HPTLC, HPTLC-MS/MS and HPTLC-DPPH methods for analyses of flavonoids and their antioxidant activity in Cyclanthera pedata leaves, fruits and dietary supplement

Abstract

HPTLC silica gel, amino and C18 plates in combination with different developing solvents were explored for the first HPTLC chemical profiles of flavonoids and sugars in crude extracts from leaves and fruits of caigua (Cyclanthera pedata Scrabs) harvested in Slovenia and Italy. New HPTLC and HPTLC-MS methods for analyses of flavonoids are based on HPTLC silica gel (preconditioned with water) or C18 plates in combination with developing solvents ethyl acetate–water–formic acid (17:3:2, v/v) or 5% formic acid in methanol-water (7:3, v/v), respectively. Detection was performed before (for flavonoids) and/or after post-chromatographic derivatization with Natural product (NP) reagent for flavonoids and diphenylamine-aniline-phosphoric acid (DAP) reagent for sugars. HPTLC–MS/(MSⁿ) analyses on silica gel and C18 stationary phases enabled tentative identification of several compounds in crude extracts from leaves and fruits. HPTLC-DPPH^• assay combined with image analyses and HPTLC–MS/(MSⁿ) analyses, applied for direct screening of antioxidant activity of separated chromatographic zones (compounds) on HPTLC silica gel plates, confirmed activity in several zones of all crude extracts, in which some active compounds were tentatively identified. Apigenin 6-C-glucoside (isovitexin) and luteolin 8-C-glucoside (orientin) were found in all crude extracts, but the last was the most active free radical scavenger. Both compounds were also identified in dietary supplement product produced from caigua fruits. Crude extracts from leaves showed much higher antioxidant activity than those from fruits. The highest antioxidant activity was determined for crude extract from leaves harvested in Slovenia, while among crude extract from fruits the highest activity was determined for the extract from fruits harvested in Italy.

GRAPHICAL ABSTRACT

Keywords:

• Antioxidant activity
• Cucurbitaceae
• DPPH
• flavone C-glycosides
• flavones
• flavonoid glycosides
• flavonoids
• HPTLC
• HPTLC–MS

Acknowledgments

The authors want to express their gratitude to Merck company for the donated HPTLC plates used in the study. Francesca Orsini would like to gratefully acknowledge the financial support from the Campus Bio-Medico University of Rome, in particular for the opportunity to obtain the “Erasmus Plus” grant for a training period of nine months (from October 2016 to June 2017) at the Department of Food Chemistry of the National Institute of Chemistry (Ljubljana, Slovenia). Urška Jug is a student at the doctoral programme in Chemical sciences at the Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.

Additional information

Funding

The presented research was performed in the Department of Food Chemistry at the National Institute of Chemistry with the financial support from the Slovenian Research Agency [research core funding No. P1-0005 and “Young Researchers” programme – Urška Jug] and EN-FIST Centre of Excellence infrastructure (TLC-MS interface).