Isolation and characterisation of bioactive compounds from Antidesma venosum E.Mey. ex Tul. and Euphorbia cooperi N.E.Br. ex A.Berger
Theses submitted in accordance with the requirements for the degree
Master of Science
By
Sbonelo Sanele Hlengwa
School of Chemistry and Physics University of KwaZulu-Natal Pietermaritzburg
Supervisor: Professor Fanie R. van Heerden
November 2018
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Abstract
The need for the discovery of new pharmacologically active compounds is a global priority as new diseases and drug resistance mechanisms are emerging and spreading throughout the world. In the past natural products have played an important role in drug discovery. Plant- derived natural products are important in the discovery of new drugs for treatment of infectious diseases and cancer.
Many biologically active compounds with diverse structures have been isolated from the Euphorbiaceae family. In South Africa, Euphorbiaceae is the largest flowering plant family and some species within the family have not been investigated for unique secondary metabolites. However, many species have not been researched. This project aimed to identify bioactive compounds in South Africa Euphorbiaceae species. Two species within the Euphorbiaceae family, Euphorbia cooperi N.E.Br. ex A.Berger and Antidesma venosum E.Mey. ex Tul. were selected for investigation.
The crude extracts were fractionated using column chromatography and centrifugal thin-layer chromatography to yield single compounds. A mixture of hexane, dichloromethane, chloroform, ethyl acetate and methanol were used as eluting solvents. The structures of the compounds isolated were elucidated by 1D and 2D NMR experiments (1H, 13C NMR, DEPT- 135, COSY, HSQC, HMBC and NOESY).
The compounds isolated from A. venosum were identified as loliolide, β-sitosterol 3-O-β-D- glucoside, lutein, 4-hydroxyphenylethyl trans-ferulate as well as pheophytin A and pheophytin B. Although these compounds are known compounds, only pheophytin A has previously been identified in A. venosum.
A phytochemical study of the aerial part of E. cooperi has led to the isolation of a novel norsesquiterpenoid named euphorbilactone and its glucoside, arachiside A, along with a triterpenoid, glutinol, a known phorbol ester, 16-angeloyloxy-13α-isobutanoyloxy-4β,9α,20- trihydroxytiglia-1,5-diene-3,7-dione, and a novel phorbol ester, 20-acetoxy-16-angeloyloxy- 13α-isobutanoyloxy-4β,9α,20-tetrahydroxytiglia-1,5-diene-3-one. This is the first report on the isolation of these compounds from E. cooperi.
The crude extracts and loliolide, p-hydroxyphenylethyl trans-ferulate and β-sitosterol 3-O-β- D-glucoside, were assayed for antimicrobial activities against one Gram-positive (Staphylococcus aureus ATCC 12600), two Gram-negative (Pseudomonas aeruginosa ATCC
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10145, Klebsiella pneumoniae ATCC 13883), and one drug-resistant Gram-negative (Escherichia coli ATCC 25218) bacteria. The crude extracts of the plants showed moderate but promising antimicrobial activity. The pure compounds did not show any antimicrobial activity.
This investigation has shown that South African Euphorbiaceae contains novel bioactive compounds. The two usual phorbol esters isolated from E cooperi warrant further investigation of the bioactivity of these compounds.
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Declaration
The experimental work described in this dissertation was carried out in School of Chemistry and Physics, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Pietermaritzburg, under the supervision of Professor Fanie R. van Heerden.
I hereby declare that these studies represent original work by the author and have not otherwise been submitted in any form for any degree or diploma to any tertiary institution. Where the use of published information from other authors has been made and it dully acknowledged in the text
Signed ………………………….
Sbonelo Sanele Hlengwa
I hereby certify that this statement is correct
Signed ………………………….
Professor Fanie R. van Heerden (Supervisor)
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Plagiarism
I, Sbonelo Sanele declare that:
1. The research reported in this thesis, except where otherwise indicated, is my original research.
2. This thesis has not been submitted for any degree or examination at any other university.
3. This thesis does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons.
4. This thesis does not contain other persons' writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then:
a. Their words have been re-written but the general information attributed to them has been referenced
b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced.
5. This thesis does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References sections.
Signed………………………… Date……………………… (Sbonelo Sanele Hlengwa)
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Acknowledgements
I would like to express my sincere graduate to:
My supervisor Professor Fanie van Heerden for her encouragements, advice guidance and support throughout this project, without you this theses would never have been a reality.
My lovely Family for always being there for me through tough times and supporting me
The Natural Products Research Group for creating a good atmosphere
The academic and the technical staff in the School of Chemistry and Physics for their assistance
National Research Funding for financial assistance
All friends for creating a good environment for me
“Ngiyabonga”
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Table of Contents
Isolation and characterisation of bioactive compounds from Antidesma venosum E.Mey. ex Tul. and Euphorbia cooperi N.E.Br. ex A.Berger
Abstract ...... i
Declaration ...... iii
Plagiarism...... iv
Acknowledgements ...... v
Table of Contents ...... vi
List of Figures ...... ix
List of Tables ...... xiii
List of schemes...... xv
Abbreviations ...... xvi
CHAPTER 1 ...... 1
Introduction and Aims of the Project ...... 1
1.1 African traditional medicine ...... 1
1.2 Natural products and drug discovery ...... 2
1.3 Problem statement ...... 4
1.4 Aims and objectives ...... 5
CHAPTER 2 ...... 6
A Literature Review on the Euphorbiaceae and Diterpenoids ...... 6
2.1 Euphorbiaceae (Spurge family) ...... 6
2.2 Tigliane diterpenes isolated from Euphorbiaceae plant species ...... 9
CHAPTER 3 ...... 16
Phytochemistry of Antidesma venosum E.Mey. ex Tul...... 16
3.1 Introduction ...... 16
3.1.1 Antidesma Genus ...... 16
3.1.2 Bioactive compounds from Antidesma ...... 16
3.1.3 Antidesma venosum E.Mey. ex Tul...... 18
3.2 Results and discussion ...... 19
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3.2.1 Structural elucidation of loliolide (37) ...... 20
3.2.2 Structural elucidation of lutein (38) ...... 25
3.2.3 Structural elucidation of pheophytin A (39) and pheophytin B (40) ...... 32
3.2.5 Structural elucidation of β-sitosterol 3-O-β-D-glucoside (41) ...... 34
3.2.4 Structural elucidation of compounds p-hydroxyphenylethyl trans-ferulate (42) and p-hydroxyphenylethyl cis-ferulate (43) ...... 39
3.3 Biological activity ...... 46
3.4 Conclusion ...... 47
3.5 Experimental ...... 48
3.5.1 General experimental procedure ...... 48
3.5.2 Plant material ...... 49
3.5.3 Extraction and isolation of compounds ...... 49
3.5.4 Biological assay ...... 51
3.5.5 Physical data of isolated compounds ...... 53
CHAPTER 4 ...... 54
Phytochemistry of Euphorbia cooperi N.E.Br. ex A.Berger ...... 54
4.1 Introduction ...... 54
4.1.1 Euphorbia subg. Euphorbia (E. subg. Euphorbia) ...... 54
4.1.2 Euphorbia cooperi N.E.Br. ex A.Berger ...... 56
4.3 Results and discussion ...... 57
4.3.1 Structural elucidation glutinol (47) ...... 58
4.3.1 Structural elucidation of compounds 48 and 49 ...... 63
4.3.4 Structural elucidation of compounds 51 and 52 ...... 71
4.4. Biological activity ...... 78
4.5 Conclusion ...... 79
4.6 Experimental ...... 79
4.6.1 General experimental procedure ...... 79
4.6.2 Plant material ...... 79
4.6.3 Extraction and isolation of compounds ...... 79
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4.6.4 Biological assay ...... 83
4.6.5 Physical data of isolated compounds ...... 83
CHAPTER 5 ...... 84
Conclusion and Future Work ...... 84
REFERENCES...... 86
APPENDIX ...... 96
Plate 1. 13C and DEPT-135 NMR spectra of compound 39...... 97
Plate 2. COSY NMR spectrum of compound 39...... 98
Plate 3. HSQC NMR spectrum of compound 39...... 99
Plate 4. HMBC NMR spectrum of compound 39...... 100
Plate 5. 13C and DEPT-135 NMR spectra of compound 40...... 101
Plate 6. COSY NMR spectrum of compound 40...... 102
Plate 7. HSQC NMR spectrum of compound 40...... 103
Plate 8. HMBC NMR spectrum of compound 40...... 104
Plate 9. COSY NMR spectrum of compound 47 ...... 105
Plate 10 COSY NMR spectrum of compound 50 ...... 106
Plate 11 HSQC NMR spectrum of compound 50 ...... 107
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List of Figures
Figure 1.1 Chemical structures of compounds used for medication 3
Figure 2.1 Some plant species of the Euphorbiaceae family 6
Figure 2.2 Sticky, irritant, milky sap latex 8
Figure 2.3 The core framework of diterpenoids from Euphorbiaceae 9
Figure 2.4 Skeletal structure of tigliane 9
Figure 2.5 12-Deoxyphorbol derivatives with antileukemic activity 12
Figure 2.6 12-Deoxyphorbol esters with potent cytotoxic activity against Ramos B 12
cells
Figure 2.7 The chemical structures of prostratin (13) and 2-deoxyphorbol 13- 13
phenyl-acetate (14)
Figure 2.8 Chemical structure of ingenol mebutate (15) and 3-caproyl ingenol (16) 13
Figure 3.1 The structures of antidesmanin A (24), antidemanin B (25) and 18
antidesmanin C (26)
Figure 3.2 Antidesma venosum E.Mey. ex Tul. 20
Figure 3.3 HR-ESIMS (positive mode) of compound 37 21
Figure 3.4 1H NMR spectrum of compound 37 21
Figure 3.5 13C NMR and DEPT-135 spectra of 37 22
Figure 3.6 The COSY and HSQC NMR spectrum of 37 23
Figure 3.7 Observed COSY correlations of compound 37 23
Figure 3.8 HMBC NMR spectrum of 37 24
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Figure 3.9 HR-ESI-(-)-MS spectrum of compound 38 26
1 Figure 3.10. H NMR spectrum of compound 38 in CDCl3 26
Figure 3.11. 13C and DEPT-135 NMR spectra of compound 38 (400 MHz) 27
Figure 3.12. HSQC and COSY NMR spectra of compound 38 (400 MHz) 27
Figure 3.13 Correlations observed in the 1H-1H COSY NMR spectrum of 28
compound 38
Figure 3.14. Long-range 1H-13C HMBC correlations of compound 38 (400 MHz) 28
Figure 3.15. HR-ESI-(+)-MS for compound 39 29
1 Figure 3.16. H NMR spectrum of compound 39 in CDCl3 30
1 Figure 3.17. H NMR spectrum of compound 40 in CDCl3 32
Figure 3.18. HR-ESI-(-)-MS of compound 41 33
1 Figure 3.19. H NMR spectrum of compound 41 in CDCl3 and CD3OD (400 MHz) 33
Figure 3.20. 13C and DEPT-135 NMR spectra of compound 41 34
Figure 3.21. Correlations observed in the COSY NMR spectrum of compound 40 35
Figure 3.22. Long-range 1H-13C HMBC NMR spectrum of compound 40 35
Figure 3.23: HSQC NMR spectra of compound 40 36
Figure 3.24: 1H NMR spectrum of compound 41 36
Figure 3.25: 13C and DEPT-135 NMR spectrum of compound 41 37
Figure 3.26: HSQC and COSY NMR spectrum of compound 41 37
Figure 3.27 Correlations observed in the 1H-1H COSY NMR spectrum of 37
compound 41
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Figure 3.28. Long-range 1H-13C HMBC correlation of compound 41 40
Figure 3.29. HR-ESIMS for compound 42 40
Figure 3.30. 1H NMR spectrum of compound 42 41
Figure 3.31. 1H NMR spectrum of compound 43 42
Figure 3.32. LCMS chromatogram of A. venosum leaves crude extract 43
Figure 3.33. LCMS chromatogram of A. venosum branches crude extract 44
Figure 3.34. Flow diagram for the isolation of compound 37, 41, 42 and 43 44
Figure 3.35. Flow diagram for the isolation of compound 38 and 40 45
Figure 3.36 HMBC correlations observed for compounds 42 and 43 45
Figure 3.37 Flow diagram of the isolation of compound 37, 41, 42 and 43 50
Figure 3.38 Flow diagram of the isolation of compound 38 and 40 52
Figure 4.1. Growth forms of Euphorbia subgenus 55
Figure 4.2. Euphorbia cooperi N.E.Br. ex A.Berger 56
Figure 4.3. HR-ESIMS (positive mode) of compound 47 58
Figure 4.4 1H NMR spectrum of compound 47 59
Figure 4.5 COSY correlations observed for compound 47 59
Figure 4.6 13C and DEPT-135 NMR spectrum of compound 47 60
Figure 4.7 HSQC NMR spectrum of compound 47 60
Figure 4.8 HMBC NMR spectrum of compound 47 61
Figure 4.9 1H NMR spectrum of compound 48 63
Figure 4.10 COSY NMR spectrum of compound 48 64
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Figure 4.11 13C and DEPT-135 NMR spectra of compound 48 64
Figure 4.12 HSQC NMR spectrum of compound 48 65
Figure 4.13 HMBC NMR spectrum of compound 48 65
Figure 4.14 HMBC NMR spectrum of compound 48 66
Figure 4.15 HR-ESI-()-MS of compound 50 67
Figure 4.16 1H NMR spectrum of compound 50 68
Figure 4.17 HMBC NMR spectrum of compound 50 69
Figure 4.18 HR-ESI-()-MS of compound 51 71
Figure 4.19 HSQC NMR spectrum of compound 51 71
Figure 4.20 HSQC NMR spectrum of compound 51 72
Figure 4.21 HMBC spectrum of compound 52 73
Figure 4.22. HR-ESI-()-MS of compound 52 73
1 Figure 4.23 H NMR spectrum of compound 52 in CDCl3 (400 MHz) 74
Figure 4.24 HSQC NMR spectrum of compound 52 74
Figure 4.25 COSY NMR of compound 52 75
Figure 4.26 HMBC NMR spectrum of compound 52 75
Figure 4.27 NOESY NMR spectrum of compound 52 76
Figure 4.28 Flow diagram of the isolation of compounds 47, 48, 50, 51 and 52 82
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List of Tables
1 13 Table 3.1. H (400 MHz) and C (100 MHz) NMR data of compound 37 in CDCl3 25
Table 3.2. 1H (400 MHz) and 13C (100 MHz) NMR data of compound 38 in CDCl3 30
1 13 Table 3.3. H (400 MHz) and C (100 MHz) NMR data of compound 41 in CDCl3 38
Table 3.4. 1H (400 MHz) and 13C (100 MHz) NMR data of compound 42 and 43 in 46
CDCl3
Table 3.5. Antibacterial activity results for A. venosum 47
Table 3.6. Fractions obtained by column chromatography of the DCM leaves crude 49 extract (A. venosum)
Table 3.7. Fractions obtained by column chromatography of the branch crude extract (A. 51 venosum)
1 13 Table 4.1. H (400 MHz) and C (100 MHz) NMR data for compound 47 in CDCl3 61
1 13 Table 4.2. H (400 MHz) and C (100 MHz) NMR data for compound 48 in CDCl3 and 70
50 in CD3OD
Table 4.3. 1H (400 MHz) and 13C (100 MHz) NMR data of compounds 51 and 52 in 77
CDCl3
Table 4.4. Antibacterial activity results 78
Table 4.5. Fractions obtained from the VLC fractionation of the crude extract of E. 79 cooperi
Table 4.6. Subfractions obtained from fractionation of the combined fractions F and G 80
Table 4.7. Sufbractions obtained by centrifugal thin-layer chromatography of subfraction 80
SSH-2-6D
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Table 4.8. Subfractions obtained from fractionation of subfration SSH-2-6E 80
Table 4.9. Subfractions obtained from the fractionation of the combined fractions 3D and 81
3E
Table 4.10. Subfractions obtained from fractionation of subfration SSH-15D 82
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List of schemes
Scheme 3.1 Photo-isomerisation of compounds 42 to 43 46
Scheme 4.1 Proposed biosynthetic pathway of compounds 48 and 50 69
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Abbreviations
AIDS Acquired immunodeficiency syndrome
13C Carbon-13
CAM Complementary alternative medicine
Calcd Calculated
COSY Correlation spectroscopy
C Degrees Celsius
CDCl3 Deuterated chloroform
CD3OD Deuterated methanol
DCM Dichloromethane d Doublet
DMSO Dimethyl sulfoxide
DEPT Distortionless enhancement by polarisation transfer
EtOAc Ethyl acetate g Grams
IR Infrared spectroscopy h Hours
Hex Hexanes
HIV Human immunodeficiency virus
HMBC Heteronuclear multiple-bond correlation
HR-ESIMS High-resolution electrospray ionisation - mass spectrometry
HPLC High-performance liquid chromatography
HSQC Heteronuclear single-quantum correlation
Hz Hertz
INT Iodonitrotetrazolium chloride
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LR-ESIMS Low-resolution electrospray ionisation mass spectrometry
MeOH Methanol
µg/mL Microgram per millilitre m Multiplet
MHA Mueller Hinton Agar
MHB Mueller Hinton Broth
1H Proton s Singlet
TB Tuberculosis
TM Trade mark t Triplet
UV Ultraviolet-visible
VLC Vacuum liquid chromatography cm-1 Wavenumber
WHO World Health Organisation
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CHAPTER 1
Introduction and Aims of the Project
1.1 African traditional medicine
The term traditional medicine1 and complementary alternative medicine2 are used interchangeably, depending on the cultural or ethnic groups interacting with traditional health practitioners. Complementary alternative medicine is mainly used in Western countries, whereas traditional medicine is mostly used in China and developing countries such as African countries.3 The World Health Organisation (WHO) defines traditional medicine as “Health practices, approaches, knowledge and beliefs incorporating plants, animals and mineral based medicine, spiritual therapies, manual techniques and exercises, applied singular or in combination, to treat, diagnose and prevent illnesses or maintain well-being”.4-5 The South African Traditional Health Practitioners Act 22 of 20076 describes traditional medicine as “An object or a substance that is used in traditional health practice for (i) the diagnosis, treatment or prevention of a physical or mental illness and (ii) any curative or therapeutic purpose, including the maintenance or restoration of physical or mental health or well-being in human beings but does not include a dependence-producing or dangerous substance or drug”.
