| 1Background and objectiveWuji Wan has a significant effect in the treatment of irritable bowel syndrome(IBS), which is a prescription of a traditional Chinese medicine(TCM) consisting of three herbs:Rhizoma Coptidis (Ranunculaceae), Fructus Evodiae Rutaecarpae (Rutaceae) and Radix Paeoniae Alba(Paeoniaceae). Rhizoma Coptidis is the king in the prescription and the other drugs are the ministers including Fructus Evodiae and Radix Paeoniae Alba. In our previous study, the pharmacokinetics of Wuji Wan was only conducted in normal animals and not involved in pathological animals. It is more significant to study the pharmacokinetics of drugs in the pathological states than normal states because the patients are the ultimate consumer of drugs. Moreover, IBS is a kind of disease, which has functional disorder of the gut but without obvious structural abnormalities of the gastrointestinal tract. In addition, it is no answer to the question whether the intestine under IBS pathological state affect drug absorption. Therefore, in the current study, the pharmacokinetics of representative constituents in rat plasma are compared between normal and IBS model rats including post inflammation irritable bowel syndrome rats(PI-IBS) and chronic visceral hypersensitivity IBS (CVH-IBS) after single oral administration of berberine hydrochloride, Rhizoma Coptidis and Wuji Wan. The representative constituents of Wuji Wan are berberine, palmatine, evodiamine, rutacarpine and paeoniflorin. That of berberine hydrochloride is berberine and Rhizoma Coptidis is berberine and palmatine. Quantification of representative constituents in rat plasma are achieved by using a sensitive and rapid UPLC-MS/MS method. Then, PK differences of the representative constituents are compared in normal and IBS model rats after single and multiple oral administration of Wuji Wan. Finally, the reason for the above differences are investigated by detecting the content of tight junction proteins (Occludin and ZO-1), myosin light chain kinase (MLCK) and transport proteins (P-gp, MRP1and MRP2) in the colon of rats by the immunohistochemistry and immunofluorescence.2Methods 2.1The simultaneous determination of the representative constituents of Wuji Wan including berberine, palmatine, evodiamine, rutacarpine and paeoniflorin in rat plasma by UPLC-MS/MS methodThe method of protein precipitation was adopted for the pretreatment of plasma sample. Then UPLC-MS/MS method of simultaneous determination of the representative constituents of Wuji Wan including berberine, palmatine, evodiamine, rutacarpine and paeoniflorin in rat plasma was established.2.2Comparing the PK of representative constituents after single oral administration of berberine hydrochloride, Rhizoma Coptidis and Wuji Wan between in normal and PI-IBS model ratsPI-IBS model rats were established by intracolonic instillation of acetic acid with restraint stress. After that, PI-IBS model rats were evaluated by observing the colonic motility, the number of the fecal pellet output and the time of the glass bead output in2hours, the amount of mast cells and the extent of colonic inflammation by toluene ammonia blue and hematoxylin-eosin staining. After the success of the model, the rats were fasted overnight and then subjected to the following surgeries under anesthesia condition by intraperitoneal injection of chloral hydrate at150mg/kg. A polyethylene catheter (0.50mm i.d.,1.00mm o.d.,) was cannulated into the right jugularvein. The distal end of the catheter was led under the skin and exteriorized at the back of the neck. After surgery, the rats were then allowed to recover for12h and fasted overnight prior to drug administration. Before drug administration, the blood samples(200μL) were collected from the catheter into heparinized centrifuge. Then berberine hydrochloride (25mg/kg, equivalent to berberine22mg/kg), Rhizoma Coptidis (96mg/kg, equivalent to berberine22mg/kg and palmatine5.3mg/kg), Wuji Wan (157.4mg/kg, equivalent to berberine22mg/kg, palmatine5.3mg/kg, evodiamine0.055mg/kg, rutacarpine0.07mg/kg and paeoniflorin6.265mg/kg) were i.g. to rats. After drug administration, the blood samples(200μL) were collected from the catheter into heparinized centrifuge tubes at appropriate intervals (5min,15min,30min,1h,1.5,2,3,4,6,8,10,12,24,36h). After centrifugation at3500rpm for15min,100μL of plasma was collected and stored at-80℃until analysis. After each blood collection, 200μL of normal saline