UNIVERSITY OF CAPE COAST
PHYTOCHEMISTRY, ANTI-INFLAMMATORY AND ANTIOXIDANT ACTIVITIES OF THE ROOT BARK OF ANTHOSTEMA AUBRYANUM (BAILL)
PATRICK MALCOLM FYNN
2016
Digitized by UCC, Library
© Patrick Malcolm Fynn
University of Cape Coast
Digitized by UCC, Library DECLARATION
Candidate’s Declaration
I hereby declare that this thesis is the result of my own original research and that no part of it has been presented for another degree in this university or elsewhere.
Candidate’s Signature:...... Date:......
Name: Patrick Malcolm Fynn
Supervisors’ Declaration
We hereby declare that the preparation and presentation of the thesis were supervised in accordance with the guidelines on supervision of thesis laid down by the University of Cape Coast.
Principal Supervisor’s Signature:...... Date:......
Name: Prof. Yaw Opoku-Boahen
Co-Supervisor’s Signature: ...... Date:......
Name: Dr. (Mrs) Genevieve Adukpo
ii
Digitized by UCC, Library ABSTRACT
The work presented in this thesis involves the scientific investigation into the traditional uses of the root bark of Anthostema aubryanum (Baill., family, Euphorbiaceae) as an anti-inflammatory and antioxidant agent. It also describes the isolation and characterization of two compounds from the alkaloid extract of the root bark of Anthostema aubryanum Baill. The anti-inflammatory activity was investigated using the acute carrageenan – induced foot pad edema model in six weeks old rats. The extracts were given orally to the rats at 30, 100 and 300 mg/kg body weight, 1 hour after induction of oedema with carrageenan using diclofenac as the reference drug. All extracts of the root bark were demonstrated to display a time-and dose-dependent anti-inflammatory effects in rats with methanolic extract showing the highest activity (ED50 = 5.29± 0.02
BDW) compared to the standard drug, diclofenac (ED50 = 1.99± 0.01). The antioxidant properties of the extracts were investigated using three assays; total antioxidant capacity, total phenolic content and DPPH scavenging activity. The antioxidant activity of the methanolic crude extract (IC50=8.84±0.02 µg/ml) was equivalent to the standard vitamin E (IC50=8.61±0.01 µg/ml) with total phenolic content of 74.53±0.004. Comprehensive chromatographic and spectroscopic analyses of the alkaloid extract led to the isolation and characterization of two major anti-inflammatory and antioxidant agent as 5-methoxycanthin-6-one and canthin-6-one with the former showing the highest pharmacological activity
(ED50=60.84±0.01, IC50=27.62±0.010 and ED50=96.64±0.01, IC50=33.60±0.01 respectively). This is the first report of the isolation of these compounds from the family Euphorbiaceae.
iii
Digitized by UCC, Library ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my supervisors,
Professor Yaw Opoku-Boahen and Dr (Mrs) Genevieve Adukpo, both of the
Department of Chemistry, for their professional guidance, advice, encouragement and the goodwill with which they guided this work. I am really very grateful.
I am again grateful to my good friend Dr Francis Armah for providing us with the plant sample and assisting in the pharmacological activities.
I also express my appreciation to the laboratory technicians of the
Departments of Chemistry, University of Cape Coast, Biomedical and
Forensic Sciences, University of Cape Coast and Pharmacognosy, Kwame
Nkrumah University of Science and Technology, Kumasi, for their excellent technical assistance. I am forever grateful.
I would like to thank Professors Solomon Habtemariam of the
Department of Pharmacognosy Research Laboratories, Medway School of
Science, University of Greenwich, United Kingdom and Baldwyn Torto,
Chemical and Behavioral Ecology Department, International Centre for Insect
Physiology and Ecology, Kenya for generously running and providing us with the NMR and MS spectra of the isolated compounds.
I would like to thank Rev. Sr. Elizabeth Amoako-Arhen, the Principal of OLA College of Education, Cape Coast for her unflinching support throughout the programme. The sponsorship from the Ghana Education Trust
Fund (GETFUND) is gratefully acknowledged.
Finally, I wish to thank my family and friends for their support, especially, my friend, Justice Owuraku Addo.
iv
Digitized by UCC, Library DEDICATION
To my lovely wife, Naomi Arthur Fynn (Mrs) and children, Nhyiraba, Nyameyie, Judalyn and Jedida
v
Digitized by UCC, Library TABLE OF CONTENTS
Page
DECLARATION ii
ABSTRACT i ii
ACKNOWLEDGEMENTS iv
DEDICATION v
TABLE OF CONTENTS vi
LIST OF TABLES xiii
LIST OF FIGURES x i v
LIST OF ABBREVIATIONS xviii
CHAPTER ONE: INTRODUCTION
Background to the Study 1
The Plant Anthostema aubryanum (Baill) 3
Botanical Description of Plant Species 4
Ethnomedicinal Uses 5
Statement of the Problem 6
Justification of the Study 8
Main Objectives of the Study 11
Specific Objectives of the Study 11
CHAPTER TWO: LITERATURE REVIEW
Introduction 12
The Family Euphorbiaceae 12
Ethnomedicinal Uses of Euphorbiaceae 14
Phytochemistry of Euphorbiaceae 16
Diterpenes 17
vi
Digitized by UCC, Library Triterpenes 22
Alkaloids 23
Flavonoids and other phenolic compounds 25
Tannins 28
Coumarins 30
Cyanogenic Glycosides 31
Fatty Alcohols 33
Other Classes of Compounds 34
Alkaloids 35
Properties of Alkaloids 3 6
Structure and Classification of Alkaloids 37
Biosynthetic Classification 37
Chemical Classification 38
Pharmacological Classification 39
Taxonomic Classification 39
Types of Alkaloids 40
True Alkaloids 40
Protoalkaloids 42
Pseudoalkaloids 4 2
Nomenclature of Alkaloids 43
Pharmacological Uses of Alkaloids 44
Distribution of Alkaloids 44
The Family Euphorbiaceae 46
The Family Apocynaceae 47
The Family Asteraceae 4 8
vii
Digitized by UCC, Library The Family Loganiaceae 49
The Papaveraceae Family 50
The Family Rutaceae 51
The Family Solanaceae 5 3
The Family Erythroxylaceae 54
The Family Boraginaceae 55
The Family Fabaceae 56
The Family Menispermaceae 57
The Family Berberidaceae 59
The Family Ranunculaceae 60
The Family Liliaceae 61
The Family Rubiaceae 62
The Family Amaryllidaceae 64
The Family Elaeagnaceae 65
The Family Zygophyllaceae 65
Mushroom 66
Moss 67
Fungi and Bacteria 68
Animals 69
Tests for Alkaloids 73
Extraction and Isolation of Alkaloids 76
Acidic Water Extraction 76
Aqueous-Alcohol Extraction 77
Organic Solvent Extraction 77
Beta-carboline Alkaloids 78
viii
Digitized by UCC, Library Nomenclature of Beta-carboline Alkaloids 78
Distribution of Beta-carboline Alkaloids 78
Biosynthesis of Beta-carboline Alkaloids 82
Synthesis of Beta-carboline Alkaloids 82
Pharmacological Uses of Beta-carboline Alkaloids 86
Inflammation 94
Inflammatory Pathway 98
Experimental Models of Inflammation 99
Models of Acute Inflammation 99
Carrageenan-induced Paw Edema 100
Oxidative Stress 101
Antioxidants 103
Determination of Antioxidant Properties 104
Total Antioxidant Capacity 105
DPPH radical scavenging activity 106
Total Antioxidant Activity by the Phosphomolybdenum Method 107
Total Phenolic Activity by Folin-ciocalteau Method 107
CHAPTER THREE: MATERIALS AND METHODS
Chemicals 109
General Experimental Procedures 109
Collection and Authentication of Plant Sample 110
Processing of Plant Material 110
Phytochemicals Screening of Crude Plant Extract 110
Extraction of Plant Material 116
Anti-Inflammatory Assay of Extracts 117
ix
Digitized by UCC, Library Experimental Animals 117
Carrageenan-Induced Edema in Rats 117
Anti-inflammatory Assay of Crude Methanolic Extract 118
Anti-inflammatory Assay of Crude Alkaloid Extract 119
Antioxidant Assay of Extracts 119
Total Phenolic Content Assay 119
Total Antioxidant Capacity Assay 119
In Vitro Qualitative DPPH Test 120
Quantitative Antioxidant Assays of Extracts 120
Statistical Analysis of Data 121
Fractionation of Alkaloid Extract 122
Chromatographic Materials 122
Detection for Analytical thin Layer Chromatography 122
Column Chromatography 123
Preparative-Layer Chromatography 123
Development of Thin Layer Chromatogram 124
Isolation of Compounds from the Crude Alkaloid Extract 125
Column chromatographic separation 0f the crude alkaloid extract 125
Isolation of Compound M1 128
Isolation of Compound M2 and M3 128
Isolation of Compounds M4 and M5 129
Anti-inflammatory Activity of Isolated Compounds 130
In Vitro DPPH Radical Scavenging Activity of Isolated Compounds 130
CHAPTER FOUR: RESULTS AND DISCUSION
Introduction 131
x
Digitized by UCC, Library Characterization and Identification of Isolated Compounds 133
Identification of M1 as 5-Methoxy-Canthin-6-one (1) 133
Identification of M5 as Canthin-6-one (2) 138
Bioassays 142
Anti-inflammatory Activity of Root Bark Extract 142
Anti-inflammatory Activity of Crude Alkaloid Extract 147
Anti-inflammatory Activity of the Isolated Compounds 147
Antioxidant Activity of Extracts 151
Antioxidant Activity of Crude Extracts and Isolated compounds 151
Quantitative Antioxidant Assay of Extracts 152
Total Phenolic Content 152
Total Antioxidant Capacity 153
DPPH Radical Scavenging Activity of Extracts of A. Aubryanum 156
Antioxidant Activity of Isolated Compounds 157
Quantitative DPPH Radical Scavenging Test 157
CHAPTER FIVE: SUMMARY, CONCLUSIONS AND
RECOMMENDATIONS
Introduction 161
Summary 161
Conclusions 163
Recommendations 165
Suggestions For Further Research 167
REFERENCES 1 68
xi
Digitized by UCC, Library 1 APPENDIX A: H-NMR of M1 in MeOD at 500 MHz 197
1 APPENDIX B: Integrated H-NMR of M1 in MeOD at 500 MHz 198
13 APPENDIX C: C-NMR of M1 in MeOD at 500 MHz 199
13 APPENDIX D: Expanded C-NMR of M1 in MeOD at 500 MHz 200
APPENDIX E: Mass spectrum of M1 201
APPENDIX F: Elemental analysis of M1 202
1 APPENDIX G: H-NMR of M5 in MeOD at 500 MHz 203
1 APPENDIX H: Integrated H-NMR of M5 in MeOD at 500 MHz 204
13 APPENDIX I: C-NMR of M5 in MeOD at 500 MHz 205
13 APPENDIX J: Expanded C-NMR of M5 in MeOD at 500 MHz 206
APPENDIX K: Mass spectrum of M5 207
CURRICULUM VITAE
LIST OF PUBLICATIONS
xii
Digitized by UCC, Library LIST OF TABLES
Table Page
1 Phytochemical Analysis of A. aubryanum 132
2 1H-NMR and 13C-NMR spectral data and 1H-13C long-range
correlations of M1 in MeOD at 500 MHz 137
3 13C-NMR Chemical shifts (ppm) of Canthin-6-one and
Compound M5 140
4 1H-NMR and 13C-NMR spectral data and 1H-13C long-range
correlations of M5 in MeOD at 500 MHz 141
5 Effect of Crude Extracts and Standard Drug on Carrageenan-
induced Edema 143
6 Effect of M1 and M5 on Carrageenan-induced Edema 148
7 Total Phenolic Content of Root Extract 152
8 Total Antioxidant Capacity of Root Extract 153
9 DPPH Scavenging Activity of Root Extract 156
10 DPPH Scavenging Activity of M1 and M5 158
xiii
Digitized by UCC, Library LIST OF FIGURES
Figure Page
1 Diseases with Chronic Inflammation 2
2 Photograph of A. aubryanum 5
3 Examples of Diterpenoids Isolated from the Family
Euphorbiaceae 21
4 Examples of Triterpenoids Isolated from the Family
Euphorbiaceae 22
5 Examples of Alkaloids Isolated from the Family Euphorbiaceae 25
6 Examples of Flavonoids Isolated from the Family
Euphorbiaceae 27
7 Examples of Tannins Isolated from the Family Euphorbiaceae 29
8 Examples of Coumarins Isolated from the Family Euphorbiaceae 31
9 Examples of Cyanogenic Glycosides Isolated from the Family
Euphorbiaceae 33
10 Examples of Fatty Alcohols Isolated from the Family
Euphorbiaceae 34
11 Examples of Phenylbutanoid isolated from the Family
Euphorbiaceae 35
12 Examples of True Alkaloids 41
13 Examples of Protoalkaloids 42
14 Examples of Pseudoalkaloids 43
15 Alkaloids of Euphorbiaceae 47
16 Alkaloids of Asteraceae 49
xiv
Digitized by UCC, Library 17 Alkaloids of Loganiaceae 50
18 Alkaloids of Papaveraceae 51
19 Alkaloids of Rutaceae 53
20 Alkaloids of Solanaceae 54
21 Alkaloids of Erythroxylaceae 55
22 Alkaloids of Boraginaceae 56
23 Alkaloids of Fabaceae 57
24 Alkaloids of Menispermaceae 59
25 Alkaloids of Berberidaceae 60
26 Alkaloids of Ranunculaceae 61
27 Alkaloids of Liliaceae 62
28 Alkaloids of Rubiaceae 63
29 Alkaloids of Amaryllidaceae 65
30 Alkaloids of Elaeagnaceae 65
31 Alkaloids of Zygophyllaceae 66
32 Alkaloids of Mushroom 67
33 Alkaloids of Moss 68
34 Alkaloids of Fungi and Bacteria 69
35 Alkaloids of Animals 73
36 Biosynthesis of Simple Beta-carboline Alkaloids 82
37 Thermolysis of Tryptophan (1) to Form Tryptamine (2) 85
38 By-products of the thermolysis of tryptophan to form tryptamine 86
39 Pathways for the Generation of the Various Mediators of
Inflammation 99
40 Pathway for the Detoxification of Reactive Oxygen species by
xv
Digitized by UCC, Library Superoxide Dismutase, Catalase and Peroxidases 104
41 Schematic Representation of the Isolation of Alkaloid 126
42 TLC Analysis of Crude Alkaloid Extract 127
43 Schematic Representation of the Isolation of M1 128
44 Schematic Representation of the Isolation of M2 and M3 129
45 Schematic Representation of the Isolation of M4 and M5 130
46 Fragmentation Pattern of Compound M1 136
47 The structure of compound M5 139
48 Time-course Oedema Development Following Carrageenan
Injection into Rat Paws and Dose (mg/Kg-)-dependent anti-
inflammatory Effect of the Standard Positive Controls, 144 Diclofenac
49 Effect of the Methanol Root Bark Extract (30-300 mg/kg Oral),
on Time Course Curve (a) and Total OedemaResponse
(Expressed as AUC, b) for 5 Hours, in Carrageenan –Induced
Paw Edema in Rats. .***p<0.0001; ***p<0.001; ***p<0.01
compared to vehicle-treated group 145
50 Effect of crude alkaloidal extract (30-300 mg/Kg oral), on time
course curve (a) and the total oedema response (expressed as
AUC, b) for 5 hours, in carrageenan-induced paw oedema in rats.
