Effect of glycyrrhizic acid on the oral absorption of paeoniflorin in rats in vivo

Abstract

Paeoniflorin (PF) and glycyrrhizic acid (GL) are the major active components in the peony liquorice decoction, which has been widely used clinically in China for more than one thousand years. The available reports on the pharmacokinetic behaviour of the two compounds in the presence of each other still are not consistent and are sometimes even contradictory. The aim of this study was to investigate the effect of GL on the absorption of PF administrated orally with or without pre- or co-administration of GL at different dosages in rats. The results indicated that GL has effects on the absorption extent of PF, however, without any significant impact on the absorption rate and excretion of PF. Furthermore, the effect of GL on the absorption of PF was GL dosage dependent and the concentration of GL in the small intestinal tissue should be a decisive factor for the effect. GL (300 or 900 mg kg⁻¹ BW) showed an inhibition effect on the absorption of PF (300 mg kg⁻¹ BW) when the two drugs were co-administrated orally, while the effect was reversed when GL at a higher dosage of 2700 mg kg⁻¹ BW. The present study explained the contradiction of varying reports on the effect of GL on the absorption of PF and will provide important information for the rational design of peony and liquorice based formula in TCM.

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1. Introduction

Traditional Chinese Medicine (TCM) has been used for the treatment and prevention of diseases for more than one thousand years in China and the other Asian countries.^1,2 In clinical practice, TCMs are often prescribed to patients in the form of single herbs or herbal formulas (combination of several herbs). For better therapy efficacy and less side effects, a formula formed by several herbs is often used for treating chronic and complicated diseases, which were developed based on the clinical efficacy and Chinese philosophy.^2,3 With the development of modern techniques, more and more of the active components from TCM are identified and characterized.² The studies on the active components from TCM make it possible for us to understand the action mechanism of TCM and develop and optimize TCMs. Meanwhile, ADME properties evaluation and the interaction study between the active components from TCM are very attractive for many researchers nowadays.^4–7

Peony liquorice decoction was first recorded in “Treatise on Febrile Diseases” written by Zhang Zhong-jing, which has been widely used to treat inflammation, pain, spasms, cough, asthma and ulcers for thousands of years.⁸ This decoction is composed of two Chinese medicines: peony and liquorice. Paeoniflorin (PF) and glycyrrhizic acid (GL) is the major active component in peony and liquorice, respectively.^9,10 As reported, PF shows a very poor bioavailability (F) of 3%, which might be caused by the first-pass effect in the gut wall or liver, metabolism or decomposition in the intestine by bacterial microflora and/or poor absorption from gastrointestinal tract.^11,12 However, the oral availability of PF in rats could be improved significantly by the co-administration of sinomenine or Shao-yao Gan-chao Tang.¹³ As reported, PF is a typical substrate of P-gp and the transport activity of P-gp to PF should be responsible for the poor bioavailability of PF.^14–16 Sinomenine is an inhibitor of P-glycoprotein. Therefore, the bioavailability (F) of PF could be improved significantly through the transport inhibition by sinomenine to P-gp. As well-matched partner compounds in decoctions of TCM, PF and GL interacts with each other in multi-aspects to achieve better clinical efficacy. The pharmacokinetic properties of PF and GL have been widely reported.^15,17 However, the available reports on the pharmacokinetic behaviour of the two compounds with the presence of each other still are not consistent and sometimes even contradict each other.^{7,8,16,18–20}

In the present study, we investigated the effect of GL on the absorption of PF in rats by pre- or co-administration orally of GL at different dosages with PF. The results indicated that the effect of GL on the absorption of PF was GL dosage dependent. GL showed inhibition effect at low dosages (300 or 900 mg kg⁻¹ BW), while the effect was reversed when GL at a higher dosage of 2700 mg kg⁻¹ BW. The present study explained the contradictory nature of varying reports on the effect of GL on the absorption of PF.

2. Experimental

2.1 Chemicals and animals

PF and geniposide were purchased from Zelang Pharmaceutical Technology Co. Ltd. (Nanjing, China). GL was purchased from Baoji Guokang Bio-Technology Co., Ltd. (Baoji, China). Orthophosphoric acid was obtained from Zhiao Chemical Reagents Research Institute (Anshan, China), and HPLC-graded methanol and acetonitrile were purchased from Kemiou Technology Co. Ltd. (Tianjin, China). All other chemicals and solvents used were of reagent grade.

Sprague-Dawley rats in all experiments (220–250 g, male) were supplied by Dalian Medical University (Dalian, China). The rats were housed in a room with controlled temperature and humidity, and were allowed to freely access the water and standard laboratory diet. They were fasted overnight before drug administration. All animal studies were performed in accordance with the experimental protocols approved by the Animal Care Committee of Dalian Medical University.

