Effects Of Total Extracts Of Glycyrrhiza Uralensis And Glycyrrhizic Acid And Glycyrrhetinic Acid On P - Glycoprotein And Drug Metabolizing Enzymes

Chinese medicine Radix Glycyrrhiza comes from dry root and rhizome of leguminous plant licorice, its property is mild and taste is sweet, it goes to twelve meridians and has a reputation as "the old" in medicines based on traditional Chinese medicine theory. With detoxification, spasmolysis, pain relief,eliminating phlegm and anticancer pharma-cological effects, Radix Glycyrrhiza is the most commonly used drug in clinical. Because licorice can "reconcile the medicine", it has been so widely used in traditional medicine that there is a "ten prescriptions nine grass" theory. As one of the most important function of licorice, detoxification has been described by previous dynasties classics of traditional Chinese medicine.There are studies which have found that licorice can adjust the liver CYP3A enzyme to induce poisons’metabolism, and some studies have pointed out that P-glycoprotein also participated in the detoxification mechanism of licorice. P-glycoprotein is a kind of drug efflux pump dependent on energy, it transports the substrate from intracellular to the outside of the cell against a concentration gradient depending on the energy released from hydrolysis of ATP. We can conjecture that once the efflux function of P-glycoprotein has been inducted, it can promote the efflux of internal toxic substances, realize the goal of protection and reduce its poison on body. Metabolism is one of major pathways of remanent drug’s elimination in vivo, and this process requires a variety of drug metabolism enzymes involved in catalysis. As known, hepatic Cytochrome P45O (CYP450) enzymes and glutathione-S-transferase (GST), respectively, is the most important phase Ⅰ enzyme and phase Ⅱ enzyme in drug metabolism, most drugs’s metabolism are associated with them, thus their contents and activities can be induced or inhibited to achieve detoxification and synergies.Therefore, we chosen the total extract of glycyrrhiza and glycyrrhizic acid, glycyrrhe-tinic acid as experimental drugs, to study their influences on P-glycoprotein of Caco-2cells, as well as CYP3A4and GST drug metabolic enzymes in HepG2cells, in order to explore the possible detoxification mechanisms of glycyrrhiza.The main results were as follows:1. Cell toxicity experiment, the results showed that the growth inhibition rate of Caco-2cells was20%or less when the concentration of glycyrrhizic acid was not greater than40μM, glycyrrhetinic acid’s was not greater than20μM and glycyrrhiza extract’s was not abundance than10μg/mL, these concentrations were non-cytotoxic; When the concentration of glycyrrhizic acid was not greater than20μM, glycyrrhetinic acid’s was not extra than10μM and glycyrrhiza extract’s was not greater than40μg/mL, the growth inhibition rate of HepG2cells was20%or less, they were non-cytotoxicity concentrations. So the concentration gradient of glycyrrhizic acid was selected as1.6μM,8μM,40μM;glycyrrhetinic acid0.8μM,4μM,20μM; glycyrrhiza extract0.4u g/mL,2μg/mL,10μg/mL, for the next research of P-glycoprotein. The concentration gradient of glycyrrhizic acid was0.8μM,4μM,20μM, glycyrrhetinic acid’s concentration gradient was0.4μM,2μM,10μM, glycyrrhiza extract’s was1.6μg/mL,8μg/mL,40μg/mL for drug metabolism enzymes research.2. Effect of drugs on P-glycoprotein function test, ADR accumulation test results showed that ADR mean fluorescence intensity (MFI) in blank control and positive control cells was12.54and19.96;when cells were processed by the low. medium and high concentration group of glycyrrhizic acid, the intracellular adriamycin MFI were9.85,9.06,8.84, respectively; the intracellular adriamycin MFI were9.56、9.15、8.82after treated by glycyrrhetinic acid; when processed by total licorice extract, the intracellular adriamycin MFI were8.61,7.88,7.52, respectively.Rho123accumulation test results showed that the intracellular rhodamine MFI were27.99,19.89.13.6when Caco-2cells were processed by the low、medium and high concentration group of glycyrrhizic acid; the intracellular rhodamine MFI were29.02,20.68,12.29in glycyrrhetinic acid group respectively; the intracellular rhodamine MFI were23.55,20.75,15.35in total licorice extract group; the blank control and positive control group were28.9and42.68. Rho123efflux test results showed that rhodamine MFI in cells processed by the low、medium and high concentration group of glycyrrhizic acid were19.24、15.34、13.56,respectively; the intracellular rhodamine MFI were18.2、15.07、12.9in glycyrrhetinic acid group; the intracellular rhodamine MFI were17.28、13.2、10.67in total licorice extract group; the blank control and positive control group were18.3and36.68.These results showed that all the test medicines could significantly inhibit the accumulation of adriamycin and rhodamine123in Caco-2cells, and significantly promote the efflux of rhodamine123also(P<0.05, P<0.01or P<0.001) concentration-dependently. Because adriamycin and rhodamine123were substrates of P-glycoprotein which was a efflux pump on membrane, so,glycyrrhizic