Combining DNA Barcoding and HPLC Fingerprints to Trace Species of an Important Traditional Chinese Medicine Fritillariae Bulbus

Abstract

Fritillariae Bulbus is a precious Chinese herbal medicine that is grown at high elevation and used to relieve coughs, remove phlegm, and nourish the lungs. Historically, Fritillariae Bulbus has been divided into two odourless crude drugs: Fritillariae Cirrhosae Bulbus and Fritillariae Thunbergii Bulbus. However, now the Chinese Pharmacopoeia has described five Fritillariae Bulbus—the new additions include Fritillariae Pallidiflorae Bulbus, Fritillariae Ussuriensis Bulbus, and Fritillariae Hupehensis Bulbus. Because the morphology of dried Fritillariae Bulbus is similar, it is difficult to accurately identify the different types of Fritillariae Bulbus. In the current study, we develop a method combining DNA barcoding and high-performance liquid chromatography (HPLC) to help distinguish Fritillariae Cirrhosae Bulbus from other Fritillariae Bulbus and guarantee species traceability of the five types of Fritillariae Bulbus. We report on the validation of an integrated analysis method for plant species identification using DNA barcoding that is based on genetic distance, identification efficiency, inter- and intra-specific variation, calculated nearest distance, neighbour-joining tree and barcoding gap. Our results show that the DNA barcoding data successfully identified the five Fritillariae Bulbus by internal transcribed spacer region (ITS) and ITS2, with the ability to distinguish the species origin of these Fritillariae Bulbus. ITS2 can serve as a potentially useful DNA barcode for the Fritillaria species. Additionally, the effective chemical constituents are identified by HPLC combined with a chemical identification method to classify Fritillaria. The HPLC fingerprint data and HCA (hierarchical clustering analysis) show that Fritillariae Cirrhosae Bulbus is clearly different from Fritillariae Thunbergii Bulbus and Fritillariae Hupehensis Bulbus, but there is no difference between Fritillariae Cirrhosae Bulbus, Fritillariae Ussuriensis Bulbus, and Fritillariae Pallidiflorae Bulbus. These results show that DNA barcoding and HPLC fingerprinting can discriminate between the five Fritillariae Bulbus types and trace species to identify related species that are genetically similar.

Antitumor chemical constituents of Toddalia asiatica (Linn) Lam root bark and its rational alternative medicinal parts by multivariate statistical analysis

“…1∼18 of T. asiatica were calculated by the cosine value method of vectorial angle, and the results were detailed below: 0.9507-0.9765 of samples no. [16][17][18]. The degree of chromatographic fingerprint similarity between T. asiatica root bark and T. asiatica stem bark was much higher than that of other parts.…”

“…High performance liquid chromatography (HPLC) fingerprint technology as an important analytical method had got quick development in many areas such as TCMs, food and biological samples. It can be a relatively effective method to evaluate the quality of complex TCMs as a whole [16][17][18]. In order to further explore the differences of all medicinal parts of T. asiatica, the obtained data were statistically processed by multivariate statistical analysis including bivariate analysis, hierarchical clustering analysis (HCA) and principal component analysis (PCA).…”

Natural drug sources for respiratory diseases from Fritillaria: chemical and biological analyses

“…The overall quality evaluation was established via the combination strategy between HPLC fingerprint and chemometrics [ 155 ]. What’s more, there are both HPLC fingerprints and DNA barcoding combined to analyze Fritillaria species [ 156 ]. In general, ELSD is not a commonly utilized detector, whereas the qualitative and quantitative analyses of isosteroidal alkaloids by conventional LC with DAD detector is difficult given the lack of chromophore groups.…”

Integrated Approach for Species Identification and Quality Analysis for Labisia pumila Using DNA Barcoding and HPLC

“…Usage of the dual methods of DNA barcoding and chemical analysis (HPLC) is, thus, beneficial in screening the authenticity and quality of HMPs. Previous reports showed that a combination of DNA barcoding and HPLC successfully identified five Fritillariae bulbus species from TCM (Traditional Chinese Medicine) and its effective chemical constituents [49]. The authenticity of Eurycoma longifolia HMPs has been tested by DNA barcoding using the ITS2 marker, and its quality was verified using HPLC [50].…”