***p<0.0001; ***p<0.001; ***p<0.01 compared to vehicle-
treated group. 146
51 Effect of 5-methoxy-canthin-6-one (3-30mg/Kg; i.p) on time
course curve (a) and the total edema response (expressed as AUC,
b) in carrageenan-induced paw oedema in rats. ***p<0.0001;
xvi
Digitized by UCC, Library ***p<0.001; ***p<0.01 compared to vehicle treated group 149
52 Effect of Canthin-6-one (3-30 mg/kg; i.p) on time Course
Curve (a) and the total edema Response (Expressed as AUC,
b) in Carrageenan-induced Paw Edema in Rats.*** p<0.0001;
***p<0.001; ***p<0.01compared to vehicle-treated group
53 Dose Response Curves for Crude, Alkaloidal, M1, M5 and 150
Diclofenac on Carrageenan-induced Foot Edema in Rats 151
54 Absorbance against Concentration of Vitamin E Used in the
calibration curve 152
55 Concentration Response Curves for Standard Drug, Extracts and
Isolated Compounds 158
Plot of56 Percent Inhibition Against Concentration of Extracts and Isolated Compounds 159
57 DPPH Absorption Spectra of Extracts and Isolated Compounds 159
xvii
Digitized by UCC, Library LIST OF ABBREVIATIONS
ANOVA Analysis of variance
ASTM American Standard Test Method
C Carbon
CC Column Chromatography
13C-NMR Carbon-13 Nuclear Magnetic Resonance
COX Cyclooxygenase
COSY Correlation Spectroscopy
DEPT Distortionless Enhancement by Polarization Transfer
DMARDS Disease modifying antirheumatic drugs
DMSO Dimethyl sulfoxide
DPPH 2, 2-diphenyl-1-picrylhydrazyl
EI Electron Impact ionization eV Electron Volt
GC Gas Chromatography
H Proton
Hz Hertz
1H-NMR Proton Nuclear Magnetic Resonance
HMBC Heteronuclear Multiple Bond Correlation
HPLC High Performance Liquid Chromatography
HSQC Heteronuclear Single Quantum Coherence
IR Infrared
J Coupling constant
LT Leukotrienes
MS Mass Spectrometry
xviii
Digitized by UCC, Library m/z Mass-to-Charge Ratio
NMR Nuclear Magnetic Resonance
NOE Nuclear Overhauser Effect
NOESY Nuclear Overhauser Enhancement Spectroscopy
NSAIDS Non-steroidal anti-inflammatory drugs
PAF Platelet Activation Factor
PGs Prostaglandins ppm Parts Per Million
PTLC Preparative Thin Layer Chromatography
Rf Retardation factor s Singlet t Triplet
TLC Thin layer chromatography
2D Two Dimensional
UV Ultraviolet
WHO World Health Organization
xix
Digitized by UCC, Library
CHAPTER ONE
INTRODUCTION
Plant-based remedies have proved to be useful in the treatment and management of diseases and are used extensively in ethnomedical and ethnoveterinary practices (Dangarembizi et al., 2013). The prohibitive cost of conventional medicines and their limited availability especially to rural communities in Africa and other developing countries have driven the continued dependence on traditional therapeutics. About 75-90% of the world population still relies on plant and plant extracts as a source of primary health care (Bruno,
2012). This widespread use of plant derived extracts in disease management has led to an interest in the identification and characterization of the active compounds which give the extracts their therapeutic potential. The active compounds have provided significant leads in the development of more effective synthetic molecules.
Background to the Study
Pain and inflammation are the major conditions associated with various diseases (Agnihotri et al., 2010). Typical inflammatory diseases such as meningitis, rheumatoid arthritis, asthma, colitis and hepatitis are the leading cause of disability and death (Amponsah, 2012) and chronic inflammation has been implicated in the pathogenesis of cancer, cardiovascular, pulmonary and neurodegenerative diseases (Amponsah, 2012). Inflammation activates neutrophils and macrophages to produce free radicals such as reactive oxygen species and reactive nitrogen (ROS/RNS species as well nitric oxide (NO)
1
Digitized by UCC, Library
which deregulate cellular function causing tissue damage leading to chronic inflammatory diseases (Wu et al., 2006) and also inhibit wound healing.
Various molecules have been isolated from plant drugs which have been proven to be effective in such conditions. For example; aspirin, a potent anti- inflammatory analgesic molecule was developed from salicin, a compound isolated from the bark of Salix alba Linn (Agnihotri et al., 2010).
Cancer Diabetes Cardiov ascular
Neurological Alzheimer’s diseases INFLAMATION diseases
Autoimmun Pulmonary e diseases diseases Arthritis
Figure 1: Diseases with chronic inflammation
About 25 % of the drugs prescribed worldwide come from plants, with
121 of such active compounds being in current use (Rates, 2001). Of the 252 drugs considered as basic and essential by the World Health Organization
(WHO), 11 % are exclusively of plant origin and a significant number are semi-synthetic drugs obtained from natural precursors and that about 60% of
2
Digitized by UCC, Library
anti-tumor and anti-infectious drugs in use or under clinical trials are of natural origin (Amponsah, 2012).
The vast majority of these drugs cannot be synthesized and are still obtained from wild or cultivated plants. Natural compounds can thus be lead compounds, allowing the design, development and the discovery of new therapeutic agents (Hamburger & Hostettmann, 1991). A search in the natural product alert data base suggest that only about 15% of all plant species had been studied to some extent for their phytochemistry and only about 5% for one or more biological activities (Amponsah, 2012). Although extensive research on medicinal plants is published every year, only a few plants have been comprehensively studied for their pharmacological properties. Thus traditional medicines and medicinal plants obviously represent a great source of novel medicines and leads for drug development.
The Plant Anthostema aubryanum (Baill)
Anthostema aubryanum (Baill,) is a flowering plant in the family
Euphorbiaceae (Spurge family) and Anthostema was first described as a genus in 1824 (A. Juss 1824). The genus is native to Africa and consists of only three species, Anthostema aubryanum (Baill), Anthostema senegalense (A. juss) and
Anthostema madagascariense (Baill). The genus is related to the genus
Dichostema. The genus can be found in humid evergreen forest from sea level up to 900-1700 metres high in altitude, sometimes in swamps. Geographically,
Anthostema aubryanum (Baill) can be found in Gabon, Guinea-Bissau, Ghana,
Cote d’Ivoire, DR Congo and Madagascar.
3
Digitized by UCC, Library
In Ghana, it is found in the swampy surroundings of Axim and Abora, all in the
Western region.
Botanical Description of Plant Species
Anthostema aubryanum (Baill) is an evergreen monoecious shrub to medium-sized tree up to 30 metres tall with succulent white latex in all parts
(Hawthorne & Jongkind, 2006). Branches have layers with evenly spaced leaves. The leaves are rounded at the base; young leaves reddish which are ten in pairs and laterally meeting near the margin. The leaves have finer veins which tend to run parallel. The leaves are alternate, simple and entire. Stipules are small, petiole up to 1.50 cm long and groved. Bole is branchless, up to 15 metres high, 50 cm in diameter and is cylindrical. The bark surface is densely fissured or smooth, reddish to blackish. The blade is elliptical to oborate, 5-13 cm x 2.5-5 cm, cuneate at base, acuminate to obtuse at apex, leathery, glaborous, pinnately veined with 10-15 pairs of lateral veins. Inflorescence on axillary cyme with apex of each cyme-branch having common involucres composed of four small partly fused bracts with glandular margins enclosing a female flower surrounded by involucres, each containing several male flowers.
Flowers are unisexual. Male flowers have short pedicel, 3-4 toothed perianth with a single stamen. Female flowers have short, stout-pedicel, 3-4 lobed perianth, ovary superior and glaborous, 3-celled, styles short and spreading.
Fruit has 3-lobed capsule, 3 cm in diameter, and green turning brown at dehiscence with persistent style, 3-seeded. Seeds are ovoid, 12 mm long, laterally compressed, brownish and shiny (Govaerts et al., 2000).
4
Digitized by UCC, Library
Figure 2: Photograph of A. aubryanum
Ethnomedicinal Uses
Anthostema aubryanum Baill (Euphorbiaceae) is a tropical wild plant which is commonly used in African ethnomedicine for treating a number of disease conditions which include inflammation, malaria, urinary tract infections, mental illness, wounds (especially post abortion or after delivery) and other disease conditions like pregnancy troubles (Abbiw, 1990;Muganza et al., 2012).
In Democratic Republic of Congo, it is used to treat infections of the gastrointestinal tract, constipation, diarrhoea and dysentery (Muganza et al.,
2012; Bruno, 2012). In DRC, it is called Assogo. In Ghana, the Nzemas called it
“Sese” and the Ahantas called it “kyirikasa” (hate talking).
In Senegal, a bark maceration is drunk to treat and manage intestinal infection, kidney problems, edema, impotence and as a laxative (Bruno, 2012). The bark
5
Digitized by UCC, Library
is also used as a fish poison to catch small fish in Senegal. Just like Anthostema senegalense, it used to treat leprosy, menstrual problems and help with the expulsion of the afterbirth (Abreu et al., 1999).
The latex is toxic, acrid and vesicant and can cause blindness. The latex is used as a drastic purgative and is applied externally to sores. The latex is used in traditional medicine as glue and the smoke from the wood is reportedly used to drive away animals
Like many woody trees, A. aubryanum is commonly used in homesteads for fencing, firewood and construction.
Statement of the Problem
The stem and root bark of Anthostema aubryanum are routinely employed in the West African ethnomedicine to treat inflammation and a variety of other disease conditions. Although the chemistry and pharmacology of different classes of phytochemicals from the family Euphorbiaceae are fairly established, the plant has not yet been investigated phytochemically.
Majority of human population worldwide is getting affected by the inflammation related disorders. The excessive production of free radicals by phagocytic leucocytes during the inflammatory process, as part of host defence, deregulates cellular function causing tissue injury which in turn augments the state of inflammation leading to chronic inflammatory diseases (Amponsah,
2012). Known treatments against inflammation include the use of corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), disease modifying anti-rheumatic drugs (DMARDS) and the opiates (Amponsah, 2012).
6
Digitized by UCC, Library
However, common side effects of these synthetic drugs include gastrointestinal ulceration, haemorrhage, erectile dysfunction, kidney dysfunction (nephrotoxicity), hypertension, liver toxicity and liver failure
(hepatotoxicity), etc. There is also tolerance and dependence induced by the opiates. The use of these drugs also produces free radicals which cause tissue damage. A number of immuno-suppressing agents have been developed based on their inhibition of cyclooxygenase-1 (COX-1), but they cause detrimental side effects on long term administration. Accordingly, selective inhibitors of cyclooxygenase-2 (COX-2) were developed to avoid side effects of COX-1 inhibitors. However, one of these inhibitors has been reported to increase the risk of myocardial infarction and atherothrombotic conditions. Thus, it is likely that COX-2 inhibitors will not be suitable for the treatment of chronic inflammatory diseases, such as rheumatoid arthritis (Agnihotri et al., 2010).
Drug therapy for rheumatoid arthritis is based on the principal approaches of symptomatic treatment with non-steroidal anti-inflammatory drugs (NSAIDs) and disease modifying antirheumatic drugs (DMARDs). However, most of the currently available drugs primarily target the control of pain and/or the inflammation associated with joint synovitis, but do little to interfere with the underlying immuno-inflammatory condition, and hence do little to block the disease progression and reduce cartilage and bone destruction of joints
(Agnihotri et al., 2010). As a result, therapeutic agents suitable for the treatment of chronic inflammatory diseases are highly desirable, which has led to an increased interest in complementary and alternative medicines
7
Digitized by UCC, Library
Many synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoulene (BHT), tertiary hydroquinone (TBHQ), etc are commonly used as additives or preservatives by the pharmaceutical, cosmetic and food industries (Esa, et al., 2013).These antioxidants have toxic and/or carcinogenic and mutagenic effects..
Therefore, new drugs are needed to augment or replace the currently available therapeutics.
The crude water extract of the stem bark of Anthostema senegalense showed strong anthelmintic activity against the larvae of Haemonchus contortus in vitro (Abreu et al., 1999). A crude stem bark extract exhibited significant activity against Leishmania donovani with IC50 of 9.10 μg/mL as well as moderate antibacterial and antifungal activities in vitro (Tandon et al., 2011).
