Absorption And Metabolism Of Polyphenols In The Intestine

Purpose Polyphenols, a group of complex naturally occurring compounds, arewidely distributed throughout the plant kingdom and thus are readily consumed byhumans. As a member of polyphenols family, dietary anthraquinone have receivedmuch attention as potential protectors against a variety of human diseases, inparticular cardiovascular disease and cancer. Epidemiological evidence has longsuggested that dietary polyphenols, which are abundant in fruits and vegetables,can reduce the risk of cancer. Such diets are well known to contain a variety ofchemicals that can affect the carcinogenic process in many ways. A large numberof mechanisms of action have been investigated, including antioxidant propertiesand effects on enzymes and signal transduction pathways. Increasing evidence forthe possible effects of plant polyphenols on human health have been obtained in invitro and in vivo systems. Intestinal absorption is a prerequisite for a possiblecausal relationship between polyphenols intake and its proposed chemopreventiveaction. However, data on their absorption from the gastrointestinal tract are stillscarce. To address this issue, we analysed the pharmacokinetics of polyphenols in rat plasma and evaluated the contribution of the small intestine to the absorptionand first-pass metabolism of polyphenols using an isolated rat small intestineperfusion model, the human Caco-2 cell model.Methods (1) The selected polyphenols, including apigenin, chrysophanol, emodinand resveratrol, were suspended in 0.5% CMC-Na and administered at a dose of50 =mol/kg to rats in experiments involving oral administration. Blood sampleswere collected from the vena abdominalis under light ether anesthesia at differenttimes(0.08,0.17,0.5,1,2,4,8,12,16,24,36h). The samples were immediatelycentrifuged for 10 min at 3000 rpm to obtain plasma, which was kept frozen at -70°C until analysis. We developed a high-throughput and sensitive bioanalyticalmethod using liquid chromatography/electrospray ionization tandem massspectrometry (LC/ESI-MS/MS) equipped with an electrospray ionization interfaceused to generate negative ions [M-H]– for the estimation of the concentration ofthe selected polyphenols and their metabolites. Quantitation was performed bymultiple reaction monitoring (MRM) of the deprotonated precursor ion and therelated product ion for the selected polyphenols after treatment with the?-glucuronidase and sulfatase, respectively. Pharmacokinetic parameters ofdifferent polyphenols and their metabolites were calculated by the 3p97 softwareafter oral administration of the drugs to rat. (2) The rats were anesthetized with anintra-abdominal injection of a mixture containing 40 mg/kg phenobarbital sodium.The animals were then heparinized (90 U/ kg–1) via the vena caudalis. A 7- to11-cm-long segment of the intestine was identified and separated. Silicone tubingwas placed inside both ends of the segment, and the tube at the proximal side wasconnected to a peristaltic pump for luminal perfusion. A polyethylene cannula, connected to a peristaltic pump for the vascular perfusion, was inserted into thesuperior mesenteric artery. The solutions on both sides were circulated by gas liftwith 95% O2 and 5% CO2 throughout the transport studies. Samples obtainedfrom the outlet of the mesenteric vein and luminal aliquots were collected into thepreweighted microtubes every 10 min and stored at -70°C until analysis. Wedeveloped a high-throughput and sensitive bioanalytical method using liquidchromatography/electrospray ionization tandem mass spectrometry(LC/ESI-MS/MS) equipped with an electrospray ionization interface used togenerate negative ions [M-H]– for the estimation of the concentration of theselected polyphenols and their metabolites. Quantitation was performed bymultiple reaction monitoring (MRM) of the deprotonated precursor ion and therelated product ion for the selected polyphenols after treatment with the?-glucuronidase and sulfatase, respectively. (3) For all cellular uptake studies ofpolyphenols, Caco-2 cells were seeded at a cell density of 5×104cells/cm2 ontwenty-four well plastic plates. Cells were used 14 to 21 days after seeding. Freshculture medium was replaced 24 h before uptake experiments. The selectedpolyphenols at various concentrations was added to evaluate the uptakecharacteristics at different times(5,10,30,60,120 min). To determine theintracellular polyphenols concentration, the cell lysate was obtained by subjectingthe drug-containing cells to three freeze-thaw cycles in liquid nitrogen. Proteinconcentrations were measured by the method of Bradford with bovine serumalbumin as a standard. For transport experiments, Caco-2 cells were seeded at acell density of 1×105cells/cm2 on Millicells. Cells were used 19 to 21 days afterseeding and the value of TEER should more than 300·cm2. Fresh culture medium was replaced 24 h before transport experiments. The selectedpolyphenols at various concentrations was added from the apical or basolateralside to evaluate the transport characteristics at different times(5,10,30,60,120min). The samples were analyzed by reverse phase HPLC system after treatmentwith the ?