SIMULTANEOUS METABOLITE PROFILING OF HYDROPHILIC AND LIPOPHILIC COMPOUNDS IN ECHINACEA PALLIDA BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH PHOTODIODE ARRAY AND ELECTROSPRAY IONIZATION-MASS SPECTROMETRY DETECTION

Abstract

Echinacea spp. (family Asteraceae) herbal medicines and dietary supplements are traditionally used as immunostimulants in the prevention and treatment of inflammatory and viral diseases . Despite of many studies that have shown the chemical composition of alkamides in medicinally important species such as E. purpurea and E. angustifolia, in this study the attention was focused on secondary metabolites contained in E. pallida. In particular, caffeic acids derivatives, that have demonstrated to possess antioxidant, antiviral and anti-inflammatory activities, have been identified from the hydrophilic fractions of E. pallida extracts, while polyacetylenes and polyenes, that have shown interesting cytotoxic activities against a number of solid and leukemic cancer cell lines, have been isolated and characterized from lipophilic extracts of E. pallida roots.In this study, a detailed phytochemical characterization of E. pallida extracts and herbal medicines was carried out for the first time by developing advanced chromatographic techniques, based on RP-HPLC coupled with diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection, for the simultaneous analysis of the hydrophilic and lipophilic secondary metabolites. In the context of mass spectrometry detection, the performance of two mass analyzers, such as an ion trap and a triple quadrupole, were compared. The HPLC analyses were carried out on an Ascentis C18 column (250 mm × 4.6 mm I.D., 5 um), with a mobile phase composed by 0.1% aqueous formic acid and ACN with 0.1% of formic acid, under gradient elution. The flow rate was 1.0 mL/min and the column temperature was set at 30 °C. The UV spectra, in combination with MS and MS/MS data, allowed the identification of ten compounds, including caffeic acids derivatives, polyacetylenes and polyenes, in the analyzed samples. MS and MS/MS data were discussed in details and the typical fragmentation patterns of each class of secondary metabolites were identified.The RP-HPLC method was then fully validated in agreement with the ICH (Q2-R1) guidelines and then applied to real samples. The reference compounds used for quantification were isolated from the plant material and their structures were determined on the basis of the analysis of UV, IR, NMR and MS data. The quantitative analysis indicated that there was a great variability in the amount of the active compounds in the herbal medicines: the content of total caffeic acid derivatives ranged from 2.02 to 11.05 mg/g and the level of total polyacetylenes and polyenes from 9.62 to 39.12 mg/g. In the analyzed samples, the most abundant caffeic acid derivative was found to be echinacoside. Regarding polyacetylenes and polyenes, the most representative compounds were found to be pentadec-8-en-2-one, pentadeca-8,11-dien-2-one and tetradec-8-ene-11,13-diyn-2-one .The chemical standardization of E. pallida extracts is necessary to guarantee the quality, efficacy and safety of the corresponding herbal medicines. In this context, the proposed method, allowing the simultaneous determination of both the hydrophilic and lipophilic compounds contained in E. pallida, can be considered a useful and reliable tool for the metabolite profiling of plant material and the quality control of natural product