acid, glycyrrhetinic acid and total licorice extract had induction effects on the efflux function of P-glycoprotein.3. Utilizing Semi-quantitative reverse transcription PCR and real-time quantitative PCR technologies to determine the regulation of test medicines on P-glycoprotein ABCB1gene expression. According to the results of semi-quantitative reverse transcription PCR, glycyrrhizic acid, glycyrrhetinic acid and glycyrrhiza extract could increase ABCB1gene transcription level in Caco-2cells. The ratio of ABCB1grey value in control cells/β-actin grey value was7.3%; after treatment with glycyrrhizic acid, the ratios were8.5%,9.7%,10.7%; after treatment with glycyrrhetinic acid, the gray ratios were higher and were21.1%,22.9%,30.4%; After treatment with total glycyrrhiza extract, gray ratio were20.5%,24.4%,35.7%. Real time quantitative PCR results were basically identical with the results of semi-quantitative RT-PCR, after drugs treatment.BCB1gene mRNA expression levels were increased significantly than the untreated group (P<0.05, P<0.01), the medium, high concentration groups of glycyrrhizic acid, glycyrrhetinic acid, and glycyrrhiza extract in all concentration gradients presented a concentration dependence, showed that experimental drugs possessed an inductive effect on P-glycoprotein ABCB1gene transcription in cells.4. Flow cytometry in combination with immunofluorescence method were used to detect the expression of P-glycoprotein, results showed that when processed by the low, medium and high concentration of glycyrrhizic acid, glycyrrhetinic acid and glycyrrhiza extract, P-gp expression on membrane were11.91,12.27,13.19;12.12,12.49,13.48;13.0,13.69,15.15;respectively. P-gp expression in control group was7.33.These results showed that glycyrrhizic acid, glycyrrhetinic acid and total glycyrrhiza extract could significantly increase the expression of P glycoprotein (P<0.05), and had a concen-tration dependence.5. The results of P-glycoprotein ATPase activity detection showed that after processed by the low, medium and high concentration of glycyrrhizic acid, glycyrrhetinic acid and glycyrrhiza extract, P-gp ATPase activity in Caco-2cells were3.25,10.47,15.33U/mgprot;3.31,12.45,25.62U/mgprot;3.38,13.67,21.44U/mgprot; P-gp ATPase activity was3.12U/mgprot in control group. These data showed that glycyrrhizic acid, glycyrrhetinic acid and glycyrrhiza extract could significantly induce P-gp ATPase activity (P<0.01or P<0.001), to provide enough energy for P-glycoprotein and to achieve for the promotion of its efflux function. 6. By detection of CYP450enzyme CYP3A4and GST enzyme activities in HepG2cells, the results showed that when processed by the low、medium and high concen-tration of glycyrrhizic acid, glycyrrhetinic acid and glycyrrhiza extract, CYP3A4enzyme activity in HepG2cells were1.59,1.56,1.51nmol/mg·min;1.58、1.49、1.38nmol/mg·min;1.31、2.14、2.37nmol/mg·min;respectively. GSTs enzyme activity in HepG2cells were3.91、6.94、9.61U/mgprot;9.09、12.34、13.63U/mgprot;13.66、16.62、22.17U/mgprot.CYP3A4enzyme activity in control group was1.75nmol/mg·min, and GSTs enzyme activity in control group was3.97U/mgprot. These results showed that glycyrrhizic acid and glycyrrhetinic acid of high, medium and low dose, and low dose of glycyrrhiza extract could always inhibit CYP3A4enzyme activity, and had significant differences. However, medium and high concentration groups of total glycyrrhiza extract could significantly increase the activity of CYP3A4(P<0.01). And glycyrrhizic acid, glycyrrhetinic acid and glycyrrhiza extract in low, medium and high concentration groups could significantly raise GSTs enzyme activity (P<0.05or P<0.01), and had a concentration dependence. Glycyrrhiza extract induced GST enzyme activity more significantly than glycyrrhizic acid and glycyrrhetinic acid.Conclusion:glycyrrhiza extract, glycyrrhizic acid and glycyrrhetinic acid could concentration-dependently induce the efflux function of P-glycoprotein on the membrane of Caco-2cells by up-regulating the expression levels of ABCB1gene and P-glycoprotein,and increasing the acticity of P-gp ATPase significantly to ensure the energy supply for P-glycoprotein. Thus it could be speculated that glycyrrhizic acid and glycyrrhetinic acid may be the main ingredients to induce the efflux function of P-glycoprotein, thereby promoting the poison excretion. Glycyrrhiza extract of medium,high concentration could significantly enhance the activity of drug metabolism phase I enzyme CYP3A4(P<0.01), while glycyrrhizic acid and glycyrrhetinic acid inhibited the activity of CYP3A4concentration-dependently. That glycyrrhizic acid and glycyrrhetinic acid may not be the main components in inducing CYP3A4activity.And glycyrrhiza as a CYP3A4inducer, may be related to its detoxification. Glycyrrhizic acid, glycyrrhetinic acid and glycyrrhiza extract could induce phase II drug metabolism enzymes GST activity to speed up the efflux of poison, and achieve the detoxification function in vivo. Research results of this experiment could provide certain theoretical basis to study the detoxification mechanism of Glycyrrhiza.