Effects of Glycyrrhizic Acid on Solubility, Pharmacokinetics and Liver Distribution of Osthole in Rats
doi: 10.14148/j.issn.1672-0482.2023.1076
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摘要:
目的 研究甘草酸对蛇床子素溶解度和生物利用度的影响, 并探讨甘草酸影响蛇床子素溶解度、改善其药代动力学的作用机制。 方法 雄性SD大鼠单独口服蛇床子素(20 mg·kg-1)和配伍甘草酸(45 mg·kg-1)给药前后, 在特定时间点采集血液和肝脏样本并用LC-MS/MS方法测定蛇床子素浓度配伍前后的变化。通过X射线衍射(XRD)、傅立叶变换红外光谱(FT-IR)等物理表征, 研究甘草酸对蛇床子素溶解度的影响, 揭示甘草酸的增溶机理。采用Caco-2细胞单层模型, 研究蛇床子素在体外的吸收转运过程, 以及甘草酸及其活性代谢物甘草次酸(GC)对蛇床子素在Caco-2细胞吸收的影响。采用大鼠肠上皮细胞S9和肝细胞S9孵育系统, 探讨甘草酸和甘草次酸对蛇床子素体外代谢的影响。 结果 大鼠体内药代动力学结果表明甘草酸配伍蛇床子素可显著提高蛇床子素大鼠体内生物利用度。大鼠肝组织样本实验结果表明, 甘草酸增加蛇床子素在肝组织中的分布。体外溶解度实验研究发现, 甘草酸能显著提高蛇床子素在水中的溶解度, 推测可能的原因为蛇床子素结晶度的降低以及蛇床子素与甘草酸之间形成氢键所致。Caco-2细胞单层模型结果显示甘草酸和甘草次酸均不能促进蛇床子素的吸收, 体外孵育研究表明, 甘草酸与甘草次酸对蛇床子素的体外Ⅰ相代谢的影响较小。 结论 通过甘草酸和蛇床子素二者配伍机制的探讨, 配伍后蛇床子素生物利用度的提高和肝脏浓度的增加可能是由于甘草酸提高了蛇床子素的溶解度。对进一步研究以溶解度作为胃肠道吸收限速步骤的难溶性药物与天然增溶剂甘草酸配伍使用的合理性具有重要意义。 Abstract:OBJECTIVE To investigate the potential of glycyrrhizic acid (GL) to improve the solubility and bioavailability of osthole (Ost), and to explore the underlying mechanism of the potential solubility and pharmacokinetic interactions. METHODS Male Sprague-Dawley rats were orally given osthole (20 mg·kg-1) alone or with glycyrrhizic acid (45 mg·kg-1). Blood and liver samples were collected at specific time points and determined by an LC-MS/MS method. The effect of glycyrrhizic acid on the solubility of osthole, and physical characterizations, including X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy, were performed to reveal the mechanism of the solubilization of GL. In addition, bidirectional transport study using Caco-2 cell monolayer model was employed to study the effects of glycyrrhizic acid and its active metabolite glycyrrhetinic acid (GC) on the absorption of osthole. In incubation studies, rat enterocyte S9 and liver S9 were used to explore the potential effect of GL and GC on the metabolism of osthole. RESULTS The results of pharmacokinetics in rats showed that co-administration of glycyrrhizic acid could significantly increase the AUC of osthole as compared with osthole administered alone. The results of rat liver tissue samples indicated that glycyrrhizic acid increased the distribution of osthol in liver tissue. Solubility study found that glycyrrhizic acid could significantly enhance the solubility of osthole in water due to the decreased crystallinity of osthole and the formation of hydrogen bonding between Ost and GL. Caco-2 cell monolayer model demonstrated that neither GL nor GC could enhance osthole absorption. Further in vitro incubation study revealed that there was little metabolic interaction between osthole and GL or GC in phaseⅠmetabolism. CONCLUSION Increased AUC and liver concentration of osthole are probably due to the increased solubility of osthole by GL. The current study has significant implications for further investigation on the potential combination use of natural solubilizing agent GL and drugs with solubility as the rate-limiting step to absorption from the gastrointestinal tract. -
Key words:
- osthole /
- glycyrrhizic acid /
- glycyrrhetinic acid /
- solubility /
- pharmacokinetics
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Figure 1. Chemical structures of osthole (A), imperatorin (internal standard) (B), glycyrrhizic acid (C) and glycyrrhetinic acid (D)
Figure 2. (A) Solubility data of osthole singly and with glycyrrhizic acid (GL) at different concentrations at room temperature and 37 ℃. (B) Solubility data of osthole singly and with glycyrrhetinic acid (GC) at low, middle, high concentrations at 37 ℃
Figure 3. X-ray diffraction analysis of (a) osthole, (b) glycyrrhizic acid, (c) physical mixture, (d) lyophilized sample
Figure 4. Fourier transform infrared spectra of (a) osthole, (b) glycyrrhizic acid, (c) physical mixture, (d) lyophilized sample
Figure 5. Representative MRM chromatograms of (A) a blank rat liver sample (non-spiked), (B) a blank rat liver sample spiked with IS (180 ng·g-1)and osthole (100 ng·g-1), and (C) a rat liver sample obtained 10 h after oral administration of osthole (20 mg·kg-1)
Note: 1. IS; 2. osthole
Figure 6. (A) The mean plasma concentration-time profiles of osthole in rats after oral administration of osthole (20 mg·kg-1) without and with glycyrrhizic acid (45 mg·kg-1) (B) The osthole concentration in the liver of rats at different time points after oral administration of osthole (20 mg·kg-1) without and with glycyrrhizic acid (45 mg·kg-1).
Note: *P < 0.05 and * *P < 0.01 compared with the osthole group.x±s, n=6.
Figure 7. (A) Effects of glycyrrhizic acid (GL) and glycyrrhetinic acid (GC) at various concentrations on absorptive permeability parameter (Papp(A-B)) of osthole in Caco-2 cell in vitro model (B) Effects of glycyrrhizic acid and glycyrrhetinic acid at various concentration on secretory permeability parameter (Papp(B-A)) of osthole in Caco-2 cell in vitro model
Note: *P < 0.05 and **P < 0.01 compared with the control group. x±s, n=6.
Figure 8. (A) Effects of glycyrrhizic acid (A1) and glycyrrhetinic acid (A2) at various concentrations on the metabolic reduction of osthole in the rat intestine S9. (B) Effects of glycyrrhizic acid (B1) and glycyrrhetinic acid (B2) at various concentrations on the metabolic reduction of osthole in the rat liver S9 fraction
Note: *P < 0.05 and * *P < 0.01 compared with the ost group. x±s, n=6.
Table 1. The pharmacokinetic parameters of osthole in rats after oral administration of osthole (20 mg·kg-1) without and with glycyrrhizic acid (45 mg·kg-1) (x±s, n=6)
Parameters Osthole Osthole+Glycyrrhizic acid Tmax/min 62.00±43.82 94.00±43.36 Cmax/(ng·mL-1) 19.23±6.18 45.04±16.28* AUC(0-t)/(ng·min·mL-1) 8 247.09±1 627.12 14 099.57±3 657.95* AUC(0-∞)/(ng·min·mL-1) 9 069.39±1 708.37 14 522.62±3 698.25* MRT(0-t)/min 416.03±59.17 342.01±46.33 T1/2/min 469.96±178.22 301.76±55.11 CL/(L·min-1·kg-1) 2.27±0.40 1.43±0.30** Note: *P < 0.05, * *P < 0.01. 
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