槲皮素在Caco-2单层细胞模型上的跨膜吸收和甲基化代谢
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摘要
目的基于人小肠吸收模型,研究槲皮素的跨膜吸收和甲基化代谢。方法人小肠吸收模型选用Caco-2单层细胞,分别于基底侧或腔侧加入槲皮素9.0和18.0 mg·L-1(终浓度),于30,60,90,120和150 min采样,采用高效液相色谱-质谱(LC-MS)法测定加载侧和透过侧样品中槲皮素、异鼠李亭和柽柳黄素的含量。先于腔侧加入外排蛋白P-糖蛋白(P-pg)或多药耐药蛋白2(MRP2)的特效抑制剂环孢菌素A(CysA)10 mmol·L-1或MK571 1 mmol·L-1(pH=7.2),孵育15 min后,再于腔侧加入槲皮素(终浓度9.0和18.0 mg·L-1),同法测定透过侧样品中槲皮素含量。结果双向转运过程中,在150 min孵育时间内,加载侧槲皮素剩余量的动态变化表现为随孵育时间持续下降(相邻时间点之间比较P<0.05);透过侧槲皮素透过量则表现为先升后降的趋势特征,120 min达峰,150 min下降(相邻时间点之间比较P<0.05);在透过侧和加载侧均检测到槲皮素的甲基化产物异鼠李亭和柽柳黄素,两侧的动态变化特征与槲皮素相似;不同的是,无论腔侧还是基底侧加载槲皮素,在30～60 min后,腔侧异鼠李亭和柽柳黄素含量均明显高于基底侧。分析双侧槲皮素、异鼠李亭和柽柳黄素占槲皮素加载量的百分比,发现孵育30 min时,加载侧槲皮素剩余量不足加载量的20%～25%,透过侧槲皮素含量仅为加载量的约1%;150 min时加载侧槲皮素剩余量下降至不足加载量的10%,而透过侧槲皮素含量仅为加载量的6%～7%;槲皮素甲基化产物异鼠李亭和柽柳黄素在两侧的产生量也仅为槲皮素加载量的0.1%～0.3%。与槲皮素对照组比较,预先加入CysA或MK571,均明显增加槲皮素从腔侧到基底侧的转运(P<0.05)。结论槲皮素在Caco-2单层细胞模型上的转运具有先升后降的趋势特征,并发生甲基化代谢;P-pg和MRP2的外排作用对槲皮素跨膜转运可能具有一定影响。
OBJECTIVE To study transmembrane transport and methylation metabolism of quercetin based on the human intestinal absorption model. METHODS Caco-2 cell monolayer was used as the human intestinal absorption model. The incubation concentration of quercetin was 9.0 and 18.0 mg·L-1.Samples were collected at 30, 60, 90, 120 and 150 min, and the contents of quercetin, isorhamnetin and tamarixetin on the loading side and receiver side were determined by high performance liquid chromatography-mass spectrometry(LC-MS) method. Bupropion was used as the internal standard, and the injection volume was 20 μL. The specific inhibitors of P-glycoprotein(P-pg) or multidrug resistant protein 2(MRP2), cyclosporins(CysA) 10 mmol·L-1 or MK571 1 mmol·L-1(final concentration), were added to the apical side. After 15 min incubation, quercetin 9.0 or 18.0 mg·L-1(final concentration) was added to the apical side, respectively. The quercetin content on the receiver side was determined by the same method. RESULTS During bi-directional transport, the dynamic change of quercetin residues on the loading side showed a continuous decline within 150 min(P<0.05 between adjacent time points),while the amount of quercetin on the receiver side tended to increase before decreasing, reaching the peak at 120 min and falling at 150 min(P<0.05 between adjacent time points). Isorhamnetin and tamarixetin could be detected on both the loading side and receiver side. But the difference was that when quercetin was loaded on the apical side or the basolateral side, there was much more isorhamnetin and tamarixetin on the apical side than on the basolateral side after 30-60 min. Analysis of the percentage of quercetin,isorhamnetin and tamarixetin on the loading side and receiver side found that when incubated for 30 min, the residual quercetin on the loading side was less than 20%-25%, and quercetin on the receiver side was only about 1% of the loading amount. At 150 min, the residual quercetin decreased to <10%,while quercetin in the receiver side was only 6%-7% of the loading amount, and isorhamnetin and tamarixetinin on both sides were only 0.1%-0.3% of loading quercetin. Compared with the quercetin control group, the addition of CysA or MK571 in advance significantly increased the transport of quercetin from the apical side to the basolateral side(P<0.05). CONCLUSION The transport of quercetin on the Caco-2 monolayer cell model shows a trend of rise and fall, accompanied by methylation metabolism. The efflux of P-pg and MRP2 may have an effect on the transmembrane transport of quercetin.
