HPTLC-ESI-MS and HPTLC-fluorescence methods for identification and quantification of darutoside in Sigesbeckia orientalis leaves extracts

Sigesbeckia orientalis L. (Asteraceae) is a small shrub native to India and widely distributed in tropical and temperate parts in the world. In some countries, this herb is an important traditional medicine to treat skin disorders. In Malagasy Pharmacopeia, the leaves are used externally as protective covering for wounds and burns to stimulate wound healing. We confirmed the real interest of this plant for cosmetic application by the development of an extract of leaves of Sigesbeckia orientalis really efficient for sensitive skins. Our phytochemical investigations showed that diterpenoids are one of the main groups of secondary metabolites. Thus, we developed a convenient and reliable analysis method to assess the quality control of the leaves and darutoside was selected as marker. RP-18 HPLC with UV detection at 210nm was often used to identify terpenoids. Nevertheless, the methodology is limited in its ability to separate all components of interest in hydroethanolic extracts of Sigesbeckia due to the presence of a resin-gum (mix of oligosaccharides and terpenoids). The aim of our study was to develop a quantitative analysis of darutoside in leaves extracts by HPTLC-fluorescence. The separation was performed on Si60 HPTLC plates. The mobile phase was chloroform/methanol/water in the ratio 65/25/4 (v/v/v). For revelation, the plates were sprayed with primuline reagent and then scanned in the fluorescence mode in a TLC scanner at 366nm. Darutoside was previously identified by ESI-MS with TLC interface. We applied our method for quantification of darutoside on our leaves samples collected in Madagascar. We observed that the amount of darutoside is variable depending on the date of harvest. April to June is the best period to harvest the leaves because darutoside reaches up to 1.5% of dry extract. The present study described for the first time the quantification of darutoside in Sigesbeckia orientalis leaves extracts by HPTLC-fluorescence method.