International Journal of Food Microbiology 92 (2004) 181 – 187 www.elsevier.com/locate/ijfoodmicro Antimicrobial and antioxidative enrichment of oak (Quercus robur) bark by rotation planar extraction using ExtraChromR Samo Andrenšek a, Breda Simonovska a, Irena Vovk a, Pia Fyhrquist b, Heikki Vuorela b, Pia Vuorela c,* a Laboratory for Food Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia Department of Pharmacy, Division of Pharmacognosy, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland c Department of Pharmacy, Viikki Drug Discovery Technology Center, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland b Received 22 July 2003; received in revised form 15 September 2003; accepted 16 September 2003 Abstract The multifunctional ExtraChromR instrument was used in the extraction of antimicrobial and radical scavenging components from oak (Quercus robur L.) bark. Milled and sieved oak bark was extracted with 80% (v/v) methanol solution in water on the ExtraChromR instrument using step-gradient in the preparative separation. Extracts were tested using agar diffusion method on Staphylococcus aureus, Enterobacter aerogenes and Candida albicans. Some extracts showed moderate bactericidal, fungicidal, bacteriostatic and fungistatic activity. The composition related to activity of the fractions and extracts was screened simultaneously by thin-layer chromatography (TLC) detected by UV and by spraying the plate with radical scavenging reagent 1,1-diphenyl-2-picrylhydrazyl (DPPH) to detect antioxidant activity. Thus, we could demonstrate the antiradical and antimicrobial activity of oak beneficial in the storage of wine against the oxidation and human microbial exposure. D 2004 Elsevier B.V. All rights reserved. Keywords: Quercus robur L. cortex; Rotation planar extraction; Staphylococcus aureus; Candida albicans; Enterobacter aerogenes; Radical scavenging activity; Thin-layer chromatography 1. Introduction The antimicrobial and antioxidant properties of plant-derived phenolic substances have been the subject of keen interest (a.o. Murakami et al., 1996; Rauha et al., 2000). The oak wood barrels have been found as a source of phenolic substances during wine and cognac production and storage (Puech et al., 1999; * Corresponding author. Tel.: +358-9-191-59178; fax: +358-9191-59138. E-mail address: pia.vuorela@helsinki.fi (P. Vuorela). 0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2003.09.009 Tedesco et al., 2000; Da Porto et al., 2000). Once the juice is released from the grapes by cracking and pressing, it becomes a new environment for microorganisms. The juice is exposed to the microorganisms on the grape skins, grape stems, leaves, equipment surfaces and hands. After alcoholic and malolactic fermentation, the winemaker must control the growth of other yeast and bacteria to protect the wine against spoilage. Wine after completion of fermentation can be microbially stabilized by removal of microorganisms by filtration, and pasteurization. Sulfite is also used as a preservative to protect wines from oxidation and microbial spoilage (Jackisch, 1985); The influence of 182 S. Andrenšek et al. / International Journal of Food Microbiology 92 (2004) 181–187 oak wood barrel on the flavor of wine must also be taken into account during storage. A variation among individual oak trees can be detected (Mosedale et al., 1999; Sauvageot and Feuillat, 1999). The spoilage and poisoning of foods by oxidation and/or microorganisms is still a problem that is not yet overcome despite of the range of robust preservation techniques available. The screening of plant extracts and natural products for antioxidative and antimicrobial activity has revealed the potential of higher plants as a source of new agents (Rios et al., 1988), to serve the processing of natural products. ExtraChromR instrument is a multifunctional instrument, where solid – liquid extraction and off-line analytical and micropreparative, as well as online preparative solid – liquid chromatographic methods can be carried out (Nyiredy, 2001a,b). Rotation planar extraction (RPE) is an on-line extraction technique in which the material to be extracted is placed in a closed chamber, and the flow is forced by centrifugal force (Hostettmann et al., 1997; Yrjönen et al., 2001). This paper reports the application of ExtraChromR on the extraction of oak bark in the evaluation of active components in the plant material against representative microorganisms in human skin or hands. The composition related to the activity of the extracts was screened simultaneously by TLC detected by fluorescence and by spraying the plate with radical scavenging activity reagent 1,1-diphenyl2-picrylhydrazyl (DPPH). 