containing50units/mL of heparin was immediately injected back into the body to flush the catheter and prevent coagulation. The amounts of representative constituents in plasma were estimated by UPLC-MS/MS analysis as described previously. The curve of drug concentration vs time was plot and the PK parameters(T1/2, Tmax, Cmax, AUC0-t, Vd/F and Cl/F) of representative constituents were calculated by the software of WinNonlin6.3. Finally, the PK differences of the representative constituents were compared in normal and PI-IBS model rats.2.3Comparing the PK of representative constituents after single oral administration of berberine hydrochloride, Rhizoma Coptidis and Wuji Wan between in normal and CVH-IBS model ratsFirstly, CVH-IBS model rats were established by mechancial irritation of the neonatal rat colon with using PTCA balloons. After that, CVH-IBS model rats were evaluated by observing the change of rats in body weight, the threshold of rat lifting its abdomen off the platform and its pelvic structures off the platform, the amount of mast cells and the extent of colonic inflammation by toluene ammonia blue and HE staining, the expression of proto-oncogene c-fos by immunohistochemistry. After the model was successful, the method of administration, blood collection and the determination of content of representative constituents was the same as the section2.2. After that, the curve of drug concentration vs time was plot and the PK parameters(T1/2, Tmax, Cmax, AUC0-t, Vd/F and Cl/F) of representative constituents were calculated by the software of WinNonlin6.3. Finally, the PK differences of the representative constituents were compared in normal and CVH-IBS model rats.2.4Comparing the PK of representative constituents after single and multiple oral administration of Wuji Wan in normal and PI-IBS model rats and investigating the reason for the difference of PKPI-IBS model rats were established and evaluated according to the method of the section2.2. After the model was successful, they were used to study the pharmacokinetics of representative constituents after single and multiple oral administration of Wuji Wan in normal and PI-IBS model rats. For single administration, Wuji Wan(629.6mg/kg, equivalent to berberine88mg/kg, palmatine 21.1mg/kg, Evodiamine0.22mg/kg, Rutacarpine0.28mg/kg and paeoniflorin25.1mg/kg) were i.g. to rats. For multiple administration, Wuji Wan were administered continuously to rats for6days, once a day with the same dose as the single dose administration. After12h in the sixth day, the rats were carried out jugular vein catheterization. After surgery, the rat was then allowed to recover for12h and fasted overnight prior to drug administration. Before drug administration, the blood samples(200μL) were collected from the catheter into heparinized centrifuge. Then Wuji Wan(629.6mg/kg, equivalent to berberine88mg/kg, palmatine21.1mg/kg, Evodiamine0.22mg/kg, Rutacarpine0.28mg/kg and paeoniflorin25.1mg/kg) were i.g. to rats. After that, blood collection was the same as the section2.2and the determination of content of representative constituents adopted the section2.1method. The curve of drug concentration vs time was plot and the PK parameters(T1/2, Tmax, Cmax, AUCo-t, Vd/F and Cl/F) of representative constituents were calculated by the software of WinNonlin6.3. Then, the PK differences of the representative constituents were compared in normal and CVH-IBS model rats.After time point of blood collection36h was completed, the rats were killed. Then, the colon was quickly taken out and fixed by using4%paraformaldehyde, paraffin-embedded sections. Finally, the content of tight junction proteins (Occludin and ZO-1), myosin light chain kinase (MLCK) and transport proteins (P-gp, MRP1and MRP2) in the colon of rats were detected by the immunohistochemistry and immunofluorescence in order to investigate the reason for the differences of PK of representative constituents.2.5Comparing the PK of representative constituents after single and multiple oral administration of Wuji Wan in normal and CVH-IBS model rats and investigating the reason for the difference of PKCVH-IBS model rats were established and evaluated according to the method of the section2.3. After the model was successful, the PK of representative constituents were compared after single and multiple oral administration of Wuji Wan in normal and CVH-IBS model rats and the reason for the difference of PK was investigated in accordance with the method of the section2.4. 