Berry phenolics: isolation, analysis, identification, and antioxidant properties Petri Kylli ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in lecture hall B2, Viikki, on August 26th 2011, at 12 o’clock noon. University of Helsinki Department of Food and Environmental Sciences Food Chemistry Helsinki 2011 Custos: Professor Vieno Piironen Department of Food and Environmental Sciences University of Helsinki Helsinki, Finland Supervisor: Professor Marina Heinonen Department of Food and Environmental Sciences University of Helsinki Helsinki, Finland Reviewers: Ph.D. Pirjo Mattila MTT Agrifood Research Finland Jokioinen, Finland Ph.D. Claudine Manach INRA, Nutrition Humaine Saint-Genes-Champanelle, France Opponent: Professor Anne S. Meyer Department of Chemical and Biochemical Engineering Technical University of Denmark Kgs. Lyngby, Denmark ISBN 978-952-10-7114-0 (paperback) ISBN 978-952-10-7115-7 (pdf; http://ethesis.helsinki.fi) ISSN 0355-1180 Cover picture: Tuuli Koivumäki Unigrafia Helsinki 2011 Kylli, P 2011. Berry phenolics: isolation, analysis, identification, and antioxidant properties (dissertation). EKT-series 1502. University of Helsinki. Department of Food and Environmental Sciences. 90+62 pp. ABSTRACT The main objectives in this thesis were to isolate and identify the phenolic compounds in wild (Sorbus aucuparia) and cultivated rowanberries, European cranberries (Vaccinium microcarpon), lingonberries (Vaccinium vitis-idaea), and cloudberries (Rubus chamaemorus), as well as to investigate the antioxidant activity of phenolics occurring in berries in food oxidation models. In addition, the storage stability of cloudberry ellagitannin isolate was studied. In wild and cultivated rowanberries, the main phenolic compounds were chlorogenic acids and neochlorogenic acids with increasing anthocyanin content depending on the crossing partners. The proanthocyanidin contents of cranberries and lingonberries were investigated, revealing that the lingonberry contained more rare A-type dimers than the European cranberry. The liquid chromatography mass spectrometry (LC-MS) analysis of cloudberry ellagitannins showed that trimeric lambertianin C and sanguiin H-10 were the main ellagitannins. The berries, rich in different types of phenolic compounds including hydroxycinnamic acids, proanthocyanidins, and ellagitannins, showed antioxidant activity toward lipid oxidation in liposome and emulsion oxidation models. All the different rowanberry cultivars prevented lipid oxidation in the same way, in spite of the differences in their phenolic composition. In terms of liposomes, rowanberries were slightly more effective antioxidants than cranberry and lingonberry phenolics. Greater differences were found when comparing proanthocyanidin fractions. Proanthocyanidin dimers and trimers of both cranberries and lingonberries were most potent in inhibiting lipid oxidation. Antioxidant activities and antiradical capacities were also studied with hydroxycinnamic acid glycosides. The sinapic acid derivatives of the hydroxycinnamic acid glycosides were the most effective at preventing lipid oxidation in emulsions and liposomes and scavenging radicals in DPPH• assay. In liposomes and emulsions, the formation of the secondary oxidation product, hexanal, was inhibited more than that of the primary oxidation product, conjugated diene hydroperoxides, by hydroxycinnamic acid derivatives. This indicates that they are principally chain-breaking antioxidants rather than metal chelators, although they possess chelating activity as well. The storage stability test of cloudberry ellagitannins was performed by storing ellagitannin isolate and ellagitannins encapsulated with maltodextrin at different relative vapor pressures. The storage stability was enhanced by the encapsulation when higher molecular weight maltodextrin was used. The best preservation was achieved when the capsules were stored at 0 or 33% relative vapor pressures. In addition, the antioxidant activities of encapsulated cloudberry extracts were followed during the storage period. Different storage conditions did not alter the antioxidant activity, even though changes in the ellagitannin contents were seen. The current results may be of use in improving the oxidative stability of food products by using berries as natural antioxidants. ACKNOWLEDGEMENTS This study was carried out at the Department of Food and Environmental Sciences, Food Chemistry Division, at the University of Helsinki during the years 2005-2011. The work was funded by The Finnish Graduate School on Applied Bioscience, The University of Helsinki Funds, The National Technology Agency (TEKES), and The Finnish Cultural Foundation. Their financial support is gratefully