Traditional medicine systems across the world are dynamic and variable because of different regions and countries of origins and the various agricultural systems in which they exist.5 The “Primary Health Care Declaration of Alma-Ata” identified primary health care as an important part of “Health for All” and, for the first time, traditional medicine was included as part of primary health care. Traditional medicine was subsequently recognised by the “Traditional Medicine Programme” of the WHO.6-7
In African countries, many people are treated with plant remedies by traditional healers. It is reported that in South Africa between 60% and 80% of the people consult traditional practitioners for primary health care.8-10 The reasons may be because some Western drugs are too expensive for many people to afford and in some areas, people must travel long distances to reach the nearest health facilities. The African continent has a vast variation in climate, plant species and cultures and has the human and natural resources to become leaders in the production of natural plant product and medicines.7
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1.2 Natural products and drug discovery
The use of natural products for medicinal purposes dates back practically to the start of human civilisation. Despite the advent of high-throughput synthesis and screening, and the post- genomic era, natural products will remain an important source of future drugs.11-15
Before the 20th century, the medications used to treat human illnesses were based on semi-pure and crude extracts from plants, animals, microbes and minerals. In the 20th century, it was discovered that chemical compounds in extracts were the main factors responsible for biological activity. This led to the hypothesis that the effect of a drug in the human body is mediated by a specific interaction of the molecule with biological macromolecules (proteins and nucleic acids).14
The unique chemical structures of natural products have inspired medicinal chemists to use them as scaffolds for the design of drugs and chemical probes.15 Natural products have been successfully employed in the discovery of new drugs and have exerted a significant impact in chemico-biology (the chemistry of metabolic processes). The efficacy of natural products is related to the complexity of the unique three-dimensional structures as well as steric properties, which offer many benefits in terms of efficiency and selectivity of molecular targets.16
Plants have been an important source of novel bioactive compounds with a diversity of pharmacologically active drugs being derived directly or indirectly from them. Despite the current progress by synthetic chemistry towards the discovery and manufacturing of drugs, the contribution of plants to disease treatment and prevention is still substantial. The pharmacological, clinical, and chemical studies of traditional medicines, which were predominantly derived from plants, led to the development of medications such as the painkiller drugs aspirin and morphine, the antimalarial drugs quinine and artemisinin, the glaucoma drug pilocarpine, the cardiac glycoside digitoxin, the anti-inflammatory, analgesic and antipyretic drug salicin, and the anticancer drug taxol. Natural products from microorganisms led to drugs such as the hypolipidemic agent mevastatin (Figure 1.1).14,17-23
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Figure 1.1. Chemical structures of natural products used for medication.
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1.3 Problem statement
Antimicrobial resistance occurs when a microorganism (virus, bacteria and some parasites) mutate when exposed to antimicrobial drugs. This way the medication becomes inactive and infections persist in the body. Antimicrobial resistance is a global concern as new resistance mechanisms are emerging and spreading globally, threatening the current treatment and thus resulting in prolonged illnesses of influenza, HIV, malaria and tuberculosis (TB), which eventually causes death. In South Africa, TB continues to be the leading cause of death. About 25 000 deaths caused by TB were recorded in 2015 by the WHO, and these excluded those people who had both HIV and TB infections when they died, as they were considered to have been killed by HIV.24-25 This urges the discovery of new antimicrobial agents.
The human immunodeficiency virus (HIV) is a retrovirus that attacks human immune cells called CD4+ T cells and causes HIV infections and over time acquired immunodeficiency syndrome (AIDS). HIV/AIDS has killed 1 million people in 2016 worldwide.26 South Africa has the biggest HIV epidemic in the world, with about 7.1 million people living with HIV and a prevalence rate of approximately 18.9% among the general population.27 Accordingly, many efforts have been made to develop anti-HIV drugs. Although some drugs have been used successfully in the treatment of HIV infections, such as antiretroviral therapy,28 there are still problems with side effects and drug resistance. Furthermore, the current drugs can keep the viral load of a patient low but cannot eradicate the virus completely. Therefore, new and more potent anti-HIV agents are still needed.
Cancer is an abnormal multiplication of cells in an uncontrolled manner. South Africa is ranked 50th on the World Cancer Research Fund’s list of countries with the highest cancer prevalence rates. More than a 100 000 patients in South Africa are diagnosed with cancer each year, and recent studies predict that South Africa could see an increase in the number of cancer cases by 78% in 2030.29 Treatments for cancer include chemotherapy, radiotherapy and surgery. However, the prevention of tumour recurrence and the spread of cancer cells is difficult to achieve using these methods. Furthermore, tumour resistance to chemotherapeutic drugs is a serious problem and requires the discovery of novel adjuvant therapies.
With the goal of discovering new lead compounds that can be developed into new bioactive drugs that can be used for the treatment of life-threatening diseases, we investigated some plant species within the Euphorbiaceae family, indigenous to South Africa. This plant family is known to contain diterpenes, amongst other bioactive compounds, as the major metabolites
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with different unique structures of which some display a range of biological activities including anti-inflammatory, cytotoxic, antimicrobial, anti-HIV, and modulation of multidrug-resistance activities. Two plants species within this family were selected based on the mentioned phytochemical studies and the ethnopharmacological studies mentioned in the literature review, namely, Antidesma venosum E.Mey. ex Tul and Euphorbia cooperi N.E.Br. ex A.Berger.
The research questions that I would like to address are:
Which bioactive compounds are present in South African Euphorbiaceae species? What are the structures of these compounds? How much of these compounds can be extracted or isolated from a given mass of plant material? Can these compounds be used for the treatment for HIV, cancer and other diseases?
1.4 Aims and objectives
This project aimed to isolate bioactive compounds from indigenous Euphorbiaceae species and to investigate the structures and biological activities of these compounds.
The objectives of this project were
To briefly review Euphorbiaceae species and diterpenoids present in this family Identify species that have not been extensively investigated and of which plant material are readily available Isolate compounds from the plant extracts and elucidate the structures Investigate antibacterial activities of the crude extracts.
In this theses, a literature review on the Euphorbiaceae family and diterpenoids is given in Chapter 2. Chapters 3 and 4 discuss the isolation and characterisation of compounds from A. venosum and E. cooperi, respectively. The overall conclusion and future work is presented in Chapter 5.
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CHAPTER 2
A Literature Review on the Euphorbiaceae and Diterpenoids
2.1 Euphorbiaceae (Spurge family)
Euphorbiaceae (Spurges) is the sixth largest flowering plant family consisting of more than 300 genera and about 7 500 species, Orchidaceae being the largest flowering plant with more than 19 000 species.30-32 This family has been admired for its life (growth) forms, including a variety of xerophytic species. Despite the great diversity of plants within the family, the plant members are united morphologically by the possession of a cyathium, a highly reduced inflorescence that resembles a single flower.33
Spurges are an extraordinarily diverse group of dioecious or monoecious herbs, shrubs, geophytes, understory and canopy trees, vines, sometimes succulent or trees as well as an array of succulent and xerophytic forms. The leaves are generally alternate. The flowers are unisexual with 0-5 petals and 2-6 sepals; usually three carpels (tricarpellate ovary), three styles (sometimes divided), one ovule per locule, inflorescences often highly modified, fruits are schizocarp and seeds are usually arillate.32,34
This family is known to have a wide range of beautiful and useful succulent plant species, including lawn weeds, cactus-like plant and poinsettias (Euphorbia pulcherrima); ornamental trees such as Codiaeum, sandbox tree (Hula crepitans and H. polyandra), copperleaf (Acalypha hispida), Phyllanthus, redbird cactus (Pedilanthus tithymaloides) and Jatropha. Some of the plant members of the family are of economic importance, such as castor-oil plant (Ricinus communis), croton (Croton tiglium), Omphalea, cassava (Manihot esculenta), rubber (Hevea brasiliensis), tung tree (Aleurites fordii) and tallow tree (Sapium sebiferum) (Figure 2.1.).35
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http://www.llifle.com/Encyclopedia/SUCCULENTS/Family/Euphorbiaceae/24098/Pedilanth https://www.rhs.org.uk/Plants/88057/Euphorbia-pulcherrima/Details https://en.wikipedia.org/wiki/Acalypha_hispida us_tithymaloides
Euphorbia pulcherrima Acalypha hispida Pedilanthus tithymaloides
https://www.helios.co.uk/shop/croton-tiglium https://www.ebay.co.uk/itm/Tapioca-Tree-Cassava-Manihot-dulcis-12-Fresh-Seeds- http://www.horizon-custom-homes.com/HeveaBrasiliensis.html /380743890184
Croton tiglium Hevea brasiliensis Manihot esculenta
https://plants.ces.ncsu.edu/plants/all/aleurites-fordii/ https://www.ebay.com/itm/SAPIUM-Sebiferum-Chinese-Tallow-Tree-Seeds-ES-33- http://www.ethnopharmacologia.org/recherche-dans-prelude/?plant_id=4560 /221989637711
Aleurites fordii Phyllanthus niruri Sapium sebiferum
Figure 2.1. Some plant species of the Euphorbiaceae family.
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Despite the vegetative variation, the plant family is united by a distinctive morphological synapomorphy, the cyathium, a pseudanthial inflorescence that looks typically like a dicot flower. This structural form is an intermediate between a flower and an inflorescence in development terms.33
One of the significant characteristics of spurges is the sticky, irritant, milky sap called latex and common names are milky mangrove, buta-buta, and gewa (Figure 2.2).36-37 It is poisonous in the sub-family Euphorbioideae, and innocuous in the sub-family Crotonoideae.33 This type of fluid is found in trees, shrubs, lianas, both xerophytic succulents and water plants, it is more typically contained in tubes and cells which are collectively known as laticifer.36 The plants containing this latex use it as a defence mechanism for repelling browsing animals and insects, as well as killing and controlling the growth of microbial phytopathogens, and sealing wounded areas. It also contains low molecular weight rubber.38 For many plants like E. tirucalli and E. cooperi the latex is poisonous and possesses irritant properties toward the skin and mucous membranes. An example of a latex that is used medicinally is Euphorbium. Euphorbium is the air-dried latex of E. resinifera Berg. and is an over-the-counter drug used to suppress chronic pain, treat dental cavities to mitigate toothache, and treat tuberculosis, amongst others.37
Euphorbiaceae latex contains a wide variety of classes of compounds including flavonoids, phenolic acids,39-40 tannins,41 diterpenoids, and triterpenoids, with diterpenoids being the dominant class of compounds.42 It has been shown that the highly unsaturated irritant phorbol esters are the main constituents responsible for the cytotoxicity of the latex.42
Figure 2.2. Sticky, irritant, milky sap latex.
The diterpenoids found in the Euphorbiaceae (also in the Thymelaeaceae) can be divided into six classes (based on the core framework), which are the tiglianes, ingenanes, daphnanes, lathyranes, jatrophanes, and abietanes (Figure 2.3).43
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Figure 2.3. The core framework of diterpenoids from Euphorbiaceae.
These classes of diterpenoids display a wide variety of biological activities, including anti-HIV, tumour-promoting, skin irritant, cytotoxic, and proinflammatory activities. The tiglianes are the compounds with the most exciting and diverse biological activities.44-46
2.2 Tigliane diterpenes isolated from Euphorbiaceae plant species
The core structure of the tigliane diterpenoids isolated from Euphorbiaceae has a tetracyclic ring system consisting of a five-membered ring A, a seven-membered ring B, a six-membered ring C and a cyclopropane ring D. The skeleton system contains twenty carbons which consist of five methyls, five methylenes, nine methines and one quaternary carbon (Figure 2.4). Generally, compounds belonging to this structural class of compounds contain two double bonds, one in ring B and another one in ring A. Ring A also contains an α,β-unsaturated ketone.47
Figure 2.4. The skeletal structure of tigliane.
A common tigliane diterpenoid, phorbol (1), was initially isolated from Croton tiglium.48 There are many tigliane diterpenoids derivatives derived from phorbol, including 4-deoxyphorbol, 4,12-dideoxyphorbol, 4,20-dideoxyphorbol, 4,12,20-trideoxyphorbol, and 12-deoxyphorbol. The derivatives generally have hydroxy groups at C-12, C-13, C-16, and C-20, which can be readily esterified during biosynthesis. Tigliane derivatives derived from phorbol occur naturally as 13-monoesters, 12,13-, 13,16-, or 12,20-diesters, and 12,13,20- or 13,16,20-
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triesters, which have been esterified with acetic acid, isobutyric acid, tiglic acid, 2- methylbutyric acid, benzoic acid, 2-methylaminobenzoic acid or saturated and unsaturated long-chain aliphatic fatty acids.47
Phorbol (1)
Some of the reported tigliane diterpenoids exist as aglycons. Only three tigliane glycosides, 2, 3 and 4, have been isolated so far from plant species belonging to the Euphorbiaceae and Thymelaeaceae families.49
Tigliane diterpenoids possess a wide variety of biological activities including anti-HIV, anticancer, tumour-promoting and pro-inflammatory activities.47 It has been reported that the active principles responsible for the irritancy of skin and mucous membranes caused by plant members of the Euphorbiaceae family are tigliane diterpenoids. Unlike other tigliane diterpenoids, phorbol (1) did not have irritant properties when tested in the mouse ear assay.50 In this study, the measures of irritancy towards the skin of a mouse ear were in irritant dose 50
(ID50) units. Phorbol 12-tetradecanoate 13-acetate (TPA) (5) displayed the highest irritant activity of (ID50 = 0.016 nmol/ear) in this assay. The ID50 results of other related phorbol derivatives showed that an increase in the number of carbon atoms or an increase in the number of double bonds in a fixed number of carbon atoms in the acyl moiety leads to an increase in irritant activity of the investigated compounds.50
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Phorbol 12-tetradecanoate 13-acetate (TPA) (5)
There is a significant interest in tigliane diterpenes because of their tumourigenic and cocarcinogen activities. Some of the naturally occurring diterpenoids cocarcinogens are potent skin blistering and reddening agents. Some are classified as tumour promoters, but that does not imply that these are also cocarcinogens. Only after the initiating agents have initiated carcinogenesis can the tumour promoters function, whereas cocarcinogens enhance tumour yield when applied together with carcinogens.51 TPA was reported not to be a carcinogen, but a cocarcinogen that was classified as a tumour promoter. It has been reported that of all the 13- monoester derivatives of 12-deoxyphorbol tested so far, only 12-deoxyphorbol 13- tetradecanoate (6) has shown a level of cocarcinogenic activity similar to that of TPA. In contrast to TPA, 12-deoxyphorbol esters possess intermediate hydrophobicity with tigliate, isobutyrate, and phenylacetate esters and these are regarded as inflammation agents but only weakly tumour promoting or nonpromoting. Esters of 12-deoxy-16-hydroxy-phorbol have been shown to be weaker tumour promoters than TPA.52
12-Deoxyphorbol 13-tetradecanoate (6)
Although tigliane diterpenoids exhibit tumour-promoting activities, studies have revealed that some derivatives show significant anticancer activity. 12-Deoxyphorbol derivatives 7-9, isolated from Baliospermum montanum, exhibited antileukaemic activity with ED50 values ranging from 0.4 to 3.4 μg/mL in a P-388 lymphocytic leukaemia system in vitro.53
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Figure 2.5. 12-Deoxyphorbol derivatives with antileukaemic activity.
