Elsevier

Journal of Ethnopharmacology

Volume 150, Issue 2, 25 November 2013, Pages 553-559
Journal of Ethnopharmacology

Elucidation of the transport mechanism of baicalin and the influence of a Radix Angelicae Dahuricae extract on the absorption of baicalin in a Caco-2 cell monolayer model

https://doi.org/10.1016/j.jep.2013.09.011Get rights and content

Abstract

Ethnopharmacological relevance

Angelicae Dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav combined with Radix Scutellariae baicalensis Georgi has been widely used in traditional Chinese medicine (TCM) as an antipyretic analgesic and anti-inflammatory drug. Modern pharmacological studies have demonstrated that the compatible application of these two drugs is an effective treatment for hepatitis. A previous study indicated that a Radix Angelicae Dahuricae extract enhanced the intestinal absorption of the baicalin found in Radix Scutellariae; however, the underlying compatibility mechanism of these two herbs remains unknown. In this study, we further examined the effect of a Radix Angelicae Dahuricae extract on the absorption and transport properties of baicalin in a Caco-2 cell model to determine the compatibility mechanism of these two herbs.

Aim of the study

The aim of this work was to study the transport properties of baicalin in Radix Scutellariae across cell membranes and the effects of a Radix Angelicae Dahuricae extract on baicalin absorption using the well-characterized, human-based intestinal Caco-2 cell model.

Materials and methods

We assessed the absorption, bidirectional transport and toxicity of baicalin using a range of parameters, including drug concentration, pH, a P-glycoprotein (P-gp) inhibitor (Verapamil), an MRP inhibitor (MK-571) and EDTA-Na2 (tight junction modulator). Next, we studied the influence of a Radix Angelicae Dahuricae extract on the transport of baicalin under the same conditions. Drug concentration was measured by HPLC, and the apparent permeability coefficient (Papp) and apparent permeability ratio (PDR) were subsequently calculated.

Results

The results showed that baicalin is non-toxic within a concentration range of 800 µg/mL to 4800 µg/mL. The transport of baicalin showed time and concentration dependence. The absorption of baicalin was optimal at pH 7.4 in 37 °C; however, the absorption decreased at 4 °C. The Papp of baicalin transport through the Caco-2 cell monolayer model was altered when specific inhibitors of P-gp or MRP were added to the cells. However, there was no significant difference in the PDR value. The Papp of baicalin improved when it was combined with the Radix Angelicae Dahuricae extract. The influence of EDTA-Na2 on the transport of baicalin showed that the permeability of baicalin significantly increased. The result further indicated that the mechanism of baicalin intestinal absorption in the Caco-2 cell monolayer involves passive transcellular diffusion.

Conclusions

Passive diffusion is the main mode of intestinal absorption of bacalin and it involved in the efflux of proteins. The enhanced intestinal absorption of baicalin by Radix Angelicae Dahuricae can be due to opening of the tight junctions between cells and inhibition of MRP efflux protein expression or function.

Introduction

Baicalin (Fig. 1) is the main active flavone extracted from Radix Scutellariaee, a plant used in traditional Chinese medicine. This compound is also the primary quality indicator used to evaluate Radix Scutellariaee preparations. Baicalin has been shown to have anti-oxidative (Guo et al., 2011), anti-bacterial (Hu and Feng, 2001), anti-viral (Xu et al., 2007), anti-carcinogenic (Tang et al., 2007) and anti-allergic (Gao et al., 1998) properties; furthermore, studies have shown that baicalin has anti-inflammatory, anti-thrombotic, vasodilator and antipyretic effects in multiple peripheral tissues and organs (Cheng et al., 2006, Li and Ge, 2010). Similar to other flavones, baicalin has low water solubility and permeability that limits its absorption and bioavailability (Liu et al., 2006, Che et al., 2001). In general, the intestinal absorption barrier is a major factor that limits the absorption and oral bioavailability of drugs. Therefore, research into novel methods and the compatibility of additional compounds that can overcome the barrier of intestinal absorption are needed to study the intestinal transport mechanism of a drug and to increase its bioavailability.

