The Absorption And Metabolism Of Emodin And Rhein In The Intestine

Emodin-type anthraquinones are widely distributed, with a variety of pharmacological effects. Recent studies found that these compounds has a strong cytotoxic-like effects on a variety of tumor cells, therefore, emodin-type anthraquinones are a type of candidate compounds with development prospects. As these compounds have good pharmacological activities, most researches are focused on their pharmacological effects were reported, while the studies related to their absorption and metabolic mechanism are still unclear. Therefore, the studies focusing on absorption and transportation mechanism of emodin-type anthraquinones in intestinal have great significances for their exploitation and utilization.On the other hand, chronic exposure of drugs which containing emodin-type anthraquinones frequently produce liver and kidney damages. In addition, many reports shown that some organ and system damages occurred were mainly due to the chronic exposure of tranditional Chinese medicines. Therefore, the researches on emodin-type anthraquinones related to the mechanism of absorption and metabolic will provide important scientific basis for their application and development in the future.The Caco-2 cells is one of the most commonly models used in vitro which is an effective tool to elucidate drug absorption, metabolism and transport mechanism. Caco-2 cells derived from human colon epithelial cells, in vitro culture conditions can be differentiated into intestinal epithelial cells. Cultured mature Caco-2 cells not only have the structure of the microvilli, but also process the stable expression of a variety of drug metabolic enzymes and transporters. In recent years, the Caco-2 cell model has been widely used in drug absorption and metabolism at intestinal both at home and abroad.Emodin and rhein are two typical emodin type anthraquinone compounds, their absorption characteristics significant difference. According to our previous study results showed that the bioavailability of emodin is quite low, in male SD rats is 7.5%, just 5% of the female. However rhein bioavailability are relatively good, research shows that rhein in SD rats bioavailability is about 20%, and in the case of beagle dog the degree is as high as 50%.And the reasons for the differences in the body about the two same type compounds are unclear.This paper intends to make further study about the absorption and transport mechanism of emodin and rhein in Caco-2 cell model, in order to providing the experimental basis for the reasonable application and development of these two compounds. The main contents include(1)Study of solubility and stability about emodin and rhein. (2) Built and optimize the Caco-2 cell model. (3) The absorption and metabolism features of emodin in Caco-2 cells. (4) The mechanism of absorption and metabolism of emodin in Caco-2 cells (5) The absorption and transport characteristics of rhein in Caco-2 cells. (6) The mechanism of absorption and transport of rhein in Caco-2 cells1.Stability and solubility of emodin and rheinIn order to carry out experiments in Caco-2 cell model of emodin and rhein, we needed clear understanding of stability and solubility of them.We determined the compounds’ stability and solubility by using UPLC method. The results showed that emodin and rhein can better dissolved in the HBSS solution, and stable within 24 h under pH 7.4, to carry out the in vitro Caco-2 transporter experiment.In addition, we also investigated the suitable container collection, found that there was no Significant effects in EP tube.2. Establishing the Caco-2 cell model and optimizing the related experimental conditionsIn our laboratory culture conditions, after 21 days training, Caco-2 cells can form similar cell polarity about the epithelial cells of the small intestine,and its transendothelial electrical resistance is more than 300Ω/4.2 cm2.At the same time use genistein as the control drug to detect the integrity of the cells and the metabolic enzymes and transporters function. The results are in accordance with reported. So the Caco-2 cell model in our laboratory can be used as an in vitro model to study the absorption and metabolism in small intestine.3. Investigating the absorbtion and metabolism characteristics of emodin in Caco-2 cellsWe tested four different concentrations of emodin such as 2.4,4,9 and 13 μmol/L across the Caco-2 monolayer to study the absorption and metabolism processes of emodin in the intestinal as anticipated, we observed concentration-dependent increase in the transport of aglycone across the Caco-2 monolayer for both A-B and B-A transport studies. The result indicated that the passive diffusion is the dominant transport way for emodin in Caco-2 cell monolayers. In Caco-2 cells Emodin was quickly metabolized into of glucuronide.The formation and metabolic rate of its metabolites were concentration-dependent, proving that emodin metabolite transported by the A side and B side efflux transporter..No matter loading emodin at Apical side or Basolateral side, the metabolite efflux to the BL side was significantly higher than to the AP side, this was consistent with our previous experimental results.4. The mechanism of absorption and transport of emodin in Caco-2 cellsIn order to further study of emodin on the Caco-2 cell model absorption and metabolism mechanisms,we