2. Materials and methods 2.1. Plant material Quercus robur L. cortex (oak bark) purchased from University Pharmacy (Quercus cortex Lot no. 65/02000; Helsinki, Finland) was milled and successively sieved (medium particle size 0.6 mm; between 0.75 and 0.45 mm). 2.2. Chemicals and standards Ethyl acetate, n-hexane and formic acid were of analytical grade, methanol was of HPLC grade from Merck (Darmstadt, Germany). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was from Sigma (St. Louis, MO, USA). Water was purified by Milli-Q system, Millipore (Bedford, MA, USA). 2.3. Rotation planar extraction (RPE) by ExtrachromR The planar column was filled with 40.0 g of oak bark using rotation speed of 1700 rpm. The volume of material to be extracted stays constant along the separation distance due to the special design of the chamber (Nyiredy, 2001b). The bark material was wetted with 135 ml of extraction solvent at a rotational speed of 1400 rpm and at a solvent flow of 6.0 ml min 1 (600E pump, Millipore-Waters, USA). The extracts were collected after a 60-min equilibrium time using a rotational speed of 1700 rpm. Extracts were evaporated to dryness under reduced pressure (Büchi Rotavapor R-114, Büchi Labortechnik, Switzerland) at 35jC and stored at 4jC in darkness for TLC screening and bioactivity test. Successive extraction was performed using six extraction solvents of different polarity. The same volume (135 ml) of each extraction solvent was applied three times successively before the extraction solvent of higher polarity was used as shown in Table 1. 2.4. Thin-layer chromatography (TLC) Thin-layer chromatography (TLC) was performed on 10  20-cm Kieselgel 60 TLC plates (Merck Art. 105626). The developing solvent consisted of a mixture of ethyl acetate –formic acid – water (85 + 10 + 15, v/v). Four microliters of each eluate from ExtraChromR was applied to TLC plates as 4-mm bands, 4 mm apart, 15 mm from the bottom edge of the plate, with the spraying speed of 10 s/Al using Linomat IV applicator (Camag, Switzerland). Plates were developed to a distance of 6.0 cm in a normal unsaturated chamber for 20  20-cm plates. Detection was carried out using the natural fluorescence at 366 nm. Documentation of all TLC plates was performed by Camag Video Documentation System (Camag), coupled to a Reprostar 3 transilluminator and a frame grabber system equipped with a 3  1/2W CCD camera (model HV-C20, Hitachi Denshi, Japan). The Video Documentation System was operated with VideoStore 2 V2.30 software (Camag). 183 S. Andrenšek et al. / International Journal of Food Microbiology 92 (2004) 181–187 Table 1 Antimicrobial effects of Q. robur extracts obtained with the agar diffusion method Extract numbera Extract mass (g) 1 3 4 6 7 9 10 12 13 15 16 18 Ampicillin Streptomycin Amphotericin B DMSO MeOH 0.198 0.029 0.017 0.006 0.046 0.039 0.820 0.121 0.260 0.121 0.434 0.222 S. aureus E. aerogenes C. albicans Inhibition zones (mm) RSD%b Inhibition zones (mm) RSD% Inhibition zones (mm) RSD% 0.0 0.0 0.0 0.0 0.0 0.0 15.1 12.7 0.0 0.0 14.3 14.4 57.2 31.9 0.0 0.0 0.0 0.0 0.0 0.0 2 1 0.0 0.0 3 2 2 3 17.1 13.6 13.4 13.6 14.1 19.4 S-14.8 0.0 0.0 0.0 0.0 0.0 35.3 44.3 17 2 4 4 7 9 7 0.0 0.0 0.0 0.0 0.0 5 5 0.0 Sc-13.1 S-12.0 S-12.4 S-13.6 13.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3 3 4 3 8 0.0 0.0 0.0 0.0 0.0 0.0 14.9 8 0.0 0.0 0.0 0.0 0.0 0.0 a Extractant 1, 75% ethyl acetate in n-hexane, extracts 1,3, Extractant 2, 100% ethyl acetate, extracts 4,6, Extractant 3, 5% MeOH in ethyl acetate, extracts 7,9, Extractant 4, 50% MeOH in ethyl acetate, extracts 10,12, Extractant 5, 100% MeOH, extracts 13,15, Extractant 6, 75% MeOH in water, extracts 16,18, (% = v/v). b RSD = relative standard deviation. c S = bacteriostatic zone. 2.5. Antimicrobial activity determinations 2.5.1. Microorganisms The following strains of microorganisms were used: the Gram-positive bacteria Staphylococcus aureus FOMK, (Division of Pharmacognosy, University of Helsinki, Finland), the Gram-negative bacteria Enterobacter aerogenes FOMK and the yeast Candida albicans ATCC 10231. 