| Background and ObjectivesNatural products appear to possess a variety of pharmacological effects, which plays a crucial role in disease treatment. The flavonoid, a typical class of natural polyphenols, is widely distributed in the nature with abundant pharmacological properties including anti-tumor, anti-oxidant and anti-inflammatory effects.The structure of flavonoids contains free hydroxyl groups, which can be easily conjugated by UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), forming the metabolites with higher polarity. Metabolites can be excreted by efflux transporters including multidrug resistance proteins (MRPs) and breast cancer resistance protein (BCRP). The bioavailability barrier consisting of drug metabolizing enzymes and efflux transporters could hinder the development of flavonoids into chemopreventive or therapeutic agents. Hence, understanding the mechanism for the disposition of flavonoids mediated by drug metabolizing enzymes and efflux transporters would help to weaken the bioavailability barrier and enhance the pharmacological effects.Besides, intestinal recycling schemes including enterohepatic recycling, enteric recycling and local recycling, are proposed to be involved in the disposition of flavonoids, which impacts the systemic and local bioavailability. Enterohepatic recycling involves the action of liver to excrete the conjugated metabolites (i.e., glucuronides) and action of bacterialβ-glucuronidase (P-gus) to release the aglycones (from the glucuronides), which could subsequently enter the enterocytes and form the glucuronides to complete the recycling loop. Enteric recycling refers to the process that glucuronides excreted by the enterocytes, is hydrolyzed to aglycones by bacterial β-gus for reabsorption into colon, completing the recycling scheme. Local recycling is similar with enteric recycling with some difference. The glucuronides are excreted by the enterocytes, and once again, hydrolyzed by the enterocyte-derived β-gus to release aglycones, which could be reabsorbed into the small intestine, completing the recycling.Some flavonoids, such as apigenin, genistein and wogonin, are demonstrated to be involved in enterohepatic recycling, enteric recycling or local recycling. These recycling schemes would prolong the half-lives and retention times of flavonoids, increasing the enterohepatic bioavailability, which might finally help to enhance the pharmacological activities of target tissue (i.e., gut and liver). For the present, there is no literature reported on the simultaneous presence of triple recycling (i.e., enterohepatic recycling, enteric recycling and local recycling). Therefore, two bioactive flavonoids tilianin and acacetin were chosen as model compounds for the investigation of the coexistence of triple recycling.Tilianin, a flavonoid glucoside, is the most active component of Dracocephalum moldavica L. (DML) and has been reported to have anti-hypertensive and anti-atherosclerotic effects. The aglycone of tilianin, acacetin, has potentials for the treatment of breast cancer, liver cancer, prostate cancer and stomach cancer. The bioavailability of tilianin and acacetin is reported to be fairly low. However, few studies have been reported on the metabolism and efflux process of tilianin and acacetin.Therefore, the objectives of our study are to characterize the in vitro glucuronidation of the flavonoid glucoside tilianin and its aglycone acacetin. Caco-2 cell model was used to determine the efflux transporters and glucose transporters involved in the recycling schemes. Tilianin and acacetin were also selected as model compounds to investigate whether enterohepatic, enteric and local recycling could simultaneously mediate the disposition of flavonoids, as determined by in situ rat intestinal perfused model.Methods1. In vitro UGT metabolism of tilianin and acacetinIn vitro glucuronidation incubation via human liver microsomes (HLM), human intestinal microsomes (HIM) and human UGT isoforms were investigated for the glucuronidation characteristics of tilianin and acacetin. A quadrupole-time of flight (Q-TOF) tandem mass spectrometry was used for the identification for the structures of metabolites. In addition, enzyme kinetics of the main UGT isoform, HLM and HIM were conducted as well as species and gender difference.2. The effects of transporters on the transport of tilianin and acacetinEnzyme kinetics of tilianin and acacetin in Caco-2 cell lysates were conducted. Besides, the Caco-2 cell line was also used to determine the