University of Bath PHD The biotransformation of diosgenin and its precursors extracted from Trigonella foenumgraecum L. seed. Saunders, Roger Award date: 1982 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 08. Oct. 2021 THE BIOTRANSFORMATION OF DIOSGENIN AND ITS PRECURSORS EXTRACTED FROM TRIGONELLA FOENUMGRAECUM L. SEED Submitted by Roger Saunders for the degree of Doctor of Philosophy of the University of Bath 1982 COPYRIGHT "Attention is drawn to the fact that copyright of this thesis rests with its author. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the prior written consent of the author." "This thesis may not be consulted, photocopied or lent to other libraries without the permission of the author for 5 years from the date of acceptance of the thesis". ProQuest Number: U641690 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U641690 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 UNIVERSITY OF BATH LIBRARY 23. 1 8 MAY m 2 .. .. 1 . SUMMARY In the introduction, the sources and use of diosgenin and other steroid raw materials in the Pharmaceutical Industry are reviewed. The biotransformations of steroids, and in particular diosgenin, are described. Microorganisms, selected from natural sources and from culture collec­ tions, were screened for activity against diosgenin. Species were found which had not been reported to attack the steroid. Biotransformation products of diosgenin were isolated and their structures elucidated. The major products were diosgenone and 1-dehydrodiosgenone; androstenes were also formed. The formation of diosgenone from diosgenin was shown to be mediated by a cholesterol oxidase enzyme which was inducible in growing cells. Methods were investigated to prevent the total degradation of the diosgenin steroid nucleus by Mycobacterium phlei. Addition of the chelating agent, ct, a' -bipyridyl, resulted in a 7% yield of androstenes. However, I mutagenesis of ^ phlei with N-methyl-N -nitro-N-nitrosoguanidine was shown to be of more promise. To optimise the biotransformation process, the effects of the physical form of the diosgenin and the physiological condition of the microorganisms (including their immobilisation in calcium alginate) were studied. Addition of anethanolic solution of diosgenin to stationary phase cells in a rich medium, was found to give the best rate of biotransformation. Methods were investigated to increase the solubility of diosgenin in the fermentation medium. Water-immiscible organic solvents allowed an 11. increased initial biotransformation activity with Nocardia rhodochrous and ^ phlei. The use of crude water-soluble glycosidic precursors of diosgenin resulted in a different pattern of biotransformations. Fusarium solani produced diosgenin (84% efficiency), thus avoiding an acid hydrolysis step. Silastic resin was used in microbial fermentations. It provided a novel and convenient method for the collection of hydrophobic products from biotransformations. Diosgenin, produced from its glycosidic precursors by solani, was collected by the resin and transferred in it to a culture of ^ phlei which further transformed the diosgenin to androstenes. These remained within the resin and could be readily recovered from it with solvents Silastic resin offers considerable advantages in its retention of intermediate and then final product, in a sequence of biotransformations with successive organisms. Ill. ACKNOWLEDGEMENTS I would like to thank my supervisors, Dr.P.S.J.Cheetham (Tate &, Lyle Ltd.) and Dr.R.Hardman (University of Bath) for their aid and advice throughout my work. The research topic was originated by Dr.Hardman. I am indebted to Mr.R.Sadler for his valuable technical assistance I would also like to thank Dr.A.J.Floyd and Dr.A.F.Casy for their help in the interpretation of spectroscopic data. This work was carried out with a C.A.S.E. Award funded jointly by the Science and Engineering Research Council and Tate & Lyle Ltd. IV CONTENTS Page CHAPTER 1 INTRODUCTION Section A Diosgenin - sources, biosynthesis and uses 1 Major sources of diosgenin 1 Other sources of diosgenin 3 Biosynthesis of diosgenin 5 Processing for diosgenin 9 Diosgenin in the Pharmaceutical Industry 12 Alternatives - total synthesis 13 Alternative raw materials to diosgenin: Sterols 13 Hecogenin 19 Bile acids 19 Section B Analytical techniques 20 Section C Microbiological transformation of steroids History 24 Microbiological transformation of diosgenin: Hydroxylation 26 Ring A oxidation 27 Removal of rings E and F, and total degradation 28 Removal of rings A and B 31 Saponin hydrolysis 33 Aims 35 Page CHAPTER 2 GENERAL MATERIALS AND METHODS Extraction of diosgenin from fenugreek seed 36 Quantitative analysis by gas-liquid chromatography 36 Diosgenone and 1-dehydrodiosgenone calibrations 38 Qualitative analysis of diosgenin and products by thin- layer chromatography 38 Microbiological Materials and Methods 43 Pipettes 43 Glassware 43 Culture maintenance 43 Extraction of steroids after fermentation 43 Culture media 47 Isolation of organisms using media with a limited carbon source 49 Growth and transformation experiments - general methods 49 Cell dry weight determinations 50 Viable count procedure 50 Collection, purification and structural determination of products 51 Separation and purification of products i) Separation by column chromatography 51 ii) Separation by preparative thin-layer chromatography 52 Spectroscopic methods Ultraviolet spectroscopy 53 Infra-red spectroscopy 53 Nuclear magnetic resonance spectroscopy 53 VI Page Mass spectrometry 53 Gas chromatographic mass spectrometry 54 CHAPTER 3 SCREENING OF MICROORGANISMS AND THE STRUCTURAL DETERMINATION OF PRODUCTS FROM DIOSGENIN Introduction Screening techniques 55 Results Screening in liquid media 58 Identification of an unknown bacterium 65 Separation of transformation products of diosgenin i) Column chromatography 66 ii) Preparative plate thin layer 66 chromatography Structural analysis of products Product 1 (25R) and (25S)-Spirosta-1,4- diene-3-one (1-dehydrodiosgenone) 70 Product 2 (25R) and (25S)-Spirost-4-en-3-one (Diosgenone) 79 Product 3 (25R) and (25S)-5a-Spirostan-3-one (Tigogenone) 82 Other products - Androstanes 83 Discussion General techniques 85 Screening 85 vil Page CHAPTER 4 THE OPTIMIZATION OF THE MICROBIOLOGICAL TRANSFORMATION OF DIOSGENIN: THE EFFECTS OF VARYING THE PHYSICAL FORM OF THE REACTANTS AND THE PHYSIOLOGICAL STATE OF THE CELLS Introduction Modes of substrate addition 8 8 Cell immobilisation 90 Pseudo-crystallofermentation 92 Specific Materials and Methods Sonication of diosgenin particles and measurement of particle size 94 Whole cell immobilisation with calcium alginate 94 Results Rates of diosgenin transformation by several microorganisms 95 Modes of diosgenin addition i) The effect of ethanol concentration Nloccvr<l\cK rhodochrous viable cell count lOO ii) The effect of addition of diosgenin in solution and in suspension to rhodochrous cultures 100 ill) The effect of sonication on diosgenin crystal size and its transformation by 2^. rhodochrous 103 Transformation of diosgenin by growing, resting and calcium alginate immobilised cells 106 The effects of media, pH and culture age on the transformation of diosgenin by Corynebacterium mediolanum 113 Vlll Page The effect of culture age on the diosgenin transforming ability of rhodochrous 116 Transformation of diosgenin by N. rhodochrous in a defined medium 118 The effect of carbon source on the rate of transformation of diosgenin by N. rhodochrous 121 The effect of diosgenin concentration on its rate of transformation by rhodochrous and 126 Discussion Rates of diosgenin transformation by several microorganisms 128 Modes of diosgenin addition i) The effect of ethanol concentration on rhodochrous viable cell count 129 ii) The effect of addition of diosgenin in solution and in suspension to rhodochrous cultures