| Objective: To investigate the pharmacokinetics of puerarin after intragastric administration of puerarin solid lipid nanoparticles (Pue-SLN), and compare with that of intragastric administration of puerarin suspension or intravenously administration of puerarin injection.Methods: Rats were administered puerarin injection intravenously or puerarin suspension intragastricly or Pue-SLN intragastricly, respectively. puerarin and its metabolites in biological samples were detected by rapid resolution liquid chromatography- electrospray ionization- tandem mass spectrometry (RRLC- ESI- MS/MS).Results:1. Pharmacokinetic parameters of puerarin after intragastric administration of Pue-SLN were: AUC0→t:2.48±0.30 mg?h /L,AUC0→∞:2.64±0.39 mg?h /L,Cmax:0.33±0.05μg/ml,Tmax:40 min;t1/2 5.60±1.00 h,MRT:9.34±1.40 h,Cl:1.49±0.24 L/h,Vz:11.83±1.02 L , Vss : 13.71±1.29 L. The relative bioavailablity was 310%, the absolute bioavailability was 5.8%, and the results indicated that it was not bioequivalent to puerarin suspension. Compared with puerarin suspension administered intragasticly, the time of peak concentration of puerarin after intragastric administration of Pue-SLN was earlier significantly (40min vs 1.5～2 h), and the elimination half time was prolonged significantly (5.6 h vs 3.27 h).2. After puerarin was administered intragastricly to rats, the time of peak concentration of puerarin in rats'tissues was 2.5 h post-dosing, and the area under the curve (AUC) of puerarin in tissues was in the order of kidney>liver>lung>spleen>heart>brain (AUC in liver and lung, AUC in heart and brain was similar, respectively ). The AUC in tissues after administration of puerarin injection was in the order of liver>kidney>lung>heart>spleen>brain (AUC in lung and heart was similar). Compared with administration of puerarin suspension, the concentration of puerarin in tissues was higer after administration of Pue-SLN, the highest concentration of puerarin in liver, kidney, lung and spleen appeared at 1h post-dosing, and the highest concentration of puerarin in heart and heart appeared at 2.5h and 6h post-dosing, respectively. The AUC in tissues was in the order of liver>kidney>spleen>lungt>brain>heart (AUC in lung and spleen, AUC in lung and brain and heart was similar, respectively). The concentration of puerarin in heart and brain was lower, but it was increased when puerarin was incorporated into SLNs, especially the concentration in brain.3. The total excretion of puerarin in feces in 24 h was 34.17% of the dose after administration of Pue-SLN intagastricly, the percentage of unchanged puerarin excreted in feces was higher during 4-8 h and 8-12 h, accounting for 74.76% and 20.32% of the total excretion in feces, respectively. The total excretion of puerarin in urine in 24 h was only 1.79% of the dose, the percentage of unchanged puerarin excreted in urine was higher during 0-4 h and 4-8 h, accounting for 30.11% and 59.79% of the total excretion in urine, respectively. The total excretion of puerarin in the feces accounted for 95.11% of the total excretion in excreta. The total excretion of puerarin in feces in 24 h was 41.56% of the dose after administration of puerarin intagastricly, the percentage of unchanged puerarin excreted in feces was higher during 4-8 h and 8-12 h, accounting for 75.26% and 23.99% of the total excretion in feces, respectively. The total excretion of puerarin in urine in 12 h was only 0.64% of the dose, the percentage of unchanged puerarin excreted in urine was higher during 0-4 h and 4-8 h, accounting for 27.91% and 66.28% of the total excretion in urine, respectively. The total excretion of puerarin in the feces accounted for 98.40% of the total excretion in excreta. The total excretion of puerarin in urine in 24 h after administration of Pue-SLN was 2.8 fold of that after administration of puerarin suspension (1.79% vs 0.64%), which mainly due to the increment of excretion during 0-8 h, the excretion during 8-12 h increased, too, but there was no statistical significance (P=0.052). The total excretion of puerarin in feces in 24 h after administration of Pue-SLN was lower than that after administration of puerarin suspension, but there was no statistical significance (P=0.058), the excretion