DNA Barcoding, Fingerprinting and Main Components Quantification of Tibetan Medicine “Tiebangchui”

OBJECTIVE To conduct molecular identification of DNA barcoding for the polybasic proto-Tibetan medicine “Tiebangchui” and perform quality evaluation of its different origins through the combination of HPLC fingerprint and chemical pattern recognitions, along with the quantification of main components.

METHODS DNA barcoding was used to study the molecular identification of 2 basigens of the Tibetan medicine “Tiebangchui”, namely Aconitum pendulum Busch and Aconitum flavum Hand-Mazz.. HPLC fingerprinting was performed on a ZORBAX Eclipse XDB-C₁₈ column with 0.01% acetic acid(pH=6.5 adjusted by triethylamine)(A)-acetonitrile(B) as the mobile phase in a gradient elution with the detection wavelength of 235 nm, the flow rate was 1.0 mL·min⁻¹, the injection was 10 μL, and the column temperature was 30 ºC. Fingerprint were analyzed using the “Similarity Evaluation System for Traditional Chinese Medicine Chromatography Fingerprint(2012Edition)”. Based on the fingerprint, principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were performed, and the contents of the main components were quantified.

RESULTS  The molecular identification of DNA barcodes could be based on the variant sites in the ITS2 sequences, genetic distances, and whether the phylogenetic neighbour joining tree was clustered into a single unit as the basis for the identification of the multibasic original Tibetan medicines of A. pendulum and A. flavum. A HPLC fingerprint overlay map was established for 20 batches of “Tiebangchui” samples, and 15 common peaks were identified. Three chromatographic peaks were identified through comparison with the control samples, and the quantification method was established. The similarity of the fingerprint spectrum was 0.853–0.997. Five principal component factors were determined by PCA, and the cumulative variance contribution rate was 83.931%, and OPLS-DA results indicated that A. pendulum and A. flavum could be classified into 2 categories, and a total of 7 differential markers were screened. The contents of the 3 recognized components benzoyl aconitine, aconitine and 3-deoxyaconitine were 0.1068–1.1269, 1.8983–28.4839, and 0.2763–7.2596 mg·g⁻¹, respectively.

CONCLUSION The method established in this study is stable and feasible, and can provide a reference for the identification and quality control of Tibetan medicine “Tiebangchui”.