Scientific research has thus validated the ethnomedicinal claims that the genus
Anthostema is useful in disease management. Therefore, Anthostema aubryanum (Baill) was selected to isolate, characterize, identify and quantify the active compounds and possibly determine the mechanisms underlying its curative properties.
Justification of the Study
Ghana is an area of high biodiversity, holding a tremendous richness of as yet uninvestigated plant species. In this contemporary world, indigenous people in Ghana still rely mainly on their herbal traditional medicine. Currently there has been an increased interest globally to identify natural products from plant sources which are pharmacologically potent and have low or no side
8
Digitized by UCC, Library
effects for use in protective medicine and the food industry. These plants can promote good health and alleviate illness and have proven to be safe, better patience tolerance, relatively less expensive and globally competitive. These plants represent a potential source of new compounds with antioxidant properties. Free radicals play a role in the health of the modern era and the diseases caused by them are becoming a part of normal life. Herbal medicine and their phytoconstituents are important in managing pathological conditions of those diseases caused by free radicals such as wound. Antioxidants, which scavenge these free radicals, have been found to complement the anti- inflammatory process, promote tissue repair and wound healing. Wound is one disease condition that is causing havoc to the world population but seems to be forgotten or neglected. Wound infection is a major complication of injury and it accounts for 50-70% of hospitalized death (Barku, 2015). For instance, in
Ghana 273,346 (1.64%) of the general population suffer one or more forms of open wounds (Barku, 2015). Wound healing disorders present a serious clinical problem of medical health care in Africa and in Ghana and are associated with diseases such as diabetes, hypertension and obesity as a result of poor hygienic conditions and malnutrition (Barku, 2015). Most of these disorders lead to complications, high morbidity and mortality rates.
A number of medicinal plants have been used in treating inflammation and its related disorders. Many of them have been studied scientifically and proved to be beneficial anti-inflammatory agents and are in clinical use such as aspirin, berberine and colchicine (Agnihotri, et al., 2010). Also, Quercetin, kaempferol
9
Digitized by UCC, Library
and their derivatives have been isolated from Cistus laurifolius Linn. These natural products exhibit potent anti-inflammatory and antinociceptive activities
(Agnihotri, et al., 2010). The potency of these flavonoids was found to be equal to that of indomethacin, a well-known anti-inflammatory drug, without inducing any apparent acute toxicity or gastric damage. These compounds also possess potent antihepatotoxic activity against acetaminophen-induced liver damage in mice.
Alchornea cordifolia has been widely used throughout Africa to treat diseases like asthma, hepatitis, colitis, metritis, vaginitis, splenomegaly and dermatitis.
These reported activities are due to the presence of guanidine alkaloids and flavonoids. These natural products have been found to inhibit human neutrophil elastase (HNE), matrix metalloproteinases (MMP-2 and -9) and arachidonic acid metabolism which are associated with anti-inflammatory process in vitro studies. Curcumin isolated from turmeric is very effective in treating postsurgical inflammation and is a potent antioxidant (Agnihotri, et al., 2010).
To date, no bleeding disorders have been reported with curcumin supplementation.
In Ghana Anthostema aubryanum (Baill) is rare and near extinct due to deforestation and there is therefore the need for documentation. Hence this research sought to evaluate the biological potential of A. aubryanum that can help prevent diseases, lower health problems and probably meet man’s demands for primary healthcare.
10
Digitized by UCC, Library
Main Objectives of the Study
The research primarily seeks to evaluate the anti-inflammatory and antioxidant activities of methanolic extract of the root bark of A. aubryanum.
The study therefore seeks to achieve the following specific objectives.
Specific Objectives of the Study
1. to screen the root bark of A. aubryanum for phytochemical constituents
2. to evaluate in vivo anti-inflammatory activity of the root bark of A.
aubryanum using the acute carrageenan-induced foot edema in rats.
3. to evaluate the antioxidant activity of the root bark of A. aubryanum.
4. to isolate and purify the alkaloids present in the root bark using various
chromatographic methods.
5. to characterize and identify the isolated alkaloids using spectroscopic
methods.
6. to evaluate the anti-inflammatory and antioxidant activities of the isolated
alkaloids.
11
Digitized by UCC, Library
CHAPTER TWO
LITERATURE REVIEW
INTRODUCTION
Phytochemical screening and pharmacological activity studies on the root bark of Anthostema aubryanum (Baill) followed by comprehensive chromatographic and spectroscopic analyses of the alkaloid extract led to the isolation and characterization of two major anti-inflammatory and antioxidant
β-carboline alkaloids. In this review, we present a brief, yet comprehensive, up- to-date summary including the biochemical and pharmacological importance of
β-carboline alkaloids.
THE FAMILY EUPHORBIACEAE
The family Euphorbiaceae is the sixth largest and one of the most diversified families of angiosperms, consisting of about 300 genera and over
8000 species (Volken, 1999). The largest genus is Euphorbia consisting of over
1600 species followed by the genus Croton with nearly 700 species. Thirteen other genera contain over 100 species. These include for example Phyllanthus
(480 species), Acalypha (430 species), Glochidon (280 species), Macaranga
(240 species), Manihot (160 species), Jatropha (150 species) and Tragia (140 species). The smallest genus is the Anthostema with only three species. The
Euphorbiaceae display an extraordinary range of growth forms, ranging from large desert succulents to trees and even small herbaceous types (Volken, 1999).
The family Euphorbiaceae has provided many problems for botanists and taxonomists due to the great variation of forms exhibited. Several systematists
12
Digitized by UCC, Library
studied the classification of the Euphorbiaceae in the last 180 years. The first major milestone in the history of the taxonomy of the Euphorbiaceae was the classification of Jussieu (1824), who identified the major series of genera that
(after much later revision) correspond roughly to the current subfamilies.
Afterwards Muller provided the first detailed classification of the family into subfamilies, tribes and subtribes. Pax and Hoffmann (cited by Volken) recognized four subfamilies of very different size, the Phyllanthoideae with 65 genera, the Crotonoideae with 209, the Porantheroideae with 34, and the
Ricinocarpoideae with 5 (Volken, 1999). In all of the classifications of the
Euphorbiaceae proposed before 1975, the major criteria were drawn from details of gross morphology observable with the naked eye or a dissecting lens
(Volken, 1999).
Webster presented in 1975 a classification, grouping the 300 genera of
Euphorbiaceae into 52 tribes in the following five subfamilies: Phyllanthoideae
Oldfieldioideae, Acalyphoideae, Crotonoideae and Euphorbioideae, with several of the tribes divided into subtribes (Volken 1999). In 1994 Webster published a revised classification, suggesting five subfamilies, 49 tribes and 317 genera
(Volken, 1999). Although the taxonomic classification of Webster from 1994 is considered the actual systematic classification, critical remarks showed the difficulties in the classification of infrafamiliar relationships in the
Euphorbiaceae (Volken, 1999). It can thus be assumed, that the classification of the Euphorbiaceae has not yet been accomplished nor will be for the next future.
13
Digitized by UCC, Library
Although present worldwide, the family Euphorbiaceae is a predominantly tropical family. There are only a few exclusively extratropical genera, e.g.
Crotonopsis (North America), Mercurialis (temperate and warm temperate
Eurasia), Seidelia (South Africa), Dysopsis (temperate and Andean South
America). Only one genus, the genus Euphorbia, is cosmopolitan. In Papua
New Guinea there are only two endemic genera, namely Annesijoa and
Neomphalea (Volken, 1999).
Characteristic of the family Euphorbiaceae are the so called cyathia; mostly greenish-yellow, single flower type formations, which represent inflorescences.
Although looking like a hermaphrodite flower, male and female flowers are separate. The male flowers consist of a single petiolate stamen. They are arranged around a single, female flower, consisting of a three-celled ovary, protruding from the cyathium. The fruit is composed by a small capsule, made up of three fruitlets or “coccae" (Euphorbiaceae are therefore also known as
Tricoccae), which split explosively to release the seed (Volken, 1999).
Ethnomedicinal Uses of Euphorbiaceae
Ethnomedicinal uses of Euphorbiaceae are based on their medicinal, toxic or economically interesting properties. Medicinal purposes for euphorbiaceous plants range from treatment of tumours, migraine, parasite infestations, bacterial infections, anti inflammation, pregnancy related problems, venereal diseases, skin conditions, purgatives to their use as abortifacients
(Volken, 1999). In 1966 Farnsworth published a review on antitumor effects of traditionally used plants, mentioning 12 species of Euphorbiaceae with
14
Digitized by UCC, Library
antitumor activity, including Acalypha phleidos, Croton monanthogynos,
Euphorbia amygdaloides, and Macaranga triloba (Volken, 1999). In a survey on the medicinal use of plants Hartwell mentioned 26 different active genera of
Euphorbiaceae for the treatment of tumours, growths and warts (Volken, 1999).
Several Euphorbiaceae are used traditionally as remedies against parasite infections. Macaranga kilimandscharica and Ormocarpum trichocarpum are used against bilharziasis. Anthostema senegalense A. juss, Anthostema aubryanum Baill as well as Mercurialis annua and Acalypha indica are traditionally used as anthelmintics and as remedies against scabies (Watt and
Breyer-Brandwijk 1962). Bacterial infections such as lepra are treated by natives in Polynesia with a wood decoction of Excoecaria agallocha or leaves of Homalanthus populneus. Many euphorbiaceous plants are reported as traditional remedies against venereal diseases. Jatropha curcas is used against syphilis, Phyllanthus virgatus and Aleurites moluccana are used against gonorrhoea (Volken, 1999). Traditional uses of euphorbiaceous plants as abortifacients or purgatives are widespread. Leaves of Croton lobatus are reported to act as abortifacient. The most drastic of all purgatives known comes from the seeds of Croton tiglium. It is now generally out of use, being too toxic.
Causing violent evacuation in minutest doses, it may also cause sloughing of the intestinal lining (Volken, 1999).
Different species of this family have been noted for their toxicological effects, for example induction of inflammation of skin and mucous membranes, conjunctivitis, and strong purgative activity. Also some species such as those of
15
Digitized by UCC, Library
the Anthostema genus are used as fish poisons and as ingredients of arrow poisons. Ricinus communis (castor oil plant) is employed in medicine as a cathartic and in industry in the manufacturing processes of greases and other lubricants. It is also used in the tanning industry to preserve both the flexibility and the impermeability of leather; and it is also used in the production of soaps, glycerine, paints, enamels, varnishes, dyes, plastics, rubber, linoleum, polishes, waxes, carbon-paper, and crayons. The most well known economic plant of the
Euphorbiaceae is the rubber tree, Hevea brasiliensis, which is the main source of natural rubber. Moreover, Manioc, cassava, or tapioca plant, Manihot esculenta, is a source of a staple foodstuff for many people in many African countries. It originated from South America and from there it has been introduced into every part of the world’s tropics. A serious drawback of cassava cultivation is that it exhausts the soil in which it grows.
Phytochemistry of Euphorbiaceae
The diverse nature of this plant family is also exhibited by its secondary metabolism. The chemistry of the Euphorbiaceae is among the most diverse and interesting of flowering plant families. Many compounds from many different chemical classes have been reported from members of the Euphorbiaceae. An intense chemical work has been done largely on the genera Euphorbia (Seigler
1994) and Croton (Salatino and Negri, 2007). Most genera contain characteristic milky latex which consists of mineral salts, proteins, amino acids, terpenes and cautchouc. The composition of these latexes shows a big chemical heterogeneity and is mainly responsible for the toxic effects and biological activities
16
Digitized by UCC, Library
(Hegnauer 1989). Terpenoids are the predominant secondary metabolite constituents in Euphorbiaceae (Salatino and Negri, 2007), chiefly diterpenoids, which may belong to the cembranoid, clerodane, neoclerodane, halimane, isopimarane, kaurene, secokaurane, labdane, phorbol and trachyobane skeletal types. Triterpenoids, either pentacyclic or steroidal, have frequently been reported for Euphorbiaceae species. Volatile oils containing mono and sesquitepenoids, and sometimes shikimate-derived compounds are also common in the family. Several species have been reported as sources of different classes of alkaloids. Phenolic compounds have frequently been reported, among which flavonoids, lignoids, glycosides and proanthocyanidins predominate.
Diterpenes
Clerodane diterpenes, an extremely diverse group of terpenoids with more than 800 known compounds, seem to be one of the prevalent classes of compounds in the family, especially the Croton genus (Salatino, et al., 2007).
The furane clerodanes with a lactone ring trans-crotonin and trans- dehydrocrotonin have been isolated from the stem bark of C. cajucara, which yielded also the nor-clerodanes cajucarin A and B, cajucarin-β, cajucarinolide and sacarin (Maciel et al., 2000). Trans-crotonin and trans-dehydrocrotonin were obtained from the aerial parts of the same plant. Other sources of furano clerodanes are the stem barks of C. eluteria and C. urucurana (Salatino and
Negri, 2007). C. urucurana yielded cascallin, cascarillone, cascarillins A-D, cascarillins E-I, cascarilldione, eluterin K and pseudoeluterin B (Salatino and
Negri, 2007). Also, ten new clerodanes (eluterins A-J) have been isolated from
17
Digitized by UCC, Library
C. eluteria. The stem bark of C. urucurana yielded sonderianin, 15,16-epoxy-
3,13(16)-clerodatriene-2-one and 12-epi-methyl-barbascoate Clerodanes were obtained from the bark of C. lechleri; crolechinol and crolechinic acid, and the lactone clerodanes korberin A and B. Methylbarbascoate is a trans-clerodane found as major diterpene in leaves of C. californicus (Salatino and Negri, 2007).
