Structural characterization of metabolites of salvianolic acid B from Salvia miltiorrhiza in normal and antibiotic-treated rats by liquid chromatography–mass spectrometry

Abstract

This study was conducted to compare the in vivo metabolites of salvianolic acid B (Sal B) between normal rats and antibiotic-treated rats and to clarify the role of intestinal bacteria on the absorption, metabolism and excretion of Sal B. A valid method using LC–MSⁿ analysis was established for identification of rat biliary and fecal metabolites. And isolation of normal rat urinary metabolites by repeated column chromatography was applied in this study. Four biliary metabolites and five fecal metabolites in normal rats were identified on the basis of their MSⁿ fragmentation patterns. Meanwhile, two normal rat urinary metabolites were firstly identified on the basis of their NMR and MS data. In contrast, no metabolites were detected in antibiotic-treated rat urine and bile, while the prototype of Sal B was found in antibiotic-treated rat feces. The differences of in vivo metabolites between normal rats and antibiotic-treated rats were proposed for the first time. Furthermore, it was indicated that the intestinal bacteria showed an important role on the absorption, metabolism and excretion of Sal B. This investigation provided scientific evidence to infer the active principles responsible for the pharmacological effects of Sal B.

Introduction

The dried root of Salvia miltiorrhiza (Danshen), a commonly used traditional Chinese medicine, is widely used to treat coronary heart disease, cerebrovascular disease, hepatitis, hepatocirrhosis and chronic renal failure [1], [2], [3], [4]. Salvianolic acid B (Sal B) is the most common constituent in Salvia species and the most abundant in their aqueous extracts [5]. Different in vitro studies showed that Sal B has various biological activities of anti-oxidation, anti-inflammatory, anti-hypoxic and anti-arteriosclerotic [6], [7], [8], [9], [10], [11]. It is assumed in these in vitro studies that Sal B reaches the target inner tissues as bioavailable component. However, Sal B is poorly absorbed through the gut barrier because of their high molecular weight [12], [13]. It is possible that these biological effects may not be due to a direct action of Sal B itself but due to an effect of some more readily absorbed low-molecular-weight metabolites. Therefore it is essential to understand how Sal B is absorbed, metabolized and eliminated from the body. So far, attention has been focused on the pharmacokinetics, tissue distribution, metabolism and biliary excretion after intravenous administration of Sal B from the roots of Salvia miltiorrhiza [14], [15], [16], [17], [18], [19], [20]. In some studies, Sal B revealed extremely low bioavailability and four methylated metabolites were excreted rapidly into rat bile after oral dosing [5], [12]. Our previous study mainly focused on the urinary metabolites after oral administration of total phenolic acids [21]. However, to our knowledge, there has been no report with regard to the identification of rat urinary and fecal metabolites after oral dosing of Sal B.

In traditional Chinese medicine, most of the remedies are administered orally in the form of crude decoction and active components have to cross the intestinal barrier to reach the systemic circulation. Active components of their prescriptions are therefore brought into contact with bacterial flora in the alimentary tract [22]. Large spectrum of antibiotics have the potential to alter the content of intestinal bacteria and may interfere with the pharmacokinetics and eventually pharmacodynamics of compounds [23], therefore germ-free or antibiotic-treated animals can be used to determine the role of the intestinal microflora in metabolism of foreign compounds in vivo. The publication has shown the co-administration of kampo medicines with antibiotics or bacterial preparations [24]. Recently, the correlation of intestinal flora and activity of kampo ingredients has increasingly been recognized. Xing et al. reported the interaction of baicalin and baicalein with antibiotics in the gastrointestinal tract [25]. However, no report has been found in the literature to discuss the effect of antibiotic treatment on the absorption, metabolism and excretion of Sal B.

In recent years, liquid chromatography/mass spectrometry (LC/MS) has been proved to be a powerful and reliable analytical approach for structural analysis of chemical components in herbal extracts with high sensitivity and low consumption of samples [26], [27], [28], [29]. Furthermore, tandem mass spectrometry techniques have been playing an important role in metabolic study, such as the structural elucidation of drug metabolites, because the high sensitivity of MS as an LC detector facilitates the discovery of new active constituents which are difficult to obtain by conventional means [30], [31]. Eventually, LC–MSⁿ technique has been applied in this study to investigate the metabolites in rat biosamples after oral dosing of Sal B.

In the present study, we proposed the possible metabolic pathway of Sal B and showed that the intestinal bacteria played an important role on the absorption, metabolism and excretion of Sal B. Two urinary metabolites, four biliary metabolites and five fecal metabolites of Sal B were detected in normal rat biosamples by isolating the pure compounds or using LC–MSⁿ technique. In contrast, no metabolites were detected in antibiotic-treated rat urine and bile, while the prototype of Sal B was found in antibiotic-treated rat feces. For the first time, we investigated the differences of in vivo metabolites between normal rats and antibiotic-treated rats after oral dosing of Sal B. Two urinary and five fecal metabolites in normal rats were firstly identified in biosamples. This finding provided a scientific basis from a metabolic point of view for the clarification of action mechanism of Sal B.