Rapid identification of absorbed components and metabolites of Gandou decoction in rat plasma and liver by UPLC-Q-TOF-MSE

https://doi.org/10.1016/j.jchromb.2019.121934Get rights and content

Highlights

  • UPLC-Q-TOF-MSE was applied to identify absorbed components and metabolites of GDD.

  • 89 and 77 GDD-related xenobiotics were identified in normal and copper-laden rats, respectively.

  • The fragmentation patterns of five types of metabolites and metabolic profile were summarized.

Abstract

Gandou Decoction (GDD), a well-known traditional Chinese medicine prescription, has been widely used for decades in clinical practice to treat Wilson’s disease (WD) in China. However, due to lack of in vivo metabolism research, the absorbed components and metabolites of GDD have not been fully elucidated. In this study, a rapid and high-throughput ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MSE) was applied to rapidly identify prototypes and metabolites after oral administration of GDD. On this basis, the possible metabolic pathways of the main prototypes were proposed between normal and copper-laden rats. As a result, a total of 89 GDD-related xenobiotics were detected in normal dosed rats, including 83 (36 prototypes and 47 metabolites) in plasma and 52 (21 prototypes and 31 metabolites) in liver; a total of 77 GDD-related xenobiotics were detected in copper-laden dosed rats, including 68 (31 prototypes and 37 metabolites) in plasma and 42 (19 prototypes and 23 metabolites) in liver. Our findings showed that anthraquinones, alkaloids and protostane triterpenoids as well as a few saponins, flavonoids, tannins and curcuminoids were the main absorbed chemical components of GDD in rat plasma; anthraquinones, protostane triterpenoids and curcuminoids were the major components in rat liver. Glucuronidation and sulfation were deduced to be the predominant metabolic pathways of GDD. Methylation, acetylation, reduction, hydroxylation, demethylation and deglycosylation were often occurred in the metabolic process. Furthermore, the holistic metabolic profile of GDD revealed that copper-laden rats and normal rats had certain differences in drug absorption and metabolism. This study offered a solid basis for ascertaining bioactive components and action mechanism of GDD.

Introduction

Wilson’s disease (WD), also known as hepatolenticular degeneration, is a genetic disorder of copper metabolism due to ATP7B gene mutation. It is an autosomal recessive illness characterized by excessive accumulation of copper in the liver, brain and other tissues, leading to various neurologic, psychiatric and hepatologic symptoms [1], [2]. Typical Western medicines, such as D-penicillamine, dimercaptosuccinic acid, sodium dimercaptosuccinate and tetrathiomolybdate, can be used for treating WD, but their toxic side effects seriously affect the patient's condition and prognosis [3], [4], [5]. In China, Gandou decoction (GDD), a traditional Chinese medicine prescription (TCMP), has been widely used to treat WD for decades. Due to its remarkable clinical effect, GDD has been recommended as the basic prescription recorded in “The Diagnosis and Treatment Guidelines of WD” in China [6], [7].

GDD is composed of six medicinal materials, including Rheum palmatum L. (Da-Huang), Coptis chinensis Franch. (Huang-Lian), Curcuma longa L. (Jiang-Huang), Lysimachia christinae Hance (Jin-Qian-Cao), Alisma orientale (Sam.) Juzep. (Ze-Xie) and Panax notoginseng (Burk.) F. H. Chen (San-Qi). Modern pharmacological researches have demonstrated that GDD appears to be effective, safe, mild adverse reactions and well-tolerated [8]. Our previous studies found that GDD could reduce the hepatic copper accumulation and improve liver pathological characteristics by restoring the impaired lipid metabolism, amino metabolism and glucose metabolism [9]. For the constituent research in our lab, 96 compounds have been isolated from GDD as well as its relative individual herbs [10]. However, the absorbed components and metabolites of GDD in vivo still remain unclear. Therefore, there is an urgent need for systematic research on the metabolic profiling of GDD.

It is an arduous task to identify the prototypes and metabolites of herbal medicines and TCMP in biologic samples due to their very low levels of concentration in vivo, diverse structure types, unpredictable metabolites and the interference from endogenous substances, resulting in incomprehensive and unsystematic constituent characterization [11], [12]. In recent years, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MSE) has been used as an effective analytical tool for chemical characterization and metabolite identification because of its high resolution, sensitivity and accuracy. TOF-MSE technology uses two interleaved scan functions with different collision energies, allowing for the simultaneous acquisition of precursor ions with low collision energy and fragment ions with high collision energy [13]. In the present study, an efficient approach was applied to rapidly characterize the metabolic profiling of GDD in plasma and liver between normal and copper-laden rats based on UPLC-Q-TOF-MSE. It is expected that the current work could provide a detailed basis for revealing the potential effective materials in vivo, and contribute to the further pharmacology and curative mechanism research on GDD.

Section snippets

Chemicals and reagents

Rheum palmatum L., Coptis chinensis Franch., Curcuma longa L., Lysimachia christinae Hance, Alisma orientale (Sam.) Juzep. and Panax notoginseng (Burk.) F. H. Chen were purchased from Beijing Tongrentang Pharmacy (Hefei, China). The medicinal plants were authenticated by Doctor Rongchun Han (College of Pharmacy, Anhui University of Chinese Medicine, Hefei, China). All voucher specimens were deposited at the authors' laboratory. Eleven compounds were used as reference standards (purity > 98%),

Determination of serum and hepatic copper levels

The abnormal elevation of serum and hepatic copper levels can be used as an indicator of copper-laden rat model [[15], [18]]. As shown in Table 1, serum copper levels showed nonsignificant changes in 30-day copper-laden model group compared to those in control group, but copper levels in 60-day and 90-day model groups were significantly different from those in control group (P < 0.05). Hepatic copper accumulation increased considerably during the experiment, in which copper levels in 30-day,

Conclusion

In this study, we successfully established a mature copper-laden rat model by intragastric administration of CuSO4. A rapid and high-throughput UPLC-Q-TOF-MSE was applied for systematic research on the metabolic profiling of GDD in vivo between normal and copper-laden rats. As a result, a total of 89 GDD-related xenobiotics, including 83 (36 prototypes and 47 metabolites) in plasma and 52 (21 prototypes and 31 metabolites) in liver, were detected in normal dosed rats; a total of 77 GDD-related

CRediT authorship contribution statement

Li Xu: Data curation, Validation, Writing - review & editing. Yi Liu: Methodology, Data curation, Writing - original draft. Hongfei Wu: Resources, Supervision, Visualization, Investigation. An Zhou: Conceptualization, Supervision, Project administration, Funding acquisition.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (No: 81872976, 81873038, 81303245), Key Project of the National Science Fund of Anhui Province (No: KJ2016A398), Key Project Foundation of Oversea Visiting and Research for the Excellent Young and Middle-aged Faculties in Universities of Anhui Province (No: gxgwfx2018042), and Primary Research & Development Plan of Anhui Province (No: 201904a07020051).

References (27)

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