From shoots of C. schiedeana, Puebia et al., (2005) isolated cis- and trans- dehydrocrotonin and the new neo-clerodanes 5β-hydroxy-cis-dehydrocrotonin and (12R)-12-hydroxy-cascarillone. The acid fraction of shoot extracts of C. schiedeanus yielded two new cis-clerodanes(-)-methyl-16-hydroxy-19-nor-2- oxo-cis-cleroda-3,13-dien-15,16-olide-20-oate and (+)-15-methoxyfloridolide A
(Palmeira et al., 2005). The same authors isolated the new clerodanes crotobrasilins A and B from leaves and stems of C. brasiliensis (spreng.) Mull
Arg. The labdane crotonadiol was obtained from the stem bark of C. zambesicus
(Ngadjui et al., 2002). From the same plant, the clerodanes crotocorylifuran and crotozambefuran A-C were isolated together with the trachylobanes, 7β- acetoxy-trachyloban-18—oic acid and trachyloban-7β,18-diol (Salatino, et al.,
2007). Two clerodanes, 3α,4β-dihydroxy-15,16-epoxy-12-oxocleroda-
13(16),14-dien-9-al and 3α,4β-dihydroxy-15,16-epoxy-12-oxocleroda-
13(16)14-diene were isolated from bark of the Madagascarian C. hovarum
Leandri (Krebs et al., 1996). From leaves of the same plant, the clerodanes
3,12-dioxo-15,16-epoxy-cleroda-13(16),14-dien-9-al and 3α,4β-dihydroxy-
15,16-epoxy-nor-12-oxo-cleroda-5(10),13(16),14-triene were isolated (Krebs and Ramiarantsoa, 1997). From leaves of C. zambesicus the trachylobane, ent-
18
Digitized by UCC, Library
trachyloban--3β-ol was obtained (Thongtan et al., 2003). From the same plant, ent-18-hydroxy-trachyloban-3-one and the isopimarane-type diterpene, isomara-
7,15-dien-3β-ol were also obtained (Block et al, 2004). C. tonkinensis, a species native to Vietnam, has been a prolific source of ent-kaurane-type diterpenes.
From the leaves of this species, ent-7β-hydroxy-15-oxokaur-16-en-18-yl-acetate and ent-1α-acetoxy-7β,14α-dihydroxy-kaur-16-en-15-one were isolated (Minh et al., 2003). From the same source, the known ent--kauranes ent-7α,14β- dihydroxykaur-16-en-15-one and ent-18-acetoxy-7α-hydroxykaur-16-en-15-one plus the new compounds ent-1β-acetoxy-7α,14β-dihroxy-kaur-16-en-15-one and ent-18-acetoxy-7α,14β-dihroxykaur-16-en-15-one were isolated together with four new ent-kauranes (Salatino and Negri, 2007). Also, Giang et al.,
(2005) isolated six new ent-kauranes from the leaves of C. tonkinensis. Besides clerodane and kaurane derivatives, the leaves of C. sublyratus contain the acyclic diterpene alcohol plaunotol (Vongchareonsathit and De-Eknamkul,
1998). Leaves of this plant are the main source of this compound though it may be found in the leaf chloroplasts of C. stellatopilosus Ohba (Wungsintaweekul and De-Eknamkul, 2005). C. oblongifolius has been a prolific source of diterpenes including: (i) the clerodane 11-dehydro (-) hardwickiic acid (ii) the labanes, labda-7,12 (E),14-tiene, labda-7,12(E),14-trien-17-al (iii) the cembranoid diterpenes, crotocembranoic acid and neocrotocembranal (iv) the cytotoxic labdane diterpenoids, 2-acetoxy-3-hydroxy-labda-8(17),12(E)-14- triene and 2,3-dihydroxy-labda-8(17)12(E),14-triene were isolated
(Roengsumran et al., 1999) (v) the labdane nidorellol, the furoclerodane
19
Digitized by UCC, Library
croblongifolin and the clerodane crovatin (Roengsumran et al., 2002); (vi) the halimanes crotohalimaneic acid and 12-benzoyloxycrotohalimaneic acid; (vii) new labdane-type diterpenoids were isolated from C. californicus, C. draco and
C. aromaticus L., a species with red latex native in Sri Lanka (Bandara et al.,
1987). Secokauranes have been isolated from the leaves of C. kongensis
(Thongtan et al., 2003). A prenylbisabolone diterpene with insecticidal effect was isolated from the Jamaican C. linearis Jacq (Alexander et al., 1991). In addition to yucalexins B-6 and P-4, roots of C. sarcopetalus, a shrub native to
Bolivia and central and north-western Argentina, contain diterpenes bearing the novel skeleton sarcopetalane: sarcopetaloic acid and two sarcopetalolides (De
Heluani et al., 2000). The same plant contains junceic acid and stress metabolites. Salatino and Negri, (2007) reported of the isolation of secolabdane diterpene- saudinolide from Cluytia species. A clerodane diterpenoid, cromiargyne has also been isolated from Croton hemiargyreus (Amaral and
Barnes 1997). Many genera contain phorbol esters, tri- or tetracyclic diterpene esters, with three different structure subtypes, known as tigliane, daphnane and ingenane. A new jatrophane polyesters and 4-deoxyphorbol diesters have been isolated from Euphorbia semiperfoliata (Salatino and Negri, 2007).
Although most Euphorbiaceae are plants not known as aromatic, some
Croton species contain volatile oils. Other species have not been reported as bearing volatile oils, though they were shown to possess sesquiterpenes commonly found in volatile oils. The volatile oils of several species contain phenylpropanoids and terpenoids (mono and sesquiterpenes), while from other
20
Digitized by UCC, Library
species only terpenoids have been isolated. The volatile oils of the leaves and stem bark of C. aepetaefolius contains mono and sesquiterpenes such as 1,8- cineole and terpineol, bicyclogermacrene, respectively, and volatile phenylpropanoids (such as methylleugenol), and the acetophenone xanthoxylin
(Magalhaes et al., 1998). A volatile oil was isolated from the roots of C. sarcopetalus with trans-methylisoeugenol as the main constituent (De Heluani et al., 2000). Linalool and cineol are monoterpenoids seemingly relatively frequent in Croton. Linalool is among the major constituents of the volatile oil of C. stellulifer Hutch, an edemic species of S. Tome and Principe ; this oil contains kessane, a sesquiterpenoid oxide not found elsewhere in Croton
(Viasberg et al., 1989).
O O O H COOCH3 H OH O
O O O O Cromiargyne O H Saudinolide
R2O HO RO OiBu
H O H OH O H OR1 H BzO AcO OAc iBu : OH O OH Bz : R1: Ac O R2 : AC 4-Deoxyphorbol diesters Jatrophane polyesters
Figure 3: Examples of diterpenoids isolated from the family Euphorbiaceae
21
Digitized by UCC, Library
Triterpenes
Triterpenoids are derived biosynthetically from squalene (Harbone,
2008) and produce several pharmacologically active groups such as steroids, saponins and cardiac glycosides (Ramawat et al., 2009). These terpenes are active against bacteria, viruses, fungi and protozoa (Cowan, 1999). Many of them find applications in industries, e.g. in perfumes, mosquito repellants, starting materials for the synthesis of vitamin A, antimalarial compounds
(Artemisinin), anticancer compounds (Taxol), insect hormones, insect antifeedant and growth inhibitors, plant growth stimulators, etc.
H
OH H H
OAc
Boeticol Kamaladiol-3-acetate
HO
R= p-coumaroyl
RO
Cis/ trans Securinegin Figure 4: Examples of triterpenoids isolated from the family Euphorbiaceae
Most species of Euphorbiaceae contain triterpenes. The major triterpenes are derivatives of cycloartenol and tetracyclic triterpenes, example boeticol,
(Volken, 1999) and securinegins. There are also pentacyclic triterpenes, example kamaladiolacetate, which was isolated from a Mallotus species
22
Digitized by UCC, Library
(Volken, 1999). Also, cucurbitacines and cucurbitacin-derivatives have been reported from several Euphorbiaceae species. The aerial parts of C. draco contains β-sitosterol, stigmasterol and the new sterol ergasterol-5α-8α- endoperoxide (Salatino et al., 2007).
Alkaloids
Alkaloids are low molecular weight nitrogen containing compounds that have remarkable physiological effects (Ramawat et al., 2009). This has led to their use as pharmaceuticals, stimulants, and narcotics. They are cyclic organic compound containing nitrogen in a negative oxidation state which is of limited distribution among living organisms (Bhat, et al., 2007)
Different classes of alkaloids have been isolated from a number of
Euphorbiaceae, especially from the genera Croton, Phyllanthus and Securinega
(Volken, 1999) and the lesser known genus ; Trigonostemon. In 1970
Yamaguchi reported on the isolation of Benzylisoquinoline alkaloids aporphine and crotonosine from Croton linearis. Yamaguchi also reported of the isolation of Securinine alkaloids, a small group of compounds which only occurs in the subfamily Phyllanthoideae, example virosecurinine from Securinega virosa.
Imidazole alkaloids have been isolated from the genera Glochidion and
Alchornea. There has also been report of isolation of alkaloids derived from nicotinic acid such as ricine, isolated from Ricinus communis (Rizk and El-
Missiry, 1986).
Attioua et al., (2012) reported of the isolation of onosmin A and B, N-(2- hydroxy-1-phenylpropyl) benzamide and aurentiamide from the aerial part of
23
Digitized by UCC, Library
Croton lobatus. Glutarimide alkaloids and a new class of sesquiterpenes guaiane-type alkaloids have recently been isolated from Croton species.
Taspine, an unusual alkaloid with a dilactone structure resembling elagic acid and one nitrogen atom not included in a heterocyclic ring, was found in the red latex of three species, C. draco (Murillo et al., 2001), C. lechleri (Risco et al.,
2003) and C. palanostigma (Itokawa et al., 1991). Taspine has also been obtained from plant sources of benzylisoquinolines and biogenetically related alkaloids, such as Berberidaceae and Magnoliaceae. From the leaves of C. lechleri other alkaloids, probably related biogenetically to Taspine, have also been isolated such as glaucine, isoboldine, magnoflorine, norisoboldine thaliporphine and sinoacutine (Salatino and Negri, 2007).
Tetrahydroprotoberberine alkaloids have been reported from C. hemiargyreus
Mull. Arg, and C. flavens L. From the leaves and stems of C. hemiargyreus,
Amaral and Barnes (1998) isolated 2,10-dihydro-3,10-dimethoxy-8β- methyldibenzo[a,g]-quinolizidine (hemiargyrine), in addition to glaucine, oxoglaucine, salutaridine and norsalutaridine. The Tetrahydroprotoberberine alkaloids scoulerine and coreximine and the morphinanedienone alkaloids salutaridine and salutarine, in addition to sebiferine, norsinoacutine and flavinantine, were isolated from plants from Barbados of C. flavens by
Eisenreich et al., (2003). From shoots of C. salutaris, Barnes and Soeiro (1981) isolated salutarine and salutaridine, the latter a biosynthetically precursor of morphine. Isoboldine and laudanine were found in the ethanolic extracts of leaves and twigs of C. celtidifolius (Amaral and Barnes, 1997). Stuart and
24
Digitized by UCC, Library
Graham (1973) verified that C. linearis synthesizes crotonosine through linearisine. The β-carboline alkaloids 2-ethoxycarbonyltetrahydroharman and 6- hydroxy-2-methyltetrahydroharman were obtained from C. moritibensis, a species from north-eastern Brazil (Araujo-junior et al., 2004). Hu et al., (2009) also reported of the isolation of six β-carboline alkaloids from Trigonostemon lii. The aerial parts of C. cuneatus yielded the new glutarimidine alkaloids julocrotol, isojulocrotol and julocrotone in addition to julocrotonine (Suarez et al., 2004). Anabasine and the new guaiane-type alkaloids muscicapines A, B and C were obtained from the roots of the north-eastern Brazilian C. muscicapa
Mull. Arg. (Araujo-junior et al., 2004). O H3CO O H NH O H3CO CH3 H N H OH N O N-(2-hydroxy-1-phenylpropyl) H Crotonosine benzamide Virosecurinine O O OH OCH3 O O H N N N N O H H H O
Onosmin A Onosmin B Aurentiamide acetate Figure 5: Examples of alkaloids isolated from the family Euphorbiaceae
Flavonoids and other Phenolic Compounds
Flavones are phenolic compounds containing benzo-γ-pyrone ring with phenyl substitution at position 2 of the pyrone ring. Flavonol is a 3-hydroxy derivative of flavone. Flavonoids are also hydroxylated phenolic compounds that occur as C6-C3 unit linked to an aromatic ring. Flavonoids are known to be synthesized by plants in response to microbial infection (Cowan, 1999).
Flavonoid compounds are effective antimicrobial (Tsuchiya et al., 1996),
25
Digitized by UCC, Library
antibacterial (Borris, 1996), antiviral including HIV (Critchfield et al., 1996) and antischistosomal (Perrett et al., 1995) agents. Flavonoid compounds are also the major anti-inflammatory agents and can inhibit both cyclooxygenase and lipooxygenase pathways of the arachidonic metabolism depending upon their chemical structures (Chi et al., 2001). Flavonoids are good antioxidants which scavenge and reduce free radical formation (Grassi et al., 2010).
Flavonoids also possess cardio-suppressant and hypotensive properties
(Ramawat et al., 2009). Flavonoids have many other biological activities including: mitochondrial-adhesion inhibition, antiulcer, estrogenic, estrogen receptor binding, antiangiogenic, anticancer, protein kinase inhibition, prostaglandin-synthesis inhibition, DNA synthesis/cell cycle arrest and topoisomerase inhibition (Bhat et al., 2007).
Flavonoids, particularly flavones and flavonols occur in the family
Euphorbiaceae. They occur as O- and C-glycosides and their methyl ethers. The two most common flavonols, kaempferol and quercetin and their glycosides are widespread in different genera of the family (Rizk 1987). From the red latex of
C. draco and C. panamensis, myricithin was isolated (Kostova et al., 1999;
Tsacheva et al., 2004). Leaves of C. cajucara yielded kaempferol-3,7-dimethyl ether and 3,4,7-trimethyl ether (Maciel et al, 2000), while shoots of C. schiedeanus contain quercetin-3,7-dimethyl ether (Guerrero et al., 2002). The leaves of C. betulaster yielded 5-hydroxy-7,4’-dimethoxyflavone (Barbosa et al., 2004) and from the C. hovarum Leandri, Krebs and Ramiarantsoa (1997) isolated the flavone C-glycoside vitexin. The n-hexane extracts of C.
