Phytochemical investigation of zanthoxylum holstzianumfor antimicrobial Principles

123 Pages·2016·4.09 MB·English

PHYTOCHEMICAL INVESTIGATION OF
ZANTHOXYLUMHOLSTZIANUMFOR ANTIMICROBIAL
PRINCIPLES
BUYINZA DANIEL
156/64754/2010
UNIVERSITY OF NAIROBI
COLLEGE OF BIOLOGICAL AND PHYSICAL SCIENCES
DEPARTMENT OF CHEMISTRY
RESEARCH THESIS SUBMITTED IN PARTIAL
FULFILLMENT FOR THE AWARD OF MASTERS OF
SCIENCE IN CHEMISTRY DEGREE
JUNE 2012
DECLARATION
This thesis is my original work and has not been presented for a degree in any other
university.
.................
BUYINZA DANIEL
This thesis is submitted for examination with our approval as supervisors
...................................................................
PROF. ABIY YENESEW
DEPARTMENT OF CHEMISTRY DEPARTMENT OF CHEMISTRY
UNIVERSITY OF NAIROBI UNIVERSITY OF NAIROBI
DR. LEONIDAH KERUBO DR. AMIR O. YUSUF
DEPARTMENT OF CHEMISTRY DEPARTMENT OF CHEMISTRY
UNIVERSITY OF NAIROBI UNIVERSITY OF NAIROBI
DEDICATION
This research thesis is dedicated to my late grandfather Shadrach Kiige Musobya for his
financial support towards my education. My life shall forever be inspired by your
humility and generosity.
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ACKNOWLEDGMENTS
I would like to acknowledge the parental and professional work done by my supervisors
Dr. Solomon Derese, Prof. Abiy Yenesew, Dr. Leonidah Kerubo and Dr. Yusuf O. Amir.
Your commitment to mentoring me is expressed in this excellent write up. I am proud of
you.
My sincere thanks go to Dr. Matthias Heydenreich, University of Potsdam, for high
resolution NMR analyses, Martha Induli and Simon Mathenge, University of Nairobi, for
identifying and collecting the plant material investigated in this study, Cathrine Kitonde,
School of Biological Sciences, University of Nairobi for the technical assistance in the
anti-microbial test, to the academic and technical staff at the Department of Chemistry
University of Nairobi who did not only build my academic muscles but also provided an
enabling environment for my study, I am grateful.
The current and former masters and PhD students of the Department of Chemistry
University of Nairobi with whom the shared jokes renewed my commitment to work
even late, I say that you were such a blessing to my study.
To my family, saying thank you is not adequate, but indeed your patience can’t be
equated to any currency. My parents, I will forever be grateful to God for your love and
uncompensatable sacrifice in making me a responsible being I am today.
The German Academic Exchange Service (DAAD), without your scholarship through
NAPRECA, the postgraduate study would have remained a dream to me. You’re a real
“Daad”.
Most importantly, to GOD be the Glory and Honour for what people see as success,
which is just His sufficient Grace extended to me.
