ABSTRACT
OBJECTIVE： To investigate absorption kinetic characteristics of main active components as 4-（glucoseoxy）- glucoseoxybenzyl cinnamate （A1）， 2-isobutyl malic acid （A2）， 1，4-bis [4-（glucoxy） benzyl]-2-isobutyl malic acid ester （A3）， dihydrophenanthrenes 1 （A4） and 1，4-bis [4-（glucosoxy） benzyl]-2-isobutyl malic acid ester-2-（4-O-cinnamoyl-6-O-acetyl） glucoside （A5） from ethanol extract of Bletilla striata in the intestines of rats. METHODS： Using puerarin as internal standard， UPLC-MS/MS was used to determined the concentration of A1-A5 in intestinal circulation fluid. The determination was performed on Acquity UPLC BEH C18 column with mobile phase consisted of acetonitrile （containing 0.1％ formic acid）-water （containing 0.1％ formic acid） （gradient elution） at the flow rate of 0.35 mL/min. The column temperature was 45 ℃， and sample size was 3 μL. The positive ion and negative ion scanning were carried out in the multiple reaction monitoring mode by electrospray ion source. The ion pairs for quantitative analysis were m/z 593.2→431.1 （A1）， m/z 189.0→129.0 （A2）， m/z 725.3→457.2 （A3）， m/z 347.1→332.1 （A4）， m/z 1 059.3→793.1 （A5）， m/z 417.0→267.0 （internal standard）. In the in vivo intestinal circulation perfusion model， using accumulative absorption transfer rate （A） and absorption and transformation rate constant （Ka） as indexes， the effects of different doses of ethanol extract from B. striata （low-， medium-， high-dose were 166， 333，667 μg/mL，respectively）， bile， P-glycoprotein （P-gp） inhibitors （verapamil） and different intestinal segments on the absorption of above 5 components were investigated. RESULTS： The linear range of A1， A2， A3， A4 and A5 were 0.22-14.00， 0.34-21.75， 1.99-127.16， 0.15-9.75， 0.16-10.00 μg/mL（r＞0.99）. The limits of quantitation were 0.22， 0.34， 1.99， 0.15， 0.16 μg/mL， respectively. The lowest detection limits were 0.028， 0.085， 0.251， 0.035 and 0.010 μg/mL. RSDs of inter-day and intra-day were all lower than 10％. The recoveries ranged 83.60％-106.91％. Matrix effect did not affect the determination of the substance to be measured. A and Ka values of A1 in B. striata ethanol extract low-dose and medium-dose groups were significantly higher than high-dose group； A value of A3 in low-dose group was significantly higher than medium-dose and high-dose groups （P＜0.05 or P＜0.01）. A and Ka values of A1 and A3 in non-ligation group were significantly lower than control group， while A and Ka values of A4 were significantly higher than control group （P＜0.05 or P＜0.01）. A and Ka values of A1 and A3 in P-gp inhibitor group were significantly lower than control group （P＜0.05 or P＜0.01）. A values of A1 in jejunum group， ileum group and colon group， Ka value of A1 in colon group， A and Ka values of A2 in colon group， A value of A3 in ileum group， A and Ka values of A4 in ileum group and colon group， A values of A5 in jejunum group and ileum group as well as Ka value of A5 in jejunum group were all significantly lower than duodenum group. Ka values of A3 in jejunum group， ileum group and colon group were significantly higher than duodenum group （P＜0.05 or P＜0.01）. CONCLUSIONS： Established UPLC-MS/MS method is specific， sensitive and simple， and it can be used for quantitative analysis and pharmacokinetic study of A1-A5. The 5 active components in B. striata ethanol extract are absorbed by the whole intestine， and the intestinal segments are different. A1 and A3 are absorbed more in intestinal tract and may be saturated. Bile can inhibit intestinal absorption of A1 and A2， but promoted intestinal absorption of A4. A1-A5 may not be the substrate of P-gp.
ABSTRACT
Aim Modelorganismzebrafishwasusedto study metabolites and metabolite profile of the gallic acid and protocatechuic acid of effective fractions of Polygonum capitatum,and discuss the feasibility and rationality of the model organism zebrafish in the study ofdrugmetabolism.Methods Thetwocomponents were exposed to model organism zebrafish after 24 h of solution treatment by using ultra-high performance liq-uid chromatography-quadrupole-time-of-flight tandem mass spectrometry technology (UHPLC-Q-TOF MS ) method with mass defect filter (MDF ),and the data was treated with data mining software(Metabolite Tool-sTM).Results Afterzebrafishmetabolism,themain reactions of gallic acid and protocatechuic were methyl-ation sulfated. In addition to the two parent com-pounds,six phase II metabolites were identified,in-cluding four methyl sulfate products and two sulfation products.Conclusions Themetabolismofthegallic acid and protocatechuic acid of effective fractions of Polygonum capitatum by zebrafish presents phase Ⅱmetabolites,which is highly consistent with the meta-bolic mechanism of rats.Thus it indicates the rationali-ty of the methods.At the same time,it also provides the experimental basis for clarifying the substance basis of the drug.