Aherb-pair of traditional Chinese medicine (TCM) is a relatively fixed composition of two herbs used in a clinical treatment and represents the most basic form of compatibility application in TCM. Clinical experience and modern research has shown that the application compatibility of herb-pairs in TCM can reduce toxicity, increase drug activity and improve the pharmacological effects of the drug (Xi and Chen, 2008). Radix Scutellariae and Radix Angelicae Dahuricae are a common herb-pair (Xu, 1996). The main components of Radix Angelicae Dahuricae are coumarin and volatile oil. It has been reported that volatile oil can promote drug absorption and coumarin can inhibit the activity of metabolic enzymes (Li et al., 2007, Li et al., 2006). However, little is known about how Radix Angelicae Dahuricae influences the absorption of baicalin.

Caco-2 cells are derived from a human colorectal carcinoma and is a widely used cell line (Pinto et al., 1983). Permeability across the fully differentiated Caco-2 monolayer is considered to be a model of intestinal absorption (Hilgers et al., 1990). Indeed, the Caco-2 cell system is an in vitro absorption model that is routinely used in both industry and academia to study intestinal permeability. These cells also express nutrient and drug transporters that allow for the study of carrier mediated uptake and efflux mechanisms (Hayeshi et al., 2008). Here, we chose to utilize the Caco-2 cell monolayer model to study the absorption of baicalin.

Based on the use of the Caco-2 cell model, a P-gp inhibitor (Verapamil) and MRP inhibitor (MK-571) were chosen to investigate if the transport of baicalin was affected by two drug intestinal cellular efflux transporters (P-gp and MRP). It is well-known that the application compatibility of medicines can change the transport process of drugs. Moreover, Radix Scutellariae and Radix Angelicae Dahuricae are frequently combined for use in traditional Chinese medicine. In this study, we studied the possible mechanism behind the enhancement of baicalin absorption with a Radix Angelicae Dahuricae extract. These data can further explain the compatibility of the Radix Angelicae Dahuricae and Radix Scutellariae herb-pair.

Section snippets

Reagents and chemicals

Radix Angelica Dahuricae and Radix Scutellariae were purchased from Bozhou Baixin Pharmaceutical Co., Ltd. (Anhui, China) and both the botanical identifications were authenticated by Professor Shouwen Zhang. The voucher specimens of Radix Angelicae Dahuricae (No. 074401) and Radix Scutellariae (No. 113323) were deposited in the Herbarium of the Department of Pharmacognosy at Jiang Xi University of Traditional Chinese Medicine.

Baicalin, with a batch number of 101765–201108, was bought from the

The toxicity of a Radix Scutellariae extract on Caco-2 cell monolayers

According to the data listed in Table 1 from the MTT assay, baicalin from a Radix Scutellariae extract is non-toxic in the range of 800 µg/mL to 4800 µg/mL. Therefore, we used concentrations within this range for subsequent experiments.

The effect of pH on baicalin transport

The effects of pH on the transport of baicalin shown in Fig. 2 indicated that the membrane permeation of baicalin was significantly higher at pH 7.4 when compared with a pH of 5.0 or 6.8. These data suggest that baicalin is quite permeable in a pH 7.4 environment.

Discussion

High concentrations of drugs can often produce toxicity to Caco-2 cells. Therefore, we examined the cytotoxicity of the baicalin solution using the MTT assay. According to the result of the MTT assay, the baicalin solution is non-toxic between 800 and 4800 µg/mL.

It was reported that baicalin was absorbed better in the colon in areas with a pH of 7.4 (Gong et al., 2008, Liu et al., 2006, Zhu et al., 2011). The structure and function of caco-2 cells is similar to human colonic epithelial cells.

Acknowledgments

The work was supported by a grant from the Key Laboratory of Modern Preparation of TCM of Ministry of Education at Jiangxi University of TCM and financially supported by the National Natural Science Foundation of China (Grant no. 30960516 and 81303237) and the Youth Science Funds of Jiangxi Province, China (Grant No. 20114BAB215040).

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