Biotransformation of aesculin by human gut bacteria and identification of its metabolites in rat urine

AIM： To observe the biotransformation process of a Chinese compound, aesculin, by human gut bacteria, and to identify its metabolites in rat urine.METHODS： Representative human gut bacteria were collected from 20 healthy volunteers, and then utilized in vitro to biotransform aesculin under anaerobic conditions. At 0, 2, 4, 8, 12, 16, 24, 48 and 72 h postincubation, 10 mL of culture medium was collected. Metabolites of aesculin were extracted 3 × from rat urine with methanol and analyzed by HPLC. For in vivo metabolite analysis, aesculetin （100 mg/kg） was administered to rats via stomach gavage, rat urine was collected from 6 to 48 h post-administration, and metabolite analysis was performed by LC/ESI-MS and MS/MS in the positive and negative modes.RESULTS： Human gut bacteria could completely convert aesculin into aesculetin in vitro. The biotransformation process occurred from 8 to 24 h post-incubation, with its highest activity was seen from 8 to 12 h. The in vitro process was much slower than the in vivo process. In contrast to the in vitro model, six aesculetin metabolites were identified in rat urine, including 6-hydroxy-7-glucocoumarin（M1）, 6-hydroxy-7-sulf-coumarin （M2）, 6, 7-digluco-coumarin （M3）, 6-glc-7-gluco-coumarin （M4）, 6-O-methyl-7-gluco-coumarin （MS） and 6-O-methyl-7- sulf-coumarin （M6）. Of which, M2 and M6 were novel metabolites.CONCLUSION： Aesculin can be transferred into aesculetin by human gut bacteria and is further modified by the host in vivo. The diverse metabolites of aesculin may explain its pleiotropic pharmaceutical effects.

Distribution in tissue and excretion in urine and feces of swertisin after intravenous administration to rats

Swertisin contents in rat urine,feces and tissues were determined by reversed-phase high-performance liquid chromatography（RP-HPLC） method.Chromatographic separations were performed on a C18 column with acetonitrile-water（23：77,v/v） as the mobile phase.The calibration curves were linear over the ranges of 0.175-35.0μg/mL for rat urine,0.5-60.0μg/mL for rat feces,and 0.014 to 53.0μg/mL for all tissues.The inter-and intra-day precisions and accuracy for all measured samples were satisfactory.The fully validated method was applied for tissue distribution and excretion of swertisin in rat urine and bile after intravenous administration.The maximum level of swertisin was found in kidney,which reached 83.87± 6.36μg/g.In rat heart,swertisin was hardly detected under used experimental conditions.Swetisin level in liver,kidney,stomach,smooth muscle and skeletal muscle continued to decrease from 5 to 60min.Swertisin showed increasing tendency in intestine,spleen and testis tissues at scheduled time points.Detectable swertisin was found in brain and lung tissue.Totally 11.9% swertisin dose was cumulatively excreted from urine in 60h after intravenous administration.There was small amount of swertisin in rat feces and the cumulative excretion level reached 4.59% of intravenous dose in 60h.