PRODUCTION AND EXTRACTION OF COMMERCIALLY IMPORTANT ERGOT ALKALOIDS FROM PENICILLIUM SPECIES BY FERMENTATION PROCESS BY MEMUNA GHAFOOR SHAHID REGISTRATION NO. 14-PHD-BOT-GCU-08 GOVERNMENT COLLEGE UNIVERSITY, LAHORE i PRODUCTION AND EXTRACTION OF COMMERCIALLY IMPORTANT ERGOT ALKALOIDS FROM PENICILLIUM SPECIES BY FERMENTATION PROCESS Submitted to the Government College University, Lahore in partial fulfillment of the requirements for the award of degree of Ph.D. In BOTANY BY MEMUNA GHAFOOR SHAHID REGISTRATION NO. 14-PHD-BOT-GCU-08 DEPARTMENT OF BOTANY GOVERNMENT COLLEGE UNIVERSITY, LAHORE ii DEDICATION I DEDICATE THIS WORK TO MY PARENTS WHO ARE THE SOURCE OF MY INSPIRATION AND ENCOURAGEMENT IN ACHIEVING THE ACADEMIC TITLE OF ’DOCTOR OF PHILOSOPHY’. iii RESEARCH COMPLETION CERTIFICATE Certified that the research work contained in this thesis, titled “Production and Extraction of Commercially Important Ergot Alkaloids from Penicillium species by Fermentation Process” has been carried out in FBRC, PCSIR Laboratories, Complex, Lahore and completed by Miss Memuna Ghafoor Shahid, Registration No. 0014-PHD-GCU-BOT-08, under my supervision during her Ph.D. studies in the subject of Botany. Dated: 22-09-2014 Supervisor Dr. Safdar Ali Mirza Assistant Professor (TTS) Department of Botany GC University, Lahore Submitted through: Dr. Ghazala Yasmeen Butt Controller of Examinations Chairperson & Associate Professor GC University, Lahore Department of Botany GC University, Lahore iv DECLARATION I, Memuna Ghafoor Shahid, Registration No. 0014-PHD-GCU-BOT-08, student of Ph.D. in the subject of Botany declare that the work contained in this thesis titled “Production and Extraction of Commercially Important Ergot Alkaloids from Penicillium species by Fermentation Process” is my work and has not been printed, published and submitted as research work, thesis or publication in any form in any university and research institute etc. within or without Pakistan. Dated: 22-09-2014 Memuna Ghafoor Shahid v Table of Contents Chapters Title/Subheads Page No. List of Tables viii List of Figures xi List of Plates xiv List of Abbreviations xv Acknowledgements xvi Abstract xviii 1 I Introduction 2 Review of Literature 6 2.1 Ergot and Ergotism 6 2.2 Biosynthesis of Ergot Alkaloids 7 2.3 Statistical Optimization of Culture Conditions 13 2.4 Structural Diversity of Ergot Alkaloids 15 2.5 Ergot Alkaloids Pharmacodynamics 16 2.6 Ergot Alkaloids and Analytical Methods 17 3 Materials and Methods 20 Section-I Optimization of Culture Conditions by OFAT 20 Method Section-II Response Surface Methodology 30 Section-III Strain Improvement 35 Section-IV Analytical Studies 42 vi 4 Results 47 Section-I Optimization of Culture Conditions by OFAT 47 Method Section-II Response Surface Methodology 83 Section-III Strain Improvement 104 Section-IV Analytical Studies 116 5 Discussion 150 Section-I Optimization of Culture Conditions by OFAT 151 Method Section-II Response Surface Methodology 156 Section-III Strain Improvement 159 Section-IV Analytical Studies 162 Conclusions 165 6 References 167 Annexure-I 180 vii List of Tables Table Title Page No. No. 3.1 Composition of different fermentation media for screening purpose 22 3.2 Composition of screened M5 fermentation medium 23 3.3 Composition of optimized fermentation medium and conditions 29 for Penicillium commune and Penicillium sp. IIB 3.4 Plackett-Burman Design experimental range and level for 31 screening of variables 3.5 Plackett-Burman experimental design for screening of variables 32 3.6 Experimental range and levels for optimization of significant 33 variables 3.7 Experimental design for optimization of significant variables 34 3.8 Composition of fermentation medium for the production of ergot 37 alkaloids by mutated strains of Penicillium commune and Penicillium sp. IIB 3.9 Absorbance of the dilutions of bromocriptine mesylate (BCM) and 39 dihydroergotamine methane sulfonate (DMS) salts 3.10 Screening of TLC mobile phases for the determination of ergot 43 alkaloids 3.11 Selection of TLC mobile phases for the determination of ergot 44 alkaloids 4.1 Screening of fungal organisms and fermentation medium for 48 production of ergot alkaloids 4.2 Effect of different carbon sources on the production of ergot 50 alkaloids 4.3 Analysis of variance for the effect of carbon sources 50 4.4 Effect of different nitrogen sources on the production of ergot 52 alkaloids 4.5 Analysis of variance for the effect of nitrogen sources 52 4.6 Effect of concentration levels of sucrose on the production of ergot 54 alkaloids 4.7 Analysis of variance for the effect of concentration levels of sucrose 54 4.8 Effect of concentration levels of yeast extract on the production of 56 ergot alkaloids 4.9 Analysis of variance of the effect of concentration levels of yeast 56 extract 4.10 Effect of concentration