The 12-deoxyphorbol esters 10-12, isolated from Euphobia fischeriana, showed potent cytotoxic activity against Ramos B-cells. The activity was enhanced by the presence of the long acyl chain at C-13 and a free hydroxyl group at C-20 when compared to the other compounds tested on the same cells. These phorbol esters also exhibited non-tumour- promoting activity.54-55
Figure 2.6. 12-Deoxyphorbol esters with potent cytotoxic activity against Ramos B cells.
Tigliane diterpenoids possess remarkable anti-HIV activity. Current HIV medication leads to a substantial reduction of actively replicating viruses but cannot eradicate reservoirs with latent HIV and patients need to use these drugs on a continuing basis. Prostratin (13) is a protein kinase C (PKC) activator isolated from the bark of mamala tree of Samoa, Homalanthus nutans (Euphorbiaceae). Prostratin is a preclinical candidate that has the potential to eliminate latently infected CD4+ T cells through a virus-induced cytopathic effect or host anti-HIV immunity. Derivatives of 13 are more potent than the preclinical lead (prostratin) in binding to cell-free PKC and inducing HIV expression in latently infected cells; 2-deoxyphorbol 13- phenylacetate (14) is one of them. Recent studies have shown that this compound is ten-fold more potent than 13 in the PKC binding assays, 20-40 times more potent at inducing viral production in latently infected cells and at least five times more potent at inducing viral expression from human peripheral blood mononuclear cells.17,43,47,56-58
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Figure 2.7. The chemical structures of prostratin (13) and 12-deoxyphorbol 13-phenylacetate (14).
Other classes of diterpenoids possess some interesting biological activities. Ingenol mebutate (ingenol 3-angelate) (15), an ester of the diterpene ingenol and angelic acid, was extracted from the latex of E. peplus.59 Several controlled studies have shown that 15 can be used in the treatment of basal cell carcinoma, non-melanoma skin cancer, and actinic keratoses and for the reactivation of latent HIV at a nanomolar level in primary CD4+ T cells from HIV infected individuals receiving ART.59-60 Ingenol mebutate (15) has been approved by the United States Food and Drug Administration (FDA) and the European Medicines Agency for the topical treatment of actinic keratosis.
Figure 2.8. Chemical structure of ingenol mebutate (15) and 3-O-caproyl-ingenol (16).
Another semi-synthetic ingenol ester is 3-O-caproylingenol (16), which is more potent in reactivating latent HIV than known activators such as ingenol 3,20-dibenzoate (17), hexamethylene bisacetamide (18) and suberoylanilide hydroxamic acid (19).61
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A bioactive compound, euphol (20), which was extracted from E. tirucalli latex, is an euphane- type triterpene alcohol with anti-inflammatory activities on human gastric cancer cells. It exhibits cytotoxic effects against a number of cancer cell lines, with most prominent effects against oesophageal squamous cells, pancreatic cell carcinomas, and human gastric cancer. The literature reveals that a topical application of euphol suppresses tumour-promoting effect in 2- stage carcinogenesis in mouse skin. However, the mechanisms explaining this effect as well as the antitumour properties of euphol remain to be evaluated.62-65
Euphol (20)
Recently, a novel tetracyclic triterpenoid with an unusual spiro scaffold, spiropedroxodiol (21), was isolated from Euphorbia pedroi. The compound displayed strong multidrug-resistance reversal activity in both L5178Y-MDR and human colo320 cells. In combination assays, compound 21 enhanced synergistically the cytotoxicity of doxorubicin (22), which was the positive control in the assay.66
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It is clear that the plant members of the Euphorbiaceae have the potential of producing interesting biologically active compounds. The family contain diterpenoids as the major metabolites and the activities of these compounds include anti-HIV, anti-cancer and antimicrobial activity. The Euphorbiaceae are well presented in South Africa, with at least 188 Euphorbia species recorded as indigenous to the country. Furthermore, there are other genera in the family, such as Antidesma, Acalypha and Croton, amongst others, that are indigenous to South Africa. The phytochemistry of many Euphorbiaceae has not been recorded. Furthermore, the biological activities of many of the diterpenoids that have been isolated, are not known.
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CHAPTER 3
Phytochemistry of Antidesma venosum E.Mey. ex Tul.
3.1 Introduction
3.1.1 Antidesma Genus
Antidesma L. is a genus that belongs to the subtribe Antidesminae in the subfamily Phyllanthoideae within the Euphorbiaceae family, and the plants are often found in the understorey of tropical rain forests or shrubby vegetation. Its distribution ranges from West Africa to the Pacific Islands and from the Himalayas to Northern Australia with the centre of diversity in Southeast Asia (Malaysian region). The genus comprises approximately 110 species worldwide.67
The species of the genus are either short and shrubby or tall and erect and can be up to 30 metres in height. The large, oval-shaped, leathery and evergreen (sometimes deciduous) leaves are up to 20 centimetres long and 7 centimetres wide with fine hairs underneath. The leaves grow in an alternate arrangement, with entire, symmetrical blades connected to the stem with petioles and stipules. Flowers of this genus have a strong and unpleasant scent, and the colours are light yellow-greenish, which may turn red when mature. This species is unisexual, with male and female flowers on separate plants. The staminate flowers are arranged in small bunches, and the pistillate flowers grow on long racemes with a single bract per flower on a short pedicel which becomes the strands of fruits. The spherical fruits are less than one centimetre wide, hanging singly or paired in long, heavy bunches. The fruits are white when immature and gradually turn red and then black when fully matured.68-69
3.1.2 Bioactive compounds from Antidesma
Plant species belonging to this genus contain a variety of bioactive compounds including phenolic compounds, coumarins, tannins, and alkaloids, amongst others, of which some displayed antioxidant, antimicrobial and cytotoxic activities.70-72
Acidumonate (23), isolated from Antidesma acidum Retz., is a monophenolic compound comprising of two ketone groups, two methoxy groups and one methyl ester moiety. It was reported that this compound displays promising cytotoxicity against both Hb and HeLa cancer 73 cells with IC50 values of 2.60 and 3.80 µg/mL, respectively.
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Acidumonate (23)
Antidesmanin A (24), antidesmanin B (25) and antidesmanin C (26) are coumarinolignans that were isolated from Antidesma pentandrum var. barbatum (C.Presl) Merr. These compounds showed marginal cytotoxicity against MCF-7 (breast) and SF-268(CNS) cancer cells in vitro.74
Antidesmone (27) is a unique tetrahydroquinoline alkaloid that was isolated from Antidesma membranaceum Müll.Arg.75 This alkaloid was also found in the stems of Waltheria douradinha A.St.-Hil.76 and Waltheria indica L.77 (Malvaceae) and is used for the treatment of respiratory disorders, urinary problems and wounds. Antidesmone is also present in the roots of Melochia chamaedrys (Sterculiaceae), which is commonly used in South Africa as a herbal medicine.78
Antidesmone (27)
Two alkaloids, clauszoline B (28) and 7-methoxymukonal (29), were recently isolated from the leaves of A. acidum. These compounds are significantly cytotoxic towards HL-60 cancer cells with IC50 values of 4.8±0.2 and 8.0±0.9 μM, respectively, but the compounds did not inhibit the growth of HEL-99, a normal cell line. Compound 28 induced apoptosis via alteration
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of expression of apoptosis-related proteins and decreased phosphorylation of AKT in HL-60 cells.79
3.1.3 Antidesma venosum E.Mey. ex Tul.
Antidesma venosum E.Mey. ex Tul. (Euphorbiaceae) (Figure 3.1) is known as isiBangamlotha (isiZulu), tassel-berry (English), voëlsitboom (Afrikaans).44 A. venosum is an evergreen tree that can grow up to 15 m in height. The oval to elliptical leathery leaves, up to 150 mm long and 70 mm broad, are bright glossy green above and hairy below. The reddish flowers have an unpleasant smell and attract insects and insect-eating birds. Fruits are tiny, almost oval, about 8-10 mm in diameter, whitish when young but turn green to red and finally black when matured.70,80 This plant species occurs in coastal areas of some tropical countries with sandy soils. In South Africa, the plant is found in Eastern Cape, KwaZulu-Natal, Mpumalanga and Limpopo.80
Figure 3.1. Antidesma venosum E.Mey. ex Tul.
A. venosum is used for decorative purposes in large gardens and bird parks. The wood of the tree is used for building huts, for firewood, knife sheaths and tool handles. The leaves and roots are used for treating snakebites, abnormal pain, hookworm and heart diseases.70
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The literature reveals only one report on the phytochemistry of the plant81 and five reports on the biological screening of the plant extract.71-72,82-84 According to the phytochemical report,81 eight compounds, friedelin (30), epifriedelanol (31), betulinic acid (32), toddaculin (33), α- tocopherol (34), pheophytin A, presqualene alcohol (35), and its acetate derivative (36), were isolated from the root bark of the plant. Qualitative phytochemical screening of A. venosum revealed the presence of four classes of compounds namely, alkaloids, flavonoids, saponins and tannins from the stem bark of the plant. The stem bark extract of the plant showed strong antimicrobial activity.71
3.2 Results and discussion
Based on the literature reports on the plant,71-72,81-84 only the roots were previously investigated for secondary metabolites. The leaves and the stems were only assayed for biological activity, and based on that it was decided that both the leaves and stems should be further investigated secondary metabolite. The leaves of A. venosum should be extracted separately with DCM and MeOH. The leaves were extracted with DCM. MeOH extraction of the leaves was omitted due to time constraints. The stems were extracted with a DCM-MeOH mixture (1:1, v/v). The crude extracts were subjected to column chromatography using a mixture of Hex and EtOAc for the leaves and DCM with MeOH for the stems as eluents. Further purification of the fractions
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obtained from column chromatography and centrifugal thin-layer chromatography afforded compounds 37 - 40 from the leaf extract, and 41 and 42 from the branch extract. The structures were elucidated using 1H NMR, 13C NMR, DEPT-135, COSY, HSQC, HMBC and TOF-MS.
3.2.1 Structural elucidation of loliolide (37)
37
Compound 37 was isolated as a white powder. In the HR-ESI-(+)-MS spectrum (Figure 3.2) a sodium adduct of the molecular ion was observed at m/z 219.0992, corresponding to a molecular formula of C11H16O3 (calcd for C11H16O3Na, 219.0997).
Figure 3.2. HR-ESI-(+)-MS spectrum of compound 37.
The 1H NMR spectrum of compound 37 (Figure 3.3) showed the presence of a vinylic proton at δH 5.69 (H-3, s) and two pairs of diastereotopic methylene protons at δH 2.46 (1H, dt, J = 14.1 and 2.7 Hz, H-5ax) and δH 1.72 (1H, dd, J = 13.4 and 4.1 Hz, H-5eq), as well as δH 1.98 (1H, dt,
J = 14.4 and 2.5 Hz, H-7ax) and δH 1.53 (1H, dd, J = 14.5 Hz, 3.5 Hz, H-7eq) (the latter resonance was partly obscured by a residual water resonance). Three methyl proton resonances were observed as singlets at δH 1.27 (H-9, s), 1.47 (H-10, s) and 1.78 (H-8, s). A residual resonance for DCM was observed at δH 5.30.
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1 Figure 3.3. H NMR spectrum of compound 37 in CDCl3 (400 MHz).
The 13C NMR and DEPT-135 spectra (Figure 3.4) showed twelve carbon signals. Three sp2 carbon resonances were observed at δC 182.6, δC 172.1 and δC 113.2, corresponding to a carbonyl 13 and two alkene carbons. A C chemical shift of δC 172.1 is in agreement with a lactone carbonyl. The other two resonances were assigned to an α,β-unsaturated carbonyl moiety. The chemical shift of the carbon signal resonating at δC 86.8 indicated a carbon bearing an electronegative atom, most likely oxygen. The last quaternary carbon resonating at δC 36.1 was assigned to a carbon bonded to alkyl groups. Two methylene carbons, resonating at δC 47.6 and δC 45.9 were observed as negative signals in the DEPT-135 spectrum. A methine carbon resonating at δC 67.1 and three methyls at δC 30.9, δC 27.2 and δC 26.7 were observed in the spectrum.
13 Figure 3.4. C NMR and DEPT-135 spectra of 37 in CDCl3 (100 MHz).
The HSQC NMR spectrum (Figure 3.5) was useful in unravelling the C-H framework of compound 37. From the spectrum the assignment of three methyl groups (δH 1.27, 1.47 and 1.78;
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δC 30.9, 26.2 and 27.2, respectively), two methylene groups (δH 2.46, 1.72 and δH 1.98, 1.53; δC
45.9 and 47.6, respectively), one vinylic group (δH 5.69, δC 113.2) and one oxygen-bearing C-H 1 1 group (δH 4.32, δC 67.1) was possible. The H- H COSY NMR spectrum (Figure 3.6) showed the correlations of the diastereotopic methylene protons (δH 2.46 (Heq) and δH 1.72 (Hax), δH 1.98
(Heq) and δH 1.53 (Hax)) with the proton at δH 4.32 (red). Each pair of diastereotopic protons showed a correlation with the geminal proton and, in addition, a W-coupling (4-bond coupling) was observed between the two equatorial protons (green). The chemical shift of the resonance at
δH 4.32 indicated that the proton was attached to an oxygen-bearing carbon atom. The splitting pattern is a quintet with a coupling constant of J = 3.4 Hz, characteristic of an equatorial proton. The coupling constants and the differences in chemical shifts observed in the 1H NMR spectrum as well as the COSY correlations of the diastereotopic protons indicated that these protons were most likely part of a six-membered ring in a chair conformation (Figure 3.7).
Figure 3.5. HSQC NMR spectra of compound 37.
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Figure 3.6. COSY NMR spectrum of compound 37
.
Figure 3.7. Observed COSY correlations for compound 37.
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Figure 3.8 HMBC NMR spectrum of 37.
A 1H-13C HMBC NMR spectrum (Figure 3.8) was useful in assembling the final pieces of the structure of compound 37. 2J and 3J C-H couplings were observed in this spectrum. Correlations between H-3 and the four quaternary carbons, δC 182.6, 172.1, 86.8, and 36.1 (assigned to C-3a,
C-2, C-7a and C-4, respectively) were observed (blue circles). The proton resonating at δH 4.32
(H-6) (δC 67.1, C-6) showed correlations to two quaternary carbons (δC 36.1 and δC 86.8, C-4 and C-7a, respectively) (orange circles). Correlations between the two pairs diastereotopic protons (H-5 and H-7) and multiple carbons in the compound were observed (green and red circles). The 1 13 H- C correlations of the two methyl resonances at δH 1.47 (δC 26.7) and δH 1.27 (δC 30.9) to the same quaternary carbon nuclei (δC 36.1) (purple circles) and to each other, confirmed the presence of a geminal dimethyl moiety. The structure was assigned as a six-membered ring attached to a five-membered lactone ring. Thus, 37 was identified as loliolide and the spectroscopic data were in agreement with the literature values.85-87 The NMR data are collated in Table 3.1
Loliolide (37) is one of the degradation products of carotenoids and was first isolated from Fumaria officinalis in 193888 but the chemical structure was only elucidated in 1964 by Hodges and Porte89 and it was synthesised for the first time by Marx and Sondheimer in 1966.90 This compound is also present in marine organisms and algae. In marine algae, it is reported that 37 is a photo-oxidation product of algal carotenoids and as such, it is used by phytoplankton in sediments as a biomarker for photo-oxidative alterations and is regarded as a phytotoxic compound with various effects such as inhibition of algal growth and germination, as well an
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ant-repelling activity.91 Recent studies have classified loliolide as a potent inhibitor of hepatitis C virus entry.92 Loliolide has showed an affinity to the serotonin transporter in a study to identify natural products with anti-depression activity.93
1 13 Table 3.1. H (400 MHz) and C (100 MHz) NMR data of compound 37 in CDCl3.