added the BCRP transporter inhibitor dipyridamole, MRPs transporters inhibitors MK-571 and MRP-2 exclusive inhibitors of LTC4 to examine the bidirection transporter of emodin,respectively. No significant effect was observed in the efflux of emodin and the excretion of emodin glucuronide for A-B or B-Atransport compare with control group. Furthermore, the apparent permeability numbers for A-B or B-A were both decreased compared to control group and the efflux ratio was nearly to 1 in the presence of dipyridamole This suggested emodin aglycone as a possible weaker substrate of BCRP efflux transporter. LTC4, a specific MRP-2 efflux transporter chemical inhibitor at 0.1 μmol/L concentrations showed significantly higher bidirectional transport of emodin (13μmol/L) aglycone than for control study. On the other hand, emodin glucuronide excretion was especially lower on the apical side (>2 folds) than on the basolateral side (< 2 folds) for both A-B and B-A emodin(13μmol/L) transport across Caco-2 cell monolayer. During the LTC4 chemical inhibition studies, the intracellular concentrations of emodin aglycone as well as emodin glucuronide were not significantly different as compared to the control studies. MRP efflux transporter LTC4 chemical inhibition study suggested emodin glucuronide rather than emodin aglycone as a possible but weak substrate of MRP2 transporter in Caco-2 cells. In case of MK-571, a well known MRP1,2,3 and 4 efflux transporter chemical inhibitor, the bidirectional transport of emodin (13 μmol/L) was significantly higher for 10 and 20 μM where as no significant difference was observed for MK-571 (5μmol/L) concentration as compared to control studies across Caco-2 cell monolayer. There was no different intra-cellular emodin and its glucuronide concentrations for MK-571 (5,10 and 20 μmol/L) A-B studies with control study and significant different for MK-571 (20 μmol/L) B-A studies with control study suggesting possible emodin glucuronide formation inhibition effect of MK-571 (20 μmol/L). The results indicated that MRP2 in Apical side and MRP3/MRP4 in Basolateral side might the most important efflux for emodin metabolite.5. Investigate the absorbtion and transport characteristics of rhein in Caco-2 cellsFour different concentrations of rhein were also tested such as 2.5,5,10 and 20 μmol/L across the Caco-2 monolayer to study the bidirectional transport process of rhein. We observed concentration-dependent increase in the transport of rhein across the Caco-2 monolayer for both A-B and B-A transport studies. In Caco-2 cell model, usually with the apparent permeability (Papp) to evaluate the absorption of drugs in the small intestine.The apparent permeability values for the apical (AP) to basolateral (BL) and from BL to AP were more than 10-6 cm/s, this was consistent with reported.The Efflux Ratio were 4.04、3.58、3.56、3.06. Generally think Papp(B-A) significantly greater than Papp(A-B) is the drug was discharged by the Apical side transporters of the small intestine membrane. According to FDA guiding principle. Efflux Ratio value in the greater than 2, suggest that efflux transporters involved in the transport of rhein..6. The mechanism of absorption and transport of rhein in Caco-2 cellsIn order to further prove the absorption transport mechanism about rhein and which transporters participation in the efflux of rhein,We tested three different transporters inhibitors,such as P-gp inhibitors verapamil, MRPs inhibitors MK-571 and BCRP exclusive inhibitors Ko143 to study the influence on the bidirection transport of rhein. Add 50 μmol/L verapamil to the apical side, compared with 10μ mol/L as control group.We found that verapamil reduced the bidirectional transport, the apparent permeability. The Efflux Ratio from 3.56 lower to 2.56, increased the intracellular concentrations suggested that rhein as a possible weak substrate of P-gp efflux transporter. By adding 50μ mol/L MK-571 significantly inhibit the B-A transport, reduced the Papp(B-A) from 2.98±0.24×10-5 cm/s to 1.84±0.04×10-5 cm/s.During the MK-571 chemical inhibition studies, the intracellular concentrations of rhein was significantly different as compared to the control studies (p< 0.05).While 10 μmol/L Ko143 increased the A-B transport. On the contrary, decreased the B-A transport and significant increase the intracellular concentration (p < 0.05).The efflux ratio was nearly to 1 in the presence of Ko143 this suggested rhein as a possible strong substrate of BCRP efflux transporter. Overall, the efflux transporter of rhein include P-gp, MRPs, and BCRP. And BCRP play a major role in this process.To sum up, the main reason due to low bioavailability of emodin might be easily etabolize into primary metabolite by UGT enzymes in intestine. The dominant transport way for emodin in Caco-2 cell is the passive diffusion. In Caco-2 cells cell, emodin was quickly metabolized into of glucuronide, while the metabolite as substrate of the MRPs were efflux from Caco-2 cell leading to the bioavailability reduced. We observed concentration-dependent increase in the transport of rhein across the Caco-2 monolayer for both A-B and B-A transport studies. This indicated that the passive diffusion is the dominant transport way for emodin in Caco-2 cell monolayers.Rhein as possible substrate of P-gp, MRPs and BCRP efflux transporter, among them BCRP transporter plays a main role.