J. of Supercritical Fluids 60 (2011) 28–37 Contents lists available at ScienceDirect The Journal of Supercritical Fluids j ourna l ho me p ag e: www.elsevier.com/lo cate/supflu Potential allelopathic of the fractions obtained from sunflower leaves using supercritical carbon dioxide Z. El Marsni a , L. Casas a , C. Mantell a,∗ , M. Rodríguez a , A. Torres b , F.A. Macias b , E.J. Martínez de la Ossa a , J.M.G. Molinillo b , R.M. Varela b a Department of Chemical Engineering and Food Technology, Faculty of Science, University of Cadiz, Box 40, 11510 Puerto Real, Cadiz, Spain b Department of Organic Chemistry, Faculty of Science, University of Cadiz, Box 40, 11510 Puerto Real, Cadiz, Spain a r t i c l e i n f o Article history: Received 11 January 2011 Received in revised form 9 May 2011 Accepted 10 May 2011 Keywords: Supercritical fluid extraction Allelopathy Bioactive compounds Herbicides a b s t r a c t The application of supercritical carbon dioxide in the extraction of bioactive compounds from three vari- eties of Helianthus annuus L. (Stella, Arianna and Hilian) has been investigated. The influence of different variables, including pre-treatment of the sample, temperature and pressure, was analyzed. The best extraction yields and activity profiles were obtained for the Arianna variety. Several sets of conditions for fractionations using two cyclonic separators were studied and the bioactivities and extraction yields of the fractions were compared. The best fractionation conditions were obtained at 90 bar and 40 ◦ C. During the fractionation process it is possible to obtain an extract that is free of chlorophylls, a situation that makes the purification process and the identification of secondary metabolites easier. © 2011 Elsevier B.V. All rights reserved. 1. Introduction From an economic point of view, the importance of the sun- flower cultivar is related to the production from the seed of high quality oil that is suitable for human consumption. The waste obtained after the extraction process is used for the preparation of cakes that are used as forage for livestock. The raw seed is also used to feed birds and can be toasted for human consumption. Spain is one of the ten main producers of sunflower oil in the world. In Andalusia this cultivar is widespread (43.88% of the total Spanish crop) [1]. Certain favorable characteristics of this cultivar have contributed to its wide dissemination: the crop is simple, does not require specialized equipment, is rustic, has a short vegetative cycle and is adaptable to soil conditions and unfavorable weather. The sunflower is one of the most widely studied plants in terms of bioactivity [2,3]. A large number of toxins are present in this plant and they are located principally in the leaves. The leaves are discarded in sunflower oil production and it is therefore possible to increase the value of this cultivar by developing new applications for substances derived from the leaves. The first reference concerning the allelopathic effects of this plant was published in 1931 [4]. Numerous examples of allelo- pathic activity in wild and agricultural ecosystems have been ∗ Corresponding author. Tel.: +34 956 016 579; fax: +34 956 016 411. E-mail addresses: zouhir.elmarsni@uca.es (Z. El Marsni), casimiro.mantell@uca.es (C. Mantell). reported: Helianthus rigidus exhibits autotoxicity and the sunflower (Helianthus annuus) has great allelopathic potential and inhibits seedling growth in weeds, including velvet leaf, apple morning glory and wild mustard, among others [5]. The results of field stud- ies have demonstrated that weed biomass is equally reduced in sunflower plants with or without herbicides [6,7]. According to the definition given by the International Allelopa- thy Society (IAS), allelopathy concerns the study of ‘any process involving secondary metabolites produced by plants, algae, bac- teria and fungi that influence the growth and development of agricultural and biological systems’ [8]. The definition includes both beneficial and detrimental effects caused by these allelo- chemicals in the surrounding organisms. The definition also covers co-evolutionary aspects as such interactions have led some species’ to overcome the deleterious effects of defence compounds and turn them into beneficial effects. Allelopathy was initially considered to be a branch of the ecological sciences and research focused on qualitative descriptions of predominant or invasive plant species [9,10]. Allelopathy today encompasses the wide range of disciplines related to the study of plants: ecology, biochemistry, chemistry (natural product isolation and synthesis), plant physiology (includ- ing mode-of-action studies), technical agriculture, forestry, genetic breeding, soil studies and, more recently, proteomics and genomics [11–14]. The bioassay is a widely applied test in research into the pro- duction of herbicides [15,16]. This test provides a measure of the biological response of a living organism and is related to the pres- ence of a particular family of compounds. The bioassay must be 0896-8446/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2011.05.007