| Objective:To establish the specific method for extraction and purification of rhoifolin from Citrus grandis ’Tomentosa’,and investigate its intestinal absorption pattern and relevant pharmacokinetic characteristics in rat blood and tissues.,which can provide experimental basis for further research of the compound and total flavonoids of Exocarpium Citrus Grandis and thus promotes reasonable use of resources.Method:1.Extraction and purification of Rhoifolin were extracted and purified by percolation method combined with recrystalization method.The sample of Rhoifolin were identified by TLC、IR、LC-MS/MS、UV and other methods and its purity detected by UPLC.2.The concentration of Rhoifolin in the rat intestinal perfusion fluid was measured by UPLC-UV.ACQUITY BEH C18 column(3.0mm × 100mm,1.7 μm);mobile phase:methanol(A):0.5%acetic acid aqueous solution(B)(48:52);the flow rate is 0.3ml/min;column temperature is 30 ℃;detection wavelength:337nm;injection volume:2uL.3.The absorption of Rhoifolin in the rat’ s duodenum,jejunum,Ileum and colon was studied using one-way intestinal perfusion model in vivo and study the effects of different concentrations on the absorption from upper duodenum to bottom of jejunum.4.The concentration of Rhoifolin in rat plasma was determined using UPLC-UV,after precipitating the proteins to pretreat plasma samples.ACQUITY BEH C18 column(3.0mm×100mm,1.7um);mobile phase:methanol(A):0.5%acetic acid aqueous solution(B),gradient elution;gradient elution:0～2min,48%A～75%A;2～4min,75%A～48%A;4～5min,48%A～48%A;the flow rate is 0.3ml/min;column temperature is 30 ℃C;detection wavelength:337nm;injection volume:2uL.Internal standard is Luteolin.5.Rats were intravenous injected with high,medium and low dose group(20mg/kg,30mg/kg,40mg/kg)of Rhoifolin in and 0.5ml blood was sampled respectively from the rat retinal venous plexus after 2 min,5 min,10 min,15 min,30 min,45 min,60 min,75 min,90 min and 120min.After pretreated and concentrations of Rhoifolin in plasma at different sample point were measured using established UPLC method.And its pharmacokinetics parameters were analysed according to obtained blood drug concentration-time curve.6.After pretreatment with tissue samples by solvent protein precipitation of,the concentration of Rhoifolin in rat’ s tissue were measured using UPLC-UV.ACQUITY BEH C18 column(3.0mm×100mm,1.7μm);mobile phase:methanol(A):0.5%acetic acid aqueous solution(B),gradient elution;gradient elution:1～3min,35%A～75%A;3～4min,75%A～75%A;4～6min,75%～35%;the flow rate is 0.3ml/min;column temperature is 30 ℃;detection wavelength:337nm;injection volume is 2uL.Internal standard is Luteolin.7.Intravenous administration(40mg/kg),rats were killed after administration at 10min,30min and 60min(representing the distribution,balance and elimination phase)kill snap spine,each time a group including 6 rats and male and female half and half.executed immediately after dissecting respectively to take out the heart,liver,spleen,lung,kidney,stomach and intestine,a total of seven kinds of tissue,preparation of tissue homogenate,measuring the concentration of Rhoifolin in tissue homogenate at different sampling points by established UPLC.Results:1.Extraction and purification of RhoifolinThe TCL identification of obtained Rhoifolin monomer by showed Rhoifolin had the same Rf=0.50 compared with its standard;by The infrared analyses showed IR spectra of Rhoifolin monomer were consistent with the standard.The molecular weight by mass was 578.09;Taken together,the monomer we got is rhoifolin.Different chromatographic conditions:ACQUITY BEH C18 column(3.0mm × 100mm,1.7um);mobile phase:methanol:H2O(45:55)、acetonitrile:H2O(30:70)、methanol:acetonitrile:0.5%aqueous acetic acid(30;10:60);The flow rate is 0.3ml/min;Column temperature is 30℃;Detection wavelength is 337nm,283nm and 267nm;Sample volume is 2uL.Rhoifolin was determined by HPLC and its.purity was ≥ 98%after calculated by area normalization method,which indicated that Rhoifolin we obtained can use as qualitative and quantitative reference substance,while its purity meet the requirements of the pharmacokinetics studies.2.Establish of determination methods of Rhoifolin concentration in perfusionExperimental results s.howed that with sample determination of Rhoifolin