δC (ppm) literature Position δH (ppm) (J in Hz) δC (ppm)
2 - 172.1 171.2
3 5.69 (1H, s) 113.2 112.9
3a - 182.6 182.8
4 - 36.1 36.1
5ax 1.54 (1H, dd, 13.4, 4.1) 47.6 47.2
5eq 1.98 (1H, dt, 14.1, 2.7)
6 4.32 (1H, q, 3.4) 67.1 66.7
7ax 1.79 (1H, dd, 14.5, 3.5) 45.9 45.4
7eq 2.46 (1H, dt, 14.4, 2.5)
7a - 86.8 86.9
8 1.78 (3H, s) 27.2 26.8
9 1.27 (3H, s) 30.9 30.6
10 1.47(3H, s) 26.7 26.5
3.2.2 Structural elucidation of lutein (38)
38
Compound 38 was isolated as an orange, amorphous powder. In the HR-ESI-(-)-MS spectrum (Figure 3.9), a quasi-molecular ion [M-H]- was observed at m/z 567.4180, corresponding to a
molecular formula of C40H56O2 (calcd for C40H55O2, 567.4202).
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Figure 3.9. HR-ESI-(-)-MS spectrum of compound 38.
The 1H NMR spectrum (Figure 3.10) revealed the presence of a long conjugated chain of vinylic protons resembling a carotenoid-type compound with resonances downfield between
δH 6.12-6.67. Two signals resonating at δH 4.01 and δH 4.25 were assigned to protons bonded to two oxygen-bearing carbons. Eight methyl singlets were observed upfield, two of them having similar intensities and each integrated for six protons, which gave a total of ten methyl groups.
1 Figure 3.10. H NMR spectrum of compound 38 in CDCl3 (400 MHz).
The 13C NMR and DEPT-135 spectra (Figure 3.11) showed 40 carbon resonances, which were identified by the DEPT-135 and HSQC spectra (Figure 3.12) as nine quaternary carbons,
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two methines (oxygen-bearing carbons), fifteen olefinic carbons, two methylene carbons and ten methyls.
Figure 3.11. 13C and DEPT-135 NMR spectra of compound 38 (100 MHz).
Figure 3.12. HSQC NMR spectrum of compound 38.
The 1H-1H COSY (Figure 3.13) NMR spectrum showed the correlations between protons resonating at δH 5.43 and δH 2.43 as well as δH 6.16, δH 6.64 - 6.68 and both 6.38, δH 6.12, δH
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5.55 and both δH 4.25 and δH 1.63. These correlations confirmed the presence of an extended 1 1 conjugated chain. H- H coupling was observed between the geminal protons Heq and Hax (δH
1.88 and δH 1.37) and δH 4.25, confirming the presence of a hydroxylated six-membered ring.
A similar correlation was observed between the proton resonating at δH 4.01 and a pair of
methylene protons, Hax′ and Heq′ (δH 1.48 and δH 1.77) and also with two methylene protons
resonating at δH 1.26, thus confirming the presence of a second six-membered ring (Figure 3.14).
Figure 3.13. COSY NMR spectrum of compound 38
.
Figure 3.14. Correlations observed in the 1H-1H COSY NMR spectrum for compound 38.
The long-range 1H-13C correlations observed in the HMBC NMR spectrum (Figure 3.15) was useful in the determination of the structure. Correlations between the methyl protons
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resonating at δH 0.85 and δH 1.00 and a tertiary carbon resonance at δC 55.0, a quaternary carbon resonance at δC 33.9 and a secondary carbon resonance at δC 44.5 (C-2) further confirmed the presence of a six-membered ring with 1 degree of unsaturation (Figure 3.16). The second ring which was confirmed by the correlation between the overlapping methyl protons resonating at δH 1.08 (δC 28.6 and δC 30.1), and two secondary carbons (δC 48.3 and
δC 29.6) and one quaternary carbon (δC 37.1). The difference between the two rings was the position of the double bond. The HMBC correlation of the olefinic protons and methyls in the conjugated chain confirmed the presence of a carotenoid-like conjugated chain. Thus compound 38 was identified as lutein and the spectroscopic data were in agreement with the literature values.94-95 The NMR data are collated in Table 3.2
Figure 3.15 HMBC NMR spectrum of compound 38.
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Figure 3.16. Long-range 1H-13C HMBC correlations observed for compound 38.
Lutein is a naturally occurring carotenoid that is classified as a xanthophyll and is synthesised only by higher plants. The plant uses it as a modulator of light energy and as a non- photochemical quenching agent to reduce the overproduction of triple chlorophyll during photosynthesis.96 In humans, lutein, together with zeaxanthin, accumulates in the retina of the eye as well as in the brain.97 These carotenoids play an essential role in human vision by preventing hypoxia-induced cell damage in the eye, by reducing the amount of the blue light that reaches the photoreceptors, preventing age-related macular degeneration, and has antioxidant and anti-inflammatory properties.98 Lutein induces the α-1,3-glucan accumulation on the cell wall surface of fungal plant pathogens.95
1 13 Table 3.2. H (400 MHz) and C (100 MHz) NMR data of compound 38 in CDCl3.
Position δH (ppm) (J in Hz) δC (ppm) δC (ppm) literature 1 - 33.9 34.0
2ax 1.37 (1H, d, 2) 44.5 44.7
2eq 1.84 (1H, dd, 4, 6) 3 4.25 (1H, brs) 65.0 65.9 4 5.55 (1H, brs) 124.4 124.9
5 - 126.0 126.2
6 2.43 (1H, brs) 55.0 55.0 7 5.43 (1H, dd, 10, 6) 128.6 128.7
8 6.16 (1H, d,5) 137.6 137.9
9 - 134.9 135.7 10 6.12 (1H, brs) 138.4 -
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11 6.64-6.68 (1H, m) 129.9 129.9 12 6.38 (1H, brs) 137.5 12.8 13 - 135.5 137.6 14 6.24-6.26 (1H, d, 10) 132.4 132.6 15 6.57-6.62 (1H, m) 124.8 - 16 0.85 (3H, s) 24.2 - 17 1.00 (3H, s) 29.4 30.3 18 1.63(3H, s) 22.7 22.9 19 1.74 (3H, s) 21.5 20.7 20 1.26 (3H, s) 22.7 - 1′ - 37.0 37.1 2′ 1.26 (2H, s) 29.6 - 3′ 4.01 (1H, brs) 65.9 65.9
4′ax 1.48 (1H, t, 12) - 48.3 4′eq 1.77 (1H, t, 4) - 5′ - 126.0 126.3 6′ - 137.8 137.6 7′ 6.15* 130.7 - 8′ 6.16 (1H, d, 5) 137.6 - 9′ - 136.3 137.6 10′ 6.12 (1H, brs) 125.5 - 11′ 6.64-6.68 (1H m) 124.8 124.5 12′ 6.33 (1H, d) 137.5 137.6 13′ - 136.2 137.6 14′ 6.24-6.26 (1H, m) 132.4 132.6
15′ 6.57-6.62 (1H, m) 124.8 - 16′ 1.08 (3H, s) 30.1 29.5
17′ 1.08 (3H, s) 28.6 12.8 18′ 0.88 (3H, s) 14.0 - 19′ 1.91(3H, s) 12.7 12.11 20′ 1.97 (3H, s) 13.0 12.8
* Undefined multiplicity due to overlapping
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3.2.3 Structural elucidation of pheophytin A (39) and pheophytin B (40)
Compound 39 was isolated as a dark solid. In the HR-ESI-(+)-MS spectrum (Figure 3.17), a sodium adduct was observed at m/z 893.5536, corresponding to a molecular formula of
C55H74N4O5 (calcd for C55H74N4O5Na, 893.5557).
Figure 3.17. HR-ESI-(+)-MS for compound 39.
Compound 40 was isolated as a brown solid. Three downfield singlet resonances on the 1H
NMR spectrum of 39 (Figure 3.18), δH 9.39, δH 9.49 and δH 8.55 were observed, corresponding to three aromatic methines of a porphyrin at positions C-5, C-10 and C-20. Two doublet of
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doublets signals resonating at δH 6.16 and δH 6.26, corresponding to the cis- and trans-olefinic protons at position C-3′′, and one doublet doublets resonating at δH 7.97 was assigned to H-3′.
The NH signal appeared as a broad singlet resonating upfield at δH -1.59. Similar resonances in the 1H NMR spectrum (Figure 3.19) were observed for compound 40, except that there was an additional singlet downfield at δH 11.04 corresponding to an aldehyde at position C-7. Comparing the chemical shifts of the protons at position C-5 of compounds 39 and 40, a slight shift was observed which was due to the presence of an electron withdrawing carbonyl of the ketone moiety at position C-7 (shifting from δH 9.39 to δH 10.25). The carbon resonances at δC
174.0 and δC 169.3 were assigned to the ester carbonyls C-17′′′ and C-13′′′, respectively, and a ketone carbonyl to the resonance at δC 189.5. The rest of the NMR data (Plate 1 – Plate 9 in the Addendum) were in agreement with the NMR resonances published for pheophytin A (39) and pheophytin B (40).99-101
1 Figure 3.18. H NMR spectrum of compound 39 in CDCl3 (400 MHz).
1 Figure 3.19. H NMR spectrum of compound 40 in CDCl3 (400 MHz).
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Both pheophytin A and pheophytin B are porphyrin compounds formed by the degradation of a major photosynthesis pigment of plants and marine algae, chlorophyll, with magnesium as the central atom. In marine algae, pheophytin A enhances signal transduction in the mitogen- activated protein kinase-signalling pathway and promotes the differentiation of PC12 cell.100 A study of a bioactivity-guided screening of Lonicera hypoglauca Miq identified pheophytin A as a potent anti-hepatitis C virus agent.102 Pheophytin A also has antioxidant activities.103 No activity has been reported for pheophytin B.
3.2.5 Structural elucidation of β-sitosterol 3-O-β-D-glucoside (41)
Compound 41 was isolated as a white crystalline solid from the branch extract of A. venosum. In the HR-ESI-(-)-MS spectrum (Figure 3.20) a chlorine adduct [M+Cl]- was observed at m/z
611.4071, corresponding to a molecular formula of C35H60O6 (calcd for C35H60O6Cl, 611.4078).
Figure 3.20. HR-ESI-(-)-MS of compound 41.
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The 1H NMR spectrum (Figure 3.21) showed the resonances of the protons for the glucose moiety at δH 3.18 (H-2’), 3.27 (H-5’), 3.36 (H-4’), 3.39 (H-3’), 3.84 (H-6’eq) and 3.71 (H-6’ax) and an anomeric proton resonating at δH 4.38 (d, J = 8 Hz). The chemical shift and coupling constant of the anomeric proton confirms the presence of an O-glycosidic bond and the β- anomeric proton. The spectrum reveals the presence of a steroid-type substructure with six methyl groups resonating at δH 0.68 (s), 1.00 (s), 0.83 (d), 0.93 (d) and 0.81(t). H-6 appeared as a multiplet at δH 5.39.
1 Figure 3.21. H NMR spectrum of compound 41 in CD3OD (400 MHz).
The 13C and DEPT-135 NMR spectrum (Figure 3.22) showed the presence of 35 carbon resonances, which, based on the HSQC spectrum (Figure 3.23), were assigned as six carbon resonances of a glucose moiety, and a total of 29 carbon resonances of the steroid moiety.
Figure 3.22. 13C and DEPT-135 NMR spectra of compound 41 (100 MHz).
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Figure 3.23. HSQC NMR spectra of compound 41.
1 1 In the H- H COSY (Figure 3.24) NMR spectrum the anomeric proton resonating at δH 4.38
(H-1’) correlated to another glucose proton at δH 3.18 (H-2’), which in turn correlated the proton resonance at δH 3.39 (H-3’) (purple circles). The methylene protons at δH 3.71 (H-6’ax) and 3.84 (H-6’eq) correlated to the proton resonance at δH 3.27 (H-5’) (orange circles). A correlation was observed between the olefinic proton δH 5.38 (H-6) and the methylene protons resonating at δH 1.85 (H-7) (Figure 3.25).
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Figure 3.24. The COSY NMR spectrum of compound 41.
Figure 3.25 COSY correlations observed for compound 41.
Figure 3.26 HMBC NMR spectrum of compound 41
Figure 3.27. HMBC correlations observed for compound 41.
The final confirmation of the structure of compound 41 was based on long-range 1H-13C HMBC correlations observed in the HMBC NMR spectrum (Figure 3.26). The presence of 3 an O-glycosidic bridge between a glucose moiety and steroid was confirmed by a JCH HMBC correlation between an anomeric proton δH 4.38 (H-1’) and a carbon resonating at δC 77.6 (C- 2 3) (red circle). Two JCH correlations of the methyl protons at δH 1.00 (H-19) to δC 144.4 (C- 3 5) and δC 36.9 (C-10), as well as a JCH correlation to δC 51.2 (C-8), were observed (purple
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circles). It can be concluded from long-range 1H-13C HMBC correlation of the rest of the methyl protons of the sitosterol moiety (green circles) and comparison with reported NMR spectra that the steroidal moiety was β-sitosterol. Since there was an O-glycosidic bridge present and the HMBC correlation was observed between the anomeric proton and C-3 of the steroid it was could be concluded that compound 41 is β-sitosterol 3-O-β-D-glucoside. The spectroscopic data of 41 was in agreement with the literature values.104-105 The NMR data are collated in Table 3.3.
β-Sitosterol 3-O-β-D-glucoside is a phytosterol glucoside that is a primer for cellulose synthesis.106 This compound was reported to have anti-inflammatory activity,107 and was also classified as a potent neurotoxic agent.108 β-Sitosterol 3-O-β-D-glucoside was also identified to stimulate human peripheral blood lymphocyte proliferation thus implying that it can be used as an immunomodulatory vitamin in combination with β-sitosterol.109 It also reported that this compound has anti-breast cancer activity.110
1 13 Table 3.3. H (400 MHz) and C (100 MHz) NMR data of compound 41 in CDCl3.
Position δH (ppm) (J in Hz) δC (ppm) δC (ppm) literature
1ax 1.08 (1H, t, 10.8) 38.2 37.5
1eq 1.85 (1H, dd,13.2, 3.7)
2 1.17 (2H, t, 6.8) 26.9 31.8
3 3.58 (1H, m) 79.9 79.1 4 2.03 (2H) * 40.7 42.2 5 - 144.4 140.1
6 5.38(1H, t, 7.5) 122.7 122.1
* 7 1.85 (2H) 29.1 - 8 1.36 (1H) * 51.2 -
* 9 0.94 (1H) 37.0 -
10 - 37.6 36.6
11 1.50 (2H) * 21.9 19.8 * 12ax 2.25 (1H) 39.5 38.7
* 12eq 2.41 (1H) 13 - 43.2 42.2
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14 1.12 (1H)* 57.0 56.7 15 1.30 (2H)* 34.8 - 16 1.60 (2H)* 28.1 28.1 17 1.37 (1H)* 37.0 - 18 0.68 (3H, s) 12.5 11.7 19 1.00 (3H, s) 19.9 18.7 20 1.08 (1H)* 57.8 - 21 0.93 (3H, d, 6.4) 19.4 20.9 22 1.98 (2H)* 34.8 - 23 1.24 (2H)* 23.9 - 24 1.67 (1H)* 30.1 -
* 25 0.93 (1H) 46.8 -
26 0.83 (3H, d, 1) 19.4 19.2 27 0.83 (3H, d, 1) 20.3 19.6 28 1.27 (2H)* 23.5 24.8 29 0.81 (3H, t, 1)* 19.5 - 1′ 4.38 (1H, d, 7.8) 102.1 101.0 2′ 3.18 (1H) 74.6 74.3 3′ 3.39 (1H) 77.6 76.9 4′ 3.36 (1H) 71.1 70.1 5′ 3.27 (1H) 79.2 76.8
6′ax 3.71 (1H) 62.5 62.3
6′eq 3.84 (1H)
* Undefined multiplicity due to overlapping,
3.2.4 Structural elucidation of compounds p-hydroxyphenylethyl trans-ferulate (42) and p-hydroxyphenylethyl cis-ferulate (43)
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42
Compound 42 was isolated as a white powder from the branch extract of A. venosum. In the HR-ESI-(-)-MS spectrum (Figure 3.28) a quasi-molecular ion was observed at m/z 312.0996, corresponding to a molecular formula of C18H16O5 (calcd for C18H16O5, 312.0998).
