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).

1. Sample pretreatment
<ol>
	<li>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 &#176;C for 24 h. 
	NOTE: The entire plant of BFH was collected in Sichuan Province, China.</li>
	<li>Crush dried BFH in a high-speed multifunctional crusher. Transfer the coarse powder into a 50-mesh sieve (Figure 1C). Coll...

Identifying Chemical Composition of Gynura bicolor by UPLC-TOF Mass Spectrometry

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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>chloroform</td>
			<td>Sinopharm Chemical ReagentCo., Ltd</td>
			<td>CAS 67-66-3</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>ChuandongChemical</td>
			<td>CAS 64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>ethyl acetate</td>
			<td>ChuandongChemical</td>
			<td>CAS 141-78-6</td>
			<td></td>
		</tr>
		<tr>
			<td>liquid chromatograph</td>
			<td>Waters</td>
			<td>ACQUITY Class 1 plus</td>
			<td></td>
		</tr>
		<tr>
			<td>MassLynx</td>
			<td>Waters</td>
			<td>V4.2</td>
			<td>MS control software</td>
		</tr>
		<tr>
			<td>n-butyl alcohol</td>
			<td>ChuandongChemical</td>
			<td>CAS 71-36-3</td>
			<td></td>
		</tr>
		<tr>
			<td>petroleum ether</td>
			<td>ChuandongChemical</td>
			<td>CAS 8032-32-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Quadrupole time-of-flight mass spectrometry</td>
			<td>Waters</td>
			<td>SYNAPT XS</td>
			<td></td>
		</tr>
		<tr>
			<td>UNIFI</td>
			<td>Waters</td>
			<td></td>
			<td>Data analysis software</td>
		</tr>
	</tbody>
</table>

high-resolution tandem mass spectrometry for studying chemical constituents of gynura bicolor dc

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.

Preparation of Gynura bicolor DC samples for High-Resolution Tandem Mass Spectrometry

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

Begin by sample pretreatment. 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 at 50 degrees Celsius for 24 hours.Crush the dried BFH in a high-speed multifunctional crusher. Transfer the coarse powder into a 50-mesh sieve. Collect the fine powder in an airtight bag and store it in a drying tower.Then accurately weigh six BFH samples of 0.25 grams each and place them in six conical flasks. Add 30 milliliters of organic solvent to each conical flask. Transfer the mixture to an ultrasound bath sonicator for 30 minutes at 25 degrees Celsius.Next, centrifuge the sample at 14, 000g for five minutes. Using an injection syringe and the 0.22-micrometer organic microporous membrane filter, filter all the supernatants into two-milliliter sample bottles.

The chemical composition identification of BFH was used as a model to display the representative results. Base peak chromatograms of solvent-extracted Gynura bicolor DC. are shown in Supplemental File 1-Supplemental Figure S1-S6, and the observed retention time (RT), component name, formula, mass-to-charge ratio (m/z), and mass error are listed in Tables S1-S6. In the ZBTK, all 35 kinds of compounds were identified from separated peaks in UPLC-TOF-MS. As shown in Supplemental File 1-Supp...

Read this Scientific Article Protocol about High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC at JoVE.com

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

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). ...

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237 visualizações.  Chongqing Hospital of Traditional Chinese Medicine. Comece pelo pré-tratamento da amostra. Lave toda a erva de BFH em água pura até que não haja depósitos e impurezas visíveis. Coloque o BFH limpo em um prato e leve ao forno a 50 graus Celsius por 24 horas.Esmague o BFH seco em um britador multifuncional de alta velocidade. Transfira o pó grosso para uma peneira de 50 malhas. Colete o pó fino em um saco hermético e guarde-o em uma torre de secagem.Em seguida, pesar com precisão seis amo...