2.5.2. Agar diffusion method The agar diffusion method (Barry and Thornsberry, 1991; Fyhrquist et al., 2002) was used to evaluate the antimicrobial activity of Q. robur extracts. Sterile petri dishes (d = 14 cm, Bibby sterilin, UK) containing two-layer medium [30 ml of Base agar (Difco, MI, USA) and 30 ml of Isosensitest agar (Oxoid, England) or Saboraud agar], were used for the assay. Saboraud agar was self prepared and used for the yeast: 40.0 g of glucose (Ph. Eur.), 10.0 g of Peptone (Biokar Diagnostics, France), 20.0 g of agar (Biokar Diagnostics). Bacteria and yeast were incubated at 35jC on solid agar test tubes using Nutrient agar and Saboraud agar, respectively, for 24 h. After incubation, a small amount of culture was transferred to 2.0 ml of sterilized 0.9% (w/v) NaCl solution and the absorbance of suspension was measured at 625 nm with a spectrophotometer (UV – Visible Spectrophotometer, Pharmacia LKB-Biochrom 4060). Suspensions were diluted with 0.9% (w/v) NaCl to reach absorbance of 0.1 at 625 nm. Two hundred forty microliters of the diluted suspension was evenly spread on the surface of agar in petri dish. Whatman filter papers (d = 12.7 mm) containing 200 Al of MeOH or dimethylsulphoxide (DMSO) solution were placed on the agar surface. Extract solution was prepared as follows: 50 Al of saturated extract solution in MeOH or DMSO was dissolved in 1000 Al of MeOH or DMSO, respectively. MeOH was used as a solvent for polar extracts (methanol/ethyl acetate, methanol and methanol/ water) and DMSO was used as a solvent for unpolar extracts (ethyl acetate in n-hexane and ethyl acetate). Ampicillin and streptomycin (1.0 mg/ml in sterile distilled water) were used as positive controls for Gram-negative and Grampositive bacteria and amphotericin B (1.0 mg/ml in 184 S. Andrenšek et al. / International Journal of Food Microbiology 92 (2004) 181–187 sterile distilled water) for the yeast. DMSO and MeOH were used as negative controls. Prior to incubation in dark at 35jC for 24 h, the petri dishes were kept at room temperature for 1 h in order to promote diffusion of the extracts into the agar (Rios et al., 1988). After incubation, the average of three diameters (0.1-mm precision) was measured. 2.6. Antioxidative activity determinations The detection of radical scavenging compounds on the TLC plate was carried out according to Yrjönen et al. (2003). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) as a 0.04% (w/v) solution in MeOH was sprayed evenly across the developed and dried TLC plate. Free radical scavenging zones were identified immediately as yellow areas against a light violet/purple background. 2.7. Statistical analysis The acquired data were input into SystatR 10.2 for WindowsR (SPSS, USA) statistics program in order to perform cluster and basic statistics analysis. 3. Results and discussion Rapid filling of the extraction chamber, extraction of materials of small particle size, extraction in a closed chamber, particle-free extract, and possibility to extract the material successively with solvents of different polarity were great advantages of rotation planar extraction (RPE) using the ExtraChromR instrument as also indicated by Vovk et al. (2003a,b). In the implementation of solid – liquid extraction as delineated by Nyiredy (2000, 2001b), the RPE method with the ExtraChromR seemed to be well suited for screening purposes. Extracts of oak bark were prepared using the ExtraChromR chamber for RPE so that each extraction solvent was used three times successively before the extraction solvent of higher polarity was applied. TLC experiments with different developing eluents showed that compounds present in the obtained extracts of the oak bark (phenolic acids, catechins, quercetin and its derivatives) could migrate and be separated by means of semipolar developing eluents such as the mixture of ethyl acetate – formic acid. Fig. 1A shows that the Fig. 1. Typical TLC screening of extracts obtained by successive rotation planar extraction (RPE). The lane numbers in the figures refer to the extracts listed in Table 1. Retardation factor (Rf) scale is indicated showing the starting point (Rf = 0) and the end point (Rf = 1) of the TLC development. (A) Video detection: natural fluorescence of the extracts measured at 366 nm. (B) Video detection: DPPH reagent has been sprayed onto the plate to detect compounds capable of free radical scavenging (antioxidative), measured at daylight. number of the fluorescent bands from the extracts is increasing with increasing polarity of the extraction solvent. Fifty-percent (v/v) MeOH in ethyl acetate already extracted the more polar compounds with the Rf