Figure 3.28 HR-ESI-(-)-MS of compound 42 (400 MHz).
1 Figure 3.29. H NMR spectrum of compound 42 in CD3OD (400 MHz).
1 The H NMR spectrum (Figure 3.29) showed the two vinylic protons resonating at δH 6.40
and δH 7.43. The coupling constant (J = 15 Hz) showed that the protons were in a trans conformation and the large difference in chemical shifts between the two resonances indicate
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that it is part of the α, β-unsaturated carbonyl moiety. Two pairs methylene protons resonating as triplets at δH 2.76 (H-7) and δH 3.47 (H-8) were observed in the spectrum. The downfield chemical shift (δH 3.47) (H-8) of one of the methylene protons showed that the methylene carbon is likely to be next to an electronegative atom (most likely oxygen). The phenyl region of the spectrum revealed a total number of seven aromatic protons when integrated. Two aromatic signals resonating at δH 6.72 (H-6) and δH 7.05 (H-5) as doublets integrated for two protons each, confirming the presence of a phenyl ring that is para-substituted. The remaining signals within the same region revealed the presence of the second phenyl ring. The other resonances in the aromatic region comprised doublet δH 7.12 d (J = 2 Hz, H-2’), doublet at δH
6.79 d (J = 8 Hz, H-5’) and a doublet of doublets δH 7.02 dd (J = 8, 2 Hz, H-6’). A methoxy signal resonating at δH 3.88 as a singlet was also observed.
The sample was left in the closed NMR tube dissolved in CD3OD solvent for six months by accident at room temperature (25C). It was analysed afterwards to check whether compound 42 was still present or not. 1H NMR spectroscopy was performed on the sample, and it was observed that the initial compound 42 was still present but two sets of signals were observed (Figure 3.30), similar in multiplicity but different in intensities with slight chemical shift differences. The relative percentage of the trans-isomer was calculated to be 67 % using the 1H NMR spectrum. Interestingly, the coupling constants of vinylic protons were different for the second set of signals. The J-value had changed from 15 Hz (trans-alkene) to 12 Hz (cis- alkene). This showed that both compounds were similar with the only difference being the geometry the alkene bond (cis or trans) bond conformation of the alkene bond.
Figure 3.30. 1H NMR spectrum of compound 42 (blue) and the mixture of cis (43) and trans-
isomers (red) in CD3OD (400 MHz).
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Analysis of the 13C NMR and DEPT-135 spectra (Figure 3.31) and the HSQC (Figure 3.32) NMR spectra of the sample further confirmed the presence of two compounds. All resonance signals were assigned by correlating the protons with carbons using the HSQC NMR spectrum.
Figure 3.31. 13C and DEPT-135 NMR spectrum of a mixture of 42 and 43 (100 MHz).
For compound 42, a total of eighteen carbon signals were observed. A signal resonating downfield at δC 169.5 could be assigned to a carbonyl carbon of an ester group. Six quaternary carbon signals at δC 169.5, δC 157.1, δC 150.0, δC 148.7, δC 131.5, δC 128.5, assigned to C-9’, C-1, C-4’, C-3’, C-4 and C-1’, respectively, were also observed, which were in agreement with quaternary carbons of two phenyl rings. Two methylene signals were observed upfield 1 resonating at δC 35.7 and δC 42.4, which correlated with the triplet signals observed in the H
NMR spectrum (δH 2.76 and δH 3.47) respectively. The methoxy resonances for both the cis and trans-isomers were observed at δC 56.5 and 56.6 (C-10′), respectively. The alkene carbons could be assigned to two pairs of signals at δC 141.9 and δC 121.7 (trans) as well as δC 138.2 and δC 118.9 (cis). The remaining signals downfield were assigned to the phenyl methine carbons, based on correlations observed in the HSQC spectrum (Figure 3.32).
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Figure 3.32. HSQC NMR spectrum of compounds 42 and 43.
The 1H-1H COSY (Figure 3.33) NMR spectrum displayed correlations in agreement with a cis- or trans-cinnamic acid derivative of 4-hydroxyphenyethyl ether. A 1H-1H COSY correlation of the phenyl protons is observed between, δH 7.02 and δH 6.79 as well δH 6.72 and δH 7.0, the methylene protons, δH 2.76 and δH 3.47 (yellow circles), and the trans-olefinic protons (purple circles) and the cis-isomer (orange circles). The observed correlations are illustrated in Figure 3.34
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Figure 3.33 COSY NMR spectra of compounds 42 and 43.
Figure 3.34. Observed COSY correlations observed for compound 42.
A final confirmation for the structures of 42 and 43 was obtained by the HMBC analysis
(Figure 3.35). For the cis-isomer, the HMBC correlation of the two pair phenyl protons δH 6.68
(H-6/2) and 7.00 (H-5/3) to carbons (δC 157.0, 116.6 and 131.4) (red circles) confirms the presence of a para-substituted phenol ring. Correlation of the methoxy protons (δH 3.83, H-10’) to the carbons δC 56.3 (C-10’) and δC 150.0 (C-4’) (yellow circles) as well as the HMBC correlation of aromatic protons (δH 7.35 (H-2’), 6.73 (H-5’), and 6.92 (H-6’) to the carbons resonating at δC 128.7 (C-1’), 114.1 (C-2), 149.5 (C-4’), and 124.8 (C-6’) and a cis-alkene carbon at δC 141.9 (C-7’) (purple circles) confirms the presence of a tri-substituted benzene ring. Another correlation of the methylene protons at H-8 and H-7 to carbons at δC 170.5 (C-
9’), δC 131.4 (H-1), δC 35.5 (C-7) and δC 42.2 (C-8) were observed (blue circles). Lastly, the HMBC correlation of the alkene and methylene protons was useful in connecting the phenyl rings, this correlation also shows the presence of an α,β-unsaturated ester subunit. Similar correlations are observed for the trans isomer. Thus, compound 42 and compound 43 were identified to be 4-hydroxyphenylethyl 4-hydroxyphenylethyl trans-ferulate and 4-
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hydroxyphenylethyl cis-ferulate, respectively and are in good agreement with the literature.111
The NMR data are collated in Table 3.4.
Figure 3.35. HMBC NMR of compounds 42 and 43
Figure 3.36. HMBC correlations observed for compounds 42 and 43.
Since the sample was not in an acidic solution (acidic media normally promotes cis-trans isomerisation of alkenes), photo-isomerisation is the only reaction that could explain this phenomenon.112 This process only occurs in the presence of UV light which act as a source of photons. Because the sample was kept indoors, it had limited exposure to UV light. This type of reaction is considered to be non-reversable, thus explaining why the sample still contain ~67 % of the trans compound after a long period of time, also (Scheme 3.1).
Scheme 3.1. Photo-isomerisation of compounds 42 to 43.
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1 13 Table 3.4. H (400 MHz) and C (100 MHz) NMR data of compounds 42 and 43 in CD3OD.
Position δH (ppm) (J in Hz) δC (ppm) 42* 43 42* 43 1 - - 131.5 131.4 2 6.72 (1H, d, 8.5) 6.68 (1H, d, 8.6) 116.6 116.6 3 7.05 (1H, d, 8.4) 7.00 (1H, d, 8.4) 130.8 130.9 4 - - 157.1 157.0
5 7.05 (1H, d, 8.4) 7.00 (1H, d, 8.4) 130.8 130.9 6 6.72 (1H, d, 8.4) 6.68 (1H, d, 8.6) 116.6 116.6 7 2.76 (2H, t, 7.2) 2.69 (2H, t, 7.1) 35.7 35.5
8 3.47 (2H, t, 7.2) 3.40 (2H, t, 7.5) 42.4 42.2
1′ - - 128.5 128.7 2′ 7.12 (1H, d, 1.8) 7.35 (1H, d, 1.9) 111.8 114.1 3′ - - 148.7 148.6 4′ - - 150.0 149.5
5′ 6.79 (1H, d, 8.2) 6.73 (1H, d, 8.4) 116.4 116.0 6′ 7.02 (1H, dd, 8.3, 1.9) 6.92 (1H, dd, 8.2, 1.8) 123.2 124.8 7′ 7.43 (1H, d, 15.9) 6.61 (1H, d, 12.7) 138.2 141.9 8′ 6.40 (1H, d, 15.9) 5.71 (1H, d, 12.6) 118.9 121.9 9′ - - 169.5 170.5
10′ 3.88 s 3.83 s 56.3 56.3
3.3 Biological activity
The stem and leaves crude extracts of A. venosum as well as loliolide (37), β-sitosterol 3-O-β-
D-glucoside (41) and p-hydroxyphenylethyl trans-ferulate (42) were assayed for antimicrobial activities against a Gram-positive (Staphylococcus aureus ATCC 12600), two Gram-negative (Pseudomonas aeruginosa ATCC 10145, Klebsiella pneumoniae ATCC 13883), and a drug- resistant Gram-negative (Escherichia coli ATCC 25218) strains of bacteria. The crude extracts showed moderate but promising antimicrobial activity. However, pure compounds did not
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show any antimicrobial activity (Table 3.5). The activity may be classified as significant (MIC < 100 µg/mL), moderate (100 < MIC ≤ 625) or weak (MIC > 625).113
Table 3.5. Antibacterial activity results for A. venosum. Antibacterial activity (MIC, µg/ml) Gram +ve Gram -ve Drug resistant Gram -ve S. aureus K. pneumoniae P. aeruginosa E. coli A. v/B 313 313 >5000 >5000 A.v/L 1250 1250 >5000 >5000 37 >500 >500 >500 >500 41 >500 >500 >500 >500 42 >500 >500 >500 >500 Neomycin <0.24 0.98 0.98 0.24
3.4 Conclusion
The phytochemical investigation of the leaves of A. venosum led to an isolation of loliolide (37), lutein (38), pheophytin A (39), and pheophytin B (40). β-Sitosterol 3-O-β-D-glucoside (41) and 4-hydroxyphenylethyl trans-ferulate (42) were isolated from the branches of A. venosum. These compounds have been isolated from of A. venosum for the first. These compounds are all known compounds and are occurring in several plant families. All compounds were identified as known compounds but are different from the previously isolated compounds from the plant species. The structures were confirmed by comparison of the NMR data with the literature data.Although known compounds, several of the compounds showed interesting biological activities as reported in the literature. The crude extracts showed promising antibacterial activities when assayed against Gram-positive (Staphylococcus aureus ATCC 12600) and Gram-negative (Pseudomonas aeruginosa ATCC 10145) bacteria but did not show any activity against a second Gram-negative (Klebsiella pneumoniae ATCC 13883) and a drug-resistant Gram-negative (Escherichia coli ATCC 25218) bacterial strain. The activity observed from by the crude extract may be due to the synergistic effect operating in the mixture or the unidentified active constituents present in the crude extract. The pure compounds did not show any activity.
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3.5 Experimental
3.5.1 General experimental procedure
The extraction of the plant material was performed on an Infors AG CH-4103 Bottmingeni orbital shaker at room temperature. Mass spectra (LCMS and TOF-MS) were obtained on a Shimadzu LCMS 2020 and Water Micromass LCT Premier TOF-MS instrument using MS- grade solvents (acetonitrile, methanol, water and formic acid). Both positive and negative ionisation modes were used for analysis.
The NMR spectra of isolated compounds were recorded in CDCl3 (deuterated chloroform) and
CD3OD (deuterated methanol) at 30 ºC using either a 5 mm BBOZ probe or a 5 mm TBIZ probe on Bruker Avance III (500 MHz for 1H, 125 MHz for 13C) and 400 (400 MHz for 1H, 100 MHz for 13C) spectrometers. Chemical shift (δ) signals are in units of parts per million (ppm) and are 1 13 1 referenced to residual protonated solvent peak (CDCl3: H 7.26, C 77.36 and CD3OD: H 3.31, 13C 49.03). Peak multiplicities are designated as s for singlet, d for doublet, t for triplet, q for quartet and m for multiplet. Coupling constants (J) are given in Hz. All solvents that were used in this research were of analytical grade.
Column chromatography packed with silica gel (silica gel 60, 0.063-0.2000 mm, 70-230 Mesh ASTM) was used for initial fractionation, and centrifugal thin-layer chromatography, performed on circular glass plates (coated with preparative silica gel 60 PF254 containing gypsum, particle size ≤ 45 µm either 1 mm or 2 mm thickness) on a Chromatotron™, was used for the final purification of the compounds. For qualitative analysis of the fractions obtained and compounds isolated, thin-layer chromatography (TLC) (TLC silica gel 60 F254, aluminium-backed plates) with detection under long- or short-wavelength UV radiation (λ 254 and 365 nm, respectively) or staining with p-anisaldehyde reagent was used. The stain solution was prepared by putting a 250 mL volumetric flask in an ice bath and add the following reagents sequentially, shaking the flask after each addition:
1 mL of p-anisaldehyde (98%) 20 mL of glacial acetic acid 170 mL of MeOH 4 mL of concentrated sulfuric acid
All the reagents were added in a 250 mL volumetric flask, cooled in an ice bath. After preparation, the solution was stored in a fridge (4 ºC).
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3.5.2 Plant material
Leaves and stems of Antidesma venosum were collected in May 2017 at the Botanical Garden of the University of KwaZulu-Natal in Pietermaritzburg and was identified by the curator of the garden, Ms Alison Young. A voucher specimen was deposited in the Bews Herbarium, School of Life Sciences, UKZN (Voucher No 1. NU 0048865).
3.5.3 Extraction and isolation of compounds
The plant material (leaves and stems separately) of A. venosum were dried for two weeks at room temperature and then pulverised into a powder. Based on the literature published on the plant, it was decided that the leaves of A. venosum would be extracted with DCM and MeOH separately. The pulverised leaves (1 kg) was extracted exhaustively with DCM (48 h, room temperature). MeOH extraction of the leaves was omitted due to time constraints. The powdered stem material of A. venosum (1 kg) were extracted exhaustively with DCM-MeOH (1:1, v/v) (48 h, room temperature). The extracts were filtered through a cotton wool plug in a glass funnel. The filtrates were then concentrated under vacuum using a rotatory evaporator to obtain the crude extracts (13 g DCM leaf crude extract, 10 g DCM-MeOH stem crude extract).
The DCM leaf crude extract was subjected to chromatography on a column packed with silica gel (14 x 3 cm) and eluted with a gradient method using a mixture of Hex and EtOAc as solvent systems: Hex (400 mL), Hex-EtOAc (9:1, 300 mL), Hex-EtOAc (7:3, 300 mL), Hex-EtOAc (1:1, 300 mL), Hex-EtOAc (3:7, 200 mL), Hex-EtOAc (1:9, 200 mL), then finally flushed with MeOH (200 mL) to yield 170 fractions (10 mL each. Fractions with similar TLC profiles were combined to afford ten fractions that were dried and weighed (Table 3.6).
Table 3.6. Fractions obtained by column chromatography of the crude DCM extract of the leaves (A. venosum).
Fraction Mass (g) Book code A 4 SSH-1-12A B 2 SSH-1-12B C 2 SSH-1-12C D 1 SSH-1-12D E 0.8 SSH-1-12E F 1 SSH-1-12F G 0.3 SSH-1-12G
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H 0.1 SSH-1-12H I 0.6 SSH-1-12I J 0.1 SSH-1-12J
Fractions 12A and 12B contained fats and oils and were not investigated further. Fractions 12C, 12D, 12E and 12F were analysed further based on the TLC profiles. The rest of the fractions has too low a mass for further fractionation. Pheophytin A (39, 2 g) was isolated from fraction 12C and pheophytin B (40, 1 g) was isolated from fraction 12D. Fraction 12E and 12F were combined since a similar spot was observed in both fractions when analysed by TLC. The combined fraction was further fractionated by centrifugal chromatography (1 mm thick layer) using Hex-EtOAc (1:9) as the solvent system to yield loliolide (37) (5 mg) and lutein 38, 9 mg).
Figure 3.37. Flow diagram of the isolation of compound 37, 41, 42 and 43.
