Potential of Complementary Methods for the Authentication of Herbal Substances and their Mixtures

Abstract

Herbal substances and preparations thereof play an important role in healthcare systems worldwide. An essential prerequisite for the safe and effective use of herbal medicines is the unequivocal identification of the plant species used therein. The majority of herbal substances are administered as multicomponent mixtures, especially in the field of TCM and ayurvedic medicine, but also in finished medicinal products used in Germany. Quality assessment of complex mixtures of herbal substances with conventional methods is challenging. Thus, emphasis of the present work was directed on the development of complementary methods to elucidate the composition of mixtures of herbal substances and finished herbal medicinal products, focusing on options provided by PCR-related methods. <br /> A multiplexed qPCR method in combination with TaqMan® probes has been established, enabling the reliable and precise detection of each species in a defined mixture of herbal substances composed of Quercus cortex, Juglandis folium, Matricariae flos, Salviae miltiorrhizae radix et rhizome and - due to recurrent concerns of contaminations of herbal substances with toxic plant species - the aristolochic acids containing species Aristolochia clematitis (Aristolochiae herba). This approach evolved to be highly sensitive and specific, and also suitable for minor DNA amounts yielded from processed plants or finished medicinal products. All components of the defined mixtures of herbal substances could be successfully detected in mixtures with highly variable quantities of each plant. The applicability of the method was also tested successfully for the finished medicinal product Imupret®, achieving limits of detection of 20 pg DNA for the component Quercus cortex. <br /> An additional objective of the present thesis was further testing of special applications of an ITS barcoding approach (Kersten, 2013) and a 1H-NMR fingerprinting technique (Daniel, 2009) for authentication of medicinal plants derived from the Lamiaceae family, Asian therapeutic systems (TCM herbal substances), the herbal substance Cimicifugae racemosae rhizoma and the plant-derived product propolis. The ITS barcoding approach proved to be a valid and quite robust method for the sensitive and unambiguous authentication of individual components of herbal substances. However, the method encountered some problems regarding highly processed herbal substances, especially those from TCM, and ethanolic plant extracts. The 1H-NMR fingerprinting approach was evaluated for its applicability to discriminate different plant genera and different plant species. For this application, there are limitations due to the presence of similar compounds occurring in different species or even different genera. It could be demonstrated that the application of this method should be restricted to a defined area and must be based on comprehensive, valid reference samples. <br /> Conventional analytical methods often have some limitations with respect to mixtures of herbal substances and finished herbal medicinal products. Minor amounts of sample material, processed herbal substances and complex mixtures place high demands on efficient quality control. Such prerequisites in many cases are hardly compatible with analytical methods that mostly are focusing on the detection of marker compounds. Results from the present work indicate that DNA-based methods can provide a suitable complementary approach for the sensitive and unambiguous authentication of herbal substances and their mixtures.