Chemical characterization and metabolic profiling of Xiao-Er-An-Shen Decoction by UPLC-QTOF/MS

Abstract

Background: Xiao-Er-An-Shen decoction (XEASD), a TCM formula composed of sixteen Chinese medicinal herbs, has been used to alleviate tic disorders (TD) in clinical practice for many years. However, the chemical basis underlying the therapeutic effects of XEASD in the treatment of TD remains unknown. Purpose: The present study aimed to determine the major chemical components of XEASD and its prototype compounds and metabolites in mice biological samples. Methods: The chemical constituents in XEASD were identified using ultra-high Performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Following this, XEASD was orally administered to mice, and samples of plasma, urine, feces, bile, and tissue were collected in order to identify effective compounds for the prevention or treatment of TD. Result: Of the total 184 compounds identified to be discriminated in the XEASD, comprising 44 flavonoids, 26 phenylpropanoids, 16 coumarins, 16 triterpenoids, 14 amino acids, 13 organic acids, 13 alkaloids, 13 ketones, 10 cyclic enol ether terpenes, 7 citrullines, 3 steroids, and 5 anthraquinones, and others. Furthermore, we summarized 54 prototype components and 78 metabolic products of XEASD, measured with biological samples, by estimating metabolic principal components, with four prototype compounds detected in plasma, 58 prototypes discriminated in urine, and 40 prototypes identified in feces. These results indicate that the Oroxylin A glucuronide from Citri reticulatae pericarpium (CRP) is a major compound with potential therapeutic effects identified in brain, while operating positive effect in inhibiting oxidative stress in vitro. Conclusion: In summary, our work delineates the chemical basis underlying the complexity of XEASD, providing insights into the therapeutic and metabolic pathways for TD. Various types of chemicals were explored in XEASD, including flavonoids, phenylpropanoids, coumarins, organic acids, triterpenoid saponins, and so on. This study can promote the further pharmacokinetic and pharmacological evaluation of XEASD.

Keywords: UPLC-QTOF/MS; Xiao-Er-An-Shen Decoction; chemical characterization; metabolic profiling; tic disorder; traditional Chinese medicine.

FIGURE 1

Base peak chromatogram (BPC) of XEASD.

FIGURE 2

Representative structures of each medicine of XEASD.

FIGURE 3

A representative MS/MS fragmentation spectrums and major fragmentation pattern of XEASD. (A) P38 Chlorogenic acid*; (B) P41 Sibiricose A1; (C) P68 Sibiricaxanthone B; (D) P147 Citrusin III; (E) P57 Vicenin-2/Saponarin; (F) P189 Limonin*; (G) P48 Oxyphyllenone A; (H) P72 Psoralen; (I) P87 Liquiritin*; (J) P145 Uralsaponin F; (K) P172 Pinicolic acid E; (L) P53 Loganin*; (M) P166 Pinellic Acid.

FIGURE 4

Identification of prototypes in bio-samples, and P53 Loganin is taken as an example. (A) multiple XICs of Loganin in XEASD and bio-samples, A- Administration,B-Blank.; (B) MS/MS spectrum of Loganin in XEASD; (C) MS/MS spectrum of Loganin in urine.

FIGURE 5

Identification of metabolites in bio-samples. Metabolite identification and matching process based on the similarity of the cleavage pattern and profile of the P20 N-Methyltyramine-M1 secondary mass spectrum.

FIGURE 6

Structural association of N-Methyltyramine with metabolites.

FIGURE 7

Correlation between prototype and metabolites.

FIGURE 8

Herbal ingredients form XEASD and major compounds with potential therapeutic effects identified in various organs.

FIGURE 9

Effect of Oroxylin A glucuronide on the proliferation and apoptosis in H9C2 cells. (A) H9C2 cells were treated with Oroxylin A glucuronide (1, 10, and 100 µm) for 12 h and cell viability was measured by MTT assay. (B) The apoptosis-inducing effect of Oroxylin A glucuronide on H9C2 cells was tested by Annexin-V FITC/PI double-staining assay. Each experiment was done independently at least three times and all the data is quantified as the mean ± SEM. ** p-value < 0.01 and *** p-value < 0.001 compared with the model group and ### p-value < 0.001 compared with the control group.