OBJECTIVE To study transmembrane transport and methylation metabolism of quercetin based on the human intestinal absorption model. METHODS Caco-2 cell monolayer was used as the human intestinal absorption model. The incubation concentration of quercetin was 9.0 and 18.0 mg·L-1.Samples were collected at 30, 60, 90, 120 and 150 min, and the contents of quercetin, isorhamnetin and tamarixetin on the loading side and receiver side were determined by high performance liquid chromatography-mass spectrometry(LC-MS) method. Bupropion was used as the internal standard, and the injection volume was 20 μL. The specific inhibitors of P-glycoprotein(P-pg) or multidrug resistant protein 2(MRP2), cyclosporins(CysA) 10 mmol·L-1 or MK571 1 mmol·L-1(final concentration), were added to the apical side. After 15 min incubation, quercetin 9.0 or 18.0 mg·L-1(final concentration) was added to the apical side, respectively. The quercetin content on the receiver side was determined by the same method. RESULTS During bi-directional transport, the dynamic change of quercetin residues on the loading side showed a continuous decline within 150 min(P<0.05 between adjacent time points),while the amount of quercetin on the receiver side tended to increase before decreasing, reaching the peak at 120 min and falling at 150 min(P<0.05 between adjacent time points). Isorhamnetin and tamarixetin could be detected on both the loading side and receiver side. But the difference was that when quercetin was loaded on the apical side or the basolateral side, there was much more isorhamnetin and tamarixetin on the apical side than on the basolateral side after 30-60 min. Analysis of the percentage of quercetin,isorhamnetin and tamarixetin on the loading side and receiver side found that when incubated for 30 min, the residual quercetin on the loading side was less than 20%-25%, and quercetin on the receiver side was only about 1% of the loading amount. At 150 min, the residual quercetin decreased to <10%,while quercetin in the receiver side was only 6%-7% of the loading amount, and isorhamnetin and tamarixetinin on both sides were only 0.1%-0.3% of loading quercetin. Compared with the quercetin control group, the addition of CysA or MK571 in advance significantly increased the transport of quercetin from the apical side to the basolateral side(P<0.05). CONCLUSION The transport of quercetin on the Caco-2 monolayer cell model shows a trend of rise and fall, accompanied by methylation metabolism. The efflux of P-pg and MRP2 may have an effect on the transmembrane transport of quercetin.
引文
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[14] Rich GT,Buchweitz M,Winterbone MS,Kroon PA,Wilde PJ. Towards an understanding of the low bioavailability of quercetin:a study of its interaction with intestinal lipids[J/OL]. Nutrients,2017,9(2):111(2017-02-05). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331542/
[15] Gonzales GB,Smagghe G,Grootaert C,Zotti M,Raes K,Van Camp J. Flavonoid interactions during digestion, absorption, distribution and metabolism:a sequential structure-activity/property relationship-based approach in the study of bioavailability and bioactivity[J]. Drug Metab Rev,2015,47(2):175-190.
[16] Sharma H,Kanwal R,Bhaskaran N,Gupta S.Plant flavone apigenin binds to nucleic acid bases and reduces oxidative DNA damage in prostate epithelial cells[J/OL]. PLoS One,2014,9(3):e91588(2014-03-10). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948873/
[17] Zheng Y,Scow JS,Duenes JA,Sarr MG. Mechanisms of glucose uptake in intestinal cell lines:role of GLUT2[J]. Surgery,2012,151(1):13-25.
[18] Yang C,Li Z,Zhang T,Liu F,Ruan J,Zhang Z.Transcellular transport of aconitine across human intestinal Caco-2 cells[J]. Food Chem Toxicol,2013,57:195-200.
[19] Walgren RA,Karnaky KJ Jr,Lindenmayer GE,Walle T. Efflux of dietary flavonoid quercetin 4′-betaglucoside across human intestinal Caco-2 cell monolayers by apical multidrug resistance-associated protein-2[J]. J Pharmacol Exp Ther,2000,294(3):830-836.