The DCM-MeOH crude extract of the stem (8 g) was applied to a column packed with silica gel (12 x 3 cm) and eluted with a mixture of DCM and MeOH as solvent systems: DCM (300 mL), DCM-MeOH (9.5:0.5, 500 mL), DCM-MeOH (9:1, 300 mL), DCM-MeOH (8.5:1.5, 200 mL). A hundred fractions (10 mL each) were collected, then finally flushed with MeOH (400 mL). A sticky sludge remained on the column and could not be eluted with MeOH. Distilled water mixed with 10 % of acetic acid was then used to elute out the sludge. The fractions were spotted on a TLC plate and viewed under long and short-wave UV lamps then stained with anisaldehyde. Fractions with similar TLC profiles were combined and dried afford ten fractions dried in the fume hood overnight and weighed (Table 3.7).
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Table 3.7. Fractions obtained by column chromatography of the branch crude extract (A. venosum).
Mass (mg) Book code Fraction A 38 SSH-1-37A B 109 SSH-1-37B C 452 SSH-1-37C D 54 SSH-1-37D E 61 SSH-1-37E F 16 SSH-1-37F G 26 SSH-1-37G H 125 SSH-1-37H I 90 SSH-1-37I 48 J SSH-1-37J 0 MeOH flush SSH-1-37k 6 g Sludge
Based on the TLC profile, fraction 37D was further fractionated on a small column (6 x 2 cm), with the eluting solvent system EtOAc-Hex (7:3). Five subfractions were obtained, and subfraction 38C was identified 4-hydroxyphenylethyl trans-ferulate (42, 5 mg). Fraction 37J was fractionated further with a small column (6 x 2 cm), and the eluting solvent system used was DCM-MeOH (8:2), four subfractions were obtained. Of the four subfractions, subfraction 27B was analysed and identified to be β-sitosterol 3-O-β-D-glucoside (41, 14 mg). (Figure 3.38).
3.5.4 Biological assay
Preparation of Microorganisms The bacteria used in this study were one Gram-positive (Staphylococcus aureus ATCC 12600), two Gram-negative (Pseudomonas aeruginosa ATCC 10145, Klebsiella pneumoniae ATCC 13883), and one drug-resistant Gram-negative (Escherichia coli ATCC 25218) bacterial strains. Preserved cultures of bacteria on Mueller Hinton Agar (MHA) slants at 4 °C were used as stock cultures throughout the study.
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Figure 3.38. Flow diagram of the isolation of compound 38 and 40.
Minimum Inhibitory Concentration (MIC) determination A rapid p-iodonitrotetrazolium chloride (INT) colourimetric assay was used to determine the MIC (minimum inhibitory concentration) values of samples against the bacterial strains.114 Samples of extracts and isolated compounds (100, 50, and 10 mg/mL) were prepared in DMSO. Stock solutions of samples were initially diluted to concentrations of 20, 5 and 2 mg/mL with sterilised distilled water. The samples were then mixed with Mueller Hinton Broth (MHB), and sequentially diluted 2-fold in a 96-well-microplate to a final concentration range of 5000 – 39 and 500 – 3.9 µg/mL, for the extracts and single compounds, respectively. Bacterial strains were cultured overnight at 37 °C on MHB and adjusted to a final density of 106 cfu/mL with MHB. These were used as inocula. An inoculum of 100 µL was added to each well (200 µL). The plates were covered with a sterile plate sealer and then incubated at 37 °C for 20 h. Neomycin was used as a positive control while wells containing 10% DMSO, MHB and 100 µl of inoculum served as the negative controls. The MICs of samples were observed after 20 h of incubation at 37 °C, followed by 30 min of incubation after the addition 40 µl of 0.2 mg/mL INT. The inhibition of bacterial growth would be indicated by clear wells with INT after incubation. Minimum inhibitory concentration (MIC) values were recorded as the lowest concentration of the sample that completely inhibited bacterial growth. The results are given in Table 3.5.
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3.5.5 Physical data of isolated compounds
Loliolide (37) was isolated as a white powder. 1H and 13C NMR data are collected in Table 3.1.
HR-ESI-(+)-MS, m/z 219.0992 (calcd for C11H16O3Na, 219.0997).
Lutein (38) was isolated as an orange powder. 1H and 13C NMR data are collected in Table
3.2. HR-ESI-(-)-MS, m/z 567.4180 (calcd for C40H55O2, 567.4202).
13 Pheophytin A (39) was isolated as a dark solid. C NMR (400 MHz, CDCl3) δC 189.6 (C-13′),
δC 172.9 (C-17′′′), δC 172.2 (C-19), δC 169.6 (C-13′′′), δC 161.2 (C-16), δC 155.6 (C-6), δC 150.9
(C-9), δC 149.7 (C-14), δC 145.17 (C-8), δC 142.8 (C-p3), δC 142.0 (C-1), δC 137.9 (C-7), δC
136.5 (C-3), δC 136.3(C-4), δC 136.2(C-7), δC 131.8 (C-2), δC 129.1(C-3′), δC 129.0 (C-12), δC
128.9 (C-13), δC 122.7 (C-3′′), δC 117.8 (C-p2), δC 105.3 (C-15), δC 104.4 (C-10), δC 97.5(C-5),
δC 93.1 (C-20), δC 64.7 (C-13′′), δC 61.5 (C-p1), δC 52.8 (C-13′′′), δC 51.2 (C-17), δC 50.1 (C-
18), δC 39.8-24.1 (C-p4-p14), δC 31.2 (C-17′′), δC 29.8 (C-17′), δC 27.9 (C-p15), δC 23.1 (C-18′),
δC 22.7 (C-p16), δC 22.6 (C-p15′), δC 19.7 (C-p11′), δC 19.6 (C-p7′), δC 19.4 (C-8′), δC 17.4 (C-
8′′), δC 16.2 (C-p3′), δC 12.1 (C-12′), δC 12.0 (C-2′), δC 11.2 (C-7′). HR-ESI-(+)-MS, m/z + 893.5536 [M+Na] (calcd for C55H74N4O5Na, 893.5557).
1 Pheophytin B (40) was isolated as a brown solid. H NMR (400 MHz, CDCl3) δH 9.39, δH 9.49 13 and δH 8.55 are observed. C NMR (400 MHz, CDCl3) δC 12.2 (C-12′), 14.1 (C-17′′), 16.3 (C- p3′), 19.1 (C-7), 19.3 (C-8′′), 19.6 (C-8′), 19.7 (C-11′′), 22.6 (C-p15′), 22.7 (C-p16), 23.0 (C- 18′), 24.4 (methylenes), 24.7 (methylenes), 25.0 (methylenes), 27.9 (C-p15), 29.7 (C-17′′), 31.2 (C-13′), 31.9 (methylenes), 32.6 (C-p5), 32.7 (methylenes), 36.6 (methylenes), 37.3 (methylenes), 37.4 (methylenes), 39.3 (methylenes), 39.7 (C-p4), 50.1 (C-18), 51.4 (C-17), 52.9 (C-13′′), 61.5 (C-13′′), 64.6 (C-17′′), 93.3 (C-20), 101.6 (C-5), 105.0 (C-10), 106.9 (C- 15), 123.5 (C-3′′′), 128.6 (C-12), 129.7 (C-3′), 132.4 (C-13), 132.9 (C-2), 137.1 (C-3), 137.7 (C-4), 138.0 (C-11), 143.5 (C-1), 147.1 (C-8), 150.7 (C-9), 151.2 (C-141), 159.3 (C-6), 164.0
(C-16), 169.3 (C-19), 172.8 (C-13′′), 174.0 (C-17′′CO2), 187.6 (C-7′), 189.5 (C-13′).
β-Sitosterol 3-O-β-D-glucoside (41) was isolated as a yellow-white solid. 1H and 13C NMR data are collected in Table 3.3. HR-ESI-(-)-MS, m/z 611.4071 (calcd for C35H60O6Cl, 611.4078) 1H and 13C NMR data are collected in Table 3.3.
4-Hydroxyphenylethyl trans-ferulate (42) was isolated as a white powder. 1H and 13C NMR data are collected in Table 3.4. HR-ESI-(-)-MS m/z 312.0996 (calcd for C11H18O5, 312.0998).
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CHAPTER 4
Phytochemistry of Euphorbia cooperi N.E.Br. ex A.Berger
4.1 Introduction
4.1.1 Euphorbia subg. Euphorbia (E. subg. Euphorbia)
Euphorbia L. is the largest genus of the Euphorbiaceae with over 2000 species and has a worldwide distribution and is diverse in arid and semiarid regions of the tropics and subtropics. Euphorbia genus consists of four subgenera namely E. subg. Esula Pers., E. subg. Rhizanthium (Boiss) Wheeler, E. subg. Chammaesyce Raf., and E. subg. Euphorbia. Of these four subgenera, Euphorbia subgenera Euphorbia (E. subg. Euphorbia) is the largest and most morphologically diverse subgenus.115-116 It includes herbs, geophytes, woody shrubs, understory and canopy tree, and the unusual growth of pencil-stemmed plants (e.g. E. decorsei, Figure 4.1. P). Woody shrubs and trees occur in both xeric and mesic forests of Madagascar and New World Tropics. Herbaceous species are found in South America, Africa, and Australia. Geophytes have evolved several times in E. subg. Euphorbia and are in Africa, Madagascar, the Arabian Peninsula, and South Asia.115
Plant species of this subgenus have succulent stems and branches, with sharp thorns on the base of each leaf. The stipules modified as prickles can be as large as the spines but can also smaller and sometimes apparently obsolete. In the leaf-axil, is the flowering-eye, from which the inflorescence grows consisting of a singular dichasial cyme. Some Madagascan species developed apparently similar horny structures bearing one or more “spines”, but these are modified stipules, each with an expanded horny base that flanks the leaf-scar but does not surround it. In related species these stipule structures are often developed into bristly fringes (Figure 4.1).117
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https://xavieraubriot.files.wordpress.com/2015/06/dorsey-et-al-2013.pdf
Figure 4.1. Examples of growth from of morphological diversity in Euphorbia subgenus. A, E. floridana; B, E. hedyotoids; C, E. cooperi; D, E. nana; E, E. horombensis; F, E. decorscei. G, E. sect.Euphorbia; H, E.sect. Goniostema; I, E. Crepidara; J, E. sect. Monodenium, E. heptropoda; K, E. sect. Brasilinses; L, E.sect.Goniostema; M, E. sect.vigureri; N, E. sect. Euphorbia, E. zoutpansbergeni; O, E. sect. Goniostema; P, E. neospinescens.
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4.1.2 Euphorbia cooperi N.E.Br. ex A.Berger
Euphorbia cooperi N.E.Br. ex A.Berger (Figure 4.2), also known as bushveld candelabra Euphorbia (Eng) and noorsdoring (Afrikaans),44 is a single-crowned, green, and succulent spiny tree that grows up to 10 m in height. It consists of branches that are restricted to the upper section of the tree with branch scars clearly visible along the length of the stems. These branches have multiple wing-like angles with segmentation along the shape of an inverted heart with the widest section near the base of the segment that tapers to its apical part. E. cooperi is native to Mozambique, the eastern parts of South Africa, south Zimbabwe, Swaziland and Zambia. Its variability mainly depends on habitat, geology, and climate.118-119
Figure 4.2. Euphorbia cooperi N.E.Br. ex A.Berger.
There are three different subtypes of E. cooperi, namely E. cooperi var. cooperi, which occurs in South Africa, E. cooperi var. ussanguensis (N.E.Br) L.C Leach, which is distributed in Mozambique, Zimbabwe, Malawi and Zambia, and E. cooperi var calidicola L.C. Leach, found in Tanzania and Zambia.118
The E. cooperi latex is considered to be very poisonous and any contact with the skin, eyes or mouth will cause severe pain and may lead to blindness.120 A search of the literature revealed that there are four reports on the phytochemistry of E. cooperi51,120-122 and one report on the cytotoxicity of the plant extract.84 According to the phytochemical reports, five compounds were isolated from E. cooperi N.E.Br., three phorbol-type diterpenoid esters, 12-deoxyphorbol 13-angelate-16-isobutyrate-20-acetate (43),121 12-deoxyphorbol 13-angelate-16-isobutyrate
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(44),121 12-deoxyphorbol-13-isobutyrate-16-angelate-20-acetate (45), and two triterpenoids, euphol (20), and obtusifoliol (46).120
Despite the toxicity associated with Euphorbia diterpenoids, recent research has showed that some of these compounds have a remarkable bioactivity, e.g. the phorbol ester prostratin (13) which in the process of being developed in an anti-HIV drug and ingenol 3-angelate (15), which has been approved as an anti-cancer drug. As a result, we decided to reinvestigate the phytochemistry of E. cooperi.
4.3 Results and discussion
The aerial parts of E. cooperi were extracted with DCM-MeOH (1:1, v/v) to afford a crude extract that were subjected to vacuum liquid chromatography (VLC) on silica gel and eluted using mixtures of Hex, DCM, EtOAc, and MeOH. The fractions were purified by column chromatography and centrifugal thin-layer chromatography to afford compounds 47 - 51. The structures were elucidated by spectroscopic analysis including 1H NMR, 13C NMR, DEPT- 135, COSY, HSQC, HMBC and TOF-MS. The structures of the known compounds were confirmed by comparison of the NMR data with those reported in the literature.
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4.3.1 Structural elucidation glutinol (47)
47
Compound 47 was isolated as a white powder. In the HR-ESI-(+)-MS spectrum (Figure 4.3), a protonated molecular ion was observed at m/z 427.3948, corresponding to a molecular formula of C30H50O (calcd for the protonated molecular ion C30H51O, 427.3940).
Figure 4.3. HR-ESIMS (positive mode) of compound 47.
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1 Figure 4.4. H NMR spectrum of compound 47 in CDCl3 (400 MHz).
1 In the H NMR spectrum of 47 (Figure 4.4), a doublet at δH 5.62 (H-6) with a coupling constant of J = 5.9 Hz was observed. The chemical shift of the proton was in agreement with an alkene proton. At δH 3.46 (H-3), a triplet with a coupling constant J = 2.6 Hz was observed and the chemical shift of this signal indicated that the proton was most likely attached on an oxygen bearing carbon. The small coupling constant showed that this proton might be in an equatorial position in a six-membered ring in the chair conformation. According to the Karplus equation, which states that the diaxial protons with a dihedral angle of 180° will have a large coupling constant, (ca. J = 11 Hz), protons with an axial-equatorial or equatorial-equatorial relationship with a dihedral angle of 60° will have a smaller coupling constant. Therefore, since J = 2.6 Hz, the protons on C-2 and C-3 have a dihedral angle close to 60°, meaning that the hydroxy group (OH) is in an axial position in a six-membered ring in a chair conformation (Figure 4.5). the COSY NMR spectrum is provided in the Addendum (Plate 9)
Figure 4.5. COSY correlation observed for compound 47.
A large number of protons were observed in the region δH 1.23-2.07. These protons had complex splittings and overlapped substantially and were difficult to analyse. In the high-field
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region eight singlets, which all integrated for three protons each, were observed. These were in agreement with eight methyl groups of a triterpenoid structure.
13 Figure 4.6. C and DEPT-135 NMR spectra of compound 47 in CDCl3 (100 MHz).
The 13C NMR and DEPT-135 NMR spectra (Figure 4.6) and the HSQC spectrum (Figure 4.7) of compound 47 showed the presence of 30 carbon signals: 7 quaternary carbons, 5 methine carbons, 11 methylene carbons and 8 methyl carbons. The carbon signals resonating at δC 141.6 and δC 122.1 were assigned to carbons at position 5 and 6 of an alkene system. The signal at δC 76.3 was assigned to an oxygen bearing carbon. The COSY spectrum of 47 is given in the Addendum
Figure 4.7. HSQC NMR spectrum of compound 47.
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The correlations observed in the HMBC spectrum (Figure 4.8) between the methyl protons and adjacent carbon resonances were crucial in constructing the triterpene structure. The methyl proton resonances at δH 1.13 (H-23) and 1.03 (H-24) both correlated to carbon signals at δC 141.6 (C-5), 76.3 (C-3) and 40.8 (C-4), as well as to each other, were assigned to the C-
23 and C-24 of a triterpenoid. The methyl singlet at δH 0.84 showed correlations to carbons at
δC 49.7 (C-10), δC 34.9 (C-9) and δC 47.5 (C-8) and was assigned to 25-Me. The methyl proton signal at δH 1.09 (26-Me) showed correlation to a carbon resonances at δC 47.5 (C-8), δC 32.1
(C-15) and δC 39.3 (C-13), the methyl proton signal at δH 1.00 (27-Me) to the carbon resonances at δC 30.4 (C-12), δC 39.3 (C-13) and δC 43.1 (C-18), and methyl proton signal at δH 1.16 (28-
Me) to the carbon resonances at δC 30.1 (C-17) and δC 43.1 (C-18) (Figure 4.7).
