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 numbers of unknown components and indistinguishable isomers in TCM, the separation and analysis of the desired chemical components are important subjects for the fundamental research of TCM4.
Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) has been applied to analyze substances in traditional Chinese medicine (TCM), which can be separated by ultra-high-performance liquid chromatography5,6. The high resolution of MS can provide extensive ion information, which is used for database analysis with error less than 5 ppm7. After turning on the collision energy, the secondary MS mode can obtain secondary fragment ions, whose intensity and number are affected by the magnitude of energy8.
Gynura bicolor DC. (BFH), a perennial stemless herb widely used for medicine and food (Figure 1A), is a rare and endangered plant unique to China9. BFH has abundant anthocyanins, polyphenols, flavonoids, and strong antioxidant capacity10. BFH has medicinal effects including clearing heat, cooling blood, moistening the lung, relieving cough, dispersing stasis, relieving swelling, relieving summer heat, and eliminating heat. Few studies have focused on the chemical composition of BFH11. Polyphenols and flavonoids contain numerous isomers, which make the identification of the complex compounds in BFH difficult. A universal method for the identification of chemical components must be developed, which can be applied to all kinds of TCM. This study aimed to report a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by the National Natural Science Foundation of China (82104881), Inheritance and Innovation Team of TCM Treatment of Immune Diseases, Chongqing Medical Scientific Research Project (Joint project of Chongqing Health Commission and Science and Technology Bureau) (2022DBXM007), A special project for performance incentive and guidance of Chongqing Scientific Research Institute (cstc2022jxjl120005), A special project for Chongqing Postdoctoral Science Foundation (2022CQBSHTB3035), Senior Medical Talents Program of Chongqing for Yong and Middle-aged, the Program for Scientific Institutions of Chongqing (independent research project No.2022GDRC015).
Materials
| 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 |
References
- Lin, P., et al. Qualitative and quantitative analysis of the chemical profile for gualou-xiebai-banxia decoction, a classical traditional chinese medicine formula for the treatment of coronary heart disease, by uplc-q/tof-ms combined with chemometric analysis. J Pharm Biomed Anal. 197, 113950 (2021).
- Hui, D., et al. Uplc-q-tof/ms-based metabolomic studies on the toxicity mechanisms of traditional chinese medicine chuanwu and the detoxification mechanisms of gancao, baishao, and ganjiang. Chinese J Nat Med. 13 (9), 687-698 (2015).
- Newman, D. J. Modern traditional chinese medicine: Identifying, defining and usage of tcm components. Adv Pharmacol. 87, 113-158 (2020).
- Liu, S., Yi, L. Z., Liang, Y. Z. Traditional chinese medicine and separation science. J Sep Sci. 31 (11), 2113-2137 (2008).
- Yuan, L., et al. Rapid classification and identification of complex chemical compositions in traditional chinese medicine based on uplc-q-tof/ms coupled with data processing techniques using the kudiezi injection as an example. Anal Methods. 7 (12), 5210-5217 (2015).
- Wang, F., et al. Hplc coupled with chemical fingerprinting for multi-pattern recognition for identifying the authenticity of clematidis armandii caulis. JoVE. (189), e64690 (2022).
- Gao, Y., et al. Ultra high-performance liquid chromatography-mass spectrometry and self-established database analysis of chinese herbal medicine components. JoVE. (201), e66091 (2023).
- Fu, X., et al. Standardized identification of compound structure in tibetan medicine using ion trap mass spectrometry and multiple-stage fragmentation analysis. JoVE. (193), e65054 (2023).
- Wang, Y., et al. Comparison of morphological and physiological characteristics in two phenotypes of a rare and endangered plant, begonia fimbristipula hance. Photosynthetica. 54, 381-389 (2016).
- Zhang, P., et al. Response of population canopy color gradation skewed distribution parameters of the rgb model to micrometeorology environment in begonia fimbristipula hance. Atmosphere. 13 (6), 890 (2022).
- Yu, L., Ning, L., Youzhen, D. Studies on microwave extraction of the pigment from begonia fimbristipula hance. Guangdong Trace Elements Science. 11 (10), 60-63 (2004).
- Xiao, Q., et al. Novel fusion protein consisting of metallothionein, cellulose binding module, and superfolder gfp for lead removal from the water decoction of traditional chinese medicine. ACS Omega. 5 (6), 2893-2898 (2020).
- Huang, H., et al. Using multiple light scattering to examine the stability of phyllanthus emblica l. Extracts obtained with different extraction methods. JoVE. (194), e65130 (2023).
- Miao, W. G., Tang, C., Ye, Y., Quinn, R. J., Feng, Y. Traditional chinese medicine extraction method by ethanol delivers drug-like molecules. Chin J Nat Med. 17 (9), 713-720 (2019).
- Olejar, K. J., et al. Ultrasonic-assisted extraction of cannabidiolic acid from cannabis biomass. JoVE. (183), e63076 (2022).
- Zhu, H. Y., et al. Sedative and hypnotic effects of supercritical carbon dioxide fluid extraction from schisandra chinensis in mice. J Food Drug Anal. 24 (4), 831-838 (2016).
- Lin, X., Wang, Y., Liu, X., Huang, S., Zeng, Q. Ils-based microwave-assisted extraction coupled with aqueous two-phase for the extraction of useful compounds from chinese medicine. Analyst. 137 (17), 4076-4085 (2012).
- Wu, T., Lv, H., Wang, F., Wang, Y. Characterization of polyphenols from lycium ruthenicum fruit by uplc-q-tof/mse and their antioxidant activity in caco-2 cells. J Agric Food Chem. 64 (11), 2280-2288 (2016).
- Distler, U., et al. Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics. Nat Methods. 11 (2), 167-170 (2014).
- Sawada, Y., et al. Widely targeted metabolomics based on large-scale ms/ms data for elucidating metabolite accumulation patterns in plants. Plant Cell Physiol. 50 (1), 37-47 (2009).
- Zhang, X. M., et al. Data-dependent acquisition based high-resolution mass spectrum for trace alternaria mycotoxin analysis and sulfated metabolites identification. Food Chem. 364, 130450 (2021).
- Downard, K. M. Indirect study of non-covalent protein complexes by maldi mass spectrometry: Origins, advantages, and applications of the "intensity-fading" approach. Mass Spectrom Rev. 35 (5), 559-573 (2016).
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