Research And Application Of The Fingerprint Of Genkwa Flos Based On The Improving Euclidean Distance Coefficient

Genkwa Flos,the dry buds of Daphne genkwa Sieb.et Zucc.(Thymelaeaceae),have been used in folk remedies to treat diarrhea.It is a toxic medicine and its toxic components have been reported as diterpenoid esters such as yuanhuacine.Its processed products were commonly used clinically due to the toxic.10 batches of Genkwa Flos were processed in accordance with Phannacopoeial method.Different factors like sample handling methods,extraction solvents and chromatography columns were systematically investigated to establish the HPLC fingerprints of raw and processed drug.Nineteen characteristic peaks were established in the HPLC fingerprint and five of them were identified as luteolin,apigenin,3’-hydroxy-genkwanin,genkwanin and yuanhuacine.The identified peaks were accounted for 59.37%of the total common peaks with strong features and representative.It was hard to distinguish the processed ones from the raw material by using similarity evaluation software issued by the State Pharmacopoeia Commission because the correlation coefficient and cosine value of the raw and processed ones were both at 0.9.A new similarity measure method called Improving Euclidean Distance(IED)was proposed to calculate the IED coefficient of the raw ones and processed.Results showed that the IED coefficients of the raw Genkwa Flos were all above 0.5 and the processed ones were all below 0.5.So it was easy to distinguish the processed ones from the raw ones by using the method of Improving Euclidean Distance.Five identified toxic and active compounds of the common peaks in the raw and processed material were determined by HPLC-MS.Results showed that the content of these compounds were all changed after being processed.The content of the luteolin,3’-hydroxy-genkwanin,genkwanin were higher and the apigenin and yuanhuacine were lower than the raw ones.At the same time,we compared the content of five compounds in the raw Genkwa Flos of different batches,finding that the content of five ingredients existed differently in each batch,especially yuanhuacine which varied 15 times.The lowest content of luteolin was 0.0693%,the highest was 0.1341%;The lowest content of apigenin was 0.2680%,the highest was 0.6471%;The lowest 3’-hydroxy-genkwanin content of was 0.2093%,the highest was 0.3008%;The lowest content of genkwanin was 0.2340%,the highest was 0.3675%;and the lowest content of yuanhuacine was 0.0018%while the highest was 0.0264%.As a toxic Chinese medcine,it is particularly important to control the content of yuanhuacine.The method of Improving Euclidean Distance coefficient was also applied to screen the best technology for processing vinegar Genkwa Flos.The amount of vinegar,moistening time,frying temperature and frying time were investigated respectively by single factor method and the levels of each factors were determined.Three levels of the amount of vinegar were 20%,30%and 40%,respectively;the levels of the moistening time were 15min,30min and 45min,respectively;the levels of the frying temperature were 130℃,160℃ and 190℃,respectively;the levels of the frying time were 6min,8min and 10min,respectively.The best processing technology was identified as A2B2C3D1 according to the orthogonal design which was take Genkwa Flos,add 30%vinegar and mix well,moisten 30 min and fry 6 min at 190℃.Analysis of variance showed that the amount of vinegar and moistening time had a significant effect on the processing technology while the frying temperature and frying time were not.