26
Digitized by UCC, Library
ciliatoglanduliferus Ori yielded the highly methoxylated flavonols retusin and pachypodol (Gonzalex-Vasquez et al., 2006). Only recently were phenyl propanoids reported for the first time in Euphorbiaceae especially in the Croton genus. From the aerial parts of C. hutchinsonianus Hos, a species native to
Thailand, two new compounds were isolated, namely 3’-(4”-hydroxy-phenyl)- propyl benzoate and 3’-(4”-hydroxy-3”,5”-dimethoxyphenyl)-propyl benzoate, together with the known 3’-(4”hydroxy-3”-methoxyphenyl)-propyl benzoate
(Athikomkulchai et al., 2006).
Lignoids are common in plant bearing benzylisoquinoline and related alkaloids (derived biosynthetically from tyrosine), such as Ranunculales and
Magnoliales. Some species of Euphorbiaceae possess this class of alkaloids.
However, only one lignoids has been found in Croton, the dihydro-benzofuran lignan 3’,4-O-dimethylcedrusin. It is interesting to note that this lignan co-occur with taspine, having been found in C. lechleri and C. palanostigma, both species with red latex (Risco et al., 2003).
OH OH OH
HO O HO O O
OH OH OH O O OH O Kaempferol Quercetin Flavone
OH OH OH HO O HO O HO O OH OH OH OH O OH O OH O Chrysin Catechin Myricetin
Figure 6: Examples of flavonoids isolated from the family Euphorbiaceae
27
Digitized by UCC, Library
Tannins
“Tannin” is a general descriptive name for a group of polymeric phenolic compounds capable of tanning leather or precipitating gelatin from solution (astringency). The term tannin can therefore be defined as chemical structure or group of chemical compounds that have tannin properties. They are found in almost every plant part: bark, wood, leaves, fruits, and roots (Scalbert,
1991). They are divided into three groups, hydrolyzable, condensed tannins and pseudotannins. Hydrolyzable tannins are based on gallic acid, usually as multiple esters with D-glucose; while the more numerous condensed tannins
(proanthocyanidins) are derived from flavonoid monomers. Pseudotannins are simpler phenolic compounds of low molecular weight co-occurring with tannins. These compounds do not give the standard test for tannins
(Goldbeater’s skin test), e.g. gallic acid, catechins, chlorogenic acid, etc.
Tannins may be formed by condensations of flavan derivatives which have been transported to woody tissues of plants or by polymerization of quinine units
(Geissman, 1963). They may also be formed by the combination of catechins monomers (the so-called proanthocyanidins), or by ester bounded units of glucose, gallic and/or elagic acid (hydrolysable tannins). So far, only proanthocyanidins have been characterized in Croton species.
Proanthocyanidins have been reported as important active principles of species containing red latex (Pieters et al., 1995).
Many human physiological activities such as stimulation of phagocytic cells, host-mediated tumor activity and a wide range of anti-infective
28
Digitized by UCC, Library
activities have been assigned to tannins (Haslam, 1996). Tannins can be toxic to filamentous fungi, yeasts and bacteria (Scalbert, 1991). Tannins also possess antidiarrheal and anti-inflammatory activities (Njoronge and
Kibunga, 2007). Tannins and tannic acid reduce secretion by denaturing proteins of the intestinal mucosa forming protein tannates which make the mucosa more resistant to chemical alteration (Dangarembizi et al., 2013).
Compounds with anti-diarrheal properties also act by decreasing intestinal motility, stimulating water absorption and reducing electrolyte secretion
(Njoronge and Bussman, 2006). Monomers such as (+)-catechin, (-)- epicatechin, (+)-gallocatechin, (-)-epigallocatechin and dimeric procyanidins B-1 and B-4 have been isolated (Salatino and Negri, 2007).
Dimers and trimers have also been isolated and characterized. The fruits of
Phyllantus emblica contain corilagen, gallic acid and elagic acid (Singh et al., 2011). OH O O OH HO O O HO OH HO OH HO OH O HO OH OH OH O OH Gallic acid Ellagic acid Gallocatechin OH OH HOH2C HO OH O
HO OH O O CO O CHOMe O O
HO O O O OH HO HO OH Corilagin OH O Furosin OH Figure 7: Examples of tannins isolated from the family Euphorbiaceae.
29
Digitized by UCC, Library
Coumarins
Coumarins are phenolic compounds made up of fused benzene and α- pyrone rings, i.e. they are 5,6-benzo-2-pyrone compounds (Bhat et al., 2007).
Coumarins are responsible for the characteristic odor of hay. More than 1350 coumarins have been isolated till 1997 (Bhat et al., 2007). They are well known for their antithrombotic (Thastrup et al., 1985), anti-inflammatory and vasodilatory (Namba et al., 1988) activities. Coumarins are known to be highly toxic to rodents especially warfarin which is used as an oral anticoagulant and a rodenticide (Keating and O’Kennedy, 1997) and may also have antiviral effects
(Berkada, 1978). Several other coumarins have antimicrobial and estrogenic activities (Cowan, 1999). Coumarins have been used to prevent recurrences of cold sores caused by HSV-1 in humans (Berkada, 1978) but are ineffective against leprosy. Also, phytoalexins, which are hydroxylated derivatives of coumarins, are produced in carrots in response to fungal infection and can be presumed to have antifungal activity (Hoult and Paya, 1996).
Coumarins particularly of the furanocoumarin type abound in
Euphorbiaceae (Seigler, 1994). Scopoletin was obtained from the wood extract of E. tirucalli and C. draco (Murillo et al., 2001). The fruits of Phyllantus emblica yielded umbelliferone and seselin (Singh et al., 2011). Daphnehtin and psolaren have been isolated from Daphnehtin tangutica and Euphorbia buxoides respectively (Pan et al., 2010).
30
Digitized by UCC, Library
H CO H3CO 3 O O O HO O H3CO O O Coumarin Scopoletin Scoparone
O O HO O O HO O O O Psoralen OH Umbelliferone Daphnethin
O O O
Seselin
Figure 8: Examples of coumarins isolated from the family Euphorbiaceae
Cyanogenic Glycosides
Cyanogenic glycosides (CGs) or cyanoglycosides account for approximately 90% of the plant toxins known as cyanogenes. The key characteristic of these toxins is cyanogenesis, the formation of free hydrogen cyanide, and is associated with cyanohydrins that have been stabilized by glycosylation to form the cyanogenic glycosides (FSANZ, 2004). The CGs are
O-β-glycosidic derivatives of α-hydroxynitriles (Poulton, 1990). Depending on their precursor amino acid, they may be aromatic, aliphatic or cyclopentenoid in nature. Most CGs are cyanogenic monosaccharides, though cyanogenic oligosaccharides also exist. Sulphated, malonylated and acylated derivatives of
CGs are also known (Poulton, 1990). The major edible plants in which CGs occur are cassava, lima beans, sorghum, almonds, stone fruits and bamboo shoots. In small quantities these glycosides do exhibit expectorant, sedative and digestive properties. However, many of these edible plants are highly cyanogenic and have caused numerous cases of acute cyanide poisoning of animals including man. Cases of acute cyanide poisoning have been associated
31
Digitized by UCC, Library
with misuse, particularly of preparations from apricot pits, bitter almonds and cyanide rich apple seeds. In areas of the world where these cyanogenic plants are the staple food, chronic cyanide poisoning and associated pathological conditions exist (Poulton, 1989). Goitre and cretinisim due to iodine deficiency can be exacerbated by chronic consumption of insufficiently processed cassava.
Neurologically, there has been report of Konzo or spastic paraparesis in children and woman of child-bearing age in East Africa in times of food shortage and is associated with a high and sustained intake of cassava in combination with a low intake of protein (Davis, 1991). Also, tropical ataxic neuropathy (TAN), which is attributed to cyanide exposure from the chronic consumption of food derived from cassava, has been reported. CGs are widely distributed among 100 families of flowering plants. They are also found in some species of ferns, fungi, bacteria and animals especially arthropods.
The family Euphorbiaceae is rich in cyanogenic glycosides, especially the genera Euphorbia and Croton. Seven cyanopyridone derivatives and one seco compound have been isolated from a methanolic extract of the inflorescences and leaves extract of Acalypha indica (Salatino and Negri, 2007).
32
Digitized by UCC, Library OH OH OCH3 O O OH OCH3 CONH2 R 4 OH O OH R3 O O N O R2 O N R1 H HO R1= CH3; R2= OH; R3= H; R4= CN- Acalyphin CH3 R1= CH3; R2= H; R3= OH; R4= CN-Epiacalyphin OH R1= H; R2= OH; R3= H; R4= CN- Noracalyphin Epiacalyphin amide ycloside R1= H; R2= H; R3= OH; R4= CN-Epinoracalyphin R1= CH3; R2= OH; R3= H; R4= CONH2- Acalypin amide
HO OCH3 OH OH O OH O O O OH OH
O N H3CO OH CN HO CH3 H N ar-Acalyphidone CH3 Seco-Acalyphin O
Figure 9: Examples of cyanogenic glycosides isolated from the family Euphorbiaceae
Fatty Alcohols
Different genera of Euphorbiaceae contain Long-chain fatty alcohols
(particularly n-octacosanol and n-hexacosanol) and hydrocarbons, especially the genus Euphorbia yielded a considerable amount of hydrocarbons and alcohols
(Rizk 1987). The dried sap of C. draco yielded 3,4,5-trimethoxycinnamic alcohol (Salatino and Negri, 2007). The polyalcohols IL-1-O-myo-inositol and neo-inositol were isolated from C. celtidifolius (Salatino et al, 2007). From the roots of the traditional Chinese medicinal plant; Phyllantus emblica L, 1,2,4,6- tetra-O-galloyl-β-D-glucose (1246 TGG) has been isolated. The less polar fractions of the latex of E. peplus were found to contain obtusifoliol, cycloartenol, 24-methylenecycloartenol and 24-methylenelanosterol in the free
33
Digitized by UCC, Library
and esterified triterpenes alcohol fractions and a new acyclic triterpenes alcohol named peplusol (Salatino and Negri, 2007).
OH HO OH R
R
C O OH O O O R= HO C O OH O O C
O Peplusol HO OH C O OH OH
HO OH OH
1246 TGG
Figure 10: Examples of fatty alcohols isolated from the family Euphorbiaceae
Other Classes of Compounds
The seeds of C. draco contains p-hydroxybenzaldehyde and p- methoxybenzoic acid (Salatino and Negri, 2007). Phenylbutanoids, an interesting class of compounds known to occur in some genera of angiosperms, were obtained from the shoots of C. schiedeanus by Puebla et al., (2005).
These authors also isolated (2S)-7,9-dimethoxyrhododendrol, (2S)-acetoxy-7,9- dimethylrhododendrol and (2S)-2,8-diacetoxy-7,9-dimethoxyrhododendrol. The formation of this class of phenolics has been proposed to occur via decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce C6C4 skeletons (Abe et al., 2001). The novel compounds 4-(2- hydroxyethyl)-benzoic acid and 2,5-dihydroxy-phenylethanol were isolated from the red sap of C. panamensis (Kostova et al, 1999). Lichexanthone was
34
Digitized by UCC, Library
obtained from the aerial parts of C. cuneatus (Suarez et al., 2004). From the same plant, Hernandez and Delgado (1992) isolated a mixture of polyprenols with castaprenol-II being the major compound. Simiarenol (a high molecular mass triterpenoid) and esters of amyrine with fatty acids containing carbon chains above 20 atoms have been isolated from the shoots of C. hemiargyreus.
Benzoyl-methylpolyols were isolated from C. betulaster and C. luetzelburgii
(Barbosa et al., 2004). Furanoarabinoid-gallactan, a polysaccharide, is the main compound found in the gum exudates of C. urucurana (Milo et al., 2002). The peptide derivatives aurentiamide acetate and N-benzoylphenylalanine were isolated from shoots of C. hieronyini (Catalan et al., 2003). Cyclopeptides were reported from the red latex of C. draco (Tsacheva et al., 2004).
MeO
HO OH
MeO 7,9-Dimethoxyrhododendrol
Figure 11: Example of phenylbutanoid isolated from the family Euphorbiaceae
ALKALOIDS
Alkaloids are a group of molecules with a relatively large occurrence in nature. They are very diverse chemicals and biomolecules, though secondary compounds and are derived from amino acids or from the transamination process. They are a large group of compounds with biological, pharmacological or physiological and chemical activities.
35
Digitized by UCC, Library
Properties of Alkaloids
In plants, alkaloids because of their basic nature occur largely as salts of organic acids like acetic, oxalic, citric, malic, lactic, tannic, aconitic, quinic acids, etc with well-defined crystalline structures. Some basic pyridine alkaloids such as nicotine, myosmine, anabasine, etc occur in free state or as N-oxides. A few alkaloids are present as glycosides of common sugars such as glucose, rhamnose, galactose (Solanum and Veratrum alkaloids), or as esters of organic acids (e.g. reserpine, hyoscyamine, cocaine). Some alkaloids are present as quaternary salts (tubocurarine hydrochloride, muscarine chloride or as tertiary amine oxides. Many neutral compounds where the nitrogen is involved in an amide group are now included as alkaloids. Examples are colchicine and piperine. In addition to the elements carbon, hydrogen and nitrogen, most alkaloids contain oxygen. A few, such as coniine and nicotine, are oxygen-free and are liquids. Although coloured alkaloids are very rare, berberine is yellow and the salts of sanguinarine are copper-red. Knowledge of the solubility of alkaloids and their salts is of considerable pharmaceutical importance. Not only are alkaloidal substances administered in solution, but also the differences in solubility between alkaloids and their salts provide methods for the isolation of alkaloids from plants and their separation from the non-alkaloidal substances also present. While the solubilities of different alkaloids and their salts show considerable variation due to their varied structures, free bases are frequently sparingly soluble in water but soluble in organic solvents; with salts the reverse is often the case. However, there are exceptions to this generalization.
