• HPLC
• extraction
• Echinacea angustifilia
• caffeic acid derivatives
• secondary metabolism

The use of medicinal plants has increased considerably in the past two decades in industrialized countries. Three species of the genera Echinacea, belonging to the Compositae family, are among the top-selling plants of pharmacological interest. The species E. angustifolia, E. purpurea, and E. pallida are recognized for their immunostimulating properties and are used for their preventative actions, but also as remedies, so as to decrease the severity and duration of the symptoms of the common cold. The pharmacological action has been ascribed to several classes of compounds: alkylamides, glycoproteins, polysaccharides and caffeic acid derivatives. The latter class of compounds, deriving from the shikimate pathway, includes some important molecules for quality control in Echinacea, such as echinacoside and cynarin, which are the most common marker compounds mentioned by the literature and are largely adopted by the industry as well. Standardization of medical products is of primary importance in order to bring on the market products that are uniform in term of active principle contents between the different production batches, and that reflect chemically the information on the etiquettes. Currently, this condition is not fulfilled in many cases for several medical plants. Evidences from the literature and from previous work in our laboratory show an elevated variability in caffeic acid derivative contents among different production batches; such a variability may result from inadequate post-harvest processing and extraction of plant samples. Therefore, a work was undertaken i) to elucidate the effect of different treatment of plant samples before extraction and HPLC analysis, and ii) to verify and eventually improve the extraction protocol. In one of the first experiments, the attention was paid to the influence of the storage temperature and/or dehydration by oven drying or lyophilization on the content of caffeic acid derivatives in roots and leaves of E. angustifolia plants grown hydroponically under greenhouse conditions. The main metabolites determined in plant tissues were echinacoside, cynarin, caftaric acid, chlorogenic acid and cichoric acid. Generally, the temperature did not influence much the content of detected phenolics in oven dryed samples, while the level of these metabolites were very low or even under detection limit in fresh samples extracted after storage at freezing temperature or in the lyophilized ones. The relatively low temperatures used for sample drying exclude the hypothesis that, in fresh tissues, caffeic acid derivatives are bound to cellular components and that the drying process makes them available to analytical detection. More likely, a degradation of metabolites of interest occurs during the extraction process in fresh samples or during freeze drying. To verify the hypothesis of sample degradation, the extraction protocol was modified in a first step by adding the extraction solvent (methanol-water, 70:30) prior to tissue homogenization. In a second step the solvent was also acidifyied by hydrochloric acid 1%. Finally, using internal standard (by adding to the sample gallic acid, a compound not detected in Echinacea plant tissues) the loss of active principles was estimated. The extraction of caffeic acid derivatives was improved by modifying the extraction protocol and showed the best results in frozen and lyophilized roots by acidification of the solvent.