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Publication type: Doctoral Thesis

Year: 2012

Author(s): Ruzicka, Joana

Title: DNA-based identification of drug material using the example of Rumex spp. and Verbena officinalis.

Other title: DNA-basierte Identifizierung von Pflanzendrogen am Beispiel von Rumex sp. und Verbena officinalis

Source: Dissertation, Vet. Med. Univ. Wien, pp. 169.


Advisor(s):

Novak Johannes

Reviewer(s):
Wagner Martin

Vetmed Research Units:
Institute of Animal Nutrition and Functional Plant Compounds


Graduation date: 07.03.13


Abstract:
Rumicis herba and Verbenae herba are widely used as herbal remedies. The authentication of the drug delivering species Rumex sp. and Verbena officinalis relied so far upon morphological and chemical analysis only. Reliability of morphological and chemical analyses, however, is limited due to processing of the drug material, high intraspecific variation of morphological characters or chemical compounds and lacking specificity of the chemical profile. For an unequivocal classification a DNA-based approach has been generated for both species in this study. The basis for setting up the identification system was direct sequencing of cp and ITS sequences in a first attempt. The cp sequences within the recent genus Verbena were nearly identical, here only ITS sequences could be used for identification on the species level. RAPD patterns were used as a second approach to identify V. officinalis. ITS sequences, RAPD-derived SCAR markers as well as markers for HRM analysis finally enabled the identification of Verbena officinalis. In contrast, in the genus Rumex, the usability of ITS sequences for species identification was problematic due to additional bands produced within species of the subgenus Acetosa. The variability of the cp regions in this genus was higher than in the recent genus Verbena (genetic distance between the subgenera of 0.034 in the trnL-trnF-IGS). The region with the highest mean sequence divergence (0.08) was the psbA-trnH-IGS. Despite of a good variability within the whole genus Rumex, the resolution within the subgenus Rumex was very low in every cp region (from 0.001 in trnL-trnF-IGS to 0.007 in the psbA-trnH-IGS) and sequences of the section Acetosa were almost homogenous. In the psbA-trnH-IGS as well as in the psbM-trnD-IGS, the taxa of the subsection Patientiae built an own clade and were found to be more distinct to the subsections Obtusifolii and Crispi. Members of the subsections Obtusifolii and Crispi are closer related but did not definitely group together. Hybridization of the highly variable R. obtusifolius and R. crispus or intraspecific sequence polymorphisms can be the reason for these discrepancies that hampered species identification. An authentication on species level for the drug deliviering species R. acetosa, R. crispus, R. obtusifolius and R. patientia was finally not possible in this approach. For the genus Rumex an ARMS with specific primers based on cp trnL-F-IGS sequences was facilitated, which proved to be a valuable, fast and robust screening method for the identification of the section Acetosa, therefore narrowing the identification of Rumicis herba. The usability of this identifcation system was successfully tested in identifying fourteen drug remedies of Rumicis herbae.

Keywords:
Rumex sp. / Verbena officinalis / DNA-based identification / trnL-trnF-IGS / psbA-trnH-IGS / psbM-trnD-IGS / ITS / ARMS / RAPD / HRM analysis


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