36
Digitized by UCC, Library
Structure and Classification of Alkaloids
Alkaloids show great variety in their botanical and biochemical origin, in chemical structure and in pharmacological action. Consequently, many different systems of classification are possible. They may be classified according to their:
(1) biological and ecological activity
(2) chemical structures
(3) biosynthetic pathway
(4) common molecular precursor used to construct the molecule.
Biosynthetic Classification
This classification is based on the types of molecular precursors or building block compounds used by living organisms from which the alkaloids are produced biosynthetically. It is therefore convenient and also logical to group all alkaloids having been derived from the same precursor but possessing different taxonomic distribution and pharmacological activities together.
Examples:
(a) Indole alkaloids derived from tryptophan
(b) Piperidine alkaloids derived from lysine
(c) Pyrrolidine alkaloids derived from ornithine,
(d) Phenylethylamine alkaloids derived from tyrosine
(e) Imidazole alkaloids derived from histidine.
The major drawback of this method is that the relationship of alkaloids to each other and to their precursors is not always apparent.
37
Digitized by UCC, Library
Chemical Classification
Chemical classification is the most widely accepted and common mode of classification of alkaloids and it depends on the type of heterocyclic ring structure present. There are two broad divisions:
1. Non-heterocyclic sometimes called ’protoalkaloids’ or biological amines.
2. Heterocyclic or typical alkaloids, divided into 14 groups according to their
ring structure. These groups are as follows:
(a) alkaloids derived from amination reactions such as acetate-derived alkaloids, phenylalanine-derived Alkaloids, terpenoid alkaloids and steroidal
Alkaloid, (b) alkaloids derived from anthranilic acid e.g. quinazoline alkaloids, quinoline alkaloids and acridine alkaloids
(c) alkaloids derived from histidine, e.g. imidazole alkaloids
(d) alkaloids derived from lysine, e.g. piperidine Alkaloids, quinolizidine alkaloids and indolizidine Alkaloids
(e) alkaloids derived from nicotinic acid such as pyridine alkaloids,
(f) alkaloids derived from ornithine, e.g. pyrrolidine alkaloids, tropane alkaloids and pyrrolizidine alkaloids
(g) alkaloids derived from tyrosine such as phenylethylamine alkaloids, simple tetrahydro iso-quinoline alkaloids and modified benzyl tetrahydro iso-quinoline
alkaloids
(h) alkaloids derived from tryptophan which include; simple indole alkaloids, simple β-carboline alkaloids, terpenoid indole alkaloids, quinoline Alkaloids, pyrroloindole alkaloids and ergot Alkaloids.
38
Digitized by UCC, Library
Pharmacological Classification
Alkaloids exhibit a broad range of very specific pharmacological characteristics which are used as a strong basis for the general classification of the wide-spectrum of alkaloids derived from the kingdom of the living organisms. These pharmacological properties include: analgesics, cardio- vascular drugs, central nervous system stimulants and depressants, dilation of pupil of eye, mydriatics, anticholinergics, sympathomimetics, antimalarials, purgatives, etc. It must be emphasized that this classification is not quite common and widely known. Examples:
(i) morphine as narcotic analgesic,
(ii) quinine as antimalarial,
(iii) strychnine as reflex excitability,
(iv) lobeline as respiratory stimulant,
(v) boldine as choleretics and laxatives,
(vi) aconitine as neuralgia,
(vii) pilocarpine as antiglaucoma agent and miotic,
(viii) ergonovine as oxytocic,
(ix) ephedrine as bronchodilator
(x) narceine as analgesic (narcotic) and antitussive, etc.
Taxonomic Classification
This classification deals with the source of compounds or alkaloids based on the taxonomy or family of the organisms. These taxa are the genus, subgenus, species, subspecies and variety. Thus, the taxonomic classification
39
Digitized by UCC, Library
deals with a large group of alkaloids based mainly on their respective distribution in a variety of plant families or ‘natural order’. Invariably, they are grouped together according to the name of the genus wherein they belong to, such as: coca, cinchona, ephedra. Examples include the following:
(i) Cannabinaceous alkaloids e.g. Cannabis sativa Linn, (hemp, marijuana),
(ii) Rubiaceous alkaloids e.g. Cinchona sp. (quinine)
(iii) Solanaceous alkaloids: e.g., Atropa belladona L. (Deadly Nightshade).
Some phytochemists also classify alkaloids based on their chemotaxonomic properties.
Types of Alkaloids
In general there are three main types of alkaloids: true alkaloids, protoalkaloids and pseudoalkaloids (Aniszewski, 1994; Jakubke, 1994).
True alkaloids and protoalkaloids are derived from amino acids while pseudoalkaloids are derived from the precursors or postcursors of amino acids
(Dewick, 2002; Hu et al., 2003).
True Alkaloids
These are alkaloids which are derived from amino acid and they share a heterocyclic ring with nitrogen (Aniszewski, 2007). They are highly reactive substances with biological activities even in low doses. They are basic, contain one or more nitrogen atoms and have a marked physiological action on man or other animals.
They generally have bitter taste and appear as white solid, except nicotine which is a brown liquid. True alkaloids form water-soluble salts and most of
40
Digitized by UCC, Library
them are crystalline. True alkaloids normally occur in plants in the free state as salts and as N-oxides. They occur in a limited number of species and families and are those compounds in which decarboxylated amino acids are condensed with a non-nitrogenous moiety. Their biological pathways are L-ornithine, L- lysine, L-tyrosine, L-tryptophan and L-histidine (Dewick, 2002). Examples include; quinine, reserpine, cocaine, atropine, adrenaline, morphine, canthinone, vinblastine, vincristine, vinorelbine,
N
N N OH N H3COOCN N H H R1 C2H5 C2H5 N R1 R2 R N OCOCH3 H R2 1 R HO COOCH3
Vinorelbine 1. R= CH 3 N Vinblastine H3CO N H H H 2. R= CHO OCH3 H Vincristine H3COOC O CO OCH3 OCH3 OCH3 Reserpine
OH N
H3CO
N Quinine
Figure 12: Examples of true alkaloids.
41
Digitized by UCC, Library
Protoalkaloids
Protoalkaloids are alkaloid-like amines in which the nitrogen atom derived from an amino acid is not a part of the heterocyclic structure (Jakubke,
1994). They are not restricted to any particular class of alkaloids and are often classified according to the amino acids from which they are derived. They lack one or more of the properties of typical alkaloids.
They are normally derived from L-tyrosine and L-tryptophan. They have a closed ring, being perfect but structurally they are simple alkaloids. Examples are yohimbine, mescaline, hordenine, protoberberine, tryptamine and the new alkaloids- stachydrine and 4-hydroxystachydrine (Aniszewski, 2007).
Figure 13: Examples of protoalkaloids
Pseudoalkaloids
In pseudoalkaloids, the basic carbon skeletons are not derived from amino acids (Jakubke, 1994). They are actually connected with amino acid pathways. Pseudoalkaloids are derived from the precursors or postcursors
(derivatives in the degradation process) of amino acids. They can also result from the amination and transamination reactions of the different pathways connected with precursors or postcursors of amino acids (Dewick, 2002). In the case of steroidal or terpenoid alkaloid skeletons, the nitrogen atom is inserted
42
Digitized by UCC, Library
into the molecule at a relatively late stage. The nitrogen atom can also be
donated by an amino acid source across a transamination reaction, if there is a
suitable aldehyde or ketone (Aniszewski, 2007). Pseudoalkaloids can be acetate
and phenylalanine-derived, terpenoid or steroidal alkaloids. Coniine, capsaicin,
ephedrine, solanidine, caffeine, theobromine, pinidine, Tomatine and jervine are
good examples of pseudoalkaloids.
H N CHOH O H3C NHCH3
Ephedrine Solasodine
CH3 N CH3 CH O H 3 H N CH H 3 H HO H Solanidine Tomatine
Figure 14: Examples of pseudoalkaloids
Nomenclature of Alkaloids
The names of the alkaloids are obtained in various ways (Bhat et al.,
2007).
(i) From the generic name of the plant producing them (e.g. berberine,
hydrastine and atropine).
(ii) From the specific name of the plant producing them (e.g. cocaine,
belladonine)
43
Digitized by UCC, Library
(iii) From the physiological activity (emetine, morphine)
(iv) Occasionally from the person who discovered it (e.g. pelletierine)
Many at times a prefix or suffix is added to the name of the principal alkaloid
to designate another alkaloid from the same source (e.g. quinine, quinidine,
hydroquinine). By convention, the names of all alkaloids must end in ‘ine’.
Pharmacological Uses of Alkaloids
Almost all alkaloids possess curative properties. Alkaloids possess a variety of pharmacological activities (Bhat et al., 2007). These activities include the following: analgesic potentiator (cocaine), antiambic (emetine), anticholinergics (atropine, hyoscyamine, scopolamine, and galanthamine), antimalarial (quinine), antihypertensive (reserpine, protoveratrine), antitussive
(codeine, noscapine), cardiac depressant (quinidine), central nervous stimulant
(caffeine), diuretic (theophylline, theobromine), gout suppressant (colchicine), local anesthetic (cocaine), narcotic analgesic (codeine, morphine), antitumor
(vinblastine, vincristine), antiglaucoma (pilocarpine), oxytocic (ergonovine), skeletal muscle relaxant (methyl lycoconitine, tubocurarine), smooth muscle relaxant (papaverine, theophylline), sympathomimetic (ephedrine), tranquilizer
(reserpine), etc.
Distribution of Alkaloids
Alkaloid-containing plants constitute an extremely varied group both taxonomically and chemically, a basic nitrogen being the only unifying factor for the various classes. For this reason, questions of the physiological role of alkaloids in the plant, their importance in taxonomy, and biogenesis are often
44
Digitized by UCC, Library
more satisfactorily discussed at the level of a particular class of alkaloid. A similar situation pertains to the therapeutic and pharmacological activities of alkaloids. As most alkaloids are extremely toxic, plants containing them do not feature strongly in herbal medicine but they have always been important in the allopathic system where dosage is strictly controlled and in homoeopathy where the dose-rate is so low as to be harmless. Some 150 years of alkaloid chemistry had resulted by the mid-1940s in the isolation of about 800 alkaloids; the new technology of the next 50 years increased this figure to the order of 10 000. In practice, those substances present in plants and giving the standard qualitative tests outlined below are termed alkaloids, and frequently in plant surveys this evidence alone is used to classify a particular plant as ‘alkaloid-containing’.
Alkaloids are most abundant in higher plants. At least 25% of higher plants contain these molecules which belong to more than 150 families. They are widely distributed in higher plants particularly the dicotyledonous of the families Euphorbiaceae, Apocynaceae, Asteraceae, Loganiaceae, Papaveraceae,
Rutaceae, Solanaceae, Erythroxylaceae, Boraginaceae, Fabaceae,
Menispermaceae, Berberidaceae, Ranunculaceae, Liliaceae, Rubiaceae,
Amaryllidaceae, Elaeagnaceae and Zygophyllaceae. Usually the occurrence of a particular alkaloid is localized to the seeds, leaves, bark or roots of the plant and each site may contain closely related alkaloids. Both the total alkaloid content and the relative proportion of the component bases may vary considerably with the stage of growth of the plant and its locality. Different species of the same family of plants may contain the same or structurally related alkaloids. For
45
Digitized by UCC, Library
example, seven different species of the family Solanaceae contain hyoscyamine.
It is also observed that simple alkaloids are often found in different plant species, while the complex alkaloids are confined in one species or genus of a family. Alkaloids also occur less frequently in lower plants and other organisms such as Mushrooms, Fungi, bacteria and Animals. In this review, major families of living organisms producing alkaloids are presented in a brief, yet comprehensive, up-to-date summary with a special emphasis on the types of alkaloids as well as their biochemical and pharmacological importance.
The Family Euphorbiaceae
Alkaloids are not common in Euphorbiaceae, but some species of the genera Croton, Phyllanthus, Securinega and Trigonostemon are notable for alkaloids. These alkaloids are benzylisoquinoline (Croton), Securinine
(Securinega), Imidazole (Glochidion and Alchornea), derivatives of Nicotinic acid (Ricinus) and β-Carboline (Croton and Trigonostemon). The β-carboline alkaloids 2-ethoxycarbonyltetrahydroharman and 6-hydroxy-2- methyltetrahydroharman were isolated from C. moritibensis (Salatino et al.,
2007) while Hu et al., (2009) isolated six β-Carboline alkaloids
(Trigonostemonines A-F) from the aerial parts of Trigonostemon lii.
46
Digitized by UCC, Library
H2N R2 N R1 N N H R1 N H O O R2 N A, R1=H, R2=OCH3 B,R1=OCH3 R2=H C,R1=OCH3 R2=H D,R1=H R2=OCH3
N N N N H H HN
H3CO N H CO E 3 F
Figure 15: Trigonostemonines A-F alkaloids of Euphorbiaceae
The Family Apocynaceae
The Apocynaceae family (Lindl. juss) is distributed worldwide, especially in tropical and sub-tropical areas (Aniszewski, 2007). It is large botanical taxa containing at least 150 genera and 1700 species (Blundell, 1987). Alkaloids are especially abundant in the following genera: Rauvolfia (devil’s-pepper),
Catharanthus G. Don (periwinkle), Tabernaemontana (milkwood), Strophantus
DC (Strophantus), Voacanga U (voacanga) and Alstoni R. Br. (alstonia)
(Endress et al., 1996). The species in these genera contain indole, terpenoid, quinoline, pyrroloindole and ergot alkaloids. Rauwolfia serpentina contains reserpine and rescinnamine, the quinine tree (R. capra) yielded quinine, and T. iboga contains iboganine. Cinchona contains around 25 closely related quinoline alkaloids, of which the most important are quinine, quinidine and cinchonidine (Bhat et al., 2007). Cinchona and its alkaloids have been used in the treatment of malaria for many years. The structure of quinine has provided
47
Digitized by UCC, Library
lead to important synthetic antimalarial drugs including chloroquine and mefloquine.