3Results3.1The simultaneous determination of the representative constituents of Wuji Wan including berberine, palmatine, evodiamine, rutacarpine and paeoniflorin in rat plasma by UPLC-MS/MS methodUPLC-MS/MS method established in this study could be used to determine simultaneously the content of five representatives ingredients of Wuji Wan in the rat plasma. Moreover, the method was specific, sensitive and accurate.3.2Comparing the PK of representative constituents after single oral administration of berberine hydrochloride, Rhizoma Coptidis and Wuji Wan between in normal and PI-IBS model ratsPI-IBS established in this study not only significantly aggravated the colonic motility but also had different influence on the function of the digestive tract and the amount of mast cell, which were the major syndromes of PI-IBS. These results point out that PI-IBS model rats established by intracolonic instillation of acetic acid with restraint stress could largely imitate the symptoms of human PI-IBS and could be used as animal models of PI-IBS for scientific research.The results of pharmacokinetic study of the representative constituents after single oral administration of berberine hydrochloride, Rhizoma Coptidis and Wuji Wan in normal rats and PI-IBS model rats showed that compared with the normal group,(1) after single oral administration of berberine hydrochloride, AUCo-t of berberine in PI-IBS model rats increased significantly and Cl/F decreased.(2) after single oral administration of Rhizoma Coptidis, Cmax and AUCo-t of berberine in PI-IBS model rats increased significantly, while Vd/F and Cl/F decreased. However, AUCo-t of palmatine in PI-IBS model rats increased significantly and other pharmacokinetic parameters had no significant differences. Additionally, the second peak at3hour for berberine and4hour for palmatine were observed in model group.(3) after single oral administration of Wuji Wan, Cmax and AUCo-t of berberine in PI-IBS model rats increased significantly, while Vd/F and Cl/F decreased. AUCo-t of palmatine in PI-IBS model rats increased significantly, while Vd/F and T1/2decreased. For paeoniflorin, Cmax and AUCo-t in PI-IBS model rats increased significantly, while and Cl/F decreased.3.3Comparing the PK of representative constituents after single oral administration of berberine hydrochloride, Rhizoma Coptidis and Wuji Wan between in normal and CVH-IBS model ratsCVH-IBS established in this study not only significantly showed visceral hypersensitivity, the increased mast cell but also significantly increased the expression of proto-oncogene c-fos, which were the major syndromes of CVH-IBS. These results pointed out that CVH-IBS model rats established by mechancial irritation of the neonatal rat colon with using PTCA balloons could largely imitate the symptoms of human CVH-IBS and could be used as animal models of CVH-IBS for scientific research.The results of pharmacokinetic study of the representative constituents after single oral administration of berberine hydrochloride, Rhizoma Coptidis and Wuji Wan in normal rats and CVH-IBS model rats showed that compared with the normal group,(1) after single oral administration of berberine hydrochloride, T/2and AUCo-t of berberine in CVH-IBS model rats increased significantly and Cl/F decreased.(2) after single oral administration of Rhizoma Coptidis, T1/2, Tmax and AUCo-t of berberine in CVH-IBS model rats increased significantly, while Vd/F and Cl/F decreased. However, all the pharmacokinetic parameters of palmatine in CVH-IBS model rats had no significant difference.(3) after single oral administration of Wuji Wan, AUCo-t of berberine in CVH-IBS model rats increased significantly, while T1/2, Cmax, Vd/F and Cl/F decreased. Cmax and AUCo-t of palmatine in CVH-IBS model rats increased significantly, while Tmax, Vd/F and Cl/F decreased. For evodiamine, Cmax in CVH-IBS model rats increased significantly, while Tmax, Vd/F and Cl/F decreased. For rutacarpine, Cmax and AUCo-t in CVH-IBS model rats increased significantly, while Vd/F decreased. Cmax and AUCo-t of paeoniflorin in CVH-IBS model rats increased significantly, while Vd/F and Cl/F decreased.3.4Comparing the PK of representative constituents after single and multiple oral administration of Wuji Wan in normal and PI-IBS model rats and investigating the reason for the difference of PK The results of pharmacokinetic study of the representative constituents after single and