Chemical evaluation of cultivated roots of Gentiana Lutea L. and of by-product obtained during bitter liqueurs production by using mass spectrometry.

Gentian (Gentiana lutea L.) is a widely distributed herbaceous species of the Gentianaceae family. It is native to the mountains of central and southern Europe, preferring calcareous soils, and is found naturally (wild) in France, Spain and the Balkan mountains. The plant is under wildlife protection in Europe, and is cultivated particularly in Germany and France [1]. The root of G. lutea is famous for its bitter properties and is often used in alcoholic bitter beverages, food products and traditional medicine to stimulate the appetite and improve digestion. This is mainly due to its content of bitter-tasting secoiridoids, in particular swertiamarin, gentiopicroside, sweroside and amarogentin that have stomachic, digestive, cholagogue, hepatoprotective and wound-healing activities, while the iridoid loganic acid and isogentisin have anti-inflammatory and MAO inhibiton activities, respectively [2]. The first aim of this study was to develop a new, simple, and fully validated HPLC-MS (ion trap) method for simultaneous analysis of iridoids (loganic acid), secoiridiods (gentiopicroside, sweroside, swertiamarin, amarogentin) and xanthones (isogentisin) in G. lutea samples that were cultivated for the first time in the Monti Sibillini National Park (Italy), collected wild in the Marche-Umbria area, or bought in herbal shops, The HPLC-MS assay was performed on RP–C18 column with aqueous–formic acid (99.9–0.1%) and acetonitrile as mobile phase. The analyzed molecules demonstrated a good linearity (R2 ≥ 0.9992) in a wide concentration range (0.1–10 µg/ml). The analysis of twenty samples of cultivated, wild and commercial G. lutea L. showed that gentiopicroside is the most dominant compound (1.85–3.97%), followed by loganic acid (0.11–1.30%), isogentisin (0.03–0.48%), sweroside (0.05–0.35%), swertiamarin (0.08–0.30%), and amarogentin (0.01–0.07%). Results confirmed that G. lutea we have succeeded in cultivation for the first time in Monti Sibillini National Park (Italy); could be an excellent source for production of high quality plant material due to its high contents of analyzed bioactive compounds compared to wild and commercial samples [3]. Furthermore, chemical analysis of the by-product obtained by processing G. lutea and other herbs during production of bitter liqueurs, has been carried out. By-products of plant food processing represent a major disposal problem for the industry concerned, but they are promising sources of compounds because of their favourable technological or nutritional properties. The second aim of this study was to develop a new, simple, selective and reliable HPLC−MS/MS (triple quadrupole) method to simultaneously estimate 18 bioactive compounds belonging to different chemical classes such as secoiridoids, alkaloids, phenolic acids, flavonoids and other polyphenolics in the waste of bitter liqueurs e.g. Amaro Sibilla®. Chromatographic separation was accomplished on a Synergi Polar-RP C18 analytical column working in the gradient mode. For the purpose of correct and unambiguous identification of the analysed compounds, the high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) analysis was performed with electrospray ionization (ESI) mode using both MRM and SIM. The method exhibited a good linearity of all 18 standards (R2 ≥ 0.9980) and an excellent sensitivity with LODs and LOQs that ranged from 0.003 to 8 ng/ml and 0.01 to 25 ng/ml, respectively. According to the results we obtained by HPLC–MS/MS, gentiopicroside, quinine, naringin and hesperidin were detected in quite higher quantities in the waste corresponding to 1200, 81.20, 104.21 and 247.97 μg/g, respectively, with respect to their quantities in the liqueur. Most of the detected analytes in this study are biologically active as antioxidant and chemopreventive agents. This indicates that the investigated waste is rich in various bioactive compounds especially secoiridoids, alkaloids, flavonoids and phenolic acids. We suggest to use and benefit this waste as a source of functional compounds e.g. gentiopicroside, quinine, naringin, hesperidin and others, since they are bioactive and expensive materials. According to the chemical analysis performed, we suggest to use and recycle bitter liqueurs by products, that represents a source of valuable bioactive compounds to be used in food, pharmaceutics and cosmetics.