Supercritical Fluid Extraction of Mogrosides from Siraitia Grosvenorii Yan Xia Department of Food Science and Agricultural Chemistry McGill University Montreal, Québec, Canada September, 2006 A thesis submitted to McGill University in the partial fulfillment of the requirement of the Degree of Master of Science Copyright © 2006 byYan Xia Library and Bibliothèque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your file Votre référence ISBN: 978-0-494-32803-3 Our file Notre référence ISBN: 978-0-494-32803-3 NOTICE: AVIS: The author has granted a non­ L'auteur a accordé une licence non exclusive exclusive license allowing Library permettant à la Bibliothèque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans loan, distribute and sell th es es le monde, à des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, électronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada Abstract Mogrosides, the main active components in S. grosvenorii SWINGLE, are considered to be sorne 250 times sweeter than sucrose and to possess several medicinal attributes. Previous isolation processes used large quantities of toxic solvent that resulted in toxic residues of organic solvent in this high value food. Supercritical fluids fulfill the requirements of non-toxicity, recycle ability, and useful solvent characteristics. The work presented in this thesis is directed to the extraction of mogrosides from the powdered S. grosvenorii concentrate (SG) and the crude extract after resin treatment (MG) with sub critical water and supercritical CO2. Because no source of mogroside V reference materiaI is available commercially, the first objective of this research was to isolate mogroside V of sufficient purity that it could be crystallized. This objective was achieved by selecting suitable eluates from resin chromatography coupled with preparative thin layer chromatography (TLC). Crystalline white isolate was further characterized by 13C-NMR and by MS and determined to he mogroside V, which was suitable as a reference material for subsequent experiments. The process variables for both sub critical water and supercritical carbon dioxide extraction were evaluated and optimized so that conclusions could be formulated regarding the relative merits of the two proposed extraction methods. The efficiency of extraction was determined spectrophotometrically based on the recovery of mogrosides from the starting material following the vanillin-HCI04 method. When compared with Soxhlet solvent extraction, supercritical fluid extraction with either sub critical water or supercritical C02 provided improved recoveries and consumed less organic solvent. In addition, the purity of the extracts differed greatly. For identical SG samples, sub critical water extraction was demonstrated to be more efficient (62.4 % recovery) compared with 37.0 % recovery by EtOH modified SCC02 extraction or 5.1 % for Soxhlet extraction with hexane. Résumé Mogrosides, le composant actif principal dans le grosvenorii SWINGLE de S., sont considérés comme environ 250 fois plus doux que le sucrose et posséder plusieurs attributs médicinaux. L'isolement précédent traite les grandes quantités utilisées de dissolvant toxique qui ont eu comme conséquence les résidus toxiques du dissolvant organique en cette haute nourriture de valeur. Les fluides supercritiques remplis les conditions de la non-toxicité, du recyclability, et des caractéristiques dissolvantes utiles. Le travail a présenté dans cette thèse a été dirigé vers l'extraction des mogrosides du concentré en poudre de grosvenorii de S. (SG) et de l'extrait brut après le traitement de résine (magnésium) avec l'eau critique secondaire et le CO2 supercritique. Puisqu'aucune source de documentation de référence du mogroside V n'est disponible commercialement, le premier objectif de cette recherche était d'isoler le mogroside V de la pureté suffisante qu'il pourrait être cristallisé. L'isolat blanc cristallin a été encore caractérisé par I3C_NMR et par la MS et déterminé pour être le mogroside V, qui convenait comme documentation de référence pour des expériences suivantes. Les variables de processus pour l'eau critique de sous-marin et l'extraction supercritique de CO2 ont été évaluées et optimisées de sorte que des conclusions aient pu être formulées concernant les mérites relatifs des deux méthodes proposées d'extraction. L'efficacité de l'extraction a été déterminée spectrophotométriquement a basé sur le rétablissement des mogrosides à partir du produit de départ après la méthode vanillin-HCI04 en comparaison avec l'extraction par solvants de Soxhlet, l'extraction liquide supercritique avec l'eau critique de sous-marin ou le CO2 supercritique a fourni des rétablissements améliorés et a consommé le dissolvant moins organique. En outre, la pureté des extraits a différé considérablement. Pour les échantillons identiques de SG; l'extraction critique de l'eau de sous-marin a été démontrée pour être plus efficace (62.4 % de rétablissement) comparée à 37.0 % de rétablissement par l'extraction scC02 modifiée EtOH ou à 5.1 % pour l'extraction de Soxhlet avec de l'hexane. Acknowledgements It is pleasant that 1 have now the opportunity to express my gratitude for many people who supported me. The flfst person 1 would like to thank is my supervisor William D. Marshall. 1 have been in his project since 2004 when 1 started my MSc assignment. During the se years, his gentlemanly personality and integral view on research has made a deep impression on me. 1 owe him lots of gratitude for gui ding my work and providing me with valuable comments on this thesis. 1 am really glad that 1 have come to know Dr. Marshall in my life. 1 would like to thank all the colleagues in our lab and department. 1 thank Kebba Sabally, for providing me brotherly advice that helped me a lot in staying at the right track. Tao Yuan has shared instruments and many experiences with me. Shiyi Gong has provided me valuable comments. Mario Rivero-Huguet has helped me translate my abstract in French. 1 thank Sadia Ehsan and Liang Yu, for being helpful in the labo 1 would also thank Muhammad Alu Datt for helping me in preparing my defense presentation. 1 also thank a nice couple, Changying Shi and Juntao Luo, for giving me lots of academic advice and emotional support. A special thanks goes to Ting Xie, whom 1 have known for more than ten years now and who showed to be a mostly helpful and trustful friend. 1 am very grateful for my husband, Xue Feng Ren, for his love and patience. He has always accompanied me and supported me since we met. Sorne of the greatest help came from my mother-in-law, Cuihua Lou, who visited us in the first term and had taken really good care ofmy daughter and my family. Finally, please allow me to devote my deepest love to my father, Zhenzhong Xia. Although he passed away last year, 1 know he's blessing me forever. III Table of Contents Abstract- ,--------------------,-1 Résumé ------, --------------II Acknowledgements ---­ ,------------III Table of Contents ------------,-------~--------rv List of Figures ---------------VII List of Charts ,--------------------VII List of Tables--------, ,-----------VIII List of Abbreviations ----------------------x Chapter 1 Introduction ...................................................................................................... 1 1.1 SeIected components of Siraitia grosvenom fruit ................................................. 1 1.1.1 Studies of S. grosvenorii ....................... , ..................................................... 1 1.1.2 Production ................................................................................................... 2 1.1.3 Main components of interest: Cucurbitane-glycosides ............................... 3 1.1.4 Other components ....................................................................................... 6 1.2 Attributes of mogrosides .................... , ........................................ , ........................ 10 1.2.1 Chemical and organoleptic properties ....................................................... 10 1.2.2 Toxicity studies ........................................................................................