| Objective:Basing traditional Chinese medicine extraction technique,combined with conventional polyamide adsorbtion method of flavonoid,innovation was made to optimize the accumulation technique of the total flavonoid aglycone from leaves of Carya cathayensis Sarg,and then column chromatography and spectroscopy methods were carried out to uncover the chemical composition of the total flavonoid aglycone.Additionaly,its acute toxicity in mice was evaluated so as to pave the way for further its pharmacological experimental research,and provide a scientific basis for the development and utilization of leaves of Carya cathayensis SargMethod:(1)Ethanol reflux extract method was used to obtain total flavonoid aglycone from leaves of Carya cathayensis Sarg;Ethanol concentration,liquid to solid ratio(V/M),extraction time and extraction frequency,above five factors were investigated,and then orthogonal experiment was carried out to optimize the extraction technique.(2)Total flavonoid aglycone from leaves of Carya cathayensis Sarg was enriched by polyamide adsorption method;polyamide type,the amount of polyamide,added water volume and other factors on polyamide adsorption capacity was investigated;eluent concentration,volume and other factors impact on the purity of the total flavonoid aglycone was investigated as well so as to optimize the enrichment processes.(3)Through the unique chemical composition chromogenic reaction to study the chemical composition of 40%ethanol elution leaves of Carya cathayensis Sar parts,and then using the traditional silica gel column chromatography,HPLC analysis and preparation techniques combined with 1H-NMR,13C-NMR spectroscopic techniques to identify the specific chemical components contained in it.(4)Gavaging(ig)the mouse with different concentrations of the total flavonoid aglycon,then observed and recorded the mouse’ diet,daily activities,and the number of death,calculate the maximum tolerated dose(MTD);additionally,mice organs were collected,histological section were prepared and stained with HE for analysis.Results:(1)Single factor and orthogonal experiment results indicated that,with 95%ethanol as the extract solvent,the volume(ml)and leaf powder(g)ratio was 25:1(V/M),reflux extracted 2 times,each time with 10min,then maximal total flavonoid aglycon from leaves of Carya cathayensis Sarg can be obtained(2)Polyamide adsorption and eluent method was optimized,the volume of added water has a greater impact on polyamide adsorption;while the ethanol concentration,elution volume,have a greater impact on the total flavonoid aglycone purity.The optimized process as following:to lOg powder of 95%ethanol extract,4g 100-200 mesh polyamide was added,following 200mL distilled water was slowly added,and then evacuated to eliminate ethanol,the suspension was loaded directly to the column and sequently eluted with 300mL 30%ethanol and 800mL 40%ethanol;the latter elunt was collected and vacuum evaporated,final product was obtained with 77.52%total flavonoid aglycon.(3)Chemical composition analysis indicated that except considerable amount of flavonoids,the 40%ethanol elution part also contains polysaccharides,glycosides,tannins,phenols,organic acids,anthraquinones and steroids et al.we also identified a new compound Tectochrysin.(4)Mice gavaging with different concentration of the total flavonoid aglycon,without abnormal activity and death was observed.The maximum tolerated dose was 1130.7 mg/kg(equivalent human dose 7 times),and HE staining showed no damage occurred in histologic level.Conclusion:Simple,stable,effective and high purity total flavonoid aglycon prepare method was established.The 40%ethanol elution part also contains polysaccharides,glycosides,tannins,phenols,organic acids,anthraquinones,steroids and triterpenoids.Acute toxicity tests in mice showed that the total flavonoid aglycon from leaves of Carya cathayensis Sarg without any acute toxic effects. |
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