| BackgroundHerbs couples as the basic composition units of Chinese Herbs is the foundation and cut-point for the investigation of prescription compatibility and drug absorption. Zhimu-huangbai is a classic clearing heat drug compatibility. In this study, the absorption kinetics of mangiferin was investigated and compared with those of mangiferin in Zhimu decoction and Zhimu-huangbai decoction. The elucidation of the influence of Zhimu and Zhimu-huangbai on mangiferin absorption will provide theory guidance for revealing the material foundation of Zhimu-huangbai in the process of clinical application.Objective1 To establish a UPLC method for simultaneous determination of three compounds (mangiferin, neomangiferin, berberine) in Zhimu-huangbai decoction, and study the changes in contents of three compounds when Rhizoma Anemarrhenae combined with different proportions of Cortex Phellodendri and the quality control method for decoction was developed.2 To develop a LC/MS/MS method for the determination of mangiferin in rat plasma for investigating the effects of Rhizoma Anemarrhenae combined with different proportions of Cortex Phellodendri on the pharmacokinetics of mangiferin in rats.3 In situ perfusion method and everted-gut technique in rats was utilized to study the absorption properties of the mangifeirn in rat intestine.Methods1 UPLC method for simultaneous determination of mangiferin, neomangif-erin and berberin:Three compounds were simultaneously determined by UPLC with WATERS ACQUITY UPLC(?)BEH C1N (2.1 mm×50 mm,1.7μm) column. The mobile phase consisted of 0.1% phosphoric acid (the PH was adjusted to 3.0 with triethylamine) and acetonitrile with gradient elution (acetonitrile:5% at 0 min,18% at 5.5 min,42% at 8 min) at the flow rate of 0.25 mL·min-1. The column temperature was set at 35℃. The determination wavelength was 260 nm.2 Determination of mangiferin in rat plasma by LC/MS/MS and its pharmacokinetic study:Chromatographic separation was carried out on a C1N column (150×2.0mm,5μm,CAPCELL) by isocratic elution with methanol-acetonitrile-l%acetic acid (40:3:57, V/V/V). Mangiferin were detected by the positive electrospray ionization-MS method under multiple reaction monitoring mode. SD rats were randomized into 6 groups and administered by single intragastric administration of mangiferin compound 17.5,35,70 mg·kg-1, Zhimu decoction, Zhimu-Huangbai 1:1 and 1:3 decoctions respectively. LC/MS/MS method was used for determination of mangiferin in rat plasma. The main pharmacokinetic parameters were calculated with DAS software by noncompartmental methods.3 Intestinal absorption properties of mangiferin:The intestine in rats was cannulated for in situ recirculation. UPLC and UV were respectively applied to measure the concentration of mangiferin in the flux and that of phenolsulfonphthalein in intestine perfusate. The absorption of mangiferin had been studied and compared with those of mangiferin in Zhimu and Zhimu-Huangbai in rat intestines.4 Study on the absorption of mangiferin in isolated rat small intestine utilizing everted-gut technique:Everted-gut technique was utilized to study the absorption properties of mangiferin which was compared with that in Anemarrhena Rhizoma and Zhimu-Huangbai. UPLC-UV method was applied to measure the concentration of mangiferin and neomangiferin in intestine perfusate.Results1 The contents determination of mangiferin, neomangiferin and berberin The linear ranges of mangiferin, neomangiferin and berberine were 3.44～220.00, 3.75～240.00,9.76～625.00μg·mL-1(r≥0.9999). The average recoveries (n=6) of the method were (95.00±3.28)%, (96.45±1.26)% and (97.78±4.84) %, respectively.2 Determination of mangiferin in rat plasma and its pharmacokinetics:The calibration curve of mangiferin in plasma were linear over the range of 3.01-600.2 ng·mL-1; the relative recovery was 100.5%～104.0%; The within-day and