acknowledged. I wish to express my deepest gratitude to Professor Marina Heinonen, supervisor of the work, for giving me the chance to work in this exciting field of berries and phenolic compounds. I thank her for her great support and advice throughout these years. I am grateful to the reviewers Dr. Pirjo Mattila and Dr. Claudine Manach for their constructive comments, and careful and thorough review of the thesis. I wish to thank Prof. Vieno Piironen for introducing me to the fascinating world of food chemistry. My sincere thanks go to docent Velimatti Ollilainen for the guidance and support with LC-MS and docent Anna-Maija Lampi for the support in the lab. I wish to thank all my present and former colleagues and coworkers in D-building. Especially I wish to thank Minnamari Edelmann, Mario Estévez, Göker Gürbüz, Kirsti Herttua, Tuuli Koivumäki, Hanna Komu, Mari Lehtonen, and Miikka Olin for their assistance and enjoyable collaboration. Prof. Maija Tenkanen, Prof. Kirsi Jouppila, M.Sc. Pia Laine, Dr. Riitta Puupponen-Pimiä, Dr. Liisa Nohynek, Dr. Benita Westerlund-Wikström, M.Sc. Paula Nousiainen, Dr. Jussi Sipilä, Dr. Tiina Leppänen, and Prof. Eeva Moilanen are acknowledged for giving valuable advice and acting as co-authors. A part of this work was done in Scotland at Scottish Crop Research Institute. I wish to thank Dr. Derek Stewart and Dr. Gordon McDougall for giving me the opportunity to visit your laboratory. My warm thanks I owe to my friends for giving me many joyful and unforgettable moments. Finally, I wish to thank my parents, sisters and brother for their interest and encouragement. Helsinki, July 2011 Petri Kylli LIST OF ORIGINAL PUBLICATIONS I Kylli P, Nohynek L, Puupponen-Pimiä R, Westerlund-Wikström B, McDougall G, Stewart D, Heinonen M. 2010. Rowanberry phenolics: compositional analysis and bioactivities. J Agric Food Chem 58: 11985-92. II Kylli P, Nohynek L, Puupponen-Pimiä R, Westerlund-Wikström B, Leppänen T, Welling J, Moilanen E, Heinonen M. 2011. Lingonberry (Vaccinium vitis-idaea) and European cranberry (Vaccinium microcarpon) proanthocyanidins: isolation, identification, and bioactivities. J Agric Food Chem 59:3373-84. III Kähkönen M, Kylli P, Ollilainen V, Salminen JP, Heinonen M. 2011. Antioxidant activity of isolated ellagitannins from red raspberries and cloudberries. J Agric Food Chem Submitted for publication. IV Kylli P, Nousiainen P, Biely P, Sipilä J, Tenkanen M, Heinonen M. 2008. Antioxidant potential of hydroxycinnamic acid glycoside esters. J Agric Food Chem 56: 4797-4805. V Laine P, Kylli P, Heinonen M, Jouppila K. 2008. Storage stability of microencapsulated cloudberry (Rubus chamaemorus) phenolics. J Agric Food Chem 56: 11251-61. The papers are reproduced with a kind permission from the copyright holder: American Chemical Society (I-V) Contribution of the author to papers I-V I The author planned the study together with the other authors. Compositional analysis, the antioxidant activity testing of rowanberry phenolics, and the writing of the manuscript were carried out by the author. He had the main responsibility for interpreting the results. II The author planned the study together with the other authors. The isolation and compositional analysis of cranberry and lingonberry proanthocyanidins and the antioxidant testing were carried out by the author. He had the main responsibility for interpreting the results and writing the manuscript. III The author participated in the LC-MS identification of the cloudberry phenolic isolate. IV The author planned the study together with the other authors. HPLC analysis, the antioxidant activity testing of hydroxycinnamic acid esters, and the writing of the manuscript were carried out by the author. He had the main responsibility for interpreting the results. V The author planned the study together with the other authors. The preparation of the phenolic extract, compositional analysis, and the antioxidant activity testing of microencapsulated cloudberry phenolics were carried out by the author. The author had the main responsibility for interpreting the results regarding phenolic composition and antioxidant activity; thus, he was the second author of the paper. LIST OF ABBREVIATIONS AS anthocyanins ASE accelerated solvent extractor DAD diode array detector EA ellagic acid EI electron ionization ESI-MS electrospray mass spectrometry ET ellagitannin FID flame ionization detector FLD fluorescence detector GC gas chromatography HPLC high performance liquid chromatography LC-MS liquid chromatography mass spectrometry LDL low density lipoprotein LOD limit of detection LOQ limit of quantification MALDI matrix assisted laser desorption ionization MC 18.5 microencapsulated maltodextrins MD18.5