Study On Chemical Constituents And In Vivo Metabolism Of Active Components Of The Leaves Of Dimocarpus Longan Lour.

Objective:（1）Study on the chemical constituents of different extracts of the leaves of Dimocarpus longan Lour..（2）To establish an HPLC wavelength switching method for simultaneous determination of five active components（gallic acid,protocatechuic acid,ethyl gallate,quercitrin and luteolin）in the ethyl acetate fraction of the leaves of Dimocarpus longan Lour..（3）Study on the best extraction process of six index components（gallic acid,protocatechuic acid,ethyl gallate,quercetin,luteolin and kaempferol）in the leaves of Dimocarpus longan Lour..（4）Preliminary study on the metabolites of the ethanol extract of the leaves of Dimocarpus longan Lour.in feces and urine of rats.Methods:（1）The leaves of Dimocarpus longan Lour.were isolated on a Thermo Hypersil Gold C₁₈column（2.1 mm×100 mm,1.9μm）.The mobile phase was acetonitrile containing 0.1%formic acid and water containing 0.1%formic acid（Containing 10 mmol ammonium acetate）by gradient elution（0～2 min,5%A;2～42 min,5%～95%A;42～47 min,95%A;47～50 min,95%～5%A）.HRMS was operated in the positive and negative ion mode with the scanning range of m/z 100～1500.The Trace Finder software was applied to analyze accurate relative molecular weight of the primary mass spectrometry and the molecular formula,then the secondary fragment ion information of target compounds was selected and compared with the fragment ion information of the compounds reported in the databases and related literature to identify these possible compounds from different extracts of the leaves of Dimocarpus longan Lour..（2）The contents of five active components in the ethyl acetate fraction of the leaves of Dimocarpus longan Lour.were determined by HPLC-wavelength switching method.The column was Waters symmetry（4.60 mm×250 mm,5μm）.The methanol（A）-0.2%phosphoric acid（B）was used as mobile phase by gradient elution（0～14 min,10%～12%A;14～16 min,12%～26%A;16～30 min,26%～28%A;30～45 min,28%～45%A;45～65 min,45%～54%A）.The detection wavelengths were210 nm（0～15 min,gallic acid and protocatechuic acid）,280 nm（15～32min,ethyl gallate）and 360 nm（32～65 min,quercitrin and luteolin）at the flow rate of 1.0 m L/min.The column temperature was 30℃.The injection volume was 5μL.（3）With the ethanol concentration,solvent volume and extraction time as independent variable,and the comprehensive scores the contents of six index components including gallic acid,protocatechuic acid,ethyl gallate,quercetin,luteolin and kaempferol in the leaves of Dimocarpus longan Lour.were taken as response values,and the extraction process of the leaves of Dimocarpus longan Lour.was optimized by Box-Behnken response surface methodology.（4）The metabolites of the ethanol extract of the leaves of Dimocarpus longan Lour.were identified in rats by UPLC-Q-TOF/MS.The ACQUITY UPLC HSS T3 column（2.1 mm×100 mm,1.8μm）was chosen.The acetonitrile（A）-0.1%formic acid（B）was used as mobile phase by gradient elution（0～8 min,10%～55%A;8～12 min,55%～63%A;12～14 min,63%～67%A;14～16 min,67%～70%A;16～17 min,70%～80%A;17～18 min,80%～90%A;18～18.5 min,90%～100%A;18.5～19 min,100%～10%A;19～20 min,10%～10%A）,and the flow rate was 0.3 m L/min.The column temperature was 35℃.Electrospray ionization（ESI）was used to collect data in the positive ion mode with the scanning range of m/z 50-1200.By comparing differences between blank samples and drug treatment samples,prototype components and metabolites of in feces and urine of rats were preliminarily identified after oral administration of the ethanol fraction of the leaves of Dimocarpus longan Lour..Results:（1）A total of 21 compounds were identified from different extracts of the leaves of Dimocarpus longan Lour.,including 7 flavonoids and flavone glycosides（quercetin,luteolin,kaempferol,quercitrin,cynaroside,astragalin and kaempferol-3-O-rutinoside）,and 5 organic acids（gallic acid,protocatechuic acid,citric acid,shikimic acid and quinic acid）,and 7 N-containing compounds（trigonelline HCl,adenosine,nicotinamide,L-tyrosine,L-leucine,L-phenylalanine and L-proline）,and2 other compounds（scopoletin and 5-hydroxymethylfurfural）.And five common compounds（cynaroside、kaempferol、luteolin、adenosine and nicotinamide）were identified in three hypoglycemic effective fractions（the ethanol fraction,the ethyl acetate fraction and the n-butanol fraction）.At the same time,kaempferol was found in six different extracts of the leaves of Dimocarpus longan Lour..（2）A method for simultaneously determining five active components in the ethyl acetate fraction of the leaves of Dimocarpus longan Lour.by HPLC-wavelength switching method was established.The linear ranges of five active components（gallic acid,protocatechuic acid,ethyl gallate,quercitrin and luteolin）were good.The average recoveries were 102.56%,99.22%,99.73%,99.68%and 100.02%,respectively.And the RSDs were 2.60%,2.88%,2.60%,2.92%and 2.29%,respectively.（3）The best extraction process was:water bath temperature 80℃,ethanol concentration 100%,ratio of solid to liquid l:8（g/m L）,extraction time 90 min.And the comprehensive score of the best extraction process was 97.54（n=3,RSD=0.33）.（4）A total of eight compounds were preliminarily identified in urine and feces of rats,including three compounds（two prototype components and one metabolite）in feces of rats and five compounds（metabolites）in urine of rats.The metabolic processes mainly included methylation,glucuronidation and oxidation and so on.Conclusion:（1）UPLC-Q-Orbitrap HRMS has the characteristics of rapidity,accuracy and high selectivity.And chemical components from different extracts of the leaves of Dimocarpus longan Lour.can be quickly separated and identified by UPLC-Q-Orbitrap HRMS,which provides a scientific basis for further quality control of the leaves of Dimocarpus longan Lour..（2）The established HPLC-wavelength switching method can simultaneously determine of five active components of the ethyl acetate fraction of the leaves of Dimocarpus longan Lour.,and the method is simple,accurate and reliable,which can be used for the quality control of the ethyl acetate fraction of the leaves of Dimocarpus longan Lour..（3）Box-Behnken response surface methodology with multi-index comprehensive scoring method could be used to optimize the extraction process of the leaves of Dimocarpus longan Lour.,and the method is simple and feasible,which could provide a reference for the extraction process of the leaves of Dimocarpus longan Lour..（4）The prototype components and metabolites of the ethanol fraction of the leaves of Dimocarpus longan Lour.in feces and urine of rats were preliminarily identified by UPLC-Q-TOF/MS,which provided a scientific basis for the study of pharmacodynamic material basis and mechanism of the leaves of Dimocarpus longan Lour..