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. 2019 Jun;9(2):11.
doi: 10.1051/bmdcn/2019090211. Epub 2019 May 24.

In vivo microdialysis and in vitro HPLC analysis of the impact of paeoniflorin on the monoamine levels and their metabolites in the rodent brain

Yuh-Tzy Lin  1 , Wei-Shih Huang  2 , Huei-Yann Tsai  3 , Min-Min Lee  4 , Yuh-Fung Chen  5
Affiliations

In vivo microdialysis and in vitro HPLC analysis of the impact of paeoniflorin on the monoamine levels and their metabolites in the rodent brain

Yuh-Tzy Lin et al. Biomedicine (Taipei). 2019 Jun.

Abstract

Background: Paeoniflorin (PF) possesses several effects such as analgesic, the anti-spasmodic effect on smooth muscle. It protects the cardiovascular system and reveals the neuroprotective effect on cerebral ischemia. Monoamine system has been identified to have complex regulatory effects in pain signaling. There are no reports regarding the impact of PF on monoamine levels in the rodent brain by microdialysis. In this study, the effects of PF on monoamines and their metabolites in the rodent brain using in vivo microdialysis and in vitro high performance liquid chromatography (HPLC) analysis.

Methods: Male S.D. rats were anesthetized, fixed onto the stereotaxic instrument to identify the positions of corpus striatum and cerebral cortex. Drilled a hole in the skull of anesthetic rats and proceeded microdialysis, and gave PF (100 μg, i.c.v.). Collected the dialysate and the concentration of monoamines and their metabolites in dialysate and analyzed with HPLC-ECD. Male ICR mice were administered with PF (96 μg, i.c.v.) and with Ringer solution as a control. After 20 mins of administration, the mice were cut off the brain immediately and separated into eight regions according to the method of Glowinski. Added extraction solution to each region, homogenized and extracted for further procedure. The extract was centrifuged, sucked the transparent layer and centrifuged once more. The transparent layer was filtered with a 0.22 μm nylon filter and analyzed with HPLC-ECD (electrochemical detection).

Results: PF increased the content of DOPAC and NE in the cortex, and increased the content of NE and decreased the content of 5-HT in the medulla of the homogenized mice brain tissue. By microdialysis, PF increased the content of DOPAC and 5-HIAA in anesthetic rat cortex and expanded the content of DOPAC, HVA, and 5-HIAA in anesthetic rat striatum.

Conclusions: It reveals that PF could activate the release of monoamines and increase their metabolites in the rodent brain.

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Figures

Fig. 1
Fig. 1
Microdialysis setup for the anesthetized rat. (1) Microdialysis syringe pump, (2) Microdialysis probe placed into the brain, (3) Stereotaxic instrument, (4) Fraction collector, (5) Temperature controller, (6) High-performance liquid chromatography/electrochemical detector (HPLC/ECD), (7) Chromatography chart. (This figure is a slightly modified adaptation of the picture of CMA product catalog with permission of the CMA representative, Anatech Co., LTD., in Taipei).
Fig. 2
Fig. 2
The analysis of a mixed standard solution of monoamines and their metabolites with chromatogram of high-performance liquid chromatography and an electrochemical detector (HPLC-ECD), and the retention time of each peak. NE: norepinephrine; EPI: epinephrine; DOPAC: 3.4-dihydroxyphenylacetic acid; DA: dopamine; 5-HIAA: 5-hydroxyindoleacetic acid; HVA: homovanillic acid; 5-HT: serotonin.
Fig. 3
Fig. 3
Multiple Regression curve. (Upper): Analytical data for the seven standards in the HPLC-ECD. X and y represent the concentration of the standard (μmol/L) and the peak current (μA), respectively. (Lower): Concentrations and AUC of the seven standards.
Fig. 4
Fig. 4
Effect of paeoniflorin (PF) on the content of monoamines and their metabolites with microdialysis of anesthetic rats in the cerebral cortex. Data are shown as mean ± S.E. (n = 8); * P < 0.05, ** P < 0.01 compared with control.
Fig. 5
Fig. 5
Effect of paeoniflorin (PF) on the content of monoamines and their metabolites with microdialysis of anesthetic rats in the corpus striatum. Data are shown as mean ± S.E. (n = 8); * P < 0.05, ** P < 0.01, *** P < 0.001
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