Figure 4.8. HMBC NMR spectrum of compound 47.
Thus, the structure of compound 47 was identified as glutinol and the spectroscopic data were in agreement with the literature values. The NMR data are collated in Table 4.1.123-124
Glutinol is a nonpolar pentacyclic triterpenoid that is known to have anti-inflammatory and analgesic activities.123-124 This compound has been isolated from plant species including Euphorbia species. But has not been reported in E. cooperi.
1 13 Table 4.1 H (400 MHz) and C (100 MHz) NMR data for compound 47 in CDCl3.
Position δH (ppm) (J in Hz) δC (ppm) δC (ppm) 1 1.23-2.32 (m) 18.2 18.2 2 1.23-2.32 (m) 27.8 3 3.46 (1H, t, 2.8) 76.3 76.4
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4 - 40.8 40.8
5 - 141.6 141.6 6 5.62 (1H, brd, 5.5) 122.1 120.1 7 1.23-2.32 (m) 23.6 23.7 8 1.23-2.32 (m) 47.5 47.5 9 - 34.9 34.9 10 - 49.7 49.7 11 1.23-2.32 (m) 34.6 32.1 12 1.23-2.32 (m) 30.4 27.8 13 - 39.3 39.3 14 - 37.8 37.9 15 1.23-2.32 (m) 32.1 30.4 16 1.23-2.32 (m) 36.0 34.6 17 - 30.1 30.1 18 1.23-2.32 (m) 43.1 43.1 19 1.23-2.32 (m) 35.1 35.1 20 - 28.2 28.2 21 1.23-2.32 (m) 33.1 33.1 22 1.23-2.32 (m) 39.0 39.0 23 1.13 (s) 25.4 25.5 24 1.03 (s) 29.1 29.0 25 0.84 (s) 16.2 16.2 26 1.09 (s) 19.6 19.6 27 1.00 (s) 18.4 18.4 28 1.16 (s) 32.0 32.0 29 0.94 (s) 34.5 34.5 30 0.98 (s) 32.4 32.4
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4.3.1 Structural elucidation of compounds 48 and 49
48
1 Figure 4.9. H NMR spectrum of compound 48 in CDCl3 (400 MHz).
The 1H NMR spectrum of compound 48 (Figure 4.9) showed two olefinic signals resonating downfield at δH 5.81(H-8) and δH 5.72 (H-7) as doublets of doublets. Tshe coupling constant (J = 15.3 Hz) was characteristic of a trans-alkene. The multiplicities (dd) also indicated that both the alkene carbons are adjacent to methine carbons. Two pairs of diastereotopic protons resonating at δH 2.16 (1H, dd, J = 13.8 and 6.7 Hz, Heq) and δH 1.62 (1H, m, Heq), as well as δH
1.84 (1H, dd, J = 13.1 and 6.8 Hz, Heq) and δH 1.58 (1H, m Heq) were observed. In the COSY spectrum (Figure 4.10), a broad multiplet at δH 3.96 correlated with the two pairs of diastereotopic methylene protons. Three methyl signals resonated upfield, two as singlets and one as a doublet at δH 1.35 (s), δH 1.10 (s), and δH 1.29 (d), respectively
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.
Figure 4.10. COSY NMR spectrum of compound 48
Figure 4.11. 13C and DEPT-135 NMR spectra of compound 48 (100 MHz).
Figure 4.11 shows a total of 13 carbon signals for compound 48. Three quaternary carbons resonating at δC 179.2, δC 85.3 and δC 46.3, five methine carbons at δC 142.2, δC 120.6, δC 68.0,
δC 65.2 and δC 57.7, two methylene carbons at δC 38.0 and δC 36.2, and three methyls at δC
23.7, δC 23.4 and δC 18.9, were observed. The two carbon signals resonating at δC 142.2 and δC
120.6 were assigned to two olefinic carbons. The signal at δC 179.2 was assigned to a carbonyl carbon, corresponding to an ester carbonyl. The chemical shift was higher than is normally observed for an ester carbon, but this can be attributed to the strained ring system in the
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compound. The chemical shifts of the methine signals at δC 68.0 and δC 65.2 indicated the attachment of an electronegative atom on the methine carbons, most likely oxygen. The carbon signals were correlated to the 1H NMR signals using a HSQC NMR spectrum (Figure 4.12).
Figure 4.12. HSQC NMR spectrum of compound 48.
The final structure of 48 was based on the HMBC NMR spectrum (Figure 4.13 and 4.14). In the spectrum, correlations between the olefinic proton at δH 5.81 and the methyl carbon at δC
23.7 as well as the methine carbon at δC 68.0 confirmed that the alkene system is attached to a methine carbon bearing an oxygen and a methyl group. The HMBC correlations of the other olefinic proton to a methine carbon signal at δC 57.7 confirms that the olefin system is also attached to a second methine carbon. The presence of the lactone ring was confirmed by the HMBC correlations between the carbonyl carbon and one of the methyl proton singlets as well as the enantiotopic protons in the axial position. Based on the NMR data, structure 48 was assigned to this compound. The NMR data are collated in Table 4.2. The absolute and relative configuration of this compound needs to be confirmed by further studies.
Figure 4.13 HMBC NMR spectrum of compound 48 (green circle – please see Figure 4.14)
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Figure 4.14. HMBC NMR spectrum of compound 48
49
An extensive search of literature revealed only one compound with a framework similar to 48, cucubalactone (49).124 The only difference was the presence of the hydroxy group at position C-6, thus compound 48 was regarded as novel and we named it euphorbilactone. Related bicyclic norsesquiterpenes (euphorbiosides A and B) were isolated from Euphorbia resinifera.125 Cucubalactone was isolated from Arachis hypogaea (Fabaceae, the plant that produce peanuts). Further investigation of the crude extract of E. cooperi led to the isolation of another known norsesquiterpenoid, arachiside A (50).
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50
Compound 50 was isolated as a yellow oil. In the HR-ESI-(+)-MS spectrum (Figure 4.15), a sodium adduct was observed at m/z 425.1793, corresponding to a molecular formula of
C19H30O9 (calcd for C19H30O9Na, 425.1788).
Figure 4.15. HR-ESI-(+)-MS (positive mode) of compound 50.
The 1H NMR spectrum of compound 50 (Figure 4.16) showed similar signals to those of
compound 48. Two olefinic signals resonated downfield at δH 5.77 and δH 5.47 as doublet of doublets. The coupling constants indicated that they were alkene protons in a trans conformation. The multiplicity (dd) also showed that each of the alkene carbons was being neighboured by a methine carbon. The equatorial protons of two pairs of diastereotopic
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protons were also observed at δH 2.28 (1H, dd, J = 14 and 7 Hz, Heq) and δH 1.90 (1H, dd, J =
13 and 7 Hz, Heq). The axial proton signals overlapped between δH 1.77 and δH 1.70. Three methyl signals resonated upfield, two as singlets and one as a doublet at δH 1.36 (s), δH 1.08
(s) and δH 1.25 (d), respectively. The change in chemical shift of the diastereotopic signals and the proton at position C-5 suggested that there was an additional group bonded to the oxygen.
Additionally, a O-glucosidic anomeric proton was observed as a doublet at δH 4.35, the coupling constant (J = 7.9 Hz) suggested that the proton is in the β-position. Based on the signals observed in the glucosidic region, the change in chemical shifts of the diastereotopic signals when compared to those of compound 48, and the presence of a β-anomeric proton being observed in the 1H NMR spectrum of compound 50, it was concluded that a β-D-glucose moiety was attached at to C-3 of the compound. This was further confirmed by the long-range HMBC (Figure 4.17) correlations of an anomeric proton of the glucose moiety to the carbon at position C-3, further confirming the presence of an O-glucosidic bond. In the 1H NMR spectrum the carbohydrate signals are overlapping but the 13C NMR chemical shifts were in agreement with a glucose moiety. The rest of the correlations observed in the HMBC spectrum as well as the COSY (Plate 10, Addendum), and HSQC NMR spectra (Plate 11) confirmed the proposed structure. Thus compound 50 was identified to arachiside A.126 The NMR data are collated in Table 4.2
Figure 4.16. 1H NMR spectrum of compound 50.
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Figure 4.17. HMBC spectrum of compound 50.
Arachiside A is a glucoside derivative of a norsesquiterpenoid, euphorbilactone (48). This compound has been isolated only once prior to the study, from Arachis hypogaea, the plant that produces peanuts. Both 48 and 50 are derived from degradation of carotenoids such as lutein (37)127 (Scheme 4.1).
Scheme 4.1. Proposed biosynthetic pathway of compounds 48 and 50
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1 13 Table 4.2. H (400 MHz) and C (100 MHz) NMR data for compounds 48 in CDCl3
and 50 in CD3OD.
Position δH (ppm, J in Hz) δC (ppm) δC (ppm) δH (ppm, J in Hz)
48 50 48 50 1 - 46.3 47.7 -
2ax 1.84 (1H, dd, 6.8, 13.1) 1.90 (1H*) 36.2 36.1 2eq 1.58(1H, dd*) 1.70-1.77(1H*) 3 3.96 (1H, m) 3.97 (1H, m) 65.2 73.6
4ax 2.16 (1H, dd, 6.7, 13.8) 2.28 (1H*) 38.0 35.8 4eq 1.62 (1H, dd*) 1.70-1.77(1H*) 5 - - 85.3 86.9 6 2.36 (1H, d, 9.2) 2.47 (1H, d, 9.0) 57.7 59.0 7 5.72 (1H, dd, 9.2, 15.5) 5.77 (1H, dd, 9.2, 15.8) 120.6 121.9 8 5.81 (1H, dd, 5.1, 15.3) 5.85 (1H, dd, 5.4, 15.2) 142.2 143.9 4.36 (1H, q, 6.0) 4.30 (1H, m) 68.0 68.9 9 10 1.29 (3H, d, 8.9) 1.25 (3H, d, 6.5) 23.7 23.9 11 - - 179.2 181.5 1.10 (3H, s) 1.08 (3H, s) 18.9 19.4 12 1.35 (3H, s) 1.35 (3H, s) 23.4 23.8 13 - 4.35 (1H, d, 7.9) - 103.1 1′ - 3.14 (1H, t, 8.4) - 75.2 2′ - 3.26-3.53 (1H*) - 78.2 3′ - 3.26-3.53 (1H*) - 71.7 4′ - 3.26-3.53 (1H*) - 78.0 5′ - 3.66 - 6′ 62.7 - 3.83 -
*undefined
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4.3.4 Structural elucidation of compounds 51 and 52
51
Compound 51 was isolated as a yellow oil. In the HR-ESI-(+)-MS spectrum (Figure 4.18) a sodium adduct was observed at m/z 553.2411, corresponding to a molecular formula of
C29H39O9 (calcd for C29H39O9Na, 553.2414).
Figure 4.18. HR-ESI-(+)-MS of compound 51.
1 Figure 4.19. H NMR spectrum of compound 51 in CDCl3 (400 MHz)
The 1H NMR spectrum of compound 51 (Figure 4.19) showed an aromatic proton resonating as a broad singlet at δH 7.65. One olefinic signal resonating as a singlet at δH 6.90 was observed.
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Two pairs of doublets resonating at δH 4.05 and δH 4.18 as well as δH 4.25 and δH 4.36 were also observed, each integrated for one proton. The signals were assigned as two methylene protons using the HSQC NMR spectrum (Figure 4.20), which were attached to an electronegative atom, most likely oxygen, based on the 1H NMR chemical shifts.
Figure 4.20. HSQC NMR spectrum of compound 51.
The observed NMR signals resembled those of phorbol esters. A pair of doublets overlapping at δH 1.15, which integrate for six protons, i.e two methyls, were easily assigned to the isobutyrate moiety, further confirmation of the group was obtained by the long-range HMBC
(Figure 4.21) correlations of both the methyls to a carbonyl carbon resonation at δC 179.0, an ester carbonyl. Assigning the position of the isobutyrate group was difficult, as there were no HMBC correlations of the core phorbol protons to this carbonyl carbon. A comparison with the literature data of phorbol esters revealed that the isobutyrate group was attached to a quaternary carbon C-13. Signals resembling an angelate moiety were observed in the 1H NMR spectrum: a broad triplet at δH 1.88 and a doublet of doublets at δH 1.96, corresponding to the two methyls, and a signal resonating as a quartet of doublets at δH 6.07, corresponding to the methine proton. The chemical shift of the methine proton indicated that it was an olefinic methine, further confirming the presence of an angelate group. The attachment of the angelate group at C-16 is based on the HMBC correlation between two diastereotopic protons on C6 (δH 4.05 and δH 4.18) and the resonance at C-1''' (167.9). In the seven-membered ring of the phorbol ester, the presence of an α,β-unsaturated carbonyl ketone (δC 201.4). The position of the double bond the terminal methanol group, was observed by an extensive comparison of NMR data with the literature data of similar phorbol esters as well as the HMBC correlations between the methylene protons to both the carbonyl and the olefinic carbons. The rest of the signals and correlations were in agreement with the phorbol ester, 16-angeloyloxy-13α-isobutanoyloxy-4β,9α,20-
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trihydroxytiglia-1,5-diene-3,7-dione, which was recently isolated from Euphorbia grandicornis.128
Figure 4.21. HMBC spectrum of compound 51.
52 Compound 52 was isolated as a yellow oil. In the HR-ESI-(+)-MS spectrum (Figure 4.22) a sodium adduct was observed at m/z 595.2545, corresponding to a molecular formula of
C31H40O10 (calcd for C31H40O10Na, 595.2519).
Figure 4.22. HR-ESI-(+)-MS of compound 52.
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Figure 4.23. 1H NMR spectrum of compound 52. The 1H NMR spectrum of compound 52 (Figure 4.23) showed signals which were similar to those of compound 51. Most of the signals resembled the signals of a tigliane phorbol ester.
Proton signals resonating at δH 7.60 and δH 6.28 could be assign to the olefinic protons at position C-1 and C-5 of a phorbol ester. The appearance of H-5 at δH 6.28 for 52 compared to
H-5 at δH 6.90 for 51 is due to the electronic effects contributed by the hydroxyl and the carbonyl groups attached at position C-20. The hydroxyl is an electron donating group when compared to the carbonyl group, thus the signal for H-5 of 51 will be slightly downfield (deshielded) and the signal for H-5 of 52 will be slightly upfield (shielded). The presence of an angelate and an isobutyrate moieties were also confirmed by the comparison of the 1H NMR data with compounds 51. For compound 52, an additional singlet resonance, integrated for three protons, at δH 2.08 was observed. This signal was assigned to an acetate methyl group. A slight shift of signals is also observed, especially the signals of the protons on the seven- membered ring when compared to compound 51. Additionally, one proton signal was observed at δH 4.82, the chemical shift indicated that it was attached to an oxygen-bearing carbon. In the
COSY NMR spectrum (Figure 4.25) the proton (δH 4.82) correlated to a proton resonating at
δH 2.66, assigned to H-8.
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Figure 4.24. HSQC NMR spectrum of compound 52.
Figure 4.25. COSY NMR spectrum of compound 52.
The HMBC (Figure 4.26) revealed that region highlighted in red was different from compound 51. At position C-7 there was hydroxy group, based on the HMBC correlations of the proton resonating at δH 4.82 to the carbons at δC 147.4 (olefin quaternary carbon), δC 72.8 (oxygen- bearing carbon), δC 45.2 (methine carbon). At position C-20, an acetate group was attached.
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Figure 4.26. HMBC spectrum of compound 52.
The configuration at position C-7 of the molecule was confirmed using the NOESY-NMR experiment (Figure 4.27). A correlation of the proton to the proton at position C-10 of the molecule confirmed that the proton at position C-7 of compound 52 is facing away, in the same plain as H-10. The coupling constant between H-7 and H-8 (J = 9.4 Hz) is characteristic of protons in a diaxial orientation. Thus, the structure of 52 was assigned as 20-acetoxy-16- angeloyloxy-13α-isobutanoyloxy-4β,7β,9α,20-tetrahydroxytiglia-1,5-diene-3-one. The NMR data of 51 and 52 are collated in Table 4.3.