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[21] Li SY,Li Z,Li JL,Gao WN,Zhang ZQ,Guo CJ.Quercitrin is absorbed and metabolized by Caco-2cells[J]. Curr Top Phytochem,2011,10:67-73
[22] Li SY,Li Z,Li JL,Gao WN,Zhang ZQ,Guo CJ.The simultaneous determination of quercitrin,quercetin and its methylated metabolites by high performance liquid chromatography-mass spectrometry[J]. Acta Nutr Sin(营养学报),2010,32(6):603-607.
[23] Li SY,Li Z,Gao WN,Li JL,Zhang ZQ,Guo CJ.Role of glucose transporter 1 and glucose transporter 2 in transmembrane absorption of quercirin and isoquercitrin[J]. Chin J Pharmacol Toxicol(中国药理学与毒理学杂志),2018,32(3):192-200.
[24] Li SY,Li Z,Gao WN,Chen CX,Zhang ZQ,Guo CJ.The role of glucose transporter SGLT1 and GLUT2in the intestinal absorption of quercetin[J]. Electron J Metab Nutr Cancer(肿瘤代谢与营养电子杂志),2018,5(1):33-37.
[25] Li SY,Li Z,Gao WN,Li JL,Guo CJ,Zhang ZQ.Effect of P-glycoprotein on the transmembrane absorption and metabolism of quercitrin on Caco-2 cells[J]. J Int Pharm Res(国际药学研究杂志),2017,44(12):1150-1154,1162.
[26] Li Z. P-glycoprotein drug interaction models and its application in the drug evaluation(P-糖蛋白与药物相互作用模型的建立及其在药物评价中的应用)[D].Beijing:Academy of Military Medical Sciences(军事医学科学院),2011.
[27] Guo Y,Bruno RS. Endogenous and exogenous mediators of quercetin bioavailability[J]. J Nutr Biochem,2015,26(3):201-210.
[2] Brito AF,Ribeiro M,Abrantes AM,Mamede AC,Laranjo M,Casalta-Lopes JE,et al. New approach for treatment of primary liver tumors:the role of quercetin[J]. Nutr Cancer,2016,68(2):250-266.
[3] Leyva-López N,Gutierrez-Grijalva EP,AmbrizPerez DL,Heredia JB. Flavonoids as cytokine modulators:a possible therapy for inflammationrelated diseases[J/OL]. Int J Mol Sci,2016,17(6):921(2016-06-09). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926454/
[4] Tribolo S,Lodi F,Connor C,Suri S,Wilson VG,Taylor MA,et al. Comparative effects of quercetin and its predominant human metabolites on adhesion molecule expression in activated human vascular endothelial cells[J]. Atherosclerosis,2008,197(1):50-56.
[5] Dihal AA, Woutersen RA, van Ommen B,Rietjens IM,Stierum RH. Modulatory effects of quercetin on proliferation and differentiation of the human colorectal cell line Caco-2[J]. Cancer Lett,2006,238(2):248-259.
[6] Kwon O,Eck P,Chen S,Corpe CP,Lee JH,Kruhlak M,et al. Inhibition of the intestinal glucose transporter GLUT2 by flavonoids[J]. FASEB J,2007,21(2):366-377.
[7] Dai X,Ding Y,Zhang Z,Cai X,Bao L,Li Y.Quercetin but not quercitrin ameliorates tumor necrosis factor-alpha-induced insulin resistance in C2C12skeletal muscle cells[J]. Biol Pharm Bull,2013,36(5):788-795.
[8] Kittl M,Beyreis M,Tumurkhuu M,Fürst J,Helm K,Pitschmann A,et al. Quercetin stimulates insulin secretion and reduces the viability of rat INS-1 betacells[J]. Cel Physiol Biochem,2016,39(1):278-293.
[9] de Boer VC,Dihal AA,van der Woude H,Arts IC,Wolffram S,Alink GM,et al. Tissue distribution of quercetin in rats and pigs[J]. J Nutr,2005,135(7):1718-1725.
[10] Mullen W,Edwards CA,Crozier A. Absorption,excretion and metabolite profiling of methyl-,glucuronyl-,glucosyl-and sulpho-conjugates of quercetin in human plasma and urine after ingestion of onions[J]. Br J Nutr,2006,96(1):107-116.
[11] Walle T. Absorption and metabolism of flavonoids[J]. Free Radic Biol Med,2004,36(7):829-837.
[12] Walle T,Otake Y,Walle UK,Wilson FA. Quercetin glucosides are completely hydrolyzed in ileostomy patients before absorption[J]. J Nutr,2000,130(11):2658-2661.
[13] Fang Y,Liang F,Liu K,Qaiser S,Pan S,Xu X.Structure characteristics for intestinal uptake of flavonoids in Caco-2 cells[J]. Food Res Int,2018,105:353-360.
[14] Rich GT,Buchweitz M,Winterbone MS,Kroon PA,Wilde PJ. Towards an understanding of the low bioavailability of quercetin:a study of its interaction with intestinal lipids[J/OL]. Nutrients,2017,9(2):111(2017-02-05). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331542/
[15] Gonzales GB,Smagghe G,Grootaert C,Zotti M,Raes K,Van Camp J. Flavonoid interactions during digestion, absorption, distribution and metabolism:a sequential structure-activity/property relationship-based approach in the study of bioavailability and bioactivity[J]. Drug Metab Rev,2015,47(2):175-190.
[16] Sharma H,Kanwal R,Bhaskaran N,Gupta S.Plant flavone apigenin binds to nucleic acid bases and reduces oxidative DNA damage in prostate epithelial cells[J/OL]. PLoS One,2014,9(3):e91588(2014-03-10). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948873/
[17] Zheng Y,Scow JS,Duenes JA,Sarr MG. Mechanisms of glucose uptake in intestinal cell lines:role of GLUT2[J]. Surgery,2012,151(1):13-25.
[18] Yang C,Li Z,Zhang T,Liu F,Ruan J,Zhang Z.Transcellular transport of aconitine across human intestinal Caco-2 cells[J]. Food Chem Toxicol,2013,57:195-200.
[19] Walgren RA,Karnaky KJ Jr,Lindenmayer GE,Walle T. Efflux of dietary flavonoid quercetin 4′-betaglucoside across human intestinal Caco-2 cell monolayers by apical multidrug resistance-associated protein-2[J]. J Pharmacol Exp Ther,2000,294(3):830-836.
[20] Hollman PC, de Vries JH, van Leeuwen SD,Mengelers MJ,Katan MB. Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers[J]. Am J Clin Nutr,1995,62(6):1276-1282.
[21] Li SY,Li Z,Li JL,Gao WN,Zhang ZQ,Guo CJ.Quercitrin is absorbed and metabolized by Caco-2cells[J]. Curr Top Phytochem,2011,10:67-73
[22] Li SY,Li Z,Li JL,Gao WN,Zhang ZQ,Guo CJ.The simultaneous determination of quercitrin,quercetin and its methylated metabolites by high performance liquid chromatography-mass spectrometry[J]. Acta Nutr Sin(营养学报),2010,32(6):603-607.
[23] Li SY,Li Z,Gao WN,Li JL,Zhang ZQ,Guo CJ.Role of glucose transporter 1 and glucose transporter 2 in transmembrane absorption of quercirin and isoquercitrin[J]. Chin J Pharmacol Toxicol(中国药理学与毒理学杂志),2018,32(3):192-200.
[24] Li SY,Li Z,Gao WN,Chen CX,Zhang ZQ,Guo CJ.The role of glucose transporter SGLT1 and GLUT2in the intestinal absorption of quercetin[J]. Electron J Metab Nutr Cancer(肿瘤代谢与营养电子杂志),2018,5(1):33-37.
[25] Li SY,Li Z,Gao WN,Li JL,Guo CJ,Zhang ZQ.Effect of P-glycoprotein on the transmembrane absorption and metabolism of quercitrin on Caco-2 cells[J]. J Int Pharm Res(国际药学研究杂志),2017,44(12):1150-1154,1162.
[26] Li Z. P-glycoprotein drug interaction models and its application in the drug evaluation(P-糖蛋白与药物相互作用模型的建立及其在药物评价中的应用)[D].Beijing:Academy of Military Medical Sciences(军事医学科学院),2011.
[27] Guo Y,Bruno RS. Endogenous and exogenous mediators of quercetin bioavailability[J]. J Nutr Biochem,2015,26(3):201-210.