Planar and Column High Performance Liquid Chromatography of Biologically Active Compounds Extracted from Leaves and Fruits of Cyclanthera pedata (L.) Schrab

This communication reports the results of a recent study carried out to develop straightforward methods for the identification and quantification of phenolic compounds occurring in the leaves and fruits of Cyclanthera pedata Scrabs (Caigua), as well as in food supplements produced from this plant. Caigua is an ancient Peruvian plant belonging to the Cucurbitaceae family, which is grown for its edible mature fruit in many parts of Central America, South America and the Andes. It is found in the Caribbean Islands, Colombia, Bolivia, Peru and Mexico. Caigua is also an ancient medicinal plant whose use dates back to the Inca population. Nowadays, this plant is gaining market importance around the world due to its anti-inflammatory, hypoglycaemic and hypocholesterolemic properties. Recent scientific studies attribute the anti-hyperglycemic properties of Caigua to the high content of phenolic compounds, the most abundant class of secondary metabolites, and, in particular, to the glycosilated flavonoids subclass. The occurrence of phenolic compounds in the leaves and fruits of Caigua grown in Italy and Slovenia was investigated using both high performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC). Sample detection was performed by either photodiode array (PDA) spectroscopy or mass spectrometry (MS) with electrospray (ESI) ionization, used for obtaining structural information on the separated compounds and for their quantitative analysis. A special surface sampling probe was used for extracting the analyte on-line from the HPTLC plate to the ESI-MS instrument. The separations by HPTLC were carried out in normal phase mode using silica plates and mobile phases consisting of a variety of organic solvents in mixtures at different volume rates. HPLC was performed in reversed phase mode employing a narrow-bore C-18 column and a semimicro photodiode array detector (PDA) cell of 2.5 ?L, in conjunction with a single quadrupole mass spectrometer, equipped with an electrospray ionization source (ESI-MS). The C-18 narrow bore column was eluted by a multi-segment gradient of increasing concentration of acetonitrile in water-formic acid solution that has been optimized on the basis of the results of a study carried out to evaluate the influence of mobile phase composition and gradient shape on separation performance and detection sensitivity by ESI-MS. The identification of individual phenolic compounds was performed on the basis of their HPTLC retention factor (Rf) and HPLC retention time and both UV-visible and mass spectra, acquired by PDA and ESI-MS, respectively. Different organic solvents and hydro-organic mixtures were tested for their efficiency to extract the phenolic compounds from leaves and fruits of Cyclanthera pedata grown either in Italy (Rome) or Slovenia (Ljubljana), as well as from commercially available food supplements. A HPTLC-DPPH method was developed in order to assess the antioxidant properties of the compounds occurring in the Caigua extracts and in food supplements produced from this plant. Pros and cons, as well as complementary aspects, of HPTLC and HPLC methods for the identification and quantification of biologically active compounds in plant extracts are illustrated and discussed.