Molecular identification and quantitation of Hypericum perforatum in mixed samples

The requirement for quality assurance in commercial medicinal plant products has been brought into focus as European national regulatory authorities move towards the 2011 deadline to implement the Traditional Herbal Medicines Directive (Directive 2004/24/EC. Medicinal plant products for human use in the European Union).

In this context, we report the development of a DNA-based method for the identification and authentication of plant species, based upon the economically important St John's Wort (SJW) (Hypericum perforatum L.). The ITS regions of the nuclear-encoded ribosomal RNA genes provided the target for primer design. Sequences from 91 Hypericum species were analysed in order to identify the most divergent regions, and four PCR primers were designed to anneal specifically to H. perforatum in these regions. All of these were proved empirically to be SJW-specific when compared to DNA from likely contaminant or adulterant species.

As herbal medicinal products are often sold as mixtures, a quantitative method of identification is of particular interest. Real-time PCR enables quantitation of template DNA by virtue of monitoring the reaction at every cycle, and this procedure was optimised for quantitation of SJW in a mixed preparation. A generic primer pairing was used to measure all of the amplifiable nuclear DNA within a sample; the specific primers allowed quantitation of SJW DNA. This gives a measure of the specific DNA as a proportion of total DNA, which could be used to calculate the ratio of SJW plant material in a mixed sample, or detect the presence of contaminant or adulterant plant material in pure SJW preparations.