efflux tranporter and glucose transporter involved in the transport of acacetin and tilianin.3. The effects of intestinal recyclings on the disposition of tilianin and acacetinAn in situ rat intestinal perfused model and a series of hydrolysis experiments were used for the study of absorption and metabolism of tilianin. The effects of β-gus inhibitor and lactase phlorizin hydrolase (LPH) inhibitor on the disposition of tilianin were investigated to figure out whether flavonoids could simultaneously participate in enterohepatic, enteric and local recycling.Results1. UGT metabolism of tilianin and acacetinA quadrupole-time of flight (Q-TOF) tandem mass spectrometer was used to determine the molecular weight and structure of the metabolites. For tilianin and acacetin, one or two metabolites were formed after incubation with liver microsomes. The pseudo-molecule ions and fragment ions corresponed to the mono-glucuronides, indicating that the mono-glucuronide was generated via glucuronidation incubation.12 expressed human UGT isoforms were used to determine the UGT isoform responsible for the glucuronidation of tilianin and acacetin. Our result showed that UGT1A8 had the fastest glucuronidation rate toward tilianin and acacetin. Enzyme kinetic patterns of UGT1A8, HLM and HIM were also conducted. The result showed that the enzyme kinetics of tilianin were in accordance with Michaelis-Menten. The clearance of tilianin was largest in UGT1A8, followed with HIM and HLM. The enzyme kinetics of acacetin conformed to substrate inhibition and its clearance was largest in HLM, followed with UGT1A8 and HIM.Female and male liver microsomes from humans, rats, mice, guinea pigs and dogs were used for the investigation of species and gender difference. Female species showed generally higher UGT activity than male species did. In male liver microsomes, the human glucuronidation for tilianin and acacetin was slowest while dog glucuronidation was most similar with human glucuronidation. In female liver microsomes, the human glucuronidation was most similar with guinea pigs and dogs glucuronidation for tilianin, while most similar with mice and dogs glucuronidation for acacetin. Besides, intestinal microsomes of rats for tilianin and acacetin showed higher glucuronidation rates than those of mice.2. The effects of transporters on the transport of tilianin and acacetinCaco-2 cell lysate was used for the enzyme kinetics of tilianin and acacetin. The result showed that tilianin could not be metabolized into glucuronides in Caco-2 cell lysate. Acacetin could be rapidly metabolized into two glucuronides in Caco-2 cell lysate.In the transport study of Caco-2 cell line, acacetin could be rapidly metabolized into glucuronides and then be excreted. MRP2/MRP3 inhibitor and BCRP inhibitor significantly reduced the efflux rate and Fmet of acacetin glucuronides in the Caco-2 cell line while significantly enhanced the intracellular amount of acacetin glucuronides. Our result suggested MRP2, MRP3 and LTC4 might be efflux transporter for acacetin glucuronide.In addition, tilianin could be transported across Caco-2 cell line. Approximately 10% tilianin could be absorbed into Caco-2 cell line. SGLT1 inhibitor and GLUT2 inhibitor did not significantly influence the transport or the intracellular amount of tilianin. The result indicated that tilianin could be absorbed into Caco-2 cell line, but SGLT1 and GLUT2 might not be the transporter responsible for tilianin.3. The effects of intestinal recyclings on the disposition of tilianin and acacetinTilianin was perfused in an in situ rat perfused intestinal model. Our result indicated that, except for tilianin, the aglycone acacetin and a large amount of acacetin glucuronide were found in the perfusate. In addition, tilianin glucuronide could be detected in the bile. The absorption of tilianin and metabolism of acacetin glucuronide were highest in duodenum, followed by jejunum, ileum and coln. The absorption of tilianin in duodenum and ileum reached 50% while only 15% in colon. The metabolism of acacetin glucuronide in duodenum was approximately 20% while nearly zero in colon.Tilianin was co-perfused with 20,40 and 80 mM LPH inhibitor gluconolactone, the absorption of tilianin and the excretion of acacetin glucuronide were reduced in a dose dependent manner. In addition, the excretion of acacetin glucuronide was also reduced, indicating the hydrolysis of tilianin to acacetin, catalyzed by LPH.When incubated with β-gus from Escherichia coli, acacetin glucuronides and