-glucuronidase and sulfatase, respectively.Results (1) Following oral administration apigenin, chrysophanol, emodin andresveratrol, the selected polyphanols can be absorbed rapidly, and there areobvious double peaks phenomenon in the CT curve of apigenin, emodin andresveratrol. The maximum concentration the selected polyphanols were 45±2ng/mL, 326±14ng/mL, 72±6ng/mL and 94±5ng/mL, respectively. And the AUCand the elimination half-time were 659,1721,809,98 (ng/ml)*h and 4.1,8.0,2.9,1.8h for apigenin, chrysophanol, emodin and resveratrol, respectively.Glucuronide and sulfate were identified and quantified by enzymatic hydrolysiswith glucuronidase and sulfatase using LC/MS/MS. After the full scan model, thespecial ion peak were detected, which have epactal fragment than the originaldrugs with 80 u and 176u. We presume that these fragment were theglucuronidated and sulfated parts. The selected polyphanol conjugates werequantified based on original drug concentration after enzymatic incubation ofluminal and vascular effluents. Quantitation was performed by multiple reactionmonitoring (MRM) of the deprotonated precursor ion and the related product ionfor the selected polyphenols after treatment with the ?-glucuronidase and sulfatase,respectively. We found the chromatographic peak area were more than the controlgroup. The plasma level of apigenin glucuronide was higher than that ofapigenin sulfate, the plasma levels of apigenin glucuronide reached Cmax of 51±3ng/mL within 1 hour. The plasma levels of resveratrol glucuronide rapidlyreached Cmax within 0.5 hour. (2) Glucuronide and sulfate were identified andquantified by enzymatic hydrolysis with glucuronidase and sulfatase usingLC/MS/MS. After the full scan model, the special ion peak were detected, whichhave epactal fragment than the original drugs with 80 u and 176u. We presumethat these fragment were the glucuronidated and sulfated parts. The selectedpolyphanol conjugates were quantified based on original drug concentration afterenzymatic incubation of luminal and vascular effluents. Quantitation wasperformed by multiple reaction monitoring (MRM) of the deprotonated precursorion and the related product ion for the selected polyphenols after treatment withthe ?-glucuronidase and sulfatase, respectively. We found the chromatographicpeak area were more than the control group. Approximately 28.88% of apigeninthat was administered appeared at the vascular side, chiefly as free rutin (16.39%),but also as emodin glucuronide (8.7%) and emodin sulfate (3.79%). The maincompound in the luminal effluent was apigenin, accompanied by lesser amountsof apigenin glucuronide and apigenin sulfate. Apigenin glucuronide (12.93%) andapigenin sulfate (1.83%) were found at the luminal side. For chrysophanol, thepercentage of chrysopahol glucuronide and sulfate were 13.85% and 2.48 % at thevascular side. Approximately 22.55% of emodin that was administered appearedat the vascular side, chiefly as free emodin (12.01%), but also as emodinglucuronide (8.69%) and emodin sulfate (1.84%). The main compound in theluminal effluent was emodin, accompanied by lesser amounts of emodinglucuronide and emodin sulfate. Emodin glucuronide (5.23%) and emodin sulfate(1.04%) were found at the luminal side. The mean total recovery of emodin and metabolites was 96.49±2.2%. In control perfusion experiments with the selectedpolyphanols-free basic perfusion media, no polyphanols or its conjugates weredetected. (3) The selected polyphenols can be absorpted rapidly by the caco-2cells, and the maximal concentration of chrysophanol and emodin were 414±15and 108±12 nmol/L·mg·protein, respectively. When the concentration of emodinand apigenin were increased from 2μM to 50μM, the intracellular concentrationincreased almost linearly. However, the saturation was apparent when theconcentration of was increased from 50μM to 200μM. The chrysophanol uptakecontinued to increase when the concentration of chrysophanol was increased from2μM to 200μM, and no saturation was apparent. And the saturation was 100μMfor resveratrol. After the incubation with the Caco-2 cells for 1 hour, the selectedpolyphenols were metabolized by the phase enzyme extensively, the percentageⅡof glucuronidated and sulfated resveratrol were 26.56% and 25.16%, respectively.The glucuronidated and sulfated emodin were 45.16% and 10.75%. For apigenin,the percentage of metabolites were 17.82% and 13.27%. The cellular uptake ofemodin and chrysophanol were reduced by approximately 34% and 41% in thepresence of 50 =M phloridzin, but have no obvious effect on the cellular uptake ofapigenin and resveratrol. Verapamil and cyclosporine can increase the uptake ofthe selected polyohanols in caco-2 cells.Conclusions(1)The selected polyphenols can be absorpted into bloodcirculation rapidly after oral administration.(2)The bioavailability of theseselected polyphenols was poor after oral administration.(3)Apigenin,chrysophanol, emodin and resveratrol can be metabolized by the phaseⅡenzymein rat intestine. One parts of the glucuronide or sulfate polyphenols we absorpted into blood circulation, the another were effluxed to the luminar side.The first metabolism may be the reason for the poor bioavailability in rat intestine.(4)The glucuronide chrysophanol or emodin was higher than sulfatemetabolites in caco-2 cells. There are no obvious difference between glucuronideapigenin or resveratrol and their sulfate metabolites.(5)Phloridzin can reducedthe cellular uptake of emodin and chrysophanol in caco-2 cells, which suggestedthat emodin and chrysophanol can be absorbed through the way of SGLT1.(6)Cyclosporine can increase the uptake of the selected polyohanols in caco-2 cells,which means MRP2 was the transport way for these compound.(7)The phenolichydroxyl group of these polyphenols was related to their absorption andmetabolism in the intestine possibly. The different transporters andphaseⅡenzyme can regulate the process and affect their pharmacological effect.