Figure 4.27. NOESY NMR spectrum of compound 52.
An extensive literature search revealed that 52 is a new phorbol ester. The only related compound is was isolated from Euphorbia grandicornis with a ketone at position C-7 and without an acetate group at C-20.128 Compounds 51 and 52 are classified as tigliane phorbol esters. In all the other phorbol
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esters, the double bond is between C-6 and C-7, whereas with 51 and 52, the double is between C-5 and C-6. The tigliane-type phorbol esters have a remarkable biological activity. The activity is associated with activation of protein kinase C and other structurally similar phorbol esters. These compounds mimic the action of diacyl glycerol, an activator of protein kinase C. The structural variation between C-3 and C-7 determines the activity of a compound.
Compound 51 was isolated once from Euphorbia grandicornis and no biological assays were conducted on it. As explained in the literature review (Chapter 2.2), tigliane phorbol ester are distributed in the Euphorbiaceae and Thymelaeaceae families. This class of diterpenoids possess the most exciting and diverse useful biological activities, including anti-HIV, tumour-promoting, skin irritant, cytotoxic, and proinflammatory activities. Compound 51 and 52 are structurally similar to both prostatin (13) and ingenol 3-angelate (15) and it can be concluded that compound 51 may have useful biological potential as a protein kinase C activators and modulators of the TRPV4 receptor. It would be interesting to evaluate the biological activity of 51 and 52, as the position of the double bond is unique from all known tigliane phorbol esters. Both compounds (51) and (52) have a 12- deoxy six-membered ring, so it can be concluded that both compounds should not be tumour promoters.
Table 4.3. 1H (400 MHz) and 13C (100 MHz) NMR data of compounds 51 and 52 in
CDCl3.
δH (ppm) (J in Hz) δC (ppm) Position 51 52 51 52 12 1 7.65 s 7.60 s 160.1 159.8 2 - - 135.0 134.2 3 - - 205.3 206.1 4 - - 73.1 72.3 5 6.90 s 137.1 6.28 s 127.2 6 - - 148.2 147.4 7 - 4.83 (1H, d, 9.0) 201.4 81.5
8 3.35 (1H, d, 5.3) 2.66 (1H, dd, 9.5, 5.5) 54.5 45.2 - - 73.5 72.8 9 10 3.30 (1H, t, 2.6) 3.07 s 58.6 56.9 11 2.12 s 2.55 m 38.1 37.7 12a 1.61 (1H, dd, 9.7, 2.9) 2.18 (1H, dd, 14.9, 7.4) 31.5 31.6
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12b 1.60 (1H, dd, 11.5, 3.7) 2.15 (1H, d, 6.9) 13 - - 62.9 63.3 14 1.93 (1H, d, 5.4) 1.67 (1H, d, 5.6) 24.3 26.5 15 - - 26.0 25.7 16a 4.05* 4.44 (1H, d, 11.8) 69.1 70.2 16b 4.18* 4.04 (1H, d, 11.8) 1.13 (3H, d,1.4) 1.18 s 11.5 56.3 17 0.95 (3H, d, 5.8) 0.94 (3H, d, 6.5) 18.5 18.7 18 1.82 (3H, d, 1.5) 1.81 (3H, dd, 2.7, 1.4) 10.3 10.2 19 4.25 (1H, dd, 14.6, 1.0) 2.07 s 20a 63.1 21.0 4.36 (1H, dd, 14.6, 1.0) 2.07 s 20b - - - 170.7 1′ - 2.08 s - 37.7 2′ - - 179.0 179.0 1′′ 2.53 (1H, sep, 7) 2.54 (3H, sep, 7.0) 34.2 34.2 2′′ 1.15 (1H, d, 1.0) 1.17 (1H, d, 6.5) 18.5 18.5 3′′ 1.15 (1H, d, 1.0) 1.17 (1H, d, 6.5) 18.5 18.5 4′′ - - 167.9 168.2 1′′′ - - 127.8 127.2 2′′′ 6.07 (1H, qd, 7.3, 1.4) 6.08 (1H, qd, 7.3, 1.4) 137.7 138.7 3′′′ 1.97 (3H, dd, 7.3, 1.4) 1.96 (3H, dd, 7.3, 1.5) 15.7 15.8 4′′′ 1.88 (3H, t, 1.4) 1.87 (3H, t, 1.4) 20.5 20.3 5′′′ *undefined 4.4. Biological activity
The crude extract of E. cooperi was tested for antimicrobial activities against a Gram-positive (Staphylococcus aureus ATCC 12600), two Gram-negative (Pseudomonas aeruginosa ATCC 10145, Klebsiella pneumoniae ATCC 13883), and a drug-resistant Gram-negative (Escherichia coli ATCC 25218) bacteria. The crude extracts showed moderate but promising antimicrobial activity. The single compounds were not assayed.
Table 4.4: Antibacterial activity results. Antibacterial activity (MIC, µg/ml) Gram +ve Gram -ve Drug resistant Gram -ve
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S. aureus K. pneumoniae P. aeruginosa E. coli E.cooperi 625 625 >5000 >5000 Neomycin <0.24 0.98 0.98 0.24
4.5 Conclusion
A phytochemical investigation of E. cooperi led to an isolation of a novel norsesquiterpenoid (48), its glycoside derivative, arachiside A (50), glutinol (47), a novel phorbol ester (51) and a known phorbol ester (52).
4.6 Experimental
4.6.1 General experimental procedure
Please see Chapter 3 (3.5.1).
4.6.2 Plant material
The aerial parts of Euphorbia cooperi N.E.Br. ex A.Berger were collected in May 2017 at the University of KwaZulu-Natal Botanical Garden in Pietermaritzburg and was identified by a curator, Ms Alison Young. The voucher specimen was deposited to the Bews Herbarium, UKZN, School of Life Sciences (Voucher No 2. NU0048866).
4.6.3 Extraction and isolation of compounds
The plant material E. cooperi was dried for two weeks indoors at room temperature then pulverised to a powder and extracted exhaustively with 1:1 (v/v) of DCM-MeOH for 24 h. This was followed by filtration through cotton wool in a glass funnel. The filtrate was then concentrated under vacuum using a rotatory evaporator (65 C) to afford a crude (10 g aerial crude extract).
The crude extract was then dissolved and mixed with silica gel and dry-packed on a VLC column with silica gel (88 g). The column was eluted with different solvent systems as follows: Hex (400 mL), Hex-DCM (9:1, 400 mL), DCM-EtOAc (20:1, 630 mL), EtOAc (400 mL),
EtOAc-MeOH (5:1, 480 mL), MeOH (600 mL) and distilled H2O (300 mL). The fractions were then dried in the fume-hood over night to afford dry fractions then weighed (Table 4.5).
Table 4.5. Fractions obtained from the VLC fractionation of the crude extract of E. cooperi.
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Fraction Mass (mg) Book code
1 990 SSH-2-3A 2 1330 SSH-2-3B 3 890 SSH-2-3C 4 420 SSH-2-3D 5 360 SSH-2-3E 6 645 SSH-2-3F 440 7 SSH-2-3G
Based on the TLC profiles of the fractions obtained, fractions D, E, F and G were further fractionated. Fraction F and G were combined since similar spots on TLC were observed, the combined fraction was then subjected to column chromatography (18 x 3 cm) packed with silica gel. The column was eluted with a mixture of DCM and MeOH as follows: DCM (400 mL), DCM-MeOH (9:1, 200 mL), DCM-MeOH (7:3, 200 mL), DCM-MeOH (1:1, 200 mL) and MeOH (300 mL). The obtained subfractions (8 mL each) were spotted and combined based on the TLC profile to afford 7 subfrations (Table 4.6). Subfractions 4 (SSH-2-6D) was further purified by centrifugal thin-layer chromatography on a chromatotron™ (1 mm thickness) using DCM-MeOH (9:1) as the solvent system to afford five subfractions (Table 4.7). Subfraction 5 (SSH-2-7E) was identified to be compound 48
Table 4.6. Subfractions obtained from fractionation of the combined fractions F and G.
Fraction Mass (mg) Book code
1 90 SSH-2-6A 2 32 SSH-2-6B 3 24 SSH-2-6C 4 97 SSH-2-6D 5 120 SSH-2-6E
Table 4.7. Subfractions obtained by centrifugal thin-layer chromatography of subfraction SSH- 2-6D.
Fraction Mass (mg) Book code 1 5 SSH-2-7A 2 12 SSH-2-7B
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3 6 SSH-2-7C 4 12 SSH-2-7D 5 23 SSH-2-7E 6 9 SSH-2-7F 10 7 SSH-2-7G
Subfration 5 (SSH-2-6E) was fractionated on a chromatotron™ (2 mm thickness) using DCM- MeOH (7:3) as the solvent system. Three subfrations were obtained (Table 4.8). Fraction 2 (SS-2-13B) was identified as compound 50.
Table 4.8. Subfractions obtained from fractionation of subfration SSH-6E.
Fraction Mass (mg) Book code
1 50 SSH-2-13A 2 23 SSH-2-13B 3 30 SSH-2-13C
Fractions 3D and 3E were also combined based on the TLC profile. The combined fraction was subjected to column chromatography (25 x 4 cm) packed with silica gel. The column was then eluted a mixture of Hex and EtOAc as follows: Hex (500 mL), Hex-EtOAc (9:1, 400 mL), Hex-EtOAc (8:2, 630 mL), Hex-EtOAc (7:3, 400 mL), Hex-EtOAc (6:4, 480 mL), Hex-EtOAc (1:1, 300 mL), Hex-EtOAc (4:6, 200 mL), Hex-EtOAc (1:9, 400 mL), EtOAc (200 mL), then eluted with MeOH (100 mL). Subfractions obtained (8 mL each) were combined based on the TLC profile. 15 subfractions were obtained (Table 4.9).
Table 4.9. Subfractions obtained from the fractionation of the combined fractions 3D and 3E.
Fraction Mass (mg) Book code
1 90 SSH-2-15A 2 33 SSH-2-15B 3 89 SSH-2-15C 4 60 SSH-2-15D 5 80 SSH-2-15E 6 45 SSH-2-15F 12 7 SSH-2-15G 77 8 SSH-2-15H
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89 9 SSH-2-15I 50 10 SSH-2-15J 23 11 SSH-2-15K 45 12 SSH-2-15L 76 13 SSH-2-15M 87 14 SSH-2-15N 99 15 SSH-2-15O
Subfraction 2 (SSH-2-15B) was identified as compound 47. Subfraction 4 (SSH-2-15D) was fractionated on a small column (18 x 2 cm) packed with silica gel and eluted using Hex-EtOAc (7:3) as the solvent system to afford 6 subfractions (Table 4.10). Subfration 1 (SSH-2-19A) was identified to be compound 51 and subfraction 4 (SSH-2-19D) was identified to compound 52.
Table 4.10. Subractions obtained from fractionation of subfration SSH-15D.
Fraction Mass (mg) Book code
1 21 SSH-2-19A 2 15 SSH-2-19B 3 8 SSH-2-19C 4 16 SSH-2-19D 5 6 SSH-2-19E 6 14 SSH-2-19F
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Figure 4.28. Flow diagram of the isolation of compound 47, 48, 50, 51 and 52.
4.6.4 Biological assay
4.6.5 Physical data of isolated compounds
Glutinol (47) was isolated as a white powder. 1H and 13C NMR data are collected in Table 4.1.
HR-ESI-(+)-MS m/z 427.3948 (calcd for C30H51O, 427.3940).
1 Euphorbilactone (48) was isolated as a white solid. H NMR (400 MHz, CDCl3) δH 1.84 (H-
4ax), 156-1.64 (H-4eq), 3.96 (H-5), 2.15 (H-6ax), 1.56-1.64 (H-6aq), 2.36 (H-8), 5.71 (H-9), 5.81 13 (H-10), 4.36 (H-11), 1.29 (H-12), 1.35 (H-13), 1.10 (H-14). C NMR (400 MHz, CDCl3):δC 179.2 (C-1), 46.3 (C-3), 36.2 (C-4), 65.2 (C-5), 38.0 (C-6), 85.3 (C-7), 57.7 (C-8), 120.6 (C- 9), 142.2 (C-10), 68.0 (C-11), 23.7 (C-12), 23.4 (C-13), 18.9 (C-14).
Arachiside A (50) was isolated as a yellow oil. 1H and 13C NMR data are collected in Table
4.2. HR-ESI-(+)-MS m/z 425.1793 (calcd for C19H30O9Na, 425.1788).
16-Angeloyloxy-13-isobutanoyloxy-4β,9,20-trihydroxytiglia-1,5-diene-3,7-dione (51) was isolated as a yellow oil. 1H and 13C NMR data are collected in Table 4.3. HR-ESI-(+)-MS m/z
553.2411 (calcd for C29H39O9Na, 553.2414).
20-Acetoxy-16-angeloyloxy-13α-isobutanoyloxy-4β,7β,9α,20-tetrahydroxytiglia-1,5- diene-3-one (52) was isolate as a yellow oil. 1H and 13C NMR data are collected in Table 4.3.
HR-ESI-(+)-MS m/z 595.2545 (calcd for C31H40O10Na, 595.2519).
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CHAPTER 5
Conclusion and Future Work
Plants are playing an important role in natural products chemistry, especially in the discovery of new drugs that can be used for the treatment of infectious diseases and cancer. The need for the discovery of new pharmacologically active compounds is a global priority as new diseases and drug resistance are emerging and spreading throughout the world. South Africa has a rich biodiversity of plants, many of which are used in traditional medicine. However, the phytochemistry and the bioactivity of many of the plants have not been investigated.
This project aimed at isolating and characterising bioactive compounds from Euphorbiaceae plant species indigenous to South Africa. Antidesma venosum E.Mey. ex Tul. and Euphorbia cooperi N.E.Br. ex A.Berger were investigated. The crude extracts as well as some single compounds were assayed for antimicrobial activities.
Six known compounds, (-)-loliolide, β-sitosteroln 3-O-D-glucoside, lutein, 4- hydroxyphenylethyl trans-ferulate, pheophytin A, and pheophytin B, were isolated from A. venosum. With the exception of pheophytin A, none of these compounds have previously been identified in A. venosum.
A novel norsesquiterpenoid named euphorbilactone and a known glycosyl derivative, arachiside A, a triterpenoid glutinol, a known phorbol ester, 16-angeloyloxy-13α- isobutanoyloxy-4β,9α,20-trihydroxytiglia-1,5-diene-3,7-dione, and a novel phorbol ester, 20- acetoxy-16-angeloyloxy-13α-isobutanoyloxy-4β,9α,20-tetrahydroxytiglia-1,5-diene-3-one, were isolate from E. cooperi. None of these compounds has previously been identified in E. cooperi. With phorbol ester, the structure and configuration of the five- and seven-membered ring determine the biological activity of the compounds. The position of the double bond in the seven-membered ring of two phorbol ester 51 and 52 is different to all the known phrobol derivatives and it would be interesting to see what the effect of this structural feature will be on the activity of 51 and 52.
The crude extracts of the plants showed moderate but promising antimicrobial activity. The pure compounds did not show any antimicrobial activity. Due to time limitations, further studies on bioactivity were not conducted and this should be addressed in future work.
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This investigation revealed that South African Euphorbiaceae have the potential of producing novel bioactive compounds, thus further investigation of these and related plant species within the family would be important in the discovery of new bioactive compounds.
In future work, the first priority should be to investigate the bioactivity of the isolated compounds. A second priority should be to identify readily available Euphorbia species that could yield more novel compounds.
In conclusion, twelve compounds have been isolated, two of which are new compounds. Future work should include the determination of the biological activity of these compounds.
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APPENDIX
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Plate 1. 13C and DEPT-135 NMR spectra of compound 39.
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Plate 2. COSY NMR spectrum of compound 39.
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Plate 3. HSQC NMR spectrum of compound 39.
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Plate 4. HMBC NMR spectrum of compound 39.
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Plate 5. 13C and DEPT-135 NMR spectra of compound 40.
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Plate 6. COSY NMR spectrum of compound 40.
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Plate 7. HSQC NMR spectrum of compound 40.
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Plate 8. HMBC NMR spectrum of compound 40.
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Plate 9. COSY NMR spectrum of compound 47
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Plate 10 COSY NMR spectrum of compound 50
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Plate 11 HSQC NMR spectrum of compound 50
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