tilianin glucuronides could be hydrolyzed into acacetin and tilianin, respectively. This indicated that acacetin glucuronides could be hydrolyzed into acacetin by bacterial β-gus in colon, which could be absorbed into the enterocytes. Subsequently, acacetin could be metabolized into acacetin glucuronides, then excreted from the liver into the enteric lumen via bile, participated in enterohepatic recycling.The hydrolysis experiment has demonstrated that glucuronides in the perfusate could be hydrolyzed by P-gus into aglycones. When tilianin was perfused with β-gus inhibitor saccharolactone, the colonic excretion of acacetin glucuronide was reduced. The result demonstrated that the hydrolysis inhibition of acacetin glucuronide to acacetin, caused by β-gus inhibitor, might decrease the amount of acacetin in enterocytes, leading to the less production and excretion of acacetin glucuronides into lumen. Our experiment proved again that acacetin glucuronide was hydrolyzed by β-gus from bacteria into acacetin, which could be reabsorbed into colon, participated in enteric recycling.When acacetin glucuronide was incubated in blank perfuseate, acacetin glucuronide could be hydrolyzed into aglycones, which was faster in upper small intestine than that in colon. When co-perfused with β-gus inhibitor saccharolactone, the hydrolysis could be inhibited. However, perfusate filteration and sterilization did not significantly have an impact on the hydrolysis, suggesting the bacteria-derived β-gus was not the only source of β-gus, and P-gus could be originated from enterocytes. When co-perfused with β-gus inhibitor, the excretion of acacetin glucuronide in the upper small intestine was significantly reduced, which demonstrated the hydrolysis of acacetin glucuronide into acacetin by enterocyte-derived β-gus, followed by the reabosprtion into enterocytes in the upper small intestine, involved in local recycling.Conclusions1. The flavonoid glucoside tilianin and its aglycone acacetin could undergo extensive glucuronidation in vitro. UGT1A8 was the most important isoform responsible for the glucuronidation of tilianin and acacetin. Acacetin was more likely to undergo glucuronidation than tilianin in the intestine and liver. In addition, the metabolism of tilianin and acacetin demonstrated significant species and gender differences. For these two compounds, dogs demonstrated most similar glucuronidation rate with humans did while females glucuronidated faster than males.2. Acacetin could be metabolized into glucuronides and excreted in Caco-2 cell line. The efflux transporters MRP2/MRP3 and BCRP are possibly involved in the efflux of acacetin glucuronide. Additionally, tilianin could be transported intact into the cell. However, the transporters SGLT1 and GLUT2 might not get involved in the transport of tilianin. More studies are underway to determine the transport of tilianin.3. The results of the study showed for the first time that flavonoids, including the glucosides of flavone, could be subjected to a triple recycling scheme (i.e., enterohepatic recycling, enteric recycling and local recycling). As model compounds, tilianin and acacetin could undergo extensive glucuronidation and efflux process in vivo, participated in triple recycling. This triple recycling process begins with hydrolysis of our model compound, tilianin, to its aglycone, acacetin, catalyzed by LPH. Once hydrolyzed, acacetin is rapidly absorbed into intestinal epithelial cells and hepatocytes. Acacetin will be rapidly metabolized into glucuronides in both enterocytes and hepatocytes, which were then rapidly excreted into lumen or bile. Glucuronides can be hydrolyzed back to its aglycone forms by bacteria-derived β-gus in the colon or enteric-derived β-gus in the upper small intestine. The released aglycone, acacetin could be subsequently re-absorbed in the colon (for enterohepatic and enteric recycling) or enterocytes of all the intestines (for local recycling), completing the recycling process.Therefore, the flavonoid glucoside tilianin and its aglycone acacetin could undergo extensive glucuronidation and partipated in triple recycling in vivo. Triple recycling results in prolonged residence time and longer than expected half-lives, which could enhance potential pharmacological effects. Searching for more compounds involved in triple recycling might help to increase the enterohepatic bioavailability, which is of great importance to the drugs treated for liver and gut diseases. |
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