Isolation of Phenolics from Rhizophora mangle by Combined Counter-current Chromatography and Gel-Filtration Fernanda das Neves Costa*, Marcos Daniel da Silva, Ricardo Moreira Borges and Gilda Guimarães Leitão Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, RJ 21941-590, Brazil fncosta@nppn.ufrj.br Received: April 30 th , 2014; Accepted: July 7 th , 2014 Nine phenolic compounds, quercetin, epi-catechin, catechin, 4-hydroxybenzoic acid, kaempferol 3-O-β-glucopyranoside, quercetin 3-O-β-glucopyranoside, quercetin 3-O-6"-trans-coumaroyl-β-glucoside, kaempferol 3-O-β-rutinoside and quercetin 3-O-β-rutinoside, were isolated from the EtOAc leaf extract of Rhizophora mangle (Rhizophoraceae) combining counter-current chromatography (CCC) and gel-filtration. A solvent system of n-hexane-ethyl acetate- methanol-water (1.5:6:1.5:6) was employed at the preliminary stage of EtOAc extract fractionation as it was shown to contain compounds that differed highly in their hydrophobicity. The obtained fractions were further purified by either CCC or gel-filtration depending on their complexity. The isolated compounds were analyzed by NMR spectroscopy and the proposed structures were confirmed by HRES/ESI/TOF MS. Some of these compounds were isolated and/or identified for the first time in R. mangle. Keywords: Counter-current chromatography, Gel-filtration, Phenolics, Rhizophora mangle, ESI-MS. The key to any study of material from natural sources is the availability of suitable separation methods for the isolation of pure compounds. Although several methodologies have been developed, most separations are performed by classic solid-support chromatography, which tends to adsorb compounds irreversibly. For this reason, all-liquid techniques are currently attracting considerable interest [1]. Counter-current chromatography (CCC) has become an effective alternative to the solid-support techniques because it is a high speed, high loading and high resolution, support-free liquid-liquid partition chromatography, which can eliminate irreversible adsorption of samples, having an excellent sample recovery [2-4]. Separation is based on the partitioning of solutes between two immiscible liquid phases being the relative proportions of solute in each phase determined by the respective distribution coefficient [1, 4]. Taking advantage of the all-liquid phases and its high ratio of stationary phase in the column [2, 5, 6], CCC was used in the preliminary stage of fractionation of the crude EtOAc extract of R. mangle leaves. The obtained fractions were further purified by either second step CCC or gel-filtration chromatography, showing that a combination of different techniques can increase the resolution power of separation methods in natural products chemistry. Rhizophora mangle (Rhizophoraceae) is a widespread, native Brazilian mangrove tree [7]. It occurs all along the coast, from the State of Pará until the State of Santa Catarina. It is popularly known as ‘red mangrove’ [8] and recently the anti-ulcer activity has been proved in many studies [9-11]. Previous phytochemical studies of this species described the isolation from its leaves of flavonoids [12], triterpenes [13-14] and tannins [12, 15]. The EtOAc extract of R. mangle was first analyzed by TLC followed by visualization under UV light and negative mode HRES/ESI/TOF MS direct injection. The elution profile on TLC and major ions obtained by MS analyses, combined with previous phytochemical studies of R. mangle [12], suggested the presence of flavonoids with highly different hydrophobicity in the extract, containing none, one or two sugar moieties. Figure 1: TLC of the combined fractions of the first EtOAc crude extract of R. mangle preparative fractionation of the isolated compounds and subsequent chromatographic steps. Taking into account that it is very difficult to isolate compounds from this kind of matrix in a single chromatographic step, CCC was used in the preliminary stage of crude extract fractionation, as it is an all-liquid technique that has a high load ability [2, 5, 6] to obtain less complex samples to purify. Solvent system selection: In contrast with conventional liquid chromatography that uses a single phase to elute the analytes from the solid support, the CCC technique uses a two-phase solvent system: one as the stationary phase and the other as the mobile phase [4, 16]. The selection of the solvent system is the most important step in CCC [16], as it is possible to choose both stationary and mobile phases at the same time and estimated as 90% of the entire work [4]. Without consulting the literature, the search for a two-phase solvent system for the successful separation of a particular class of compounds can be very time-consuming. In the last years, CCC has been broadly applied to the separation and purification of flavonoids and phenolics from different natural NPC Natural Product Communications 2014 Vol. 9 No. 12 1729 - 1731