Simultaneous Determination of Aesculin, Aesculetin, Fraxetin, Fraxin and Polydatin in Beagle Dog Plasma by UPLC-ESI-MS/MS and Its Application in a Pharmacokinetic Study after Oral Administration Extracts of Ledum palustre L.

A rapid, simple and sensitive ultra-performance liquid chromatography-electrospray-ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for the simultaneous determination of aesculin, aesculetin, fraxetin, fraxin and polydatin in beagle dog plasma for the first time. Plasma samples were pretreated by protein precipitation with methanol. Chromatographic separation was performed on an Acquity UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 µm) with gradient elution at a flow rate of 0.4 mL/min, using a mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). The analytes and IS were detected by multiple reaction monitoring (MRM) via negative ion mode with ion transitions of m/z 339.1⁻m/z 176.8 for aesculin, m/z 176.8⁻m/z 88.9 for aesculetin, m/z 206.8⁻m/z 192.1 for fraxetin, m/z 369.1⁻m/z 206.9 for fraxin, m/z 389.1⁻m/z 227.0 for polydatin and m/z 415.2⁻m/z 295.1 for puerarin. This method was validated according to the FDA guidelines and the results met the requirements of analysis. The calibration curves of analytes were linear with correlation coefficients more than 0.9980. The intra- and inter-day precisions were less than 15% and the accuracy was within ±15%. The maximum plasma concentration (Cmax) of aesculin, aesculetin, fraxetin, fraxin and polydatin was 46.75 ± 7.46, 209.9 ± 57.65, 369.7 ± 48.87, 67.04 ± 12.09 and 47.14 ± 12.04 ng/mL, respectively. The time to reach the maximum plasma concentration (Tmax) was 1.32 ± 0.38 h for aesculin, 1.03 ± 0.27 h for aesculetin, 0.94 ± 0.23 h for fraxetin, 0.83 ± 0.18 h for fraxin and 1.15 ± 0.15 h for polydatin. The results indicated that the absorption of aesculin might be slow in beagle dog plasma. This method was successfully applied for pharmacokinetics in beagle dog plasma after oral administration of the extracts of Ledum palustre L. at a dosage of 0.27 g/kg.

Simultaneous determination of anemoside B4, phellodendrine, berberine, palmatine, obakunone, esculin, esculetin in rat plasma by UPLC-ESI-MS/MS and its application to a comparative pharmacokinetic study in normal and ulcerative colitis rats.

A sensitive and rapid ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS/MS) method was developed for the simultaneous analysis of anemoside B4, phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, toosendanin (IS1 of anemoside B4), tetrahydropalmatine (IS2 of phellodendrine, berberine, palmatine and obakunone) and scopoletin (IS3 of esculin and esculetin) and to compare the pharmacokinetics of these active ingredients in normal and ulcerative colitis rats. After methanol deproteinization, solvents were evaporated at 40°C under a gentle stream of nitrogen. Chromatography was performed using a C18 column with a gradient elution of 0.1% aqueous formic acid and acetonitrile at 0.4ml/min. Detection and measurement were performed on a 4000 QTRAP UPLC-MS/MS system from AB Sciex in the multiple reaction monitoring (MRM) mode. Phellodendrine, berberine, palmatine, obakunone, esculin, esculetin, tetrahydropalmatine (IS2) and scopoletin (IS3) were monitored under positive ionization conditions. Anemoside B4, and toosendanin (IS1) were monitored under negative ionization conditions. The optimized mass transition ion-pairs (m/z) were 1221.1/750.7 for anemoside B4, 343.2/193.2 for phellodendrine, 337.1/321.0 for berberine, 353.0/336.9 for palmatine, 455.1/161.1 for obakunone, 341.2/179.2 for esculin, 179.1/123.0 for esculetin, 573.4/531.4 for toosendanin (IS1), 356.2/192.2 for tetrahydropalmatine (IS2) and 193.0/133.1 for scopoletin (IS3).

Aesculin possesses potent hypouricemic action in rodents but is devoid of xanthine oxidase/dehydrogenase inhibitory activity.

The natural product aesculin was demonstrated to possess potent hypouricemic effects in in vivo models of hyperuricemia in both mice and rats pretreated with oxonate. Aesculin, when administered intraperitoneally to the oxonate-induced hyperuricemic rodents, was able to elicit dose-dependent hypouricemic effects. At doses of 150 mg/kg of aesculin or above, the serum urate levels of the oxonate-pretreated mice were not different from normal mice. Such an effect in mice was observed as quick as 1.5 h after aesculin administration and was persistent for at least 5 h after aesculin administration. In rats, similar hypouricemic effects of intraperitoneally administered aesculin could also be demonstrated at doses of 100 mg/kg of aesculin or above, the serum urate levels of the oxonate-pretreated rats were not different from normal rats. Again, the effect persisted for at least 5 h after aesculin administration. In both rodents, however, oral administration at the same doses did not produce any observable hypouricemic effects. In addition, aesculin, when tested in vitro on rat and mouse liver homogenates, did not elicit any measurable inhibitory actions on the xanthine oxidase/xanthine dehydrogenase activities.

[Rectal administration of ash bark].

A TLC-densitometric method was applied to determine aesculin in rabbit blood. The best way for ash bark rectal administration was found by orthogonal design [L4(2(3))]. The two routes of administration (per rectal, P.O.) were compared by bioavailabilities and the result indicated that per rectal was better than P.O. Modified isolated rectum-bag method was applied successfully to finding the best way for rectal administration of ash bark.