Reserpine has been isolated from the roots of Rauvolfia canescens (Bhat et al.,
2005). This alkaloid gas been employed in clinical practice for the treatment of hypertension and as a tranquilizer and also as a controller of other cardiac disorders. It is known that 180 biologically active alkaloids have been isolated from the genus Alstonia and this makes it one of the most important in terms of alkaloid use (Macabeo et al., 2005; Keawpradub et al., 1999). The periwinkle
(e.g., Catharanthus roseus and Vinca spp.) have yielded potent anticancer alkaloids-vinblastine, vincristine, vindesine, vinorelbine, vindoline, vindolinine, leurosine, ajmalicine, etc. (refer to page 42 for examples drawn). All alkaloids from Apocynaceae have a strong biological and medical effect and many of them are used in cancer chemotherapy (Aniszewski, 2007).
The Family Asteraceae
This plant family is very large, containing over 900 genera and more than
20 000 species (Judd et al., 1999). They are distributed worldwide and the species are found everywhere. The genus Senecio L., (ragwort) is especially rich in pyrrolidine, tropane and pyrrolizidine alkaloid (senecivernine, sencionine, retrorsine, retronecine, senecivernine, seneciphylline, spartioidine, jaconine, adonifoline, sekirkine, jacoline, etc (Pelser et al., 2005). The genus Centaurea
L. is also rich in indole, terpenoid, quinoline and pyrroloindole alkaloids, for example afzelin and apigenin. Other alkaloids have been isolated from Senecio triangularis (9-0-acetyl-7-0-angelyl-retronecine, 7-0-angelyl-, 9-0-angelyl-, and
48
Digitized by UCC, Library
7-0-angelyl-9-0-sarracinylretronicine (Aniszewski, 2007). Cheng and Roeder
(1986) have isolated two pyrrolizidine alkaloids (senkirkine and doronine) from
Emilia sonchifolia.
HO HO HO OH O O O O O O O O O O H O O
N N N Senecovernine Senecionine Retrorsine
HO HO O O O O O O O O O O HO OH H
N N N Senkirkine Dehydrosekirkine Retronecine
Figure 16: Alkaloids of Asteraceae
The Family Loganiaceae
Thirty genera and more than 500 species belong to this family although new systematic research has proposed that Loganiaceae should be divided into several families (Struwe et al., 1994).
The Logan plant family contains plant species which are rich in pyrrolidine, tropane and pyrrolizidine alkaloids. The genus Strychnos is especially rich in alkaloids such as strychnine, brucine and curare (Frederich et al., 2000). This genus contains 190 species and more than 300 different alkaloids have been isolated. These alkaloids have important biological activities and strong medical applications (Lansiaux et al., 2002, Frederich et al., 2004). They are also used in exterminating rodents and for trapping fur-bearing animal (Bhat et al., 2007).
49
Digitized by UCC, Library
Sungucine and isosungucine have been isolated from S. icaja (Lansiaux et al.,
2002). These alkaloids interact with DNA, inhibit the synthesis of nucleic acid and induce apoptosis in HL-60 leukaemia cells. The alkaloids strychnogucine A and strychnogucine B have also been isolated from the stem bark of Strychnos mellodora, a tree growing in the mountainous rain forests of Tanzania and
Zimbabwe (Aniszewski, 2007).
N N H3CO
N H3CO N O O O Strychnine O Brucine N N N N N O N O O O O N N N N N OH N O O O Strychnogucine B Sungucine Strychnogucine A
Figure 17: Alkaloids of Loganiaceae
The Family Papaveraceae
This poppy plant family is relatively large, comprising 26 genera and about
250 species (Judd et al., 1999). The opium poppy (Papaver somniferum L.,) is a known source of opium from its latex. The family contains mainly phenylethylamino- and iso-quinoline alkaloids such as morphine, codeine, thebanine, papaverine, narcotine, Narceine, isoboldine and salsolinol. These alkaloids are strong narcotics and have strong medicinal applications
50
Digitized by UCC, Library
(Aniszewski, 2007). Many new alkaloids have also been isolated from this family. Alkaloids such as sanquinarine, cholidonine, hydrastine, berberine and chelerythine have been isolated from Chelidonium majus (Vavreckova et al.,
1996). Twenty-three iso-quinoline alkaloids have isolated from Corydalis bulleyana Diels (Hao and Qicheng, 1986). Examples of such alkaloids include protopine, corydamine, allocryptopine, corycavanine, bulleyamine, spallidamine etc. These alkaloids are well known for their biological activity and spallidamine has been found to display fungitoxic activity (Ma, et al., 1999).
H3CO HO H3CO N H3CO OCH3 O O N CH3 N CH3 Papaverine OCH 3 HO HO Morphine Codeine O O O N CH3 O O N N O O O OCH3 O OCH3
OCH3 OCH3 Thebaine Hydrastine Berberine
Figure 18: Alkaloids of Papaveraceae
The Family Rutaceae
The Citrus botanical family contains more than 150 genera and 900 species (Purseglove, 1979). Many species contain quinazoline, quinoline, acridine, canthinone and imidazole alkaloids (Aniszewski, 2007). Species such as Dictamus albus or Skimmia japonica contain quinazoline, quinoline and acridine alkaloids such as dictamine, skimmianine and also acronycine
(Acronychia baueri), melicopticine (Melicope fareana) and rutacridone (Ruta
51
Digitized by UCC, Library
graveolens). Many alkaloids with potential estrogenic activity have been reported in Haplophyllum A.juss (Nazrullaev, et al., 2001). These alkaloids include acutine, toddaliopin A, acetylfolifidine, bucharidine, fagaronine, dubinidine, dubinine, glycoperine, evoxine, ϒ-fagarine, folifidine, linarinic acid, perfamine and skimmianine. Recently, fagaronine has been isolated from
Fagara zanthoxyloides and this alkaloid induces erythroleukemic cell differentiation by gene activation (Dupont et al., 2005). Bioassay-guided fractionation has led to the isolation of three indolopyridoquinazoline alkaloids,
1-hydroxy rutaecarpine, rutaecarpine and 1-methoxyrutaecarpine from the fruit of Z. integrifolium (Sheen et al., 1996). Galipea officinalis (Hancock) is a shrub growing in tropical America and used in folk medicine as an antispasmodic, antipyretic, astringent and tonic. Nine quinoline alkaloids have been isolated from this plant, of which galipine, cusparine, demethoxycusparine and galipinine are active (Rakotoson et al., 1998). Moreover, a new carbazole alkaloid, Clausine Z, has been isolated from the stems and leaves of Clausena excavata Burm by Potterat et al (2005). This alkaloid exhibited inhibitory activity against cyclin-dependent kinase 5 (CDK 5) and showed protective effects on cerebellar granule neurons in vitro (Potterat et al., 2005). Cebrian-
Torrjon et al., (2015) have reported of the isolation of an antifungal alkaloid- canthin-6-one from the leaves of Zanthoxylum chiloperone.
52
Digitized by UCC, Library
Figure 19: Alkaloids of Rutaceae
The Family Solanaceae
The Nightshade plant family contains 90 genera and more than 2000 species distributed in all continents (Purseglove, 1979). The family contains pyrrolidine, tropane, steroidal and pyrrolizidine alkaloids especially the genus
Atropa L. (Nightshade) which contains hyoscyamine, hyoscine and cuscohygrine (Aniszewski, 2007). The genera Jimsweed (Thornapple), Datura
L. (Pitura plants) and the species Atropa belladona L. (deadly nightshade) contain tropane alkaloids (Ylinen et al, 1986). The genera Mandragora L. and
Scopolia L. also contain this type of alkaloids. Moreover, the Solanaceae family also contains both nicotinic acid- derived and phenylalanine-derived alkaloids such as anabasine, nornicotine, ricine, nicotine, arecoline, cathine, cathionine, ephedrine, etc. The Nicotiana L. genus (the tobacco plant genus) with about 45 species contains such alkaloids as nicotine and anabasine.
Capsicum L. (paprika plant) which has about 50 species and native to Central
53
Digitized by UCC, Library
and Southern America contains capsaicin as its main alkaloid. Steroidal alkaloids, such as solanidine are well known in the potato genus (Solanum L.).
The unripe fruits of Solanum lycocarpum St. Hill, also contains steroidal alkaloids as solamargine and solasodine (Schwarz et al., 2005). Solasodine is shown to penetrate animal body, the placental and hematoencephalical barrier and impact the foetuses. Tomatine, another steroidal alkaloid is common in the genus Lycopersicon L. (tomato plant genus).
CH3 N CH3 O H CH3 H N H CH3 H HO H Solanidine Tomatine CH N 3 H N N N CH OH H 2 CH3 H N O N N Nicotine Myosmine Anabasine O Hyoscyamine
Figure 20: Alkaloids of Solanaceae
The Family Erythroxylaceae
The Erythroxylaceae family is distributed in the tropics and is endemic to
South America, especially in the regions of Peru and Bolivia, where
Erythroxylum coca (coca bush) has been known for over 5000 years
(Aniszewski, 2005). The family contains pyrrolidine, tropane and pyrrolizidine alkaloids and three dominant species, E. coca, E. truxilense and E.
54
Digitized by UCC, Library
novagranatense contain cocaine, ecgonine, cinnamylcocaine, α-truxilline, truxilline, methylecgonine, tropine, hygrine, hygroline and cuscohygrine. These alkaloids are used as drugs in main stream medicine and are also the object of pathological and criminal activity (Aniszewski, 2007). New tropane alkaloids have been isolated from the root bark of Erythroxylum vacciniifolium
(catuabines H-I, three hydroxyl derivatives and vaccinines A and B). These tropane alkaloids are interesting for their ester moieties (Zanolari et al., 2005). It must be emphasized that the genus Erythroxylum contains about 250 species and apart from the cocaine-producing species, has not been examined systematically by modern analytical methods (Aniszewski, 2007)
CH3 CH3 N N COOH COOCH3 H H OCOC6H5 H OH H Ecgonine Cocaine
CH3
N CH3 N CH3 OH O
Hygrine Tropine
Figure 21: Alkaloids of Erythroxylaceae
The Family Boraginaceae
The Boraginaceae plant family (Forget-me-not family) contains pyrrolidine, tropane and pyrrolizidine alkaloids especially indicine-N-oxide in
Heliotropium indicum (heliotrope) and Cynoglosum creticum (Hound’s tongue) species. New alkaloids, europine, ilamine and their N-oxides have been isolated from another heliotrope species, Heliotropium crassifolium (Farsam et al.,
2000). These alkaloids have strong toxic effects. Bracca et al (2003) have
55
Digitized by UCC, Library
reported on the isolation of six pyrrolizidine alkaloids in Anchusa strigosa and europine N-oxide in Heliotropium esfandiarii. Alkaloids of these species have strong biological activities. Anchusa strigosa is common in the Mediterranean region. It is used in local folk medicine as a diuretic, analgesic, sedative, sudorific remedy and for treatment of stomach ulcers and externally for skin diseases (Al-Douri, 2000; Said et al., 2002). From Symphytum officinale
(common comfrey), acetyl-intermedine and acetyl-lycopsamine alkaloids have been reported.
OH OH HO OH HO H H H
N N N Heliotridine Retronecine (+)-Supinidine
OH HO OR AcO OH RO H H H
R=2-MeBut R=2-MeBut N N N 7-Acetylretronecine 7-(2-Methylbutyrl)retronecine 9-(2-Methylbutyryl)retronecine
Figure 22: Alkaloids of Boraginaceae
The Family Fabaceae
The Fabaceae plant family (Legume plant family) is the third largest botanical family with 650 genera and 18000 species in the humid tropics, sub- tropics, temperate and sub-arctic regions of the world (Aniszewski, 1995). The family is rich in indole, terpenoid, quinoline, pyrroloindole, ergot, pyrrolidine, tropane, pyrrolizidine, quinolizidine and piperidine alkaloids. The genus Crota
(Crotalaria L.) contains pyrrolidine, tropane and pyrrolizidine alkaloids such as
56
Digitized by UCC, Library
senecionine. Many species within this family are rich in quinolizidine alkaloids, including; lupinine, lupanine, angustifoline, epilupinine, anagyrine, etc.
Przybylak et al., (2005) have detected 46 compounds from six Mexican species and have been able to identify 24 of them as alkaloids from the lupanine group: sparteine, ammodendrine, epiaphyllidine, epiaphylline, tetrahydrorhombifoline, angustifoline, multiflorine, etc. The Calabar bean (Physostigma venenosum L.) contains indole, quinoline and ergot alkaloids such as eserine, eseramine, physovenine and geneserine. Lou et al., (2001) have isolated two new alkaloids;
2-methoxyl-3-(3-indolyl)-propionic and 2-hydroxyl-3-[3-(1-N-methyl)-indolyl] propionic acid from peanut skins (Arachis hypogeae L.). These alkaloids had not previously been isolated from natural sources. All alkaloids from this plant family have both biological and ecological importance.
O OH O H H H N N N NH N H H Lupine Lupanine Angustifoline
N H C H C HN O 3 N 3 N CH3 O O N H H Anagyrine Eserine
Figure 23: Alkaloids of Fabaceae
The Family Menispermaceae
This plant family is large containing about 70 genera and 450 species and is found throughout the tropics (Thanikaimoni, 1986). The family contains
57
Digitized by UCC, Library
isoquinoline and phenylethylamino alkaloids. The genus Stephania is rich in tetrandrine and stephanine and the Curare genus (Chondrodendron) contains curare and tubocurarine. All these alkaloids are of important medicinal value.