2.2 Plasma sample collection

PF and GL were both dissolved in water. Blood samples were taken from the rats before administration of drug. The animals were then given a PF dosage of 300 mg kg⁻¹ only or together with a GL dosage of 900 mg kg⁻¹ via intragastric administration. A series of blood samples were collected into heparinized Eppendorf tubes at 0.167, 0.5, 0.75, 1, 2, 4, 6 and 8 h after drug administration. The plasma was isolated by centrifugation (LG16-W, Beijing Medical Centrifuge Factory, Beijing) at 11 200g for 10 min and then stored immediately at −20 °C until analysis.

2.3 Tissue sample collection

Rats were divided into 5 groups randomly. There were 5 rats in each group. The rats from the group of control were administrated PF orally at the dosage of 300 mg kg⁻¹ BW. PF (300 mg kg⁻¹ BW) was co-administrated orally with GL at the dosage of 300 mg kg⁻¹ BW (group of co-admin 1), at the dosage of 900 mg kg⁻¹ BW (group of co-admin 2) or at the dosage of 2700 mg kg⁻¹ BW (group of co-admin 3). The rats from the group of pre-admin were pre-administrated GL orally at the dosage of 900 mg kg⁻¹ BW 45 min before the administration of PF (300 mg kg⁻¹ BW). Blood samples were collected at 10 min, 30 min, 45 min and 1 h post administration, and the rats were sacrificed immediately. The small intestine were collected. Tissue samples were washed and blotted with filter paper to remove excess fluid and stored at −20 °C for analysis.

2.4 Sample preparation

Plasma samples (50 μL) were thawed at room temperature, and each was then mixed with 50 μL methanol and 50 μL internal standard (geniposide in methanol 40 μg mL⁻¹) by vortexing for 1 min. The denatured proteins were separated by centrifugation at 11 200g for 10 min at room temperature, and 20 μL of the supernatant was analyzed by HPLC.

Tissue samples were thawed to room temperature and 1.0 g was homogenized in 2.0 mL methanol at 18 000 rpm using an adjustable speed homogenizer (FSH-2, Jinhua Honghua Instrument Factory, Zhejiang). The homogenate was centrifuged at 11 200g for 10 min and the supernatant was treated as described above for the plasma samples.

2.5 High-performance liquid chromatography analysis

HPLC was performed with a Shimadzu HPLC system (LC-2010A, LC solution workstation, Japan) and a Diamonsil™ C18 column (5 μm, 4.6 mm × 150 mm). The volume of sample injection was 20 μL. The mobile phase for PF was acetonitrile–purified water–orthophosphoric acid (16 : 84 : 0.03, v/v/v) whereas the mobile phase for detecting GL was acetonitrile–purified water–orthophosphoric acid (37 : 63 : 0.03, v/v/v). Both PF and GL were detected by UV absorption, 230 nm for PF and 250 nm for GL. During the run, the column was maintained at 30 °C and the flow rate at 1.0 mL min⁻¹.

2.6 Statistical analysis

Pharmacokinetic parameters were calculated with non-compartmental model of Topfit program (Version 2.0, Gustav Fischer Verlag, Stuttgart, Germany). All the results are presented as mean ± SD. A two-tailed Student's t-test was used to determine any significant differences. P < 0.05 was considered to be significant.

3. Results and discussion

3.1 Pharmacokinetics of PF with or without the co-administration of GL

The pharmacokinetic profiles of PF in rats after a single oral administration of PF at 300 mg kg⁻¹ with or without co-administration of GL at 900 mg kg⁻¹ are shown in Fig. 1, and the corresponding pharmacokinetic parameters of PF are shown in Table 1.

Fig. 1 The mean plasma concentration–time profiles of PF after a single oral administration of PF (300 mg kg⁻¹ BW) with or without co-administration of GL (900 mg kg⁻¹ BW). Bars represent the standard deviation (n = 5).

Table 1 Pharmacokinetic parameters of PF in rats after oral administration with or without the co-administration of GL (n = 5, mean ± SD)

Parameters	PF administrated without GL	PF co-administrated with GL
a Significant at the 0.05 level compared with PF administrated without GL.
Cmax (μg mL^−1)	3.27 ± 1.51	1.23 ± 0.47^a
Tmax (h)	0.70 ± 0.21	0.79 ± 0.42
AUC(0–t) (μg h mL^−1)	8.63 ± 2.51	5.05 ± 0.70^a
AUC(0–∞) (μg h mL^−1)	9.48 ± 2.65	5.45 ± 0.66^a
t1/2 (h)	3.69 ± 1.45	2.64 ± 1.10
MRT(0–∞) (h)	4.23 ± 1.68	4.25 ± 0.56

