The Molecular Identification Of 4 Bupleurum.L In Northwest Of Hubei Province And Quantitative Analysis Of The Active Ingredients Of Bupleurum Radix With NIR Spectroscopy

Objective: This study used the four kinds of techniques,which include DNA barcoding,high resolution melting curve analysis,HPLC and near infrared spectra,to analysis the species and quality of Bupleurum.L in northwest Hubei Province.In order to provide the basis for rapid identification and estimation of quality.Methods:(1)The ITS and psbA-trnH regions of 4 kinds of samples were amplified and sequenced.The sequence was spliced using the software Condon Code Aligner.And the genetic distances were calculated by kimura-2-parameter(K2P)model and the Neighbor-Joining(NJ)phylogenetic trees were constructed using MEGA6.06.(2)Universal ITS2 primers were selected and used HRM to establish a new methods for identification of Bupleurum.L.(3)To determine the content of saikosapoonin a,d in Bupleurum Radix from market by HPLC.(4)A fast quantitative analytical model for saikosapoonin a,d content in Bupleurum Radix was explored based on near infrared spectroscopy(NIRS).saikosapoonin a,d content in each sample determined by HPLC was taken as reference value,and then a variety of data processing methods were compared to establish a better NIRS model.58 batches of Bupleurum Radix samples were categorized into training set and validation set using K-S algorithm.The NIR spectrum data of Bupleurum Radix samples in training set were matched with the reference value ofsaikosapoonin a,d using partial least square(PLS)algorithm and Support Vector Machine(SVM)algorithm.Result:(1)The result indicated that the ITS sequence lengths of Bupleurum plant in Northwest Area of Hubei Province is about 534 to 536 bp.The maximum intra-specific K2 P distance were lower than the minimum intra-specific K2 P distance.The NJ tree based on ITS sequence indicated that4 Bupleurum.L plant could be distinguished clearly.The psbA-trnH sequence lengths of Bupleurum plant in Northwest Area of Hubei Province is about443 bp,and the maximum intra-specific K2 P distance were higher than the minimum intra-specific K2 P distance.Therefore,ITS sequences can be used as an ideal DNA barcode to distinguish 4 Bupleurum plant in Northwest Area of Hubei Province,and the psbA-trnH sequence were not suit for them(2)The results showed the Tm values of Bupleurum chinense was(89.40±0.04)℃and B.scorzonerifolium was(89.97±0.07)℃and B.marginatum was(89.28±0.04)℃and B.falcatum Linne was(89.74±0.08)℃.This method can achieve the authentification of 4 Bupleurum.L plant and is simple,fast,high-throughput,visual.(3)The experimental results showed that except for 4,9 and 30 samples,the total contents of saikosapoonin a,d in other samples were in accordance with the standards specified in the Chinese Pharmacopoeia(2015Edition).(4)With regard to PLS model,both internal cross and external validation were used to screenspectrum preprocessing methods from MSC.Finally the FD+MSV was chosed as the optimal pretreatment,the spectrum band(8750~6000 cm-1)was selected as the optimal spectrum band for modeling and used grid optimization to reduce the dimension of input data for saikosapoonin a;and the no pretreat as the optimal pretreatment,the spectrum band(8750~6000 cm-1,4500 ~ 4100cm-1)was selected as the optimal spectrum band for modeling and used GA to reduce the dimension of input data for saikosapoonin d.The RMSECV values of the two analytical modelswere 1.2471 and 1.4956,respectively.The quantitative analysis of a and d of Bupleurum Radix had a good accuracy and predictive ability.