levels of KH2PO4 on the production of ergot 58 alkaloids 4.11 Analysis of variance for the effect of concentration levels of 58 KH2PO4 viii 4.12 Effect of the different concentration levels of tryptophan on the 60 production of ergot alkaloids 4.13 Analysis of variance of effect of concentration levels of tryptophan 60 4.14 Effect of different concentrations of asparagine on the production 62 of ergot alkaloids 4.15 Analysis of variance for the effect of concentration levels of 62 asparagine 4.16 Effect of concentration levels of succinic acid on the production of 64 ergot alkaloids 4.17 Analysis of variance for the effect of concentration levels of 64 succinic acid 4.18 Effect of concentration levels of NH4Cl on the production of ergot 66 alkaloids 4.19 Analysis of variance of the effect of concentration levels of NH4Cl 66 4.20 Effect of the concentration levels of MgSO4.7H2O on the production 68 of ergot alkaloids 4.21 Analysis of variance for the effect of concentration levels of MgSO4. 68 7H2O 4.22 Effect of concentration levels of FeSO4.7H2O on the production of 70 ergot alkaloids 4.23 Analysis of variance for the effect of concentration levels of FeSO4. 70 7H2O 4.24 Effect of concentration levels of ZnSO4 on the production of ergot 72 alkaloids 4.25 Analysis of variance for the effect of concentration levels of ZnSO4 72 4.26 Effect of pH on the production of ergot alkaloids on the production 74 of ergot alkaloids 4.27 Analysis of variance of the effect of various pH levels 75 4.28 Effect of incubation temperature on the production of ergot 76 alkaloids 4.29 Analysis of variance for the effect of different incubation 77 temperatures 4.30 Effect of different incubation time periods on the production of 78 ergot alkaloids 4.31 Analysis of variance for the effect of incubation times 79 4.32 Effect of different size of inoculum on the production of ergot 80 alkaloids 4.33 Analysis of variance for the effect of various inoculum sizes 81 4.34 Production of ergot alkaloids in fermentor 82 4.35 Screening of variables for ergot alkaloids production by Penicillium 84 commune using PBD 4.36 Analysis of variance for ergot alkaloids yield by Penicillium 85 commune using PBD ix 4.37 Observed and predicted values of ergot alkaloids yield by 87 Penicillium commune using BBD 4.38 Observed and predicted values of ergot alkaloids yield by 87 Penicillium sp. IIB using BBD 4.39 Analysis of variance for alkaloids production by Penicillium 94 commune using BBD 4.40 Analysis of variance for ergot alkaloid production by Penicillium 95 sp. IIB using BBD 4.41 Survival rate of colonies of UV mutated strains of Penicillium 105 commune and Penicillium sp. IIB 4.42 Survival rate of colonies of EMS mutated strains of Penicillium 108 commune and Penicillium sp. IIB 4.43 Screening of UV mutant and comparison of ergot alkaloids 111 production with wild strains 4.44 Screening of EMS mutants and comparison of ergot alkaloids 113 production with wild strains 4.45 Comparison of the ergot alkaloids yield of selected mutant and 115 wild strains 4.46 values of culture liquid and mycelial filtrate extracts of 117 Penicillium commune 4.47 푅푓 values of culture liquid and mycelial filtrate extracts of 118 Penicillium sp. IIB 4.48 푅푓 values of selected culture liquid and mycelial filtrate extracts of 119 Penicillium commune 4.49 푅푓 values of selected culture liquid and mycelial filtrate extracts of 120 Penicillium sp. IIB 4.50 Retention푅푓 times of reference salts of ergot alkaloids 124 4.51 Retention time of culture liquid and mycelial filtrate extracts of 125 Penicillium commune 4.52 Retention time of culture liquid and mycelial filtrate extracts of 126 Penicillium sp. IIB x List of Figures Fig. No. Title Page No. 3.1 Standard curve of BCM salt 39 3.2 Standard curve of DMS salt 40 4.1 Mycelial growth of all fungal species in M5 fermentation medium 48 4.2 Effect of different carbon sources on the mycelial growth of 51 Penicillium commune and Penicillium sp. IIB 4.3 Effect of different nitrogen sources on the mycelial growth of 53 Penicillium commune and Penicillium sp. IIB 4.4 Effect of different concentrations of sucrose on the mycelial growth 55 of Penicillium commune and Penicillium sp. IIB 4.5 Effect of different concentrations of yeast extract on the mycelial 57 growth of Penicillium commune and Penicillium sp. IIB 4.6 Effect of different concentrations of KH2PO4 on the mycelial 59 growth of Penicillium commune and Penicillium sp. IIB 4.7 Effect of different concentrations of tryptophan on the mycelial 61 growth of Penicillium commune and Penicillium sp. IIB 4.8 Effect of different concentrations of asparagine on the mycelial 63 growth of Penicillium commune and Penicillium sp. IIB 4.9 Effect of different concentrations of succinic acid on the mycelial 65 growth of Penicillium commune and Penicillium sp.