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LIST OF ABBREVIATIONS AND ACRONYMS
ATCC American Type Culture Collection
AIDS Acquired Immuno Deficiency Syndrome
CC Column Chromatography
CDCI3 Deutrated trichloromethane
COSY Correlation Spectroscopy
CLSI Clinical and Laboratory Standard Institute
ID & 2D NMR 1-Dimensional and 2-Dimensional Nuclear Magnetic Resonance
DMSO Dimethylsulfoxide
DNP Dictionary of Natural Product
DEPT Distortionless Enhancement by Polarization Transfer
GC/MS Gas Chromatography/Mass Spectrometry
HIV Human Immunodeficiency Virus
HMBC Heteronuclear Multiple Bond Correlation
HMQC Heteronuclear Multiple Quantum Correlation
HPLC High Performance Liquid Chromatography
HPV Human Papilloma Virus
HCV Hepatitis C Virus
HBV Hepatitis B Virus
HSQC Heteronuclear Single Quantum Correlation
IR Infrared
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MDG Millennium Development Goals
MIC Minimum Inhibition Concentration
NCCLS National Committee for Clinical Laboratory Standard
NMR Nuclear Magnetic Resonance
NOE Nuclear Overhauser Effect
NOESY Nuclear Overhauser Effect Spectroscopy
PTLC Preparative Thin Layer Chromatography
SARS Severe Acute Respiratory Syndrome
SC Saccharomyces cerevisiae
TCM Traditional Chinese Medicine
v
TLC Thin Layer Chromatography
uv Ultra Violet
VLC Vacuum Liquid Chromatography
Maximum absorption wavelength
^ max
W.H.O World Health Organization
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Table of Contents
DECLARATION...................................................................................................................................................II
DEDICATION....................................................................................................................................................Ill
ACKNOWLEDGMENTS.....................................................................................................................................IV
LIST OF ABBREVIATIONS AND ACRONYMS.....................................................................................................V
TABLE OF CONTENTS.....................................................................................................................................VII
LIST OF FIGURES/ SCHEMES...........................................................................................................................IX
LIST OF TABLES................................................................................................................................................IX
APPENDIX.........................................................................................................................................................X
ABSTRACT.......................................................................................................................................................XII
CHAPTER ONE...................................................................................................................................................1
INTRODUCTION...............................................................................................................................................1
1.1 ManandtheHerb............................................................................................................................1
1.2 Infectious Diseases...........................................................................................................................3
1.3 Natural Antimicrobials...................................................................................................................5
1.4 Problem Statement..........................................................................................................................6
1.5 Objectives of the Research...............................................................................................................6
1.6 Justification of the Research............................................................................................................7
CHAPTER TWO..................................................................................................................................................8
LITERATURE REVIEW........................................................................................................................................8
2.1 The Genus Zanthoxylum...................................................................................................................8
2.2 Distribution of the genus Zanthoxylum............................................................................................8
2.3 Medicinal uses of Zanthoxylum species.............................................................................................9
2.3.1 Biological activities of Zanthoxylum........................................................................................11
2.4 Phytochemistry of the genus Zanthoxylum......................................................^...........................12
2.4.1 Alkaloids of Zanthoxylum..............................................................................................................14
2.4.1.1 Benzophenanthridine alkaloids of Zanthoxylum........................................................................14
2.4.1.2 Zanthoxylum Alkaloids Derived from Anthranilic Acid..............................................................16
2.4.1.3 Protoberberine alkaloids of Zanthoxylum.................................................................................17
2.4.1.4 Aporphine Alkaloids of Zanthoxylum.........................................................................................18
2.4.1.5 Bishordeninylterpene alkaloids of Zanthoxylum.......................................................................20
2.5 Amides of Zanthoxylum..................................................................................................................21
2.6 COUMARINS OF ZANTHOXYLUM...........................................................................................................23
2.7 Lignans of Zanthoxylum................................................................................................................24