of puerarin during 12-24 h was higher (P=0.002). The total excretion of puerarin in urine in 24 h was 36.15% of the dose after administration of puerarin intravenously, the percentage of unchanged puerarin excreted in urine was the highest in the first 4h, accounting for 97.43% of the total excretion in urine. The total excretion of puerarin in feces in 24 h was 9.18% of the dose, the percentage of unchanged puerarin excreted in urine was the highest during 8-12 h, accounting for 99.19% of the total excretion in urine. The total excretion of puerarin in urine accounted for 79.64% of the total excretion in excreta.4. To detect the metabolites of puerarin, the profile of plasma from rats administered puerarin injection was compared to blank plasma. In addition to the puerarin peak, two peaks were present, M1 and M2, the protonated molecular ion of both M1 and M2 was at m/z 593 in positive ionization mode,and the deprotonated molecular ion of both M1 and M2 was at m/z 591 in negative ionization mode. Incubation of the samples withβ-glucuronidase resulted in complete elimination of the two metabolite peaks and a concomitant increase of the puerarin peak.D-Saccharic acid 1,4-lactone, an inhibitor ofβ-glucuronidase, inhibited the hydrolytic role ofβ-glucuronidase. M2 was confirmed to be puerarin-7-O-glucuronide by NMR. we speculated that M1 maybe puerarin-4'-O-glucuronide. The profile of urine from rats administered puerarin injection was compared to blank urine, three different peaks were present, the first two peaks were as same as that in plasma, i.e., they were puerarin-4'-O-glucuronide and puerarin-7-O-glucuronide, respectively. In positive ionization mode, the protonated molecular ion of the third peak (M3) was at m/z 433, MS2 experiments on the ions at m/z 433 showed the product ions at m/z 415, 397, 379, 367, 351, 337, 313, 283, we speculated that M3 was monohydroxylated puerarin. After rats were administered Pue-SLN or puerarin suspension intragastricly, the metabolites puerarin-4'-O-glucuronide and puerarin-7-O-glucuronide were present in plasma and urine of rats, although the level was lower. There was no metabolites present in the feces of rats. 5. M1 and M2 were present in the plasma at 5 minutes after puerarin administration intravenously, the peak level of M1 and M2 appeared at 10-15 minutes and 15-30 minutes, respectively, hereafter, although the level of M1 and M2 in plasma had fluctuation, it had a slowly descending tendency. M2 were present in the plasma at 10 minutes after Pue-SLN administration intragastricly, and the kinetic curve of M2 in plasma was two-peak type, the first peak appeared at 2h post-dosing, the level of M2 was increasing again, and the second appeared at 6h, hereafter, the level of M2 in plasma descended slowly.6. Similar glucuronidated metabolites (M1 and M2) were obtained in both liver and intestine perfusates.Conclusion:1. The bioavailability of puerarin was increased when puerarin was incorporated into SLNs, which maybe related to the enhancement of absorption of puerarin.2. The tissue distribution of puerarin was changed when puerarin was incorporated into SLNs, and the concentration of puerarin in tissues was increased, especially in liver and spleen, which maybe due to SLN engulfed by monocyte-macrophage system. The increment of concentration of puerarin in brain maybe attribute to SLNs'transporting through the blood-brain barrier easily.3. Puerarin was excreted by intestine after administration of puerarin suspension intragasticly, Puerarin was excreted by kidney after administration of puerarin intravenously, when puerarin was incorporated into SLNs, there was little changes in excretive action of puerarin, except a higher excretive quantity of puerarin.4. Three metabolites of puerarin were present in rats, M1, M2 and M3. M2 was puerarin-7-O-glucuronide, which was the prominent metabolite. M1 and M3 were speculated to be puerarin-4'-O-glucuronide and monohydroxylated puerarin, respectively. The metabolic profiles were similar in rat liver and intestine investigated by in situ liver and intestine perfusion, indicating that no metabolic regioselectivity of puerarin occurs in the two organs.
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