Short communication
A HPLC-MS/MS method for determination of 6′′′-feruloylspinosin in rat plasma and tissues: Pharmacokinetics and tissue distribution study

https://doi.org/10.1016/j.jpba.2016.01.005Get rights and content

Highlights

  • A HPLC-MS/MS method was developed for assaying 6′′′-feruloylspinosin in rats.

  • Its pharmacokinetics and tissues distribution were studied for the first time.

  • Its sedative effect is through enhancing the mRNA expression of GABA receptors.

Abstract

A sensitive, reliable and accurate HPLC-MS/MS method was developed and validated for the quantification of 6′′′-feruloylspinosin in rat plasma and tissues with puerarin as the internal standard. The separation was performed on a Proshell 120 EC-C18 column (4.6 × 150 mm, 2.7 μm) with a mobile phase consisting of acetonitrile and 0.1% formic acid (20:80, v/v) at 0.3 mL/min. The quantification was performed by MRM with m/z [M–H] 783.3  427.2 for 6′′′-feruloylspinosin and m/z [M–H] 415.4  295.4 for the internal standard, respectively. The calibration curves covered over a concentration range of 20–2000 ng/mL in plasma and various tissues samples (heart, liver, spleen, lung, kidney, stomach, intestine, muscle, cerebrum and cerebellum) with good linearity (r2  0.9914). Both the intra- and inter-day precisions were less than 14.70%, and the accuracy (RE%) ranged from −5.80% to 4.93%. The extraction recoveries were within 75.21–92.96%, and the matrix effect ranged from 87.21% to 113.44%. Compared with spinosin, 6′′′-feruloylspinosin was distributed in rats faster whereas more slowly eliminated from the plasma. 6′′′-Feruloylspinosin could be distributed rapidly and widely in various tissues, and transfer across the blood–brain barrier. In addition, both 6′′′-feruloylspinosin and spinosin could enhance the expression of GABAAα1, GABAAα5, GABABR1 mRNA in rat hippocampal neurons significantly, indicating the bioactivity mechanism of 6′′′-feruloylspinosin was involved in the GABA receptors.

Introduction

Zizyphi spinosi semen (ZSS) is one of the most commonly used traditional Chinese medicines (TCMs) for the treatment of insomnia, fright palpitations and profuse dreaming. In the recent years, many reports have demonstrated that ZSS exhibits various bioactivities, including anxiolytic [1], hypnotic [2], and memory modulating [3].

As one of the main constituents of ZSS, flavonoids have been proved to be responsible for the anxiolytic and sedative effects [4]. Up to now, more than 20 flavonoid components have been found in ZSS [5]. Among them, spinosin and 6′′′-feruloylspinosin are the predominant C-glycoside flavonoids, which account for 0.10% and 0.04% respectively in ZSS (w/w) [6]. It has been found that spinosin plays a crucial role in ameliorating memory [7] and modulating sleep [8]. Furthermore, spinosin can be absorbed into plasma, transport through the blood–brain barrier, and distribute widely in the brain tissues [9], [10], [11]. As a result, it is predicated that spinosin may exert its effect through activation of the GABA and 5-HT1A receptors in the central nervous system [7], [8], [12].

6′′′-Feruloylspinosin is a derivative of spinosin with a feruloyl group bound to 6′′′-C of the glycoside (Fig. 1). Some reports have indicated that 6′′′-feruloylspinosin can prolong sleeping time in mice treated with hexobarbital [13]. This compound can be found in rat plasma and feces after oral administration of the flavonoid extract of ZSS, suggesting that 6′′′-feruloylspinosin may have specific bioactivity in vivo [14]. To date, the pharmacokinetic and tissue distribution of 6′′′-feruloylspinosin, particularly the capability of the molecule passing through the blood-brain barrier, are largely unknown.

In this study, a HPLC-MS/MS method was developed and validated for the determination of 6′′′-feruloylspinosin in rats with puerarin as the internal standard (IS). The pharmacokinetic and tissue distribution of 6′′′-feruloylspinosin were investigated in rats after intravenous (i.v.) administration of the compound compared to spinosin. Furthermore, the effect of 6′′′-feruloylspinosin on the mRNA expression of GABA subunits in cultured rat hippocampal neurons was determined by real time fluorescence quantitative reverse transcription polymerase chain reaction (RTFQ-PCR). Our results provide valuable information for the possible clinical applications of 6′′′-feruloylspinosin in the future.

Section snippets

Chemicals and reagents

Both 6′′′-feruloylspinosin and spinosin (purity  98%) were purified and further characterized by using UV, IR, MS and NMR (Fig. S1–S4) in our laboratory. Puerarin and naringin (purity  98%) were obtained from Shanghai Tauto Biotech Co., Ltd. (China). Acetonitrile was purchased from Merck (Darmstadt, Germany). Chromatographic pure water applied for preparing the mobile phase was from J.T. Baker (J.T. Baker Chemicals, USA). All other reagents used in the experiment were commercially available and

Specificity

Representative chromatograms of blank plasma and tissue homogenates, and the real samples collected after tail intravenous administration of 6′′′-feruloylspinosin are showed in Fig. S5. No significant endogenous interference was found in the retention times of 6′′′-feruloylspinosin (7.4 min) and the IS (2.5 min).

Linearity and lower limit of quantification

The calibration curves, correlation coefficients and linear ranges of 6′′′-feruloylspinosin in plasma and various tissue homogenates are showed in Table S1. The regression coefficients (r2

Conclusions

A quantitative HPLC-MS/MS method was developed and validated to determine 6′′′-feruloylspinosin in rat plasma and various tissues. With the established method, the pharmacokinetics and tissues distribution of 6′′′-feruloylspinosin was investigated for the first time. Compared with spinosin, 6′′′-feruloylspinosin was distributed in rats faster whereas more slowly eliminated from the plasma. Both 6′′′-feruloylspinosin and spinosin were found to increase the transcription level of GABAAα1, GABAAα5

Acknowledgments

This work was supported by the National Natural Science Foundation of China(Grant No. 31101235, No. 31000749), Tianjin High School Innovation Team Project (Grant No. TD12-5049, National Undergraduate Training Programs for Innovation and Entrepreneurship (Grant No. 201410069032).

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      The quick absorption in the present study might result from coexisting constituents in the aqueous extract. Compared to that of spinosin, the CL value (928.92 ± 309.06 L/h/kg) of 6‴-feruloylspinosin remarkably increased (P < 0.01), indicating that 6‴-feruloylspinosin might be rapidly and widely distributed in rats, aligning with the finding of a previous report [10]. Some studies reported that 6‴-feruloylspinosin was first hydrolyzed to spinosin and swertisin, and spinosin could be further metabolized to swertisin in vitro by rat intestinal bacteria [25,26].

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