| Objective:The main purpose was to clarify the flavonoids components in persimmon leaves and the absorption and metabolic processes of these flavonoids.combined with the characteristics of the persimmon leaf flavonoids,the total flavonoids of persimmon leaves phospholipids complex was prepared.Methods:(1)Column chromatographic techniques were applied to isolated constituents.A combination of physico-chemical properties and spectroscopic analysis were used to identify structures of constituents.(2)To develop a method for the determination of isorhannetin,kaempferol and quercetin by high performance capillary electrophoresis with laser induced fluorescence detection(HPCE-LIF),and to study the pharmacokinetics of flavonoids in rat after oral administration of flavonoids of Persimnon leaves.(3)Solvent method was used to prepare the total flavonoids of Persimmon leaves phospholipids complex and obtain the optimized preparation by means of single factor test and orthogonal design test with complex ratio as evaluation criterion.The formation of complex was analyzed by using the X-ray diffraction,and differential scanning calorimetry.(4)The microbial metabolic process of flavonoids,extracted from the leaves of Diospyros kaki(FLDK),by intestinal bacteria was investigated in vitro.High performance liquid chromatography/linear ion trap mass spectrometry(HPLC/LTQ-MS)technique combined with Xcalibur 2.1 software was used to analyze the metabolites of flavonoids in vivo.Results:(1)six compounds were obtained,and their structures were identified as genistein 1),isorhamnetin 2),Quercetin-3-O-?-D-rhamnoside 3),quercetin 4),quercetin-3-O-?-D-glucoside 5),rutin 6).Compounds 1,2 and 3 were isolated from Persimmon Leaves for the first time.(2)A new method has been developed by use of capillary electrophoresis with indirect laser induced fluorescence to determine isorhamnetin,kaempferol,quercetin.Three flavonoid compounds had good linear relationship,precision and accuracy,detection limit were 0.05,0.03,0.06 mg/L,which was better than that of UV detector.In the plasma sample,extraction reeovery rate is between 82.5?93.1%.Pharmacokinetic curve showed there were three peaks for isorhamnetin and kaempferol.(3)The formation of the total flavonoids of Persimmon leaves phospholipids complex was greatly influenced by the solvent and reaction ratio.The optimized preparation conditions for the total flavonoids of Persimnon leaves phospholipids complex were obtained as follows.Solvent was anhydrous ethanol.The ratio of persimmon leaves flavonoids to phospholipid was 1:2,the mass of reaction concentration was 20 mg/mL,temperature was 30oC,reaction time was 1 h.Under this condition,96.95%complex ratio was achieved.(4)Flavonoid glycosides and flavonoid aglycones was decreased rapidly in the process of microbial metabolism by Intestinal bacteria in vitro,and the metabolic rate may be related to the concentration of Intestinal bacteria in the culture solution.Flavonoid aglycones also can be metabolized by Intestinal bacteria.Persimmon leaves flavonoids after forming phospholipid complex,the metabolic rate by Intestinal bacteria was become slower.In vivo metabolites of FLDK were detected in rat plasma and urine after oral administration of FLDK,nine flavonoids were identified in urine and three flavonoids in plasma,but there is no flavonoid aglycone was found in plasma.Conclusion:In this paper,persimmon leaf flavonoids was the research object.Through chemical separation and structural identification of the ethyl acetate extract of persimmon leaves,the major flavonoids in it was clarified;This paper established a high performance capillary-laser induced fluorescence detection methods of flavonoids for the first time to,and explained the absorption metabolism of persimmon leaves flavonoid;According to the characteristics of flavonoids with polyphenols hydroxyl,persimmon leaves flavonoids phospholipid complex was prepared,then the physical properties was researched.Through the studies of intestinal bacteria metabolic features to persimmon leaves flavonoids,it was indicated that the metabolic rate of flavonoids slows down after flavonoids formed phospholipid complex. |
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