ABSTRACT
To accurately discriminate Stellariae Radix from its adulterants, four leading candidate DNA barcoding markers were evaluated. Sixty samples including Stellariae Radix and its adulterants have been newly collected and their total genomic DNA was extracted. Four DNA barcoding markers ITS, rbcL, psbA-trnH and matK were amplified and sequenced. Their sequence characteristic analyses, Kimura-2-parameter (K2P) distance calculation and Neighbor-joining (NJ) phylogenetic tree constructions were accomplished using the MEGA 7.0 software. DNA Barcoding gaps of the four DNA barcoding markers were estimated by the distributions of inter- and intra-sequence specific variations. Species identification efficiency was calculated using the BLAST method. The results showed that ITS had the highest (95.2%) while matK demonstrated the lowest (75%) PCR and sequencing efficiency. The length range of the four markers were in the ranger of 211-797 bp, and the G+C content of ITS was highest (54.35%). The identification efficiency of matK and ITS was 92% and 90% respectively. Barcoding gap could be found in ITS sequences. The NJ phylogenetic tree constructed using ITS sequences showed that samples of Stellariae Radix were separately formed into one clade, and samples of adulterants like Stellaria bistyla were clearly belong to different branches from Stellariae Radix, whereas NJ trees constructed using psbA-trnH, rbcL and matK could not differentiate Stellariae Radix from its adulterants. Therefore, ITS regions as DNA barcodes can stably and accurately distinguished Stellariae Radix from its adulterants, and provide a new technique for modern identification of Stellariae Radix.
ABSTRACT
Objective:To establish the fingerprints of standard decoction of Gentianae Macrophyllae Radix-dried products by different methods,and to evaluate the quality correlation. Method:HPLC,InertSustain C18 chromatographic column(4.6 mm×250 mm,5 μm),Gradient elution was performed for the mobile phase of acetonitrile-phospho,detection wavelength at 240 nm and flow rate of 0.8 mL·min-1,and column temperature was 40℃. The quality correlation analysis of different methods for different kinds of Gentianae Macrophyllae Radix standard decoction was carried out from the aspects of chemical composition consistency,common chemical composition consistency,main chemical composition content and transfer rate. Result:The control fingerprint of Gentianae Macrophyllae Radix standard decoction was established. According to the peak matching data,there were 10 common peaks in the fingerprint of 15 batches of Gentianae Macrophyllae Radix standard decoction.Among the 10 common peaks,5 chemical constituents of loganic acid,6'-O-β-D-glucosyl gentiopicroside,swertiamarin,gentiopicroside and swertia glycosides were identified. The results of quality correlation analysis showed that the three different drying methods were consistent with the chemical composition and quantity of Gentianae Macrophyllae Radix standard decoction. But in terms of the content consistency of common chemical components and the transfer rate of main chemical components,the quality correlation between the products obtained from vacuum drying and the standard decoction was lower than that obtained from spray drying and freeze-drying. Conclusion:The fingerprint of different method of Gentianae Macrophyllae Radix standard decoction was established. Through the analysis of the mass correlation of chemical composition consistency,common chemical composition content consistency,main chemical composition content and transfer rate,the mass correlation between them was comprehensively reflected. It is suggested that spray drying or freeze drying should be used for the key drying process of Gentianae Macrophyllae Radix granules. This study provides a reference for the preparation process and quality control of Gentianae Macrophyllae Radix granules.