2.8 Fatty acids of Zanthoxylum............................................................................................................25
2.9 Flavanoids of Zanthoxylum...........................................................................................................26
CHAPTER THREE..............................................................................................................................................28
METHODOLOGY AND MATERIALS.......................................................................•»........................................28
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3.1 General.........................................................................................................................................28
3.2 Plant material...............................................................................................................................28
3.3 Extraction and isolation................................................................................................................28
3.4 Antimicrobial Sensitivity Tests.......................................................................................................29
3.5 Spectroscopic Data of Isolated Compounds...................................................................................30
CHAPTER FOUR...............................................................................................................................................32
RESULTS AND DISCUSSION...........................................................................................................................32
4.1.0 Secondary Metabolites isolated from Zanthoxylum holstzianum..................................................32
4.1.1 a-HESPERIDINE (1)...........................................................................................................................................................33
4.1.2 Dihydrochelerythrine (2)...............................................................................................................35
4.1.3 A/-Methylflindersine (3).................................................................................................................37
4.1.4 (2£,4E)-A/-Isobutyltetradeca-2,4-dienamide (4)...................................................................................38
4.1.5 8-Acetonyldihydrochelerythrine(5)..............................................................................................40
4.1.6 Hexadecanoicacid(6).....................................................................................................................41
4.1.7 8-Oxochelerythrine (7)..................................................................................................................42
4.2.0 Antimicrobial Tests of the Secondary Metabolites Obtained from Zanthoxylum holstzianum......44
CHAPTER FIVE.................................................................................................................................................46
CONCLUSION AND RECOMMENDATIONS....................................................................................................46
5.1.0 Conclusion....................................................................................................................................46
5.2.0 Recommendations..........................................................................................................................47
REFERENCES...................................................................................................................................................48
APPENDIX.......................................................................................................................................................55
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List of Figures/ Schemes
FIGURE 1: ISOLATED COMPOUNDS FROM ZANTHOXYLUM HOLSTZIANUM............................................XIII
FIGURE: 2.0 FEATURES OF ZANTHOXYLUM....................................................................................................8
SCHEME 1: BIOSYNTHETIC PATH OF SOME ZANTHOXYLUM COMPOUNDS...............................................13
FIGURE: 2.1 BASIC SKELETON OF BENZOPHENANTHRIDINE ALKALOIDS....................................................14
FIGURE 2.3: EXAMPLES OF BENZOPHENANTHRIDINE ALKALOIDS..............................................................16
FIGURE 2.4: THE BASIC STRUCTURE (2-QUINOLONE) OF ANTHRANILIC DERIVED ALKALOIDS.................16
FIGURE: 2.5: BASIC SKELTON OF PROTOBERBERINE ALKALOIDS................................................................18
FIGURE 2.6: APORPHINE ALKALOID SKELETON...........................................................................................18
FIGURE 2.7: BASIC UNIT OF BISHORDENINYL TERPENE ALKALOIDS...........................................................20
FIGURE:2.8 BASIC SKELTON OF COUMARINS..............................................................................................23
FIGURE: 2.9 BASIC LIGNAN SKELTON...........................................................................................................24
FIGURE: 3.0 SOME FLAVONOID SUBCLASSES..............................................................................................26
List of Tables
TABLE 2.1: RECAPITULATIVE TABLE OF THE TRADITIONAL USES OF SOME ZANTHOXYLUM SPECIES.........9
TABLE 2.2: SUMMARY OF BIOLOGICAL ACTIVITIES OF ZANTHOXYLUM....................................................11
TABLE 2.3: EXAMPLES OF BENZOPHENANTHRIDINE ALKALOIDS.......................................15
TABLE 2.4: ANTHRANILIC ACID DERIVED ALKALOIDS OF ZANTHOXYLUM.................................................17
TABLE 2.5: APORPHINE ALKALOIDS..............................................................................................................19
TABLE 2.6: EXAMPLES OF BISHORDENINYL TERPENE ALKALOIDS...............................................................20
TABLE 2.7: AMIDES OF ZANTHOXYLUM......................................................................................................21
TABLE 2.8: COUMARINS OF ZANTHOXYLUM................................................................................................23