were not interfered by endogenous substance in the perfusion.The investigation of method ology showed the linear relation when Rhoifolin was in concentration range of 2～24ug/ml and the correlation coefficient R2 was 0.9999;Days precision RSD was in the range of 0.40%～0.60%,the day precision RSD was in 0· 25%～1.07%;Recovery of the method was between 96.11%～104.91%,which suggested that this method was applicable in the study of the rat intestinal absorption of Rhoifolin in vivo.3.Experimental study of the one-way intestinal perfusion in vivoThe results showed that the apparent absorption coefficient(Papp)of various intestinal segments were greater than 0.2 × 10-4cm/s;Rhoifolin was well absorbed at all segments the Papp and apparent absorption rate(Ka)in duodenum was the highest among them;there was a significant differences compared with other intestinal segments,indicating that the main absorb part of Rhoifolin was in duodenum.The study of the effect of different concentrations on Rhoifolin absorption found Ka was tend to be balanced between different concentrations,there was no significant difference(P>0.05)and Papp was not increased as increasing drug concentration,indicating the absorption mechanism of Rhoifolin may be passive diffusion.4.The establishment of UPLC measurement method of Rhoifolin in plasmaIn the chromatographic conditions,the determination of Rhoifolin sample were not interfered by endogenous substance in the plasma.The investigation of methodology confirmed the linear relation when Rhoifolin was in concentration range of 0.1～100.Oug/ml was good and the correlation coefficient R2was 0.9997:Days precision RSD was in the range of 0.01%～1.53%,the day precision RSD was in 0.44%～1.32%;Extraction recoveries is between 81.9%～85.7%.It showed that this method was applicable to pharmacokinetics studies of Rhoifolin in rats.5.Plasma pharmacokinetics studies in miceThe results showed that Rhoifolin in SD rats in vivo process conformed to the two-compartment model,T1/2 a were 3.62,3.76 and 3.71 min;T1/2β were 17.00,21.66 and 23,70 min.K12 were 0.081,0.077 and 0.080 min-1;K21 were 0.059,0.044 and 0.042 min-1;AUC(0-t)were 590.36,1362.91 and 1823.49 respectively.Pharmacokinetic parameters showed that absorption,distribution and elimination of Rhoifolin in rats was rapid,the concentrations in vivo reached its peak after intravenous Rhoifolin for 2 min,the blood drug concentration was eliminated as first-order kinetics,and it was hardly detected in blood after 120 min.6.The establishment of UPLC measurement method of Rhoifolin in tissue samplesIn the chromatographic conditions,the determination of Rhoifolin sample were not interfered by endogenous substance in the plasma,he investigation of methodology confirmed the linear relation when Rhoifolin was in various concentration range in tissues and the correlation coefficient R2 was0.9993;Days precision RSD was in the range of 0.06%～0.36%,the day precision RSD was in 0.58%～2.01%;Extraction recoveries was between 81.11%～91.49%,relative recovery was between 98.79%～101.69%;entire analysis time was 6min,showing the method was applicable to the study of Rhoifolin in vivo tissue distribution in rats.7.The study of Rhoifolin distribution in vivo tissue of ratsExperimental results show that Rhoifolin were widely distributed in various tissues and organs on rats,liver and kidneys were the highest,followed by the intestine,lung,stomach and spleen,spleen and heart was lower.Conclusion:Establishment of the UPLC method measuring Rhoifolin in biological samples is high sensitivity,good precision and high recovery rate,which are suitable for related pharmacokinetics research of Rhoifolin in rats in vivo.Meanwhile,Rhoifolin is well absorbed in the intestine,of which duodenum is the main absorption parts and its absorption mechanism of Rhoifolin may be passive diffusion.And Rhoifolin in vivo process in rats conforms to the two-compartment model,is eliminated as the first-order kinetics,and distribution and elimination in vivo are relatively fast.In addtion,Rhoifolin is mainly distributed in the liver and kidneys in rats,followed by the intestine,lung,stomach and spleen,spleen and heart drug concentration distribution are lower. |
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