PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Project Report Sheet Surname or Family name: HUTABARAT First name: LAMB OK SAORITA Other name/s: Abbreviation for degree as given in the University calendar: Ph.D School : Applied Bioscience Faculty: Life Science Title: PHYTOESTROGENS IN AUSTRALIAN AND INDONESIAN FOODS Abstract 350 words maximum: (PLEASE TYPE) ABSTRACT There is a lack of data on phytoestrogens in Australian and Indonesian foods. These data are needed to help local medical scientists, nutritionists and pharmacologists to elucidate the role of dietary phytoestrogens tn protection against certain cancers, distressing menopausal symptoms and other nutrition-related conditions. There is no international standard method for analysis of phytoestrogens in foods and thi s makes the collection of reliable analytical data on phytoestrogens very difficult. Reliable methods are also required for labelling foods with their phytoestrogens content. Two isocratic HPLC methods have been developed in this study. The first method was for the separation and quantification of daidzein, genistein and biochanin A from soya beans. The second method was for the separation and quantification of the isoflavones, daidzein, genistein, formononetin and biochanin A and the coumestan, coumestrol from soya beans. Both HPLC methods used C8 and phenyl columns, 1% acetic acid-water (33 :67, v/v) and acetonitrile-water (33:67, v/v) as eluent to separate all compounds in less than 24 minutes. The second method exhibited a high degree of precision and high linearity e > 0.999) with the delectability being 47, 82, 76, 75 and 224 nM for daidzein, coumestrol, genistein, formononetin and biochanin A, respectively, and a good recovery (- 100%) of standard comp::>unds from foods. Extraction with acid and heat was used to hydrolyse all the conjugate forms in foods with a maximum yield and a high level of efficiency. The method could identify isoflavones in a wide range of beans and bean products from Australia and Indonesia from 0.01 up to 75 mg/100 g dry weight basis. Levels varied between different brands, foods and countries. Since the method of analysis had been shown to be highly reliable, other factors such as agricultural conditions, cultivars, stage of maturation, food processing and preparation are likely to have been the dominant factors affecting the results. The high degree of variability in the levels of phytoestrogens makes the task of obtaining representative datas therefore very difficult. In the few cases where label claims were made, they could not be verified by the analytical results for the product. Declaration relating to disposition of project report/thesis I am fully aware of the policy of the University relating to the retention and use of higher degree project reports and theses, namely that the University retains the copies submitted for examination and is free to allow them to be consulted or borrowed. Subject to the provisions of the Copyright Act 1968, the University may issue a project report or thesis in whole or in part, in photostat or microfilm or other copying medium. I aiJ '"h"""' ili< ••bhc,.;oo :v University Microfilms of a 350 word abstract in Dissertation Abstracts International (applicable to doctorates only) . .._ _ l... .......... .. ... 1 L............ ....... ..t..g/.~/.C1L .. ....................... Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing to the Registrar. Requests for a longer period of restriction may be considered in exceptional circumstances if accompanied by a letter of support from the Supervisor or Head of School. Such requests must be submitted with the thesis/project report. FOR OFFICE USE ONLY Date of completion of requirements for Award: 1'-:-------;--::~~ 'l...7(3(o2_ Registrar and Deputy Principal THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS MBT 613.28 68 CERTIFICATE OF ORIGINALITY I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged. (Signed} •••ic__-----.JI - m mm PHYTOESTROGENS IN AUSTRALIAN AND INDONESIAN FOODS by Lambok Saorita Hutabarat A thesis submitted to The University of New South Wales as fulfillment of the requirements for the degree of DOCTOR OF PIDLOSOPHY August 2002 UNSW 1 7 OCT 2002 LIBRARY ii ABSTRACT There is a lack of data on phytoestrogens in Australian and Indonesian foods. These data are needed to