value around 0.5. The same plate sprayed with DPPH reagent to detect antiradical activity in the extracts (Fig. 1B) shows high radical scavenging activity for bands with high Rf value and also the extraction solvents with higher polarity, e.g. 50% (v/ v) MeOH in ethyl acetate gave also low Rf bands (Rf 0.0 –0.35) with high activity. According to preliminary experiments and the masses weighed shown in Table 1, we can conclude that three extractions using the same extraction solvent in RPE is enough to exhaustively extract the matrix. The total extractable dry matter from the bark was 8%. The most polar extraction solvent used in this study can roughly be compared to that of whiskey or cognac, i.e. containing 75% alcohol in water (extractant 6 in Table 1). However, the extraction process is dependent on several other factors than only polarities of solvents. A.o. extraction time (length of maturation in the case of wine, whiskey and cognac), amount of extraction solvent, temperature, and pH, results in a reversible differential alteration of the distribution of the components between the solid and liquid phase. This will change, e.g. the antioxidative capacities of the final S. Andrenšek et al. / International Journal of Food Microbiology 92 (2004) 181–187 product (McPhail et al., 1999; Troup and Hunter, 2002) or calcium inhibiting activity of the extracts (Härmälä et al., 1992) due to change in the composition of the extracted compounds. The antimicrobial activity of extracts obtained by step-gradient elution with different solvents by ExtraChromR was tested by agar diffusion method (Table 1). Extracts 10 and 12 [50% (v/v) MeOH in ethyl acetate] and extracts 16 and 18 [75% (v/v) MeOH in water] were bactericidal for S. aureus. The less polar extracts obtained by various concentrations of ethyl acetate (75% ethyl acetate in nhexane, 100% ethyl acetate and 5% MeOH in ethyl acetate; extracts 1 –9) showed antimicrobial activity against the gram negative bacteria E. aerogenes and the yeast C. albicans. The extract 10 (5% MeOH in ethyl acetate) was bacteriostatic against E. aerogenes. Lis-Balchin et al. (2003) showed that a distillate containing essential oils from s.c. ‘‘Royal oak’’ added to broccoli soup, which had been previously inoculated with S. aureus or E. aerogenes, could completely inhibit S. aureus, but the activity against E. aerogenes was much reduced. Whereas, residual hydrosols from the distillation of the essential oil from ‘‘Royal oak’’ showed no potential as antibacterial agents in food. This is in accordance with our study, since the different extracts, containing compounds of different polarities, showed similar tendency for antimicrobial activity. The active substances against E. aerogenes and C. albicans are in the lipophilic extracts and substances in lipophobic extracts were active against S. aureus. TLC fluorescence and DPPH chromatogram showed that extract 10 is unusually rich with relatively polar substances, which are not present in extracts up to the number 9. Fig. 2 shows the clustering analysis of the TLC analysis and antimicrobial activity using the complete linkage method. The activity in extracts is clearly not connected to the compounds with low Rf (0.0 – 0.35) values, as detected by DPPH. We can conclude that substances with high Rf (1– 0.65) in the TLC analysis are active against E. aerogenes, C. albicans and are in the lipophilic extracts, while substances with Rf values 0.35– 0.65 in lipophobic extracts are active against S. aureus. If we only consider solvent polarities, wine, which would be considered very polar in comparison to the used extractants in this study, would most likely extract 185 Fig. 2. Cluster analysis for the relationships between the antimicrobial activity and compounds of the extracts according to TLC analysis. The video detection on TLC was performed as indicated for Fig. 1A and B. The total peak area of compounds in all the extracts was measured. Explanation of the abbreviations: RF8, RF5 = total sum of peak areas of compounds in every individual extract (see Table 1) at 366 nm in the retardation factor region Rf 1.0 – 0.65 (nonpolar compounds) and Rf 0.65 – 0.35 (semipolar compounds) respectively. DPPH8, DPPH5, DPPH2 = total sum of peak areas of compounds in every individual extract (see Table 1) detected using DPPH at daylight in the retardation factor region Rf 1.0 – 0.65 (nonpolar compounds), Rf 0.65 – 0.35 (semipolar compounds) and Rf 0.35 – 0 (polar