multiple oral administration of Wuji Wan in normal rats and PI-IBS model rats showed that compared with the normal group,(1) after single oral administration of Wuji Wan, T1/2, Tmax, Cmax and AUC0-t of berberine in PI-IBS model rats increased significantly, while Cl/F decreased. AUC0-t of palmatine in PI-IBS model rats increased significantly, while Vd/F and Cl/F decreased. For evodiamine, Tmax, Cmax and AUCo-t in PI-IBS model rats increased significantly, while Cl/F decreased. For rutacarpine, AUC0-t in PI-IBS model rats increased significantly, while Vd/F and Cl/F decreased. Cmax and AUC0-t of paeoniflorin in PI-IBS model rats increased significantly, while Vd/F and Cl/F decreased.(2) after multiple oral administration of Wuji Wan, Cmax and AUC0-t of berberine in PI-IBS model rats increased significantly, while Tmax, Vd/F and Cl/F decreased. T1/2and AUC0.t of palmatine in PI-IBS model rats increased significantly, while Tmax, Vd/F and Cl/F decreased. For evodiamine, Cmax and AUC0-t in PI-IBS model rats increased significantly, while T1/2, Tmax, Vd/F and Cl/F decreased. For rutacarpine, Cmax in PI-IBS model rats increased significantly, while T1/2, Tmax, Vd/F and Cl/F decreased. T1/2, Cmax and AUC0-t of paeoniflorin in PI-IBS model rats increased significantly, while Cl/F decreased.(3) the results of transmission electron microscope showed that the colonic epithelial tight junction in normal rats had structural integrity and connection dense. However, those of in model rats had structural damage, loose connections, the widened gap and lower density. But after oral administration of Wuji Wan, the colonic epithelial tight junction in model rats became structural integrity.(4) the results of immunohistochemistry showed that: For the tight junction proteins including Occludin and ZO-1, compared with the normal group, a significant decrease in PI-IBS model rat colon. After multiple dose administration, they were significantly higher in the model group of multiple dose administration than in the untreated model group, but there was no significant difference in the expression of them between the normal group of multiple dose administration and the untreated normal group. But the expression of them in the model group of multiple dose administration was lower than the normal group of multiple dose administration; For MLCK, compared with the normal group, a significant increase in PI-IBS model rat colon. After multiple dose administration, they was significantly lower in the model group of multiple dose administration than in the untreated model group, but there was no significant difference in its expression between the normal group of multiple dose administration and the untreated normal group. But its expression in the model group of multiple dose administration was higher than the normal group of multiple dose administration; For the transporters including P-gp, MRP1and MRP2, compared with the normal group, the expression of them in PI-IBS model rats colon had no significant changes. After multiple dose administration, they was significantly higher in the model group and the normal group than the previous corresponding untreated model group and normal group. However, the expression of them had no significant difference between the model group of multiple dose administration and the normal group of multiple dose administration.(5) the results of immunofluorescence showed that:compared with the normal group, a significant decrease of the tight junction proteins and a significant increase of MLCK in PI-IBS model rat colon. However, after multiple dose administration, tight junction proteins were significantly higher and MLCK lower in the model group of multiple dose administration than in the untreated model group. But, the expression of them had no significant difference between the normal group of multiple dose administration and in the untreated normal group. However, the expression of tight junction proteins and MLCK were lower and higher in the model group of multiple dose administration than in the normal group of multiple dose administration.3.5Comparing the PK of representative constituents after single and multiple oral administration of Wuji Wan in normal and CVH-IBS model rats and investigating the reason for the difference of PKThe results of pharmacokinetic study of the representative constituents after single and multiple oral administration of Wuji Wan in normal rats and CVH-IBS model rats showed that compared with the normal group,(1) after single oral administration of Wuji Wan, for berberine and palmatine, T1/2and AUC0-t in CVH-IBS model rats increased significantly, while Cl/F decreased. For evodiamine, Cmax and AUC0-t in CVH-IBS model rats increased significantly, while T1/2, Tmax, Cl/F and Vd/F had no significant difference. However, all the pharmacokinetic parameters of rutacarpine in CVH-IBS model rats had no significant difference. Tmax and AUCo-t of paeoniflorin in CVH-IBS model rats increased significantly, while Cl/F decreased.