TABLE 2.9: LIGNANS OF ZANTHOXYLUM......................................................................................................25
TABLE 3.0: FLAVONES OF ZANTHOXYLUM...................................................................................................26
TABLE 4.1: *H (600 MHZ) AND 13C (200 MHZ) NMR DATA FOR A-HESPERIDINE (1) IN DMSO.......35
TABLE 4.2: lH (600 MHZ) AND 13C (150 MHZ) NMR DATA FOR DIHYDROCHELERYTHRINE (2) IN CD2CL2 ..37
TABLE 4.3:*H (600 MHZ) AND 13C (150 MHZ) NMR DATA FOR N-METHYLFLINDERSINE IN CD2CL2.38
TABLE 4.4: *H (600 MHZ) AND 13C (150 MHZ) NMR DATA FOR (2E, 4E)-N-ISOBUTYLTETRADECA-2, 4-
DIENAMIDE IN CD2CL2....................................................................................................................................39
TABLE 4.5: *H (600 MHZ) AND 13C (150 MHZ) NMR DATA FOR 8-ACETONYLDIHYDROCHELERYTHRINE IN
CD2CL2......................................................................................}...........................2.......................................41
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TABLE 4.6: *H (600 MHZ) AND 13C (150 MHZ) NMR DATA FOR HEXADECANOIC ACID IN CD2CL2..............42
TABLE 4.7:*H (600 MHZ) AND 13C (150 MHZ) NMR DATA FOR 8-OXOCHELERYTHRINE IN DMSO-D6......44
TABLE 4.8: ANTIMICROBIAL TEST RESULTS..................................................................................................45
Appendix
APPENDIX 1.0: SPECTRAL FEATURES OF HESPERIDINE (1)...................................................................55
1.1: STRUCTURE OF HESPERIDINE.........................................................................................................55
1.2: *H SPECTRA OF HESPERIDINE (DMSO-d6 JH AV600)......................................................................55
1.3: EXPANDED 1H SPECTRA OF HESPERIDINE.....................................................................................56
1.4: 13C SPECTRA OF HESPERIDINE (DMSO-d6 AVISO).........................................................................58
1.5: H-H COSY OF HESPERIDINE.............................................................................................................59
1.6: HMBC OF HESPERIDINE..................................................................................................................60
1.7: HSQC OF HESPERIDINE...................................................................................................................62
1.8: NOESY OF HESPERIDINE.................................................................................................................64
APPENDIX2.0: SPECTRAL DATA OF DIHYDROCHELERYTHRINE (2)......................................................66
2.1: STRUCTURE OF DIHYDROCHELERYTHRINE.....................................................................................66
2.2: JH DATA FOR DIHYDROCHELERYTHRINE (CD2CI2 *H AV600)..........................................................66
2.3:13C SPECTRA FOR DIHYDROCHELERYTHRINE (CD2CI2AV600)........................................................67
2.4: H-H-COSY OF DIHYDROCHELERYTHRINE........................................................................................68
2.5: HMBC OF DIHYDROCHELERYTHRINE..............................................................................................69
2.6: HSQC OF DIHYDROCHELERYTHRINE...............................................................................................70
2.7: NOESY OF DIHYCHELERYTHRINE.....................................................................................................71
APPENDIX3.0: SPECTRA DATA OF N-METHYLFLINDERSINE (3)............................................................72
3.1: STRUCTURE OF N-METHYLFLINDERSINE........................................................................................72
3.2: *H NMR OF N-METHYLFLINDERSINE (CD2CI2 1H AV600)................................................................72
3.3:13C NMR OF N-METHYLFLINDERSINE (CD^t^ AV600)...................................................................74
3.4: H-H COSY OF N-METHYLFLINDERSINE............................................................................................74
3.5: HMBC OF N-METHYLFLINDERSINE.................................................................................................75
3.6: HSQC OF N-METHYLFLINDERSINE...................................................................................................77
3.7: NOESY OF N-METHYLFLINDERSINE.................................................................................................77
APPENDIX 4.0: SPECTRA DATA OF (2E,4E)-N-ISOBUTYLTETRADECA-2J4-DIENAMIDE (4)...................78
4.1: STRUCTURE OF (2E,4E)-N-ISOBUTYLTETRADECA-2,4-DIENAMIDE.................................................78
4.2:1HNMR OF (2E,4E)-N-ISOBUTYLTETRADECA-2,4-DIENAMIDE (CD2CI2 JH AV600)..........................78
4.3:13C NMR OF (2E,4E)-N-ISOBUTYLTETRADECA-2,4-DIENAMIDE (CD2CI2 AV600)............................80
4.4: H-H COSY OF (2E,4E)-N-ISOBUTYLTETRADECA-2,4-DIENAMIDE.....................................................81
4.5: HMBC OF (2E,4E)-N-ISOBUTYLTETRADECA-2,4-DIENAMIDE.........................................................83
4.6: HSQC OF (2E,4E)-N-ISOBUTYLTETRADECA-2,4-DIENAMIDE...........................................................84
4.7: NOESY OF (2E,4E)-N-ISOBUTYLTETRADECA-2,4-DIENAMIDE........................................................85
APPENDIX 5.0: SPECTRAL DATA OF 8-ACETONYLDIHYDROCHELERYTHRINE (5)..................................86
5.1: STRUCTURE OF 8-ACETONYLDIHYDROCHELERYTHRINE.................................................................86
5.2 1H NMR DATA OF 8-ACETONYLDIHYDROCHELERYTHRINE (CD2CI2 1H A V600).............................86
5.3:13C NMR OF 8-ACETONYLDIHYDROCHELERYTHRINE (CD2CI2AV600).............................................88
5.4: H-H COSY OF 8-ACETONYLDIHYDROCHELERYTHRINE.....................................................................90
5.5: HMBC OF 8-ACETONYLDIHYDROCHELERYTHRINE............... 91
5.6: HSQC OF 8-ACETONYLDIHYDROCHELERYTHRINE..*......’....................... 93
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