compounds), respectively. AUREUS, AEROGENES, ALBICANS = inhibition zones produced by every individual extract for S. aureus, E. aerogenes and C. albicans, respectively. Cluster analysis is a multivariate procedure for detecting groupings in data. In this cluster analysis, squared distance metric correlation with complete linkage was used. mainly the lipophobic compounds from the oak barrel thus exhibiting antioxidative activity and antimicrobial activity against S. aureus. However, further investigation must be focused to the activity guided fractionation of the oak bark extracts on a preparative reversed phase (RP) in order to find pure, antioxidatively and/or antimicrobially active substances. Antioxidative activities in the methanol extracts of Quercus alba L. methanolic extracts support the contribution of the traditional medicines in a lifestyle of peoples of the North American boreal forest, historically low in the incidence of diabetes (McCune and Johns, 2002). It can be expected that oak barrels have been adopted into wine making during its history, also not only on its flavor basis. 186 S. Andrenšek et al. / International Journal of Food Microbiology 92 (2004) 181–187 Foliar antioxidant status of adult Mediterranean oak species (Quercus ilex L. and Q. pubescens Willd.) exposed to permanent CO2-enrichment and to seasonal water stress was studied by Marabottini et al. (2001). In oak trees permanently exposed to elevated CO2, the activities of antioxidant enzymes tended to be lower and the ascorbate pool was larger and more in reduced form, suggesting an attenuation of the oxidative risk in the CO2-enriched trees. In the enriched individuals, the imposition of water shortage significantly increased the size of the glutathione pool and the total superoxide dismutase activity in a species-specific manner. Moreover, water-stressed trees exposed to elevated CO2 tended to have higher catalase and ascorbate peroxidase activities than water-stressed control trees. Such changes may reflect the need for an enhanced compensatory effort when trees acclimated to elevated CO2 are exposed to oxidative stress-promoting environmental factors. Mallea et al. (1991) have shown that seven Epicoccum purpurascens strains isolated from evergreen oak leaves (Q. ilex) possessed antimicrobial activity. Two strains sporulated and conspicuously inhibited the growth of the Gram-positive bacterium S. aureus and the fungus Trichophyton mentagrophytes. Thinlayer chromatographic studies showed the existence of some compounds, such as flavipin, which were common to all the strains produced on the surface of the oak. Thus, the oak contains protective compounds against microbial contamination, also from earlier microbial exposure by Epicoccum. We can conclude that Q. robur cortex is a promising plant material, and should be studied further for its potential antioxidative and antimicrobial activity. The oak material may contain compounds intended to compensate the influence of environmental stress factors such as oxidative stress and microbial contamination. During the history of wine making, it has also proven the oak to be the beneficial material in the storage of wine in barrels in order to control the oxidation and to prevent the wine from excess microbial contamination. Acknowledgements This study was supported by grants from the Ministry of Education, Science and Sport of the Republic of Slovenia and the European Commission through project with a Contract no. ICA1-CT-2000-70034. References Barry, A.L., Thornsberry, C., 1991. Susceptibility tests: diffusion test procedures. In: Balows, A. (Ed.), Manual of Clinical Microbiology. American Society of Microbiology, Washington DC, pp. 1117 – 1125. Da Porto, C., Calligaris, S., Celotti, E., Nicoli, M.C., 2000. Antiradical properties of commercial cognacs assessed by the DPPH center dot test. Journal of Agricultural and Food Chemistry 48, 4241 – 4245. Fyhrquist, P., Mwasumbi, L., Häggeström, C.A., Vuorela, H., Hiltunen, R., Vuorela, P., 2002. Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania. Journal of Etnopharmacology 79, 169 – 177. Härmälä, P., Vuorela, H., Törnquist, K., Hiltunen, R., 1992. Choice of solvent in the extraction of Angelica archangelica roots with reference to calcium blocking activity. Planta Medica 58, 176 – 183. Hostettmann, K., Terreaux, C., Marston, A., Potterat, O., 1997. The role of planar chromatography in the rapid screening and isolation of bioactive compounds from medicinal plants. Journal of Planar Chromatography 10, 251 – 257. Jackisch, P., 1985. Modern Winemaking. Cornell Univ. Press, New York, p. 289. Lis-Balchin, M., Styrl, H., Krenn, E., 2003. The comparative effect of novel Pelargonium essential oils and their corresponding hydrosols as antimicrobial agents in a model food system. Phytotherapy Research 17, 60 – 65. Mallea, M., Pesando, D., Bernard, P., Khoulalene, B., 1991. Comparison between antifungal and antibacterial activities of several strains of Epicoccum purpurascens from the mediterranean area. Mycopathologia 115, 83 – 88. Marabottini, R., Schraml, C., Paolacci, A.R., Sorgona, A., Raschi, A., Rennenberg, H., Badiani, M., 2001. Foliar antioxidant status of adult mediterranean oak species (Quercus ilex L. and Q. pubescens Willd.) exposed to permanent CO2-enrichment and to seasonal water stress. Environmental Pollution 115, 413 – 423. McCune, L.M., Johns, T., 2002. Antioxidant activity in medicinal plants associated with the symptoms of diabetes mellitus used by the indigenous peoples of the North American boreal forest. Journal of Ethnopharmacology 82, 197 – 205. McPhail, D.B., Gardner, P.T., Duthie, G.G., Steele, G.M., Reid, K., 1999. Assessment of the antioxidant potential of Scotch whiskeys by electron spin resonance spectroscopy: relationship to hydroxyl-containing aromatic components. Journal of Agricultural and Food Chemistry 47, 1937 – 1941. Mosedale, J.R., Puech, J.L., Feuillat, F., 1999. The influence on wine flavor of the oak species and natural variation of heartwood components. American Journal of Enology and Viticulture 50, 503 – 512. Murakami, A., Ohigashi, H., Koshimizu, K., 1996. Anti-tumor S. Andrenšek et al. / International Journal of Food Microbiology 92 (2004) 181–187 promotion with food phytochemicals: a strategy for cancer chemoprevention. Bioscience Biotechnology and Biochemistry 60, 1 – 8. Nyiredy, Sz., 2000. Solid – liquid extraction strategy on the basis of solvent characterization. Chromatographia 51, S288 – S296. (Suppl.). Nyiredy, Sz., 2001a. The role of planar chromatography in medicinal plant research. Journal of AOAC International 84, 1219 – 1231. Nyiredy, Sz., 2001b. Rotation planar extraction (RPE)—a new exhaustive, preparative forced-flow technique: Part 1. Description of the method and practical aspects. Journal of Planar Chromatography 14, 393 – 395. Puech, J.L., Feuillat, F., Mosedale, J.R., 1999. The tannins of oak heartwood: structure, properties, and their influence on wine flavor. American Journal of Enology and Viticulture 50, 469 – 478. Rauha, J.-P., Remes, S., Heinonen, M., Hopia, A., Kähkönen, M., Kujala, T., Pihlaja, K., Vuorela, H., Vuorela, P., 2000. Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds. International Journal of Food Microbiology 56, 3 – 12. Rios, J.L., Recio, M.C., Vilar, A., 1988. Screening methods for natural products with antimicrobial activity: a review of the literature. Journal of Ethnopharmacology 23, 127 – 149. Sauvageot, F., Feuillat, F., 1999. The influence of oak wood (Querrcus robur L., Q. petraea Liebl.) on the flavor of Burgundy 187 Pinot noir. An examination of variation among individual trees. American Journal of Enology and Viticulture 50, 447 – 455. Tedesco, I., Russo, M., Russo, P., Iacomino, G., Russo, G.L., Carraturo, A., Faruolo, C., Moio, L., Palumbo, R., 2000. Antioxidant effect of red wine polyphenols on red blood cells. Journal of Nutritional Biochemistry 11, 114 – 119. Troup, G.J., Hunter, C.R., 2002. EPR, free radicals, wine, and the industry: some achievements. Annals of the New York Academy of Sciences 957, 345 – 347. Vovk, I., Simonovska, B., Andrenšek, S., Yrjönen, T., Vuorela, P., Vuorela, H., 2003a. Rotation planar extraction and medium pressure solid – liquid extraction of onion (Allium cepa). Journal of Planar Chromatography 16, 72 – 76. Vovk, I., Simonovska, B., Andrenšek, S., Vuorela, H., Vuorela, P., 2003b. Rotation planar extraction and rotation planar chromatography of oak (Quercus robur L.) bark. Journal of Chromatography A 991, 267 – 274. Yrjönen, T., Haansuu, J.P., Haahtela, K., Vuorela, H., Vuorela, P., 2001. Rapid screening of indole-3-acetic acid and other indole derivatives in bacterial culture broths by planar chromatography. Journal of Planar Chromatography 14, 47 – 52. Yrjönen, T., Peiwu, L., Summanen, J., Hopia, A., Vuorela, H., 2003. Free radical-scavenging activity of phenolics by reversed-phase TLC. Journal of the American Oil Chemists’ Society 80, 9 – 14.