Low-pressure chromatography on sephadex LH-20:treatment to separate, identify and evaluate the antioxidant activity of Eucalyptus globulus phenolic compounds

Lila Boulekbache-Makhlouf1,Emmanuelle Meudec2, Veronique Cheynier2, Jean-Paul Mazauric2, Sakina Slimani1, and Khodir Madani1

1-Faculty of Nature and Life Sciences, 3BS Laboratory, University of Bejaia06000, Bejaia, Algeria

2-INRA, UMR 1083 Sciences pour l’œnologie. 2, Place Viala-34060 Montpellier cedex, France

Abstract

The present study is focused on the treatment of Eucalyptus globulus (E. globulus) fruit extract, in order to separate, identify, and evaluate the antioxidant activity of its phenolic compounds.The treatment of crude extract was carried out by fractionation using chromatography on a Sephadex LH-20 column [1]; then a method based on high-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) has been developed for qualitative analysis of different phenolics [1]. The antioxidant activity of crude extract and its chromatographic fractions (F1, F2 and F3) was measured by ferric reducing power [2], 2, 2-diphenyl-1-picrylhydrazyl(DPPH•) radical scavenging activity [3] and neutralizing of hydrogen peroxide (H2O2) [4] assays. On the basis of the absorbance data of different phenolics, three fractions (F1-F3) were made (Figure 1). The presence of 18 gallotannins, 26 ellagitannins, and 2 flavonols was established. Most compounds described in this study have not previously detected in fruit of E. globulus. Moreover, this is the first report of methyl digalloyl diglucose, 3, 3-0 -dimethylellagic acid 4- -β-glucopyranoside, ellagic acid hexose, methyl ellagic acid pentose, methyltetragalloylglucose, and valoneic acid isomers (sanguisorbic, flavogallic acid dilactone) in the genus. Quantitatively, ellagic acid and its derivatives, including ellagitannins, are largely predominant. Results shows that extracts of E. globulus fruits exhibited a potent free radical scavenging activity, as well as higher reducing power and neutralizing capacity of hydrogen peroxide, than that of synthetic and natural antioxidants (BHA and α-tocopherol).

References

[1] Lila Boulekbache -Makhlouf, Emmanuelle Meudec, Chibane Mouhamed, Jean-Paul Mazauric, Sakina Slimani , Henry Max, Veronique Cheynier, & Khodir Madani. Journal of agricultural and food chemistry 58 (2010) 2615-12624.

[2]Hseu YC, Chang WH, Chen CS, Liao JW, Huang CJ, Lu FJ, Hia YC, Hsu HK, Wu JJ &Yang HL. Food and Chemical Toxicology46 (2008) 105-114.

[3] Suja KP, Jayalekshmy A & Arumughan C. Food Chemistry. 91 (2005) 213-219.

[4] Sfahlan AJ, Mahmoodzadeh A, Hasanzadeh A, Heidari R & Jamei R. Food Chemistry. 115 (2009) 529–533.

Figure

Figure 1. Sephadex LH-20 column chromatography profile of E. globulus fruit extract