help local medical scientists, nutritionists and pharmacologists to elucidate the role of dietary phytoestrogens in protection against certain cancers, distressing menopausal symptoms and other nutrition-related conditions. There is no international standard method for analysis of phytoestrogens in foods and this makes the collection of reliable analytical data on phytoestrogens very difficult. Reliable methods are also required for labelling foods with their phytoestrogens content. Two isocratic HPLC methods have been developed in this study. The first method was for the separation and quantification of daidzein, genistein and biochanin A from soya beans. The second method was for the separation and quantification of the isoflavones, daidzein, genistein, formononetin and biochanin A and the coumestan, coumestrol from soya beans. Both HPLC methods used C8 and phenyl columns, 1% acetic acid-water (33:67, v/v) and acetonitrile-water (33:67, v/v) as eluent to separate all compounds in less than 24 minutes. The second method exhibited a high degree of precision and high linearity (? > 0.999) with the detectability being 47, 82, 76, 75 and 224 nM for daidzein, coumestrol, genistein, formononetin and biochanin A, respectively, and a good recovery (~ 100%) of standard compounds from foods. Extraction with acid and heat was used to hydrolyse all the conjugate forms in foods with a maximum yield and a high level of efficiency. The method could identify isoflavones in a wide range of beans and bean products from Australia and Indonesia from 0.01 up to 75 mg/100 g iii dry weight basis. Levels varied between different brands, foods and countries. Since the method of analysis had been shown to be highly reliable, other factors such as agricultural conditions, cultivars, stage of maturation, food processing and preparation are likely to have been the dominant factors affecting the results. The high degree of variability in the levels of phytoestrogens makes the task of obtaining representative datas therefore very difficult. In the few cases where label claims were made, they could not be verified by the analytical results for the product. IV PUBLICATION ARISING FROM THE WORK RELATED TO THE THESIS Papers Hutabarat, L. S., Mulholland, M. & Greefield, H. (1998) Development and validation of an isocratic high-performance liquid chromatographic method for quantitative determination ofphytoestrogens in soya bean. J. Chromatogr. 795: 377- 382. Hutabarat, L. S., Greenfield, H. & Mulholland, M. (2000) Quantitative determination of isoflavones and coumestrol in soybean by column liquid chromatography. J. Chromatogr. 886: 55-63. Hutabarat, L. S. Greenfield, H. & Mulholland, M. (2001) Isoflavones and coumestrol in soybeans and soybean products from Australia and Indonesia. J. Food Comp. Analysis 14: 43-58. Abstract Hutabarat, L. S., Greenfield, H. & Mulholland, M. (1997) Prelimenary studies of phytoestrogens in Australia foods. Proceeding of The Nutrition Society ofAustralia, 2Ft Annual Scientific Meeting. Briesbane, Australia. Hutabarat, L. S., Greenfield, H. (1999) Phytoestrogens in soybean products from Indonesia.2nd South-West Pacific Nutrition and Dietetic Conference. Auckland, New Zealand. Hutabarat, L. S., Greenfield, H. & Mulholland, M. (1999) Isoflavones and coumestrol in soybeans and soybean products available in Australia and Indonesia. Third International Food Data Conference. Rome, Italy. v Hutabarat, L. S., Greenfield, H. & Mulholland, M. (1999) Isojlavones and coumestrol in soybeans and soybean products available in Australia and Indonesia. gth Asian Congress of Nutrition. Seoul, Korea. vi ACKNOWLEDGEMENT I wish to express sincere gratitude to my immediate supervisor, Associate Professor H. Greenfield, for providing invaluable encouragement, guidance and, most importantly, inspiration, to me during this project and in the production of this thesis. I also wish to thank my co-supervisor, Dr M. Mulholland, for her guidance and advice in the early stages of this project. My sincere gratitude also goes to Ms Eileen Emmerson for her invaluable assistance concerning the technical aspects of this project. I wish to express my particular appreciation to Mrs Yvonne El-Ghetany and Mr Camillo Taraborrelli for their help regarding various technical aspects, also Mrs Christine Locke and Mrs Sharon Debreczeni