More than 150 different alkaloids have been isolated from the Stephania genus
(Camacho, 2000). Some of these alkaloids include liriodenine, isocorydine, atherospermidine, stephalagine and dehydrostephalagine. Liriodenine showed strong cytotoxic activity while corydine and atherospermidine are able to damage DNA (Goren et al., 2003). Chen et al., (2000) have isolated tetrandrine from the root of a Chinese herb Stephania tetrandra S. Moore. This alkaloid showed inhibition to both culture-activation and TGF-beta (1)-stimulated activation of quiescent rat hepatic stellate cells (HSCs) in vitro (Chen et al.,
2005). The species Stephania cepharantha Hayata has yielded cepharanthine, cepharanoline, isotetrandrine and berbamine (Nakaoji et al., 1997).
Cepharanthine is an active component of hair growth (Aniszewski, 2007).
Epinetrum villosum is a twining liana found in Congo and Angola and is used traditionally for the treatment of fever, malaria and dysentery (Otshudi et al.,
2000). From this plant, cycleanine, cycleanine N-oxide, isochondodendrine, cocsoline, troupin, and quinine have been isolated. These alkaloids exhibit both antimicrobial and antiplasmodial activities (Otshudi et al., 2005). The genus
Cissampelos contains cissampareine, which has potential medicinal uses, but it’s also psychoactive (Aniszewski, 2007).
58
Digitized by UCC, Library
O OCH3 N O O O H N N O O O O O O O H Liriodenine Atherospermidine O N
Cepharantine
H3CO H3CO H3CO N H H3CO N CH3 N CH3 HO H3CO H HO HO H3CO
H3CO H CO H3CO 3 Norisocorydine Corydine Isocorydine
Figure 24: Alkaloids of Menispermaceae
The Family Berberidaceae
The Berberry botanical family (Berberidaceae Torr., Gray Juss) contains isoquinoline and phenylethylamino alkaloids especially berberine. Other alkaloids such as glaucine, hydroxyacanthin and berbamine have also been isolated from this family (Guo and Fu, 2005). Berbamine has shown to possess anti-arrhythmia, anti-myocardial, ischemia and anti-thrombosis activities
(Aniszewski, 2007). Extracts of Nandina domestica T., are widely used in
Japanese folk medicine for the treatment of whooping cough, asthma, pharynx tumours, uterine bleeding and diabetes (Aniszewski, 2007). Orallo (2004) has isolated (+)-nantenine from the extract of this plant and this natural alkaloid was first isolated by Takase and Ohasi in 1926.
59
Digitized by UCC, Library
O OO O N N O O H OCH N 3
OCH3 O OH Berberine Berbamine
CH3 O O O N
O O
O N H3CO H OCH3 Glaucine Nantenine
Figure 25: Alkaloids of Berberidaceae
The Family Ranunculaceae
The Buttercup plant family, which has 50 genera and about 2000 species, is found in the temperate regions (Judd et al., 1999). It contains isoquinoline, phenylethylamino and terpenoid alkaloids (Aniszewski, 2007). The genus
Hydrastis L., is rich in isoquinoline and phenylethylamino alkaloids such as berberine and hydrastine and the genus Aconitum L., contains terpenoid alkaloids as aconitine, aconine, benzoylaconitine and sinomontanine.
Fangcholine and fuzitine have been isolated from the genus Thalictrum orientale, growing in Turkey (Erdemgil et al., 2000). Many other alkaloids have been found in this genus. For instance, karacoline, karakanine, songorine, nepelline, cammaconine and secokaraconitine have been isolated from
Aconitum karacolicum (Rupaics) from Kyrgyzstan (Sudtankhodzhaev et al.,
2002). A new alkaloid, arcutin with antibacterial and medicinal activities has been isolated from Aconitum arcuatum Maxim (Sudtankhodzhaev et al., 200).
60
Digitized by UCC, Library
C2H5 C2H5 OH OCH OH N 3 N OCH3
OCOC H OH 6 5 OH OH OH COCH3 H3CH2CO H3CH2CO OCH OCH3 3 Aconine Aconitine
C2H5 OH N OCH3
OCOC6H5
OH OH H CH CO 2 3 OCH3 Benzoylacotinine
Figure 26: Alkaloids of Ranunculaceae
The Family Liliaceae
This plant family contains more than 200 genera and about 3500 species and it is distributed worldwide (Judd et al., 1999). It contains both isoquinoline and steroidal alkaloids. The isoquinoline and phenylethylamino alkaloids are found in the genera Kreysigia which yielded autumnaline, floramultine and kreysigine, and Colchicum L., which produced colchicine. Steroidal alkaloids are common in the Hellebore genus, example, jervine, cyclopamine, cycloposine and protoveratrine A and B (Veratrum album) and O-acetyljervine
(Veratrum lobelianum) (Suladze and Vachnadez, 2002). Four new steroid alkaloids- puqienine A and B, N-demethylpuqietinone and puqietinonoside have been isolated from Fritillaria species. The bulb of this plant is used traditionally in China as an antitussive and expectorant. All four alkaloids had established the scientific basis for the ethnomedicinal uses of the plant (Aniszewski, 2007).
61
Digitized by UCC, Library
H C CH CH CH 3 HN 3 O 3 3 H N H3C H H O CH3 H O H CH H 3 HO H H HO CH 3 H Jervin Cyclopamine N CH3 OH H O OH CH OH OCOCHCH 3 O 3 HO COOCH3 CH3 HO O COOCH3 Protoveratrine
Figure 27: Alkaloids of Liliaceae
The Family Rubiaceae
The Rubiaceae (Coffee family) contains more than 400 genera and over
6000 species (Judd et al., 1999). It is distributed in the tropics and the sub- tropics (Purseglove, 1979). Species in this family are trees, bushes and liane
(Blundell, 1987). The family contains indole, pyrrolidinoindoline, quinoline and benzoquinolizidine alkaloids (Aniszewski, 2007).
The coffee family is especially rich in purine alkaloids such as caffeine, theophylline and theobromine. Other plant families like the tea (Theaceae), the guarana (Sapinidaceae) and the cola (Sterculiaceae) contain the same or similar purine alkaloids. Purine alkaloids (especially caffeine) have positive biological and prophylactic effect in decreasing the risk of Parkinson’s disease
(unpublished). Tryptophan-derived alkaloids with important biological activities also exist in the cola family (Hoelzel et al., 2005).
The genus Psychotria is one of the largest genera of flowering plants and the largest within the Rubiaceae, with estimated 2000 species distributed worldwide
62
Digitized by UCC, Library
(Fynn, 2011). The indole alkaloids are the predominant groups of alkaloids isolated from Psychotria species. For example, the leaves of Psychotria forsteriana contains quadrigemine A and B, psychotridine and isopsychotridine
C with high cytotoxic activity on cultured rat hepatoma cells (HTC line) (Roth et al., 1986). Also, Staerk et al., (2000) reported of the isolation of corynantheidine derivatives and α-yohimbine from the bark of Corynanthe pachyceras K. Schum. All these alkaloids demonstrated powerful leishmanicidal, antiplasmodial and cytotoxic activity. Many indole alkaloids such as emetine, calycosidine and cephaeline with potent pharmacological activity occur in the Rubiaceae family.
O O O H CH3 CH3 H N H C H N N 3 N N N O N N O N N O N N
CH3 CH3 CH3 Theobromine Caffeine Theophylline
H CH C H CH C 3 2 N 3 2 N H H
H3CO H3CO NH OCH3 NH OCH3 OCH3 OCH3 H3CO HO Emetine Cephaeline
Calycosidine
Figure 28: Alkaloids of Rubiaceae
63
Digitized by UCC, Library
The Family Amaryllidaceae
The amaryllidaceae botanical family is a large family consisting of 50 genera and 850 species and is distributed throughout the world (Judd et al.,
1999). The family is rich in isoquinoline and phenylethylamino alkaloids
(Aniszewski, 2007). The genus Lycorus L., (Spider lily genus) contains lycorine and Galanthus L.(Snowdrop genus) is rich in galanthamine and galanthindole
(Unver et al., 1999, 2003) . Galanthine, haemanthine, lycorine and lycorenine have been isolated from zephyranthes citrine Baker (Aniszewski, 2007). Herrera et al., (2001) have also isolated oxomaritidine, maritidine and vittatine from the same plant species. Alkaloids of Zephyranthes citrina especially haemanthamine have inhibitory effects on the growth of HeLa cells and protein synthesis, as well as being a cytotoxic against both MOLT 4 and various human tumoural cell lines (Weniger et al., 1995). Maritidine exhibits antineoplastic activity and galanthine has a high inhibitory capacity with ascorbic acid biosynthesis in the potato (Evidente et al., 1983). Alkaloids having antiviral, antitumoural, analgesic and insecticidal activities have been isolated from Pancratium sickenbergi (Lewis, 2000; Abou-Donia et al., 2002).
These alkaloids are hippadine, pseudolycorine, tris-pheridine, norgalanthamine, haemanthidine, vittatine, pancracine, 11-hydroxyvittatine, ent-6α-6β- hydroxybuphasine, and (-)-8-demethylmaritidine. From the bulbs Leucojum vernum, two new alkaloids, leucoverine and acetylleicoverine have been isolated (Forgo and Hohmann 2005). Shihunine and dihydroshihunine exist in
Behria tenuiflora and these alkaloids have been shown to be inhibitors of
64
Digitized by UCC, Library
Na+/K+ ATPase in the rat kidney (Bastida et al., 1996). It must be emphasized that all alkaloids from Amaryllidaceae display antiviral activity (Aniszewski,
2007).
OH OCH3 O OH HO OH H H3CO O O H N N N O O CH3 Haemanthamine Galanthamine Lycorine
Figure 29: Alkaloids of Amaryllidaceae
The Family Elaeagnaceae
The Oleaster botanical family is one of the smallest families comprising
3 genera and 50 species (Aniszewski, 2007). It is found mostly in the temperate regions of the world. It contains indole (β-carboline) alkaloids especially elaeagine which is predominantly found in the Russian olive Elaeagnus angustifolia (Oleaster genus) (Aniszewski, 2007) together with harman, harmine, harmol and harmalol.
NH
N H Elaeagine
Figure 30: Alkaloids of Elaeagnaceae
The Family Zygophyllaceae
The Zygophyllaceae (the Caltrop plant family) consists of nearly 30 genera and more than 230 species, grows in the tropic, subtropics and warm regions of the world (Judd et al., 1999; Blundell, 1987). The family is very rich
65
Digitized by UCC, Library
in β-carboline alkaloids especially harman and harmine which is normally found in the Pegan genus (Peganum harmala L.). The genus Nitraria (Nitraria sibirica
Pall) contains alkaloids derived from acetate, dihydroschoberine and nitrabirine
N-oxide (Tulyaganov et al., 2001). Komavine and acetylkomavine have been isolated from Nitraria komarovii (Tulyaganov et al., 2001).
H N H N N N HO O N H Nitramine Nitraramine Nitrarine
O NH N N N N H N H H3CO H CH3 Harmaline Komavine Acetylkomavine
N HO N N H HO N CH3 H CH3 Harmol Harmolol
Figure 31: Alkaloids of Zygophyllaceae
Mushroom
Apart from the plant botanical family, alkaloids occur in many other botanical families including the mushroom (Aniszewski, 2007). The mushroom genera Psilocybe, Conocybe, Panaeolus and Stoparia are rich in the β-carboline alkaloids serotonin, psilocin and psilocybin. These alkaloids are powerful psychoactive and neurotransmitter compounds. These compounds also demonstrated a broad spectrum of pharmacological properties including
66
Digitized by UCC, Library
sedative, anxiolytic, hypnotic anticonvulsant as well as antimicrobial activities
(Cao et al., 2007) OH HO P O CH3 HO N O CH HO NH 3 2 CH3 N NH NH CH3 NH Serotonin Psilocin Psilocybin
Figure 32: Alkaloids of Mushroom
Moss
The moss (Lycopodiaceae family) contains indole and isoquinoline
alkaloids and the genus Lycopodium L., is a rich source of annotinine,
lycopodine and cernuine (Aniszewski, 2007). The genus Huperzia contains
huperzine J, K, L, A and its derivatives (Ayer and Trifonov, 1993). These
alkaloids have potential effects on Alzheimer’s disease (Aniszewski, 2007). Tan
et al (2002) have isolated phlegmariurine, 11α-hydroxy-phlegmariurine B, 7α-
hydroxyphlegmariurine B, fawcettimine and 7α11α-dihydroxyphlegmariurine
from H. serrata (Thumb.).
67
Digitized by UCC, Library
Figure 33: Alkaloids of Moss
Fungi and Bacteria
The fungus botanical family contains ergot alkaloids and the fungi
Aspergillus, Rhizopus, Penicillium and Claviceps produce parasitic ergoline and ergotamine alkaloids (Aniszewski, 2007). The ergot alkaloids derived from indole in the fungus Claviceps purpurea, are highly toxic and have been used in the development of lysergic acid diethylamine, LSD, which is hallucinogenic and, in small doses, is used in the treatment of schizophrenia (Li et al., 2005). A new alkaloid, asterrelenin, together with terretonin, territem A and B have been isolated from Aspergillus terreus (Li et al., 2005). Two new diastereomeric quinoline alkaloids have been isolated from Penicillium janczewskii obtained from a marine sample (He et al., 2005). These compounds showed a low to moderate general toxicity (Aniszewski, 2007). From the new species
Penicillium rivulum Frisvad, communesins G and H have been isolated
(Dalsgaard et al., 2005). These alkaloids however, have negative antiviral, antimicrobial and anticancer activities. A pentacyclic indolinole alkaloid,
68
Digitized by UCC, Library
citrinadin A, has been isolated from the cultured broth of the fungus Penicillium citrinum and a marine red alga (Muqishima et al., 2005). The fungus
Aspergillus echinulatus produces toxic diketopiperazine alkaloid echinuline.
Variety of other fungi produces toxic alkaloids, whereas very few alkaloids have been from bacterial cultures (Bhat et al., 2007). The bacteria Pseudomonas spp. contains the alkