Effect Of Astragalus Polysaccharides And Astragaloside On Intestinal Absorption Of Rhubarb Anthraquinones And Its Mechanism

ObjectiveIn this experiment,a rat one-way intestinal perfusion model was used to study the intestinal absorption characteristics of emodin,rhein,and aloe-emodin in different intestinal segments with concentration changes.Based on this,the effects of astragalus polysaccharide and astragaloside Ⅳ on the intestinal absorption of emodin,rhein,aloeemodin and the activity of efflux proteins MRP2 and MRP3 were discussed.The aim is to explain the mechanism of compatibility of rhubarb and astragalus from the perspective of gastrointestinal absorption,and to explain the scientific connotation of drug compatibility.Methods1.Optimized HPLC method for determination of emodin,rhein,and aloe-emodinAccurately weighed 12.4 mg,13.6 mg,and 11.6 mg of emodin,rhein,and aloe-emodin in a volumetric flask,dissolved 1,2-propanediol,and prepared a Kreb-Ringer’s test solution to prepare a standard solution.According to the 2015 Pharmacopoeia method,the HPLC method for the determination of emodin,rhein,and aloe-emodin was optimized,and the specificity,linear relationship,limit of quantification,precision,accuracy,stability,matrix effect,and absolute recovery were determined.2.Study on the absorption characteristics of emodin,rhein and aloe-emodin in rats’intestinesPreparation of low,medium and high concentrations of emodin(24.8 μg/mL.43.4μg/mL,62 μg/mL),rhein(27.2 μg/mL,47.6 μg/mL,68 μg/mL),aloe-emodin(23.2 μg/mL,40.6 μg/mL,58 μg/mL)perfusate.The duodenum,jejunum,ileum,and colon of appropriate length of the rat were selected,and perfusion was performed at a rate of 0.2 mL/min,and the receiving sample tube was replaced every 15 minutes until 90 minutes.The sample was centrifuged at 13,500 rpm for 10 min,0.75 mL of the supernatant was taken,25 μL of an internal standard was added,and the solution was filtered through a 0.45 μm microporous filter,and 10 μL was injected by HPLC for analysis.After the perfusion,the experimental intestine segment was removed,and the length(1)and cross-section circumference(d)of the duodenum,jejunum,ileum,and colon were measured,and the radius(r)was calculated based on the circumference(d).3.Study on the intestinal absorption characteristics of astragalus polysaccharide and astragaloside Ⅳ compatible with emodin,rhein and aloe-emodin,respectivelyPreparation of emodin(43.4 μg/mL),rhein(47.6 μg/mL),aloe-emodin(40.6 μg/mL),and astragalus polysaccharide(130.2 μg/mL),astragaloside IV(86.8 μg/mL)respectively compatible perfusate.The duodenum,jejunum,ileum,and colon of the rats were selected to have appropriate lengths,and perfusion was performed at a rate of 0.2 mL/min,and the receiving sample tube was replaced every 15 minutes until 90 minutes.The received samples were centrifuged at 13,500 rpm for 10 min,0.75 mL of the supernatant was taken,25 μL of an internal standard was added,and a 0.45 μm microporous filter was passed.10μL was injected by HPLC for analysis.After the perfusion,the experimental intestine segment was removed,and the length(1)and cross-section circumference(d)of the duodenum,jejunum,ileum,and colon were measured,and the radius(r)was calculated based on the circumference(d).4.Effect of Astragalus Polysaccharide and Astragaloside Ⅳ on MRP2,MRP3 efflux activity4.1 Establishment of HPLC method for pravastatin and teniposide After accurately weighing 5 mg of pravastatin and teniposide in a 50 mL volumetric flask,dissolve them with Kreb-Ringer’s test solution and methanol to prepare a standard solution.After establishing a method for determining pravastatin and teniposide by HPLC,Dilute the standard solution to determine the standard curve,and investigate the specificity,limit of quantification,precision,accuracy,stability,matrix effect,and absolute recovery.4.2 Effect of Astragalus Polysaccharide and Astragaloside Ⅳ on MRP2,MRP3 efflux activityPrepare low-,medium-and high-concentration astragalus polysaccharides(55.8μg/mL,130.2 μg/mL,186 μg/mL)and astragaloside Ⅳ(49.6 μg/mL,86.8 μg/mL,124μg/mL)of pravastatin(20 μg/mL)and teniposide(20 μg/mL)solution.According to the comprehensive analysis of the previous experimental results,the ileum was selected to measure the intestinal segment for one-way intestinal perfusion.After the perfusion,the length(1)and cross-section circumference(d)of the experimental ileum were measured.The perfusate was centrifuged,15 μL of sample was injected for HPLC analysis,Ka and Peff were calculated according to the formula,and statistics were analyzed by analysis of variance.Results1.Optimized HPLC method for determination of emodin,rhein,and aloe-emodinAfter optimization according to the 2015 Pharmacopoeia method,the determination methods of emodin,rhein,and aloe-emodin have good specificity,good linearity,precision,accuracy,stability,matrix effects,and absolute compliance with relevant regulations.2.Study on the absorption characteristics of emodin,rhein and aloe-emodin in rats’intestinesIn the mass concentration range of emodin 24.8～62 μg/mL,the Ka and Peff values in duodenum were larger than those in jejunum,ileum and colon(P<0.05).At a concentration of 43.4 μg/mL,the Ka value in the colon is lower than that of duodenum and jejunum(P<0.05),the Peff value of emodin in the colon is significantly lower than that in the duodenum,jejunum,and ileum(P<0.05).At a concentration of 62 μg/mL,the Ka and Peff values of emodin in the colon were lower than those in the duodenum and ileum(P<0.05).When the concentration of emodin in the duodenum was 43.4 μg/mL and 62 μg/mL,the Ka and Peff values were lower than when the concentration was 24.8 μg/mL(P<0.05).At the concentration of 62 μg/mL,the Peff value of emodin in the jejunum was lower than that at 43.4 and 62 μg/mL(P<0.05).The emodin Ka value in the ileum is lower than the concentration of 24.8 μg/mL at the concentration of 43.4 and 62 μg/mL(P<0.05),and the Peff value is lower than the concentration of 24.8 μg/mL at the concentration of 62□μg/mL(P<0.05).The colon emodin Peff value at the concentration of 62 μg/mL was lower than that at the concentrations of 43.4 μg/mL and 62 μg/mL(P<0.05).When the concentration of rhein was 27.2 μg/mL,the Peff value of rhein in jejunum and ileum was higher than that in duodenum and colon(P<0.05).At a concentration of 47.6μg/mL,the Ka and Peff values in the jejunum and ileum were higher than those in the duodenum and colon(P<0.05).At a concentration of 68 μg/mL,the Ka and Peff values of emodin in the jejunum and ileum were higher than those in the duodenum and colon(P<0.05),and the Ka and Peff values in ileum were higher than those in the jejunum(P<0.05).The Peff value of rhein in the duodenum was lower than 27.2 μg/mL at 68 μg/mL(P<0.05).In the colon,the rhein Ka value is lower than 27.2 μg/mL at a concentration of 68μg/mL(P<0.05).The Ka and Peff values of aloe emodin in the duodenum were higher than those in the jejunum,ileum,and colon within the range of 23.2～58 μg/mL(P<0.05).At a concentration of 40.6 μg/mL,the Ka value of aloe emodin in the colon was lower than that in the duodenum,jejunum(P<0.05),and ileum,and the Peff value was lower than that in the duodenum(P<0.05).At a concentration of 58 μg/mL,the Ka and Peff values of aloe emodin in the colon were lower than those in the duodenum and higher than those in the jejunum and ileum(P<0.05).The aloe emodin Ka and Peff values in the duodenum were higher at a concentration of 40.6 μg/mL and 58 μg/mL than at a concentration of 23.2μg/mL(P<0.05).At a concentration of 58 μg/mL,the aloe emodin Ka and Peff The value is higher than when the concentration is 40.6 μg/mL(P<0.05).The Ka value of aloe emodin in the jejunum was higher at a concentration of 40.6 μg/mL and 58 μg/mL than at a concentration of 23.2 μg/mL(P<0.05).The Peff value of aloe emodin in the jejunum was higher at a concentration of 58 μg/mL than at 23.2 and 40.6 μg/mL(P<0.05).The Ka value of aloe emodin in the ileum was higher than the concentration of 23.2 μg/mL at a concentration of 40.6 and 58 μg/mL(P<0.05),and the Ka value of aloe emodin was higher than the concentration at 40.6 μg/mL at a concentration of 58 μg/mL(P<0.05).In the ileum,the aloe emodin Peff value was higher at a concentration of 58 μg/mL than at 23.2 and 40.6 μg/mL(P<0.05).The aloe emodin Ka and Peff values in the colon at 58 μg/mL were higher than those at 23.2 and 40.6μg/mL(P<0.05).3.Study on the intestinal absorption characteristics of astragalus polysaccharide and astragaloside Ⅳ compatible with emodin,rhein and aloe-emodin,respectivelyCompatible with astragalus polysaccharides,Ka value of emodin in duodenum,jejunum,and colon decreased,and Peff value of emodin in duodenum,jejunum,and ileum decreased(P<0.05);Rhein Ka value reduced in jejunum(P<0.05);The Ka value of aloe emodin in duodenum,jejunum,ileum,colon decreased,and the Peff value of aloe emodin in duodenum,jejunum,and colon decreased(P<0.05).After compatibility with astragaloside Ⅳ,the Ka value of emodin in the duodenum,jejunum,ileum,and colon increased,and the Peff value of emodin in the duodenum and colon increased(P<0.05).The Ka and Peff values of rhein increased(P<0.05),and the Peff value of rhein in the ileum increased(P<0.05);The Ka value of aloe emodin in the duodenum increased,and the Peff value of aloe emodin in the duodenum,jejunum,ileum,and colon increased(P<0.05).4.Effect of Astragalus Polysaccharide and Astragaloside Ⅳ on MRP2,MRP3 efflux activity4.1 Establishment of HPLC method for pravastatin and teniposideThe established HPLC method for pravastatin and teniposide has good specificity and good linearity.The precision,accuracy,stability,matrix effect,and absolute recovery rate meet the relevant regulations.4.2 Effect of Astragalus Polysaccharide and Astragaloside Ⅳ on MRP2,MRP3 efflux activityIn the mass concentration of astragalus polysaccharides in the range of 55.8～186μg/mL,as the concentration of astragalus polysaccharides increased,the Ka and Peff values of pravastatin gradually decreased in the intestinal tract.In the mass concentration of astragaloside Ⅳ ranging from 49.6～124 μg/mL,as the astragaloside Ⅳ concentration increased,the Ka and Peff values of pravastatin gradually increased in the intestine.When the mass concentration of astragalus polysaccharide is in the range of 55.8～186μg/mL,as the concentration of astragalus polysaccharide increases,the Ka and Peff values of teniposide in the intestine gradually decrease.When the concentration of astragalus polysaccharide increases to 130.2 μg/mL,Ka and Peff values were less than 0 and the negative values were lower as the astragalus polysaccharide concentration increased.In the mass concentration of astragaloside Ⅳ in the range of 49.6～124 μg/mL,the intestinal Ka value of teniposide in the middle concentration(86.8 μg/mL)astragaloside group was higher than that in the low concentration(49.6 μg/mL)group.The Peff value of teniposide in the medium concentration(86.8 μg/mL)and high concentration(124μg/mL)astragaloside Ⅳ group was greater than the low concentration(49.6 μg/mL)group.The medium concentration(86.8 μg/mL)astragaloside group Compared with high concentration(124 μg/mL),the teniposide Ka and Peff values in the high concentration(124 μg/mL)group were lower than the medium concentration(86.8 μg/mL)group.Conclusion1.Optimized HPLC method for determination of emodin,rhein,and aloe-emodinThe HPLC method optimized by the 2015 Pharmacopoeia method can be used for the determination of emodin,rhein,and aloe emodin.2.Study on the absorption characteristics of emodin,rhein and aloe-emodin in rats’intestinesEmodin,rhein,and aloe-emodin may be efflux proteins-mediated in the intestine.Emodin is best absorbed in the duodenum,emodin is best absorbed in the jejunum and ileum,and aloe-emodin is best absorbed in the duodenum.3.Study on the intestinal absorption characteristics of astragalus polysaccharide and astragaloside Ⅳ compatible with emodin,rhein and aloe emodin,respectivelyAstragalus polysaccharide can inhibit the absorption of emodin in the duodenum,jejunum,ileum,and colon,inhibit the absorption of rhein in the jejunum,and inhibit the absorption of aloe emodin in the duodenum,jejunum,ileum,and colon.Astragaloside Ⅳcan promote the absorption of emodin in the duodenum,jejunum,ileum,and colon,promote the absorption of rhein in the duodenum and ileum,promote the aloe emodin in the duodenum,jejunum,ileum,Absorption in the colon.4.Effect of Astragalus Polysaccharide and Astragaloside Ⅳ on MRP2,MRP3 efflux activity4.1 Establishment of HPLC method for pravastatin and teniposideThe established HPLC method can be used to determine the content of pravastatin andteniposide.4.2 Effect of Astragalus Polysaccharide and Astragaloside Ⅳ on MRP2,MRP3 efflux activityAstragalus polysaccharide can induce the efflux activity of MRP2 and inhibit the activity of MRP3.Astragaloside Ⅳ has an inhibitory effect on the efflux activity of MRP2 and an induction effect on the efflux activity of MRP3.Both emodin and aloe emodin are substrates of MRP2 and MRP3.