Study On The Chromatographic Retention Behavious And Recovery Rate Of Thiolysis Of Hawthorn Procyanidins

Hawthorn have been widely used as medicinal and food materials in China.Polyphneols, especially procyanidins, are the main active ingredient in hawthorn fruit.Taking fresh hawthorn polyphenols as materials, studyed on the relationship between thesolute retention and the temperature changing of chromatographic column or thecomposition of mobile phase by using the method of HPLC. Using the extract of hawthornprocyanidins, the PC-thiolysis reaction conditions were optimized. And the recovery rateof thiolysis of components were investigated under the the optimized reaction conditions.And the recovery rate of thiolysis of hawthorn was calculated. The results are as follows:1. Investigating the retention rule of chromatography of hawthorn procyanidins, amethod of HPLC to analyse procyanidins was established. Chromatographic column:Hypersil BDS C18(250mm×4.6mm id,5μm); mobile phase, A:500μL/L formic acid; B:methanol/acetonitrile=5:4containing500μL/L formic acid; the gradient elution condition(min/B%) was:0/15,11/15,15/17,16/18,19/20,24/29,31/50,33/80,40/15,50/15; flowrate:0.8mL/min; detection: diode array detector (DAD) at280nm; temperature:35℃;injection quantity:5μL. The external standard calibration curves were used inquantification.2. The retention law of hawthorn polyphenols effected by changing temperature ofchromatographic column or mobile phase was handled with nonlinear fitting.Under thecondition of water methanol or acetonitrile solution, the linear relationship between thelogarithm of the capacity of hawthorn polyphenols and temperature of chromatographiccolumn was presented. However, the relationship between the logarithm of the capacityfactor (Ink) of hawthorn polyphenols and the volume fraction of the stronger solentcomposition (CB) of mobile phase accorded with quadratic polynomial. The regressioncoefficients of the quadratic polynomial equations were bigger than the equations ofdisplacement adsorption model, which were all above0.99. Based on the quadraticpolynomial equations, retention time for hawthorn polyphenols under the concentration of18%acetonitrile conditions was predicted, the relative standard deviations for eachcomponent were below7%, which were smaller than the values of the number predicted bythe equations of displacement adsorption model3. The PC-thiolysis reaction conditions were optimized. It was finally determined that: the thiolysis concentration in the reaction solution was5%; Acetic acid:1M HCl=5:1(v/v), with the reaction temperature was90℃and the reaction time was60minutes. Thiscondition could guarantee that the PC polymer in hawthorn can be completely degraded.And the recovery rate of thiolysis of D6, C3, B2, EC, C1, D1, E1, C2, D2, B5, EC-S were69.7%,72.3%,81.0%,85.8%,46.5%,61.4%,65.7%,74.3%,63.7%,90.3%,89.7%,respectively.4. According to the proportion and recovery rate of thiolysis of main component, therecovery rate of thiolysis of hawthorn was calculated. And the value was65.1%. Accordingto it, the method of thiolysis-HPLC on the quantitative evaluation of the procyanidins inhawthorn and its wine was corrected. As a result, the content of procyanidins in hawthornand its wine were4378μg/g and1457μg/g, and the mean degree of polymerization were2.0and1.7, respectively.5. The stability of preserving at room temperature after the thiolysis reaction wasanalyzed. The measured results were reliable for8h at least under this condition.