A Study On The Two-step Derivatization Method For The Determination Of The Reducing Sugars In The Decoction Of Traditional Chinese Medicine

Saccharide is not only the energy source and skeleton component of plant life activity,but also has many pharmacological functions,such as anti-radiation,anti-tumor,antiviral,immunoregulation and so on.Therefore,there are many researches focus on the qualitative,quantitative and structural analysis of carbohydrate at present.The characteristics of carbohydrates are high polarity,similar structure and no ultraviolet or fluorescence absorption groups.It is generally necessary to convert them to their derivatives with ultraviolet or fluorescence absorption group by means of chemical labeling before analysis.However,that the most reported derivatization reagents directly reacts with reducing sugar will destroy their integrity,which leads to that some reducing sugars lose their configuration and could not be separated.The aim of this study is to establish a new method for the determination of the reducing sugars with two-step derivatization to overcome the problem.It was the first time that the C-glycoside ketone were obtained after acetylacetone reacted with reducing sugar in the condition of weak base wherein the ring structure of the reducing sugar was not opened.Then the C-glycoside ketone were converted to their ketohydrazones with a slight molar excess of2,4-Dinitrophenylhydrazine in acid system,and the obtained solutions were analyzed by HPLC.The following results were obtained.(1)After the optimization of the derivatization conditions and chromatographic conditions and a series of methodological investigations,the results showed that the pre-column two-step derivatization HPLC method can be used for the analysis eight kinds of the reducing sugars in TCM,such as xylose,fructose,glucose,lactose and so on.(2)The developed method was used for determination of the type and content of the reducing sugars in 71 kinds of TCM.The relationship between the type and content of the reducing sugars in TCM and the pharmacological activities,the medicinal parts,the property and classification of TCM were explored respectively.The results showed that the type and content of the reducing sugars are related to the pharmacological activities and medicinal parts of TCM,but not related to the property and classification of TCM.(3)It is the first time to detect the distribution of the reducing sugars in the various parts of Semen Cassiae,Camptotheca acuminata Decne,Sophora japonica,Edamame and glossy privet fruit and the change of the reducing sugars contents during drying process.The results showed that glucose was the highest in the leaves,followed by the stems.And it was found that maltose and lactose were partially degraded,while glucose and galactose were increased during the drying process.(4)At the same time,the kind and content of the reducing sugars in mature persimmon fruits were also determined,and the results were consistent with the reported data.Which indicated the developed method can not only be used for the determination of the reducing sugars in the decoction of TCM,but also for persimmon fruit samples.So the application scope of method is further expanded.(5)In addition to the determination of the kind and content of the known reducing sugars,some unknown reducing sugars in the samples were also summarized,which will provide valuable references for the further research.