Agronomical and chemical studies aimed at turning Rhodiola rosea L. from Alps into a herbal medicine

The need to provide treatments for multifactorial diseases and the development of systems biology in life sciences prompted researchers to have a more holistic approach in pharmaceutical investigations. Therefore, studies in the field of medicinal plants have driven from searching for single bioactive compounds, to use as drug lead, to synergistic multiple components intervention and multi-target based strategies. Current issues of herbal medicines can be identified in: cultivation, characterization and quantification of compounds, metabolic fingerprinting, standardization of herbal preparations, quality control, ADME properties, evaluation of synergetic curative effects and safety. As part of the project named "PARMA" (Edible, Aromatic and Medicinal Alpine Plants) we chose, among others, the plant Rhodiola rosea in order to bring it into cultivation and to characterize it in terms of genetic variability and content of active principles. We present here the results obtained by LC-MSn studies and the quantitative analyses carried out on the extracts of Rhodiola rosea roots cultivated in the Trentino county. The various roseroot species are spread over the sub-arctic areas and also the mountains of Central Europe. Owing to its commercial interest and worldwide growing industrial demand, a number of studies have dealt with field cultivation. The experimental field is located at Palù del Fersina, Trento province, at an altitude of 1265 m.a.s.l. facing south. Four accessions were cultivated: two of them coming from Trento province (‘Malga Bondolo’ and ‘Adamello’), the other two respectively from Lombardy (‘Passo Gavia’) and Val d’Aosta region (‘Paradisia’). Plantlets have been obtained from seeds starting from summer 2003. To assess the metabolite production of plants, in October 2005-07 six plants for each plot were harvested. 2.0 g of dried roots were milled and exhaustively extracted with hexane (50mL) and methanol (50mL) at ambient temperature. Samples were subjected to LC-ESI/MS analysis. The HPLC separation was performed using a RP-18 column, the flow rate was 1 mL/min and the mobile phase consisted of 89% water - 10% acetonitrile – 1% formic acid (A) and methanol (B) applied in a stepped linear gradient. Three classes of secondary metabolites have been quantified : phenylpropanoids ( rosavin, rosarin and rosin ), phenylethanoids ( salidroside and tyrosol ), terpenoids ( rosiridin ). Owing to its poor ionization, tyrosol was quantified by UV detection at 280 nm. The quantitative determination of the other five marker compounds was performed using the HPLC system interfaced to a FinniganTM LXQTM Linear Ion trap mass spectrometer, operating in ESI‾ (0-15 min) and ESI+ (15-45 min) ion mode with Selected Ion Monitoring (SIM) as data acquisition mode. Salidroside was observed as [M+HCOO]- at m/z 345 ; rosarin, rosavin, rosin and rosiridin were observed as [M+Na]+ at m/z 451, 451, 319 and 355 resp. Rosarin and rosavin could be distinguished due to their different retention times. Minor constituents have also been isolated and identified : trans-cinnamic acid, cinnamyl alcohol, rosiridol, 3,7-dimethyl-2-octene-1,7-diol and rhodiocyanoside A.