between-day precisions were less than 9.1%. After single intragastric administration of free mangiferin 17.5,35,70mg·kg-1, Cmax and AUC increased but non-proportional to the dose. After intragastric administration of mangiferin, Zhimu decoction, Zhimu-Huangbai (1:1) and (1:3) decoctions to rats (the dose of mangiferin was 35 mg·kg-1), the plasma concentration level of mangiferin in Zhimu decoction group was significantly higher than that in mangiferin compound group, and was decreased by combination of Huangbai. The main pharmacokinetic parameters of mangiferin in 4 formulations were as follows: AUG0-t were 0.626,122.4,94.36,53.97μg·mL-1·h; Cmax were 0.149,21.52,16.26, 8.27μg·mL-1; Tmax were 1.0,3.0,4.0,6.0h; t1-2Z were 3.41,1.46,1.65,1.77 h.3 Intestinal absorption properties of mangiferin:Mangiferin in different concentration of 2.0,5.0,10.0,20μg·mL-1 had different the absorption rate(Ka) and the absorption percentage (P), it was 0.0541,0.0467,0.0491,0.0220 h-1 and 14.05%,13.14%,12.43%,5.82% respectively. The absorption of mangiferin increased with the increasing of pH value; there was significant difference in the absorption of mangiferin in different intetines, Ka and P drcrease from colon, duodenum, ileum to jejunum in order. In the groups of Anemarrhena Rhizoma, the mangiferin content increased along with the duration of time, while the neomangiferin content was decreased at the same time.4 Study on absorption of mangiferin in isolated rat small intestine utilizing everted-gut technique:The apparent permeability coefficients(Papp) of mangiferin in jejunum, ileum and colon were 2.0350×10-1,5.1788×10-4 3.3825×10-4cm·min-1respectively; the Papp of mangiferin in jejunum was 18.363×10-1,4.1063×10-1cm·min-1, the Papp of mangifering in ilem was 10.190×10-4 3.9650×10-1cm·min-1, respectively in the Zhimu decoction group and Zhimu-huangbai decoction group.Conclusion1 The content of mangiferin and berberin in mixed decotion is approximate or higher than the single decoction of Rhizoma Anemarrhenae or Cortex Phellodendri. The extracted quantity of berberin in Zhimu-Huangbai (1:1) was significantly higher than that of single Cortex Phellodendri or Cortex Phellodendri. The mixed decotion is favorable for the extraction of mangiferin, neomangiferin and berberin.The method is convenient, rapid and has been successfully applied to detect mangiferin, neomangiferin and berberin.2 The LC/MS/MS method is accurate, sensitive, and specific, it is suitable for the measurement of mangiferin plasma concentration. In this study, mangiferin can be quickly absorbed in vivo but the concentration of mangiferin in plasma and AUC is extremely lower than that of Zhimu decotion groups. The Tmax was prolonged to 3h and t, t1-2Z also was shorten accordingly. Following combined with Huangbai, the absorption of mangiferin from Zhimu was decreased and prolonged with the proportion increasing of Huanbai, while t1-2Z has no obvious change.3 The absorption of mangiferin is a first-order process with the passive diffusion mechanism with concentration from 2.0 to 10μg·mL-1; when the concentration increase to 20μg·mL-1, the uptake of mangiferin does not increase; Mangiferin can be better absorbed in the colon, duodenum, ileum than jejunum; Ka and P of mangiferin increased with of the increasing of pH value; the mangiferin content in intestine perfusate increased along with the duration of time, while the neomangiferin content was decreased at the same time.4 The mangiferin can be absorbed from different intestinal segments using everted gut sac. The Papp among three intestinal segments were sequenced as follows:ilem> colon> jejunum. Compared with Zhimu group, the Papp of mangiferin significantly decreased in mangiferin group and Zhimu-huangbai group in jejunum or ileum and did not have significantly change between mangiferin group and Zhimu-huangbai group.
|
PDF Full Text Request