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As Table 1 shown, all the absorption extent related pharmacokinetic parameters of PF (300 mg kg⁻¹ BW) including AUC_(0–t), AUC_(0–∞) and C_max decreased significantly (P < 0.05) when co-administered with GL (900 mg kg⁻¹ BW). However, there were no significant variations in the absorption rate and excretion related pharmacokinetic parameters of PF, such as T_max, t_1/2 and MRT_(0–∞). Thus, it could be concluded that the primary effect of GL on the PK behaviour of PF was absorption extent inhibition, however, without any significant impact on the absorption rate and excretion. These results were consistent with the findings reported by Zhao and Li.^18,19

3.2 The effects of co- or pre-administration with GL on the absorption of PF

Our study revealed that GL at the dosage of 900 mg kg⁻¹ BW could inhibit the absorption of PF. However, there are still some studies indicate that the absorption of PF could be improved by the co-administration of GL in rats,^8,20 which are in contrast with the results of Fig. 1. To investigate further on the effect of GL on the absorption of PF, PF (300 mg kg⁻¹) were co-administered with GL at the dosage of 300, 900 and 2700 mg kg⁻¹ BW in rats. Meanwhile, the concentration of GL in the small intestine tissue were determined.

As Fig. 2 shown, the effect of GL on the absorption of PF was GL dosage dependent. The C_max of PF decreased significantly (P < 0.01) (from 3.27 ± 1.51 to 0.34 ± 0.14 μg mL⁻¹) when co-administered with GL at the dosage of 300 mg kg⁻¹ BW, which means the absorption of PF was inhibited by GL at this dosage. However, the C_max of PF co-administrated with GL increased with the increase of the dosage of GL. As a result, the absorption of PF could be improved by the co-administration of GL at the dosage of 2700 mg kg⁻¹ BW. Therefore, it could be concluded that the process of oral absorption of PF was under multi-factor control and the interactions between GL and the factors showed different impact on the absorption of PF.^15,16,21 The interaction between the factors and GL at low dosage (300 and 900 mg kg⁻¹ BW) caused the inhibition of PF absorption, while caused the improvement when GL at a higher dosage (2700 mg kg⁻¹ BW). Therefore, the results of Fig. 2 explained the contradictory nature of varying reports on the effect of GL on the absorption of PF. Furthermore, as Fig. 2 shown, the intestinal concentration of GL also increased with the increase of GL dosage, which might be a decisive factor for the effect of GL on the absorption of PF.

Fig. 2 The effect of GL at different dosages on the absorption of PF in rats (note: C_in means the concentration of GL in the small intestinal tissue). Bars represent the standard deviation (n = 5).

To investigate the effect of the intestinal concentration of GL on the absorption of PF, GL (900 mg kg⁻¹ BW) was pre-administrated to the rats 45 min before PF (300 mg kg⁻¹ BW) administration. As Fig. 3 shown, the pre-administration of GL orally (group pre-admin) could improve the absorption of PF as the co-administration of GL at the dosage of 2700 mg kg⁻¹ BW (group co-admin 3) did. Meanwhile, the rats from the two groups showed similar small intestinal concentration of GL. However, the rats from the group of co-admin 2, co-administrated by GL at the same dosage of group pre-admin (900 mg kg⁻¹ BW), showed both lower C_max of PF and intestinal concentration of GL than the rats from the group of pre-admin did. Therefore, the concentration of GL in the small intestinal tissue should be decisive for the effect of GL on the absorption of PF.

Fig. 3 The C_max of PF and the concentration of GL in the small intestine tissue when GL was pre- or co-administrated at different dosages with PF. Bars represent the standard deviation (n = 5). PF was administrated to the rats from all the groups at the dosage of 300 mg kg⁻¹ BW. The rats from the group of control were administrated by PF without co-administration of GL. GL was co-administrated with PF to the rats from the group of co-admin 2 at the dosage of 900 mg kg⁻¹ BW and to the rats from the group of co-admin 3 at the dosage of 2700 mg kg⁻¹ BW. The rats from the group of pre-admin were pre-administrated by GL at the dosage of 900 mg kg⁻¹ BW 45 min before PF administration.

4. Conclusion

In summary, GL has effect on the absorption extent of PF in rats, however, without any significant impact on the absorption rate and excretion. Furthermore, the effect was GL dosage dependent and the small intestinal concentration of GL should be a decisive factor for the effect. GL (300 or 900 mg kg⁻¹ BW) showed inhibition effect on the absorption of PF (300 mg kg⁻¹ BW) in rats when the two drugs were co-administrated orally, while GL showed improvement effect at the dosage of 2700 mg kg⁻¹ BW. The present study explained the contradictory nature of varying reports on the effect of GL on the absorption of PF and will provide important information for the rational design of peony and liquorice based formula in TCM.

Acknowledgements

The research is supported by Liaoning province natural science funds (2014020015) and project of outstanding talent support program in universities of Liaoning province (LR2014002). The authors wish to thank Chonglong Song and Xiaohuan Xie for the help with sample collection and preparation.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra07194e

‡ These two authors contribute equally to this work.

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