditional Chinese medicine research.

Abstract

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.

The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

Introduction

Traditional Chinese medicine (TCM) has been clinically practiced in China for thousands of years, and it plays a vital role in maintaining the health of Chinese people¹. The composition of TCM is diverse and complex, and TCM has been widely reported in many qualitative studies focusing on the chemical composition². The chemical components in TCM can be roughly divided into the following categories such as alkaloids, organic acids, phenylpropanoids, coumarins, lignans, quinones, flavonoids, terpenoids, triterpenoid saponins, steroid saponins, cardiac glycosides, and tannins³. Given the large number....

Protocol

1. Sample pretreatment

1. Wash the whole herb of BFH in pure water until there are no visible deposits and impurities. Place the clean BFH in a dish and then place it in the oven (Figure 1B). Set the oven to 50 °C for 24 h.
   NOTE: The entire plant of BFH was collected in Sichuan Province, China.
2. Crush dried BFH in a high-speed multifunctional crusher. Transfer the coarse powder into a 50-mesh sieve (Figure 1C). Coll.......

Discussion

Besides water decoction¹², organic solvent extraction is another common method of TCM pretreatment¹³. According to the principle of similar phase dissolution, numerous components have been extracted by the combination of various organic solvents¹⁴. Ultrasonic-assisted extraction is one of the main methods used to obtain components in TCM¹⁵. Supercritical carbon dioxide extraction is good for extracting certain kinds of substan.......

Materials

Name	Company	Catalog Number	Comments
chloroform	Sinopharm Chemical ReagentCo., Ltd	CAS 67-66-3
ethanol	ChuandongChemical	CAS 64-17-5
ethyl acetate	ChuandongChemical	CAS 141-78-6
liquid chromatograph	Waters	ACQUITY Class 1 plus
MassLynx	Waters	V4.2	MS control software
n-butyl alcohol	ChuandongChemical	CAS 71-36-3
petroleum ether	ChuandongChemical	CAS 8032-32-4
Quadrupole time-of-flight mass spectrometry	Waters	SYNAPT XS
UNIFI	Waters		Data analysis software