Determination Of The Effective Components In Chinese Herbal Medicine Polygonum Cuspidatum And Radix Isatidis By Quantitative Nuclear Magnetic Resonance Spectroscopy

Traditional Chinese Medicine(TCM) contains many different complicated active compounds with low content. The determination of the effective components has always been a key point in the field of TCM research. Without reference and sample destroying, quantitative nuclear magnetic resonance spectroscopy (qNMR) can analyze sample fastly and accurately to obtain qualitative and quantitative result simultaneously. It is significant to research the determination of the components in TCM by qNMR.The thesis studied qNMR method for determination of effective components in Chinese Herbal Medicine Polygonum Cuspidatum and Radix Isatidis. The qNMR method for determination of resveratrol and polydatin in Polygonum Cuspidatum and the epigoitrin in Radix Isatidis were established, validated and application, respectively.1. qNMR method for simultaneous determination of resveratrol and polydatin in Polygonum Cuspidatum cuts and granule. The 2-step ultrasonic extraction method using 80% alcohol and acetone were used for fully extracting the two components of resveratrol and polydatin in sample before qNMR determination. The qNMR experimental conditions were investigated.The qNMR experimental conditions were selected using DMSO-D2O(10:1,V/V) as solvent,2,3,5-triiodobenzoate as internal standard which was calibrated by standard substance of potassium hydrogen phthalate, and 5mmBB0 probe, P1 (pulse width)= 14.4us, d1 (pulse delay time)=5s, NS(number of scan)=32. The’H-NMR peaks of 56.390 ppm(H-2,6,d,2H) of resveratrol and 56.326ppm(H-4,t,1H) of polydatin were chosen as the quantitative characteristic peaks. Method validation was performed in terms of precision (RSD<0.6%), linearity (correlative constants>0.999), LOD (0.23g/L and 0.24 g/L for two components respectively) and LOQ (resveratrol 0.69g/L, polydatin 1.57g/L), recovery (resveratrol 97.7%-103.5%, RSD=2.4%, polydatin 94.5%-99.2%, RSD= 1.6%, including the extraction process). The results show the reliability of qNMR for TCM assay without standard sample of TCM effective compound. The resveratrol and polydatin in Polygonum Cuspidatum samples were experimental determined as 3.57-5.69mg/g and 12.73-24.07mg/g, respectively. The results agree with the related literature of the determination of resveratrol and polydatin in Polygonum Cuspidatum cuts.2. qNMR method for determination the epigoitrin in Radix Isatidis cuts. The twice ultrasonic extraction method using pure water was used for fully extracting epigoitrin in sample and then extraction method using etlayl acetate was used for purificating the samples before qNMR determination. The qNMR experimental conditions were investigated. The qNMR experimental conditions were selected using CDCl3 as solvent, pyrazine as internal standard which was also calibrated by standard substance of potassium hydrogen phthalate.5mmBB0 probe, and P1 9.84μs, d1= 5s, NS= 16. Method validation was performed including precision (RSD<0.6%); linearity (correlation coefficient r>0.99), LOD=0.67g/L and LOQ=2.02g/L. recovery of qNMR determination (96.9%-102.9%, RSD<3%), recovery including sample pretreatment process (36.5%-45.7%, RSD=8.92%). The result shows that the extraction method is stable but need further research to improve the extraction rate. The determination of epigoitrin in a real Radix Isatidis was 0.58mg/g. It is consistent with the related literature and meets with the requirement of no less than 0.02% in China Pharmacopoeia.