Isolation, Identification and Characterisation of Antibacterial Compounds from Carissa lanceolata R

87 Pages·2004·1.34 MB·English

School of Pharmacy

Isolation, Identification and Characterisation of Antibacterial
Compounds from Carissa lanceolata R.Br. Root

Dhanushka Sugeeshwara Hettiarachchi

This thesis is presented for the Degree of
Master of Pharmacy
of
Curtin University of Technology

September 2006

i
Declaration

To the best of my knowledge and belief this thesis contains no material previously
published by any other person except where due acknowledgment has been made.

This thesis contains no material which has been accepted for the award of any other
degree or diploma in any university.

Signature: ………………………………………….

Date: ………………………...
ii
This Work is Dedicated to My Beloved Parents

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“All things which are old should not necessarily be true; all things which are new
should not necessarily be without fault. To the wise men both of them should be
acceptable only if they stand to the test. The unwise, however, gets swayed by the
current of other’s opinion”

- Kalidasa’s Malakavikagniputhra Opera

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Acknowledgements

I render my sincere gratitude to my supervisor Dr. Connie Locher for guiding me
through the research with her knowledge, experience, intellect and her well known
pleasant personality. I’m fortunate to have Dr.Locher as my mentor, whose
understanding, attention and respect I received from the beginning has been
remarkable. I thank my co-supervisor A/Prof. Bob Longmore for his valuable
guidance and advice. I also would like to acknowledge the support I received as an
international postgraduate student from Prof. Bruce Sunderland.
I pay my thanks to Susanne Schorr for her preliminary work on the project and I
appreciate the support given to me by Dr. Yandi Liu and Dr. Paul Murray throughout
the project. A special word of thank goes out to the senior technical officer of the
microbiology laboratory, Mrs. Angela Samec, for all the advice and guidance I
received during my microbial assay work, as well as to Mr. Bruce Mackinnon for
providing facilities and guidance. The laboratory managers, Mr. Michel Stack and
Mr. Michel Boddy, are also to be mentioned with credit. In essence, I would like to
thank the School of Pharmacy staff for their helpfulness and wonderful company.
I am also grateful to Mr. Kim Courtney of Kimberly TAFE in Broome for providing
me with the plant material for the research, to Dr. Lindsay Byrne (Chemistry
Department, University of Western Australia) for his help with NMR analysis and to
Mr. Todd Edwards (Department of Environmental Biology, Curtin University) for
his botanical advice.
I would like to thank my colleagues Chirag, Sarika, Gayathri, Yousuf, Goutham,
Vijay and all who were there as friends when I was in need.
I also compliment Chathura for the wonderful brotherhood shared with me, also a
salute to the Rathnasekara and Ediriweera families for their support and familiar treat
I have received during my stay.
I appreciate my brother’s motivation and support, which never let me down in my
path. Understanding and encouragement offered by my girlfriend Sonia was also a
great support to achieve my aspiration. Finally, I would like to tribute this work to
sacrifices made by my parents and both grandmothers.

