Fractionation and determination of the antitumor activity of Pinus sylvestris L. bark extracts

Abstract

This study was designed to evaluate the antioxidant and antiproliferative effects of pine bark components [1]. Pine bark was extracted with 80% methanol. Liquid-liquid partition of crude extract provided diethyl ether, ethyl acetate, n-butanol and aqueous fractions. The total phenolic content in crude extract and its fractions was determined by the Folin-Ciocalteu method. The polyphenolic profile was analyzed using HPLC-DAD and HPLC-ESI-MS. Screening for antiproliferative effects was investigated on tumour and „normal“ cells with the MTT and crystal violet incorporation assays. Antioxidant activity was determined by the DPPH assay [1,2]. The amount of total phenolics ranged from 25.25±0.05g (diethyl ether fraction) to 69.61±0.22g (ethyl acetate fraction) gallic acid equivalents per 100g extract/fraction. The ethyl acetate and the aqueous fractions showed little or no antiproliferative activity. The diethyl ether fraction was the most active as it inhibited MCF-7, PC-3, CaCo-2 and A549 tumour cell proliferation in a dose-depended manner at concentrations ranging from 1–100µg/ml. The n-butanol fraction was less active as it inhibited tumour cell growth at higher concentrations. These fractions had little effect on the growth of „normal“ cells (MCF-10A). Both crude extracts and their fractions showed strong antioxidant effects, with the most active being the ethyl acetate fraction (IC50=7.46±0.03µg/ml). These studies demonstrate that pine bark is a rich source of compounds with antioxidant and antiproliferative activity and provides leads for their isolation and characterization.