Study On The Metabolism Of Troxerutin By Rat In Vivo And In Vitro

Troxerutin, a semi-synthetic flavonoids, is made of rutin by hydroxyethylation, is also named venoruton, vitamin P4, 3′,4′,7-(-O-tri-hydroxyethyl)rutin is the chemical name. It has numbers of pharmacological actions, such as it can inhibit the cohesion function of red blood cells and platelets; protect vascular endothelial cells, reduce the permeability of blood vessel walls and eliminate edema; anti-inflammatory; protect the liver from chemical damage. Although troxerutin has been used in clinic for multiple years, but reports of its adverse reactions has been increased year by year, and currently there has few studies reported about troxerutin in vivo metabolism, just some pharmacokinetic studies of troxerutin in people and rats, the metabolism of troxerutin in vivo has not been fully understanded. In order to further reveal the metabolism and conversion situation of troxerutin in vivo and in vitro, and provide evidence for its efficacy in vivo material foundation and development of new drugs, this paper studys the metabolism in rats of troxerutin in vivo and in vitro deeply. The main research contents are summarized as follows:Part one: Study on the metabolites of troxerutinSprague-Dawley rats was administerd with troxerutin by intraperitoneal injection, thenthe 24 h urine and feces samples was collected, the samples were tested by HPLC after treated, the results showed that there was no significant difference of urine after administration and blank urine, but a significant metabolites peak was detected in feces after administration. Troxerutin was cultured by intestinal bacteria in vitro, the intestinal bacteria samples were analyzed by HPLC after treated, a significant metabolites peak was also detected, and the retention time was the same of the metabolites peak detected in the feces after administration. Plenty of troxerutin was cultured by the intestinal bacteria in vitro, and then the intestinal bacteria samples was extraction and purification, a pure metabolite was get,NMR and MS was used to determine the structure and molecular weight of the metabolite, the metabolite was the aglycone of troxerutin which was removed from its rutinose aglycone, named 3’,4’,7-tri(-o-hydroxyethyl)quercetin, formula for C12H22O10, molecular weight was 434.Part two: The establishment of analytical method of troxerutin and troxerutin aglycone in biological sampleIn this paper, a high-performance liquid chromatographic method has been established for the separation and analysis of troxerutin and troxerutin aglycone in rats intestinal bacteria, urine, feces and bile, used a liquid-liquid extraction as sample pretreatment for biological sample. The column was Shim-pack ODS C18(250mm×4.6mm,5μm). The mobile phase consisting of methyl alcohol-1.7% glacial acetic acid was run at a gradient elution(0~15min,41:59~41:59;15~30min,41:59~65:35;30~40min;65:35~41:59) with a flow rate of 1.0 m L·min-1. 350 nm was the detection wavelength. The chromatographic column temperatures was 40℃. The internal standard was rutin. The results showed that there was no interference detecting troxerutin and troxerutin aglycone in endogenous impurities. The chromatographic method used provided goodseparation and analysis condition. The linear range, sensitivity, specificity, precision and accuracy of this method were all reached the requisition of pharmaceutical analysis. The method has been successfully applied to the detection of troxerutin and troxerutin aglycone in rats intestinal bacteria, urine, feces and bile.Part three: Study on the metabolism and excretion of troxerutin and troxerutin aglycone in vivo and in vitroTroxerutin was cultured by rat intestinal bacteria in vitro and was i.p.with SD rats, and the intestinal bacteria samples, urine, feces and bile samples were collected, then studied on the metabolism and excretion situation of troxerutin and troxerutin aglycone. The results showed that, the metabolic rate of troxerutin in rat intestinal bacteria was very fast, 77.13% troxerutin was reduced at the first 6h, 99.14% was reduced in the first 12 h, and then metabolited completely at 24 h. Metabolites-troxerutin aglycone was generated from the incubation 1h, and then increased gradually, 6h concentration was the largest, and then decreased slowly. Troxerutin was with high concentration at 1h, then the concentration was decreased rapidly, then the concentration was down to the bottom after 12 h, this indicated that troxerutin was transformed rapidly by the rat intestinal bacteria, the glycoside bond was cracked quickly, most of the troxerutin was transformed into troxerutin aglycone. After i.p. troxerutin to SD rats, about 16.17% of the total dosage was excretion from urine in 24 h, nearly 0.54% of the total dosage was excretion from feces in 24 h, and 58.94% of the total dosage was excretion from bile in 24 h. at the same time, there was 22.69% trexerutin aglycone was generated in feces.