(2) after multiple oral administration of Wuji Wan, Cmax and AUCo-t of berberine and palmatine in CVH-IBS model rats decreased significantly, while T1/2, Vd/F and Cl/F increased. For evodiamine, Cmax and AUCo-t in CVH-IBS model rats increased significantly, while T1/2, Tmax and Vd/F decreased. For rutacarpine, Cmax in CVH-IBS model rats increased significantly, while Tmax decreased. However, Tmax of paeoniflorin in CVH-IBS model rats increased significantly, while other pharmacokinetic parameters of palmatine in CVH-IBS model rats had no significant difference.(3) the results of transmission electron microscope showed that the colonic epithelial tight junction in normal rats had structural integrity and connection dense. However, those of in model rats had structural damage, loose connections, the widened gap and lower density. But after oral administration of Wuji Wan, the colonic epithelial tight junction in model rats became structural integrity.(4) the results of immunohistochemistry showed that:For the tight junction proteins including Occludin and ZO-1, compared with the normal group, a significant decrease in CVH-IBS model rat colon. After multiple dose administration, they were significantly higher in the model group of multiple dose administration than in the untreated model group, but there was no significant difference in the expression of them between the normal group of multiple dose administration and the untreated normal group. But the expression of them in the model group of multiple dose administration was lower than the normal group of multiple dose administration; For MLCK, compared with the normal group, a significant increase in CVH-IBS model rat colon. After multiple dose administration, they were significantly lower in the model group of multiple dose administration than in the untreated model group, but there was no significant difference in the expression of them between the normal group of multiple dose administration and the untreated normal group. But its expression in the model group of multiple dose administration was higher than the normal group of multiple dose administration; For the transporters including P-gp, MRP1and MRP2, compared with the normal group, its expression in CVH-IBS model rats colon had no significant changes. After multiple dose administration, they were significantly higher in the model group and the normal group than the previous corresponding untreated model group and normal group. However, the expression of them in the model group of multiple dose administration were significant higher than in the normal group of multiple dose administration.(5) the results of immunofluorescence showed that:compared with the normal group, a significant decrease of the tight junction proteins and a significant increase of MLCK in CVH-IBS model rat colon. However, after multiple dose administration, tight junction proteins were significantly higher and MLCK lower in the model group of multiple dose administration than in the untreated model group. But, its expression had no significant difference between the normal group of multiple dose administration and in the untreated normal group. However, the expression of tight junction proteins and MLCK were lower and higher in the model group of multiple dose administration than in the normal group of multiple dose administration.4ConclusionThe pharmacokinetic behavior of berberine, palmatine, evodiamine, rutacarpine and paeoniflorin after single and multiple dose oral administration of Wuji Wan was significantly altered in IBS pathological conditions, which was possiblely associated with transporters and tight junction proteins. Therefore, the dosage modification of Wuji Wan was necessary and important for IBS patients to avoid side effects and increase curing effects according to the different states and medication schedules of the body. |
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