J. of Supercritical Fluids 55 (2011) 924–928 Contents lists available at ScienceDirect The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu Extraction and identification of proanthocyanidins from grape seed (Vitis Vinifera) using supercritical carbon dioxide Esen Eyiler Yilmaz ∗ , Emin Burcin Özvural, Halil Vural Hacettepe University, Departement of Food Engineering, Beytepe 06800, Ankara, Turkey article info Article history: Received 7 September 2010 Received in revised form 23 October 2010 Accepted 24 October 2010 Keywords: Supercritical carbon dioxide extraction Proantocyanidins Grape seed High performance liquid chromatography abstract In this study, supercritical carbon dioxide extraction of proantocyanidins (PRCs) was performed and the effect of different pressure, temperature and ethanol percentage was investigated. High performance liq- uid chromatography was used for the analysis of the compounds and it was found that the most effective parameter on the extraction was the amount of the ethanol percentage. Each compound was extracted from grape seeds at their maximum level when different parameters were used which was probably because of their different polarities. Gallic acid (GA), epigallocatechin (EGC) and epigallocatechingallate (EGCG) were extracted at their maximum level when the 300 bar 50 ◦ C and 20% of ethanol was used. The maximum amount of catechin (CT) and epicatechin (ECT) were obtained when 300 bar 30 ◦ C and 20% of ethanol was used for extraction, and 250 bar, 30 ◦ C and 15% of ethanol was needed to extract highest amount of epicatechingallate (ECG). © 2010 Elsevier B.V. All rights reserved. 1. Introduction Grape seed is a residue of wine and grape juice industry and is generally used as an animal feed. The investigation on grape seed has been increasing since its positive effects was shown on human health. Grape seed contains 8–15% oil and this oil contains high amounts of unsaturated fatty acids and antioxidant-rich com- pounds [1]. These antioxidants are mostly phenolic compounds like cathecins and procyanidins [2–4]. These phenols may act against the in vitro oxidation of low density lipoproteins [5], and also have anticarcinogenic, antiviral and antimutagenic [6] activities, besides these they prevent the thrombosis, reduces cholesterol and regu- lates the autonomic nerves [7]. The applications of the grape seed oil have been increasing in pharmaceuticals, medical, cosmetic and food industry due its properties. The extraction of seed oil includes many different stages like cleanup of the biomass, drying, crushing and pressing. By the appli- cation of pressing most of the oil can be extracted but nevertheless a considerable amount of oil remains in the cake, and this remaining oil can be extracted by hexane; which should be evaporated after the process [8]. The main drawback of this process is the usage of n-hexane at the last stages [1]. The methods for the extraction of phenolic compounds from grape seed include usage of methanol [3,4,9], ethanol [6,10,11] and acetone [12]. Different extraction temperatures are being used with different times that range from minutes to several hours. ∗ Corresponding author. Tel.: +90 3122977100; fax: +90 3122992123. E-mail address: eseneyiler@gmail.com (E.E. Yilmaz). Due to the complexity of the procedure to extract oil and pheno- lic compounds, newer methods are in consideration. Supercritical fluid extraction (SFE) is one of these methods, extensively studied nowadays because of its unique properties. SFE has many advan- tages, like absence of light and air, over the conventional extraction procedures. Air and light can cause degradation of both the pheno- lic compounds and unsaturated fatty acids (UFAs). Among many supercritical fluids carbon dioxide is the one that is mostly used owing to its non-toxicity, cheap and non-flammable properties [1]. Besides these properties CO 2 has moderate critical pressure (7.28 MPa) and temperature (304.1 K) which in turn prevents the thermal degradation of phenolic compounds. Supercritical carbon dioxide extraction of grape seed oil has been extensively applied [1,7,8,13], however; for the extraction of phenolic compounds a co-solvent like methanol or ethanol should be used, because carbon dioxide is a non-polar substance and polar substances like phenolic compounds cannot be extracted from grape seeds. In this study, the extraction of phenolic compounds from grape seed using supercritical carbon dioxide with ethanol as a co-solvent was performed. The effect of different temperature, 30 and 50 ◦ C, pressure, 250, 275 and 300 bar, and co-solvent percentages, 5, 10, 15 and 20 wt%, to the extraction amount has been investigated. The amounts of the phenolic compounds extracted were analyzed using RP-HPLC with a gradient program. 2. Material and methods 2.1. Material The carignan type of grape seeds used in the study was kindly given by Kavaklidere Winery. The marc given was dried 0896-8446/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2010.10.046