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Table of Content

Declaration...................................................................................................................ii
Acknowledgements......................................................................................................v
Table of Content..........................................................................................................vi
List of Figures..............................................................................................................x
List of Tables.............................................................................................................xii
List of Tables.............................................................................................................xii
Abbreviations............................................................................................................xiii
Abbreviations............................................................................................................xiii
Abstract.....................................................................................................................xiv
1 Background..........................................................................................................1
1.1 The Genus Carissa.......................................................................................2
1.1.1 Habitat......................................................................................................3
1.1.2 Morphology..............................................................................................3
1.1.3 Ethnopharmacology, Chemistry and Pharmacology................................4
1.2 Carissa lanceolata R.Br...............................................................................5
1.2.1 Habitat......................................................................................................5
1.2.2 Morphology..............................................................................................5
1.2.3 Ethnopharmacology.................................................................................6
1.2.4 Chemistry.................................................................................................6
1.3 Carissa carandus Linn.................................................................................7
1.3.1 Habitat......................................................................................................7
1.3.2 Morphology..............................................................................................7
1.3.3 Ethnopharmacology.................................................................................8
1.3.4 Chemistry.................................................................................................8
1.3.5 Pharmacology...........................................................................................9
1.4 Carissa edulis Vahl......................................................................................9
1.4.1 Habitat......................................................................................................9
1.4.2 Morpholgy................................................................................................9
1.4.3 Ethnopharmacology...............................................................................10
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1.4.4 Chemistry...............................................................................................10
1.4.5 Pharmacology.........................................................................................11
1.5 Reported compounds..................................................................................12
1.5.1 Sesquiterpenes........................................................................................12
1.5.2 Lignans...................................................................................................14
1.5.3 Other isolated compounds......................................................................15
1.6 Antibacterial activity..................................................................................17
1.6.1 Assay Methods.......................................................................................17
1.6.2 Bacterial Strains used in the Antibacterial Bioassay..............................19
2 Materials and Methods.......................................................................................21
2.1 Plant Material.............................................................................................21
2.2 Solvents......................................................................................................21
2.3 Extractions.................................................................................................21
2.3.1 Steam Distillation...................................................................................21
2.3.2 Solid Phase Micro-Extraction (SPME)..................................................22
2.3.3 Solvent Extraction..................................................................................22
2.4 Chemical Tests...........................................................................................22
2.4.1 Determination of Alkaloids....................................................................22
2.4.2 Test for Saponins...................................................................................22
2.4.3 Test for Phenolic Compounds................................................................22
2.4.4 Test for Anthraquinones.........................................................................23
2.4.5 Test for Steroids.....................................................................................23
2.4.6 Cardiac Glycosides................................................................................23
2.5 Chromatographic Methods.........................................................................24
2.5.1 Thin Layer Chromatography (TLC).......................................................24
2.5.2 Column Chromatography.......................................................................24
2.5.2.1 Dichloromethane Extracts..............................................................25
2.5.2.1.1 Fraction 2...................................................................................25
2.5.2.1.2 Fraction 3...................................................................................25
2.5.2.2 Methanol Extracts..........................................................................26
2.5.2.3 Hexane Extracts.............................................................................26
2.5.2.3.1 Root Bark Hexane Extract.........................................................26
2.5.2.3.2 Root Wood Hexane Extract.......................................................27
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2.5.3 Preparative TLC.....................................................................................27
2.5.4 Gas Chromatography.............................................................................28
2.6 Spectroscopy..............................................................................................28
2.6.1 Infrared Spectroscopy (IR).....................................................................28
2.6.2 Mass Spectroscopy (MS).......................................................................28
2.6.3 Nuclear Magnetic Resonance Spectroscopy (NMR).............................28
2.7 Antimicrobial Assay..................................................................................28
2.7.1 Microbial strains....................................................................................29
2.7.2 Assay Material.......................................................................................29
2.7.3 Assay Method........................................................................................29
3 Results & Discussion.........................................................................................31
3.1 Extraction...................................................................................................31
3.2 Preliminary phytochemical studies............................................................31
3.2.1 TLC screening........................................................................................31
3.2.2 Chemical tests........................................................................................33
3.3 Volatile oil [#1]..........................................................................................35
3.4 Dichloromethane Extracts [#4]..................................................................36
3.4.1 Fraction 2 [#4.2].....................................................................................37
3.4.2 Fraction 3 [#4.3].....................................................................................37
3.5 Methanol extract [8]...................................................................................38
3.6 Hexane extracts..........................................................................................40
3.6.1 Root bark hexane extract [#3]................................................................40
3.6.2 Root wood hexane extract [#6]..............................................................41
3.7 Isolated compounds....................................................................................42
3.7.1 2′-Hydroxy acetophenone [#1.1]............................................................42
3.7.2 Carissone [#4.2.2.1 and #6.3]................................................................45
3.7.3 Carinol [8.1.2.1.2.2]...............................................................................47
3.8 Antibacterial activity..................................................................................49
3.8.1 Antibacterial activity of extracts............................................................49
3.8.2 Antibacterial activity of isolated compounds.........................................50
4 Conclusion.........................................................................................................52
5 References..........................................................................................................54
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Appendices.................................................................................................................60
Appendix I – Spectroscopic Data of Isolated Compounds....................................60
Appendix II – Reagents..........................................................................................70
Appendix II – Reagents..........................................................................................71
Appendix III – Method Development for Anti-Microbial Assay..........................72
Appendix IV – Presentations.................................................................................73
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List of Figures
Figure 1: Geographical distribution of C. lanceolata....................................................5
Figure 2: Arial part of C. lanceolata..............................................................................6
Figure 3: Root of C. lanceolata.....................................................................................7
Figure 4: Aerial part of C. carandas..............................................................................8
Figure 5: Aerial parts of C. edulis................................................................................10
Figure 6: Biogenesis pathway of sesquiterpenes.........Error! Bookmark not defined.
Figure 7: Sesquiterpenes from Carissa spp.................Error! Bookmark not defined.
Figure 8 Lignans identified from Carissa spp.............Error! Bookmark not defined.
Figure 9: Other compounds isolated from Carissa spp.Error! Bookmark not defined.
Figure 10: Reduction path of resazurin........................................................................19
Figure 11: TLC in different solvent systems and visualisationsError! Bookmark not defined.
Figure 12: Isolation path of the C. lanceolata root......Error! Bookmark not defined.
Figure 13: Isolation path for DCM extracts.................Error! Bookmark not defined.
Figure 14: Isolation path of methanol extracts.............Error! Bookmark not defined.
Figure 15: Isolation path of root bark hexane extract..Error! Bookmark not defined.
Figure 16: Isolation path of root wood hexane extract Error! Bookmark not defined.
Figure 17: GC trace of volatile oil...............................Error! Bookmark not defined.
Figure 18: Mass spectra for the volatile compound.....Error! Bookmark not defined.
Figure 19: Fragmentation pattern of 2'-hydroxy acetophenone..................................43
Figure 20: Structure of 2′-hydroxy acetophenone........Error! Bookmark not defined.
Figure 21: Structure of Carissone................................Error! Bookmark not defined.
Figure 22: Structure of Carinol....................................Error! Bookmark not defined.
Figure 23: 1H NMR Spectrum of 2′-Hydroxy acetophenone [#1.1]............................61
Figure 24: Mass Spectrum of 2′-Hydroxy acetophenone [#1.1]..................................62
Figure 25: 1H NMR Spectrum of Carissone [#4.2.2.1]................................................63
Figure 26: Expansion of 1H NMR Spectrum of Carissone[#4.2.2.1] (2.75-2.15 ppm)64
Figure 27: Expansion of 1H NMR of Spectrum of Carissone [#4.2.2.1] (1.85-1.60
ppm).....................................................................Error! Bookmark not defined.
Figure 28: Expansion of 1H NMR of Spectrum of Carissone [#4.2.2.1] (1.55-1.20
ppm).....................................................................Error! Bookmark not defined.
Figure 29: 13C NMR Spectrum of Carissone[#4.2.2.1]Error! Bookmark not defined.
Figure 30: Infra-Red Spectrum of Carissone[#4.2.2.1]Error! Bookmark not defined.
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