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Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011ANALYSIS AND QUANTIFICATION OF TRANS-RESVERATROL INWINE FROM MUNTENIA AND OLTENIA REGION, ROMANIAELISABETA-IRINA GEANA, ANDREEA-MARIA IORDACHE, ROXANA-ELENAIONETENational R&D Institute of Cryogenics and Isotopic Technologies – ICSI Rm. Valcea, 4 Uzinei St., Rm. Valcea,RomaniaAbstract. Known for its several beneficial effects on human health (antioxidant, antibacterial,cardioprotective, antitumor, estrogenic, antiplatelet, anti-inflammatory, etc), the first supplementknown to activate a longevity gene, resveratrol, is one of the major active compounds of stilbenes. Itis a phytoalexin found in grapes, grape products, wine, peanuts, cranberries, strawberry, and someother botanical sources, in variable amounts, both as free and as piceid.The aim of this work is the development of a high performance liquid chromatographic (HPLC)method for the determination of trans-resveratrol in wines from Muntenia and Oltenia regiondelimited appellation (Romania). The method permits direct injection of the sample followed by PDAdetection, revealing to be time-saving and overcoming the need of sample pretreatment steps.Detection limit was 0.008 mg/L. Global uncertainty associated with the results, according toEURACHEM/CITAC rules was 23.5%.Resveratrol content was determined for 24 Romanian AO (appellation of origin) wines; theconcentration found ranging between 0.132 and 6.589 mg/L. The wines from Muntenia region havegrater content of trans-resveratrol than wines from Oltenia region. The levels of trans-resveratrolwere clearly higher in the red wines compared to the white wines (never exceeding 0.159 mg/L).Keywords: trans-resveratrol, wines, HPLC1. INTRODUCTIONResveratrol (3,5,4’-trihydroxystilbene) (Fig. 1) is a phytoalexin of a phenolicnature, belonging to the stilbene family. Naturally it exists in two isomeric forms (cis andtrans) and as piceid, the 3 β-glucoside of resveratrol. It occurs in grapes, grape products andsome other botanical sources (peanuts, soy, tea), in variable amounts, both as free and aspiceid [1].Figure 1. Structure of trans-resveratrol176
Proceeding of the 4 rd International Symposium“NEW RESEARCH IN BIOTECHNOLOGY” USAMV Bucharest, Romania, 2011The presence of trans-resveratrol in wine is an important qualitative parameter dueits several beneficial effects on human health: has anti-oxidant and anti-inflammatoryactivity, is in relation with anti-initiation, anti-promotion and anti-progression activities inmalignant tumors and is recognized as a chemotherapeutic agent in humans [2].Trans-resveratrol plays also an important role in the organoleptic characteristics ofwine, conferring astringency and structure to the beverage by formation of complexes withthe proteins of saliva [3].Wine, particularly red wine, is the main source of introduction of trans-resveratrolin the human diet. In grape berries, resveratrol synthesis is primarly located at the skin cellsand it is absent or low, in the fruit flesh [4].Red wine generally contains higer amounts of trans-resveratrol than white wine.This is presumably due to the longer extraction time during contact between grape skin andjuice in the production of red wine [5]. The variety of grapes also plays an important role inresveratrol synthesis which may be genetically controlled. Resveratrol concentrationincreased during fermentation of the skins but the amount extracted was depended on thevariety and enological conditions [6].Traditional methods to determine polyphenols in natural extracts are usuallyperformed by high performance liquid chromatography analysis (HPLC) andspectrophotometric measurements [7]. Methods based on gas chromatography and gaschromatography-mass spectrometry (GC-MS) have been proposed for trans-resveratrol.However, derivatization is required prior to GC analysis of this analyte to enhancevolatility; this time consuming procedure may result in some trans to cis isomerization ofresveratrol. The development of LC-MS techniques are nowadays the best analyticalapproach for the analysis of this kind of compounds in grape extract and wine, although thishyphenated techniques is very expensive and consequently not widely used in routinelaboratories of wine industry [8].This work presents a reversed phase RP-HPLC method for the determination oftrans-resveratrol in wine samples. Analyses were carried out by direct HPLC injection ofthe sample in solvent elution. A group of 24 wines from Muntenia and Oltenia Romanianregions, different cultivars and vintage year was analysed by HPLC-PDA, in order to assessthe influence of these parameters on the trans-resveratrol concentration.2. MATERIALS AND METHODSReagents and chemicals - The trans-resveratrol standard (99% GC) was purchasedfrom Sigma-Aldrich. Methanol, acetonitrile and glacial acetic acid (100% p.a.) were HPLCgrade and obtained from Merck. Twice distilled and demineralised water produced by TKAsystems was used for preparation of the aqueous solutions.Standard solutions - A stock solution of 20 mg/L of trans-resveratrol prepared in a12% alcoholic (aqueous) solution. The solid standard was initially dissolved in a minimumvolume of ethanol, before the addition of water, in order to guarantee a completedissolution. Immediately prior to analysis, a set of standards with 0.25, 0.5, 1, 1.5, 2, 3, 5and 10 mg/L were prepared from the stock solution, diluted in a 12% alcoholic (aqueous)solution. Special care was taken in relation to the degradation of the standard solutions,keeping them protected from air and light exposure.177
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