Research On Intestinal Absorption And Metabolism Of Major Bioactive Compounds Of Gegenqinlian Decoction During E-coli-induced Diarrhea In Mini-pigs

Aim To study the intestinal absorption and the intestinal enzyme metabolism features of the main ingredients of Gegenqinlian Decoction(GQD)in E.coli-induced mini-pigs.Method 1.To establish the HPLC-MS/MS determination method of the main ingredients of GQD in biological matrix K-R solution and PBS based on the biological sample testing guidelines of CFDA and carry out method validation.2 Take Drug Absorption Rate Constant(Ka)and Apparent Absorption Coefficient(Papp)as parameters to study the intestinal absorption characteristics of the main ingredients of GQD in single-pass intestinal perfusion experiments.3.To study the concentration changes of the main ingredients of GQD in physiological and pathological intestinal enzyme incubation experiments.4.Take Ka and Papp as parameters to study the intestinal absorption mechanisms of the main ingredients of GQD in everted gut sac experiments.And use Verapamil and Benemid to explore the reasons of the absorption differences.Results 1,Intestinal perfusion studies showed that the isoflavones puerarin,daidzein,daidzin,and baicalin,and the alkaloid berberine were absorbed faster in diarrheal jejunum where their Ka values were 55.96±34.95 ×10-4 min-1,31.02±28.90 ×10-4 min-1,69.20±35.93 ×10-4 min-1,61.49±31.67 ×10-4 min-1,and 47.37±41.21 ×10-4 min-1,respectively,than in normal intestines,where the corresponding Ka values were 15.49±11.98 ×10-4 min-1,-5.95±18.26 ×10-4 min-1,16.38±10.93 ×10-4 min-1,13.24±9.51 ×10-4 min-1,and 12.61±14.51 ×10-4 min-1(p<0.05).2.In intestinal mucosal enzyme metabolism experiments,isoflavones puerarin,daidzin,and liquiritin were metabolized more slowly by mucosal enzymes from diarrheal intestines,compared to that by mucosal enzymes from the normal group(p<0.05).Concentration differences(normal-diarrheal)were-6.64±1.20,-17.00±0.40 and-2.43±0.31 ?g·mL-1 respectively.The concentrations of daidzein in both perfusion and metabolism studies,and those of wogonin in metabolism studies,significantly increased(p<0.05).3.In everted gut sac experiments,peurarin and daidzin was absorbed more quickly in Normal-Jejunum-Benemid group(Ka and Papp were 0.84±0.59 ×10-3?g·min-1·cm-2,0.08±0.09 ×10-3?g·min-1·cm-2 and 13.58±9.53×10-6 cm·min-1,3.32±3.67 ×10-6 cm·min-1 respectively).Daidzein was absorbed more quickly in Model-Jejunum-Benemid group,Model-Jejunum-Verapamil group and Model-Jejunum-Benemid group and Papp were 0.57±0.24,0.45±0.31,0.66±0.25×10-3?g·min-1·cm-2 and 200.45±83.24,158.25±109.59,232.13±89.21 ×10-6cm·min-1 respectively).Berberine was absorbed more quickly in Model-Jejunum-Verapamil group and Model-Jejunum-Benemid group(Ka and Papp were 0.20±0.08,0.17±0.13× 10-3?g·min-1·cm-2 and 7.30±3.02,6.26±4.57 ×10-6cm·min-1 respectively).Wogonin was absorbed more quickly in Model-Ileum-Blank group and Normal-Ileum-Blank group(Ka and Papp were 0.10±0.07,0.12±0.08 ×1O-3?g·min-1·cm-2 and 36.77±25.06,42.18±29.41 ×10-6 cm·min-1 respectively).Conclusions 1.After the examination of the prepared herbal medicine of Gegen,Huangqin,Huanglian and Gancao,a simple and rapid high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method was developed to determine the concentrations of the main ingredients of GQD in both K-R solution and PBS.2.E.coli-induced diarrhea promoted intestinal absorption of puerarin,daidzin,berberine and baicalin.The main absorption site of these ingrediants was jejunum.3.After incubation,many ingredients(puerarin,daidzin,liquiritin and berberine)were metabolized more slowly in mucosal enzyme from diarrheal intestine.Diarrhea reduced the activity of enzymes.4.The data of the everted gut sac experiments speculated that purerarin,daidzin and daidzein were both substrates of P-gp and MRP2;Baicalin and jatrorrhizine might be substrate of MRP2,but berberine was not substrates of MRP2.What's more,berberine and palmatine might be substrates of P-gp,but wogonin or baicalin were not the substrates of P-gp.And we further found that jejunum absorbed most of the ingredients of GQD,such as puerarin,daidzin,daidzein,berberine,palmatine and baicalin.But in pathological states of diarrhea,puerarin,daidzein and berberine were absorbed more quickly by small intestine while wogonin,baicalin,jatrorrhizine or palmatine showed no significant differences.