Study of pharmacokinetic profiles and characteristics of active components and their metabolites in rat plasma following oral administration of the water extract of Astragali radix using UPLC–MS/MS

Abstract

Ethnopharmacological relevance

Astragali radix is one of the well-known traditional Chinese herbal medicine, and possesses various biological functions, such as hepatoprotective and anticancer. In present study, to investigate the metabolism and pharmacokinetics of the major constituents of A. radix, a sensitive ultra-performance liquid chromatography–electrospray ionization–mass spectrometry (UPLC–MS/MS) method with shorter chromatographic running time was developed and validated for simultaneous quantification of formononetin, ononin, calycosin, calycosin-7-β-glucoside, astragaloside IV and their glucuronide metabolites in rat plasma after oral administration of water extract of A. radix at two different doses.

Materials and methods

The chromatographic separation was achieved on a C18 column with gradient elution by using a mixture of 0.1% formic acid aqueous solution and acetonitrile as the mobile phase at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection was conducted using multiple-reaction monitoring (MRM) via electrospray ionization (ESI) source in positive ionization mode. Samples were pre-treated by a single-step protein precipitation with methanol, and erlotinib was used as internal standard (IS).

Results

The current UPLC–MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery, matrix effects and stability. The lowest limit of quantifications (LLOQ) were 1 ng/mL for all analytes. After oral administration, the plasma concentrations of the glucuronides, especially calycosin-3′-glucuronide, were much higher than the parent compounds. The mean half-life (t_1/2) was between 1 and 5 h, and the metabolites were eliminated faster than the parent constituents. The median (range) time to reach maximum plasma concentration (T_max) was between 0.5 and 1 h.

Conclusions

This is the first study of the pharmacokinetic study of bioactive compounds and their glucuronides in male rat plasma after oral administration of water extract of A. radix. The results demonstrated the biotransformation between the bioactive isoflavonoids and their glucuronides was extensive in rats and provided a significant basis for better understanding the absorption and metabolism mechanism of A. radix. Furthermore, this study could suggest that future studies should focus on the metabolites and biotransformation between the bioactive constituents when conducting a drug efficacy study.

Introduction

Astragali radix, which is a well-known traditional Chinese herbal medicine, also called Huang Qi in Chinese, is derived from the roots of Astragalus membranaceus (Fisch.) Bge. or A. membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao (The Pharmacopoeia Commission of People׳s Republic of China, 2010). Pharmacological studies and clinical practices indicate that A. radix possesses various biological functions, such as hepatoprotective properties (Gui et al., 2006, Kim et al., 2012), induces vasodilatation (Zhang et al., 2005), antioxidative effects (Chan et al., 2009), immunostimulating effects (Lee et al., 2003), antiviral (Qi et al., 2013), antimicrobial (Yan et al., 2011), immunological properties (Nalbantsoy et al., 2012), enhances cardiovascular function (Xu et al., 2008), antiaging properties (Wang et al., 2010), neuroprotective effects against ischemic brain injury (Luo et al., 2004), and induces cancer cell apoptosis (Cheng et al., 2004). For these reasons, A. radix is an important Chinese herb and is the essential ingredient in more than 200 Chinese herb formulas (Xiao-long, 2005). Clinically, A. radix is typically prepared as a water extract either alone or together with other crude drugs (such as Chuanxiong Rhizoma, Paeonia lactiflora Pall) to treat nephritis, diabetes, hypertension, cirrhosis, cancer, etc. (Xu et al., 2006).

More than 100 chemical constituents of A. radix have been isolated and identified to date. Among them, isoflavonoids and saponins are the two main types of beneficial compounds responsible for its pharmacological activities and therapeutic efficacy, and these could be the marker compounds for the chemical evaluation of A. radix (Ma et al., 2004, Qi et al., 2008, Yesilada et al., 2005). Formononetin, ononin, and calycosin and its glycoside, which boost energy, strengthen the immune system, and promote health activities and skin growth, are the major isoflavonoids in A. radix (Xiao et al., 2004). Astragaloside IV is another vital compound of A. radix and is useful for the identification of the quality of A. radix.

The therapeutic mechanism of traditional Chinese herbs is complicated owing to the multiple and complex active compounds, but their use has attracted increasing attention. The pharmacokinetic characteristics of the active ingredients in herbs are useful to predict both the efficacy and the potential toxicity of a medicine as well as to optimize dose regimens and avoid adverse effects (Wang et al., 2013, Wu et al., 2013, Zhang et al., 2014). Therefore, pharmacokinetic study of the main bioactive components of TCM is important for clinical applications and may help clarify their pharmacological action. To date, several analytical methods, including LC–MS/MS, which is being more and more widely applied in pharmaceutical research, have been used to quantify the marked compounds in biological samples of animals following oral administration of Chinese herb formulas such as A. radix and the ethanol extract of A. radix (Liu et al., 2014, Shaw et al., 2012, Shi et al., 2014, Yang et al., 2014). However, the major weakness of these methods mainly includes chromatographic running time, which at more than 14 min is considered too long. This is time-consuming and not suitable for analyzing large numbers of sample. It should be noted that the water extract of A. radix was not used in these studies. Recently, Huang Qi oral solution and injection, in which the main constituent is the water extract of A. radix, are more widely used for various diseases (Fu et al., 2014). Therefore, it is necessary to evaluate the pharmacokinetic behavior of the active compounds in the water extract of A. radix to optimize the dosage and method of administration and achieve a better understanding of the pharmacological action mechanism.

It was known that flavonoids could be extensively metabolized to their phase II metabolites, glucuronides, in the intestine and liver by UDP-glucuronosyltransferase (UGTs) after oral administration (Jeong et al., 2005, Liu et al., 2007), and that many glucuronides of flavonoids showed several pharmacological activities (Yamamoto et al., 2013, Yamazaki et al., 2014). Therefore, the real active compounds of A. radix may be the combination of absorptive constituents and their metabolites in vivo. Previous reports have focused on the metabolites of marked compounds of A. radix in vivo (Chen et al., 2011, Yang et al., 2014), but most of these studies were qualitative analyses after oral administration. To our knowledge, no one has studied the pharmacokinetic behavior of the glucuronides of isoflavonoids in A. radix. We believe that a pharmacokinetic study of these glucuronides will promote the study of the pharmacological activities of A. radix.

The aim of this study was to simultaneously characterize and determine the pharmacokinetic profiles of the active compounds and their UGTs metabolites, glucuronides, in A. radix, and to study the possible biotransformation between the main compounds in vivo. We developed a sensitive and validated ultra-performance liquid chromatography–electrospray ionization–mass spectrometry (UPLC–MS/MS) method with a short analysis time and simple sample preparation procedure to analyze large quantities of plasma samples, which were obtained in pharmacokinetic studies after oral administration of low and high doses of water extract of A. radix to rats. The pharmacokinetic study of the above components would help to clarify the absorption mechanism and elucidate the in vivo process of the water extract of A. radix for additional interpretation of traditional Chinese herbs.