| Increasing studies show that persimmon tannin has significant physiological activity, such as free radical scavenging, antioxidant, antibacterial, antitumor, hypolipidemic and so on,but the structure-activity relationship is unclear. Since the structure of persimmon tannin is complex, studdying on its structure-activity relationship is of great challenge. Study on the contribution of structural units characterized its activity helps to clarify the structure-activity relationship. At present, characterized structural units of persimmon tannin were prepared mainly by gel column chromatography and high performance liquid chromatography, but it is low yield and low efficiency. In this paper, medium-pressure preparative liquid chromatography was used for the preparation of persimmon tannin characteristic structural units and its metabolism kinetics and metabolism in mice was analyzed in order to elucidate the biological activity of persimmon tannin and provide a reference of structure-activity relationship. The main results were as follows: 1. Preparation of persimmon tannin characteristic structural units by MPLCTwo persimmon tannin characteristic structural units were separated and purified by combining use of MPLC and semi-preparative HPLC methods. Persimmon tannin as the raw material, we run a 78 min gradient separation program using reverse C18 as packing on middle pressure liquid chromatography preparation system with methanol and water as the mobile phase, and then use semi-preparative HPLC for further isolation and purification. Purified samples was confirmed and analyzed by high performance liquid chromatography and liquid mass spectrometry(LC-MS), the purity a type A type EGCG and ECG dimer-dimer was 97.2% and 95.4%. 2. Pharmacokinetic study of persimmon tannin characteristic structural units in vivo5% Tween 80 pretreatment of the ultrafiltration membrane was used to reduce the nonspecific adsorption of the drug. A final binding rate was determined to be 88.02%-92.49% for A-type EGCG dimer and plasma protein and 93.35%-96.46% for A-type ECG dimer plasma protein separately. The binding of persimmon tannin characteristic structural units with the plasma protein was concentration-dependent, the binding rate increased as increasing concentrations of structural units and the binding rate for A-type ECG dimer with plasma protein was slightly larger than that of A type EGCG dimer. We first used EGCG monomer as sample to establish convenient and accurate sample pretreating method and validated its applicability for two kinds of A-type dimers. By Pksolver software analysis, the pharmacokinetic parameters of the mice intravenous dose of 50mg/kg A-type proanthocyanidin dimer were obtained. A half-life t1/2 of intravenous injection in mice of A type EGCG and ECG dimer was 120.54 min dimer and 37.41 min, the maximum plasma concentration time Tmax was 5min, and the maximum plasma concentration Cmax was 32.56μg/m L and 55.05μg/m L, the drug area under the curve AUC(0-t) was 1949.42μg/m L*min and 2112.48μg/mL*min, the average residence time for the MRT was 170.11 min and 40.3805 min, separately. The results found that two kinds of A type dimer in vivo metabolism of mice eliminate fast and have a short duration of action. 3. Plasma and urine metabolite analysis of persimmon tannin characteristic structural unitsThe metabolites of the two A-type dimers in vivo were analyzed by high-resolution mass spectra. The results showed that metabolites of both dimers could be detected in the plasma and urine samples. Two dimers are first subjected QM degaradation to form the corresponding monomers and then continued to form derivatized metabolites. Wherein, rice urine can detect a wider variety of metabolites, primarily to small molecule acid, and also sulfated and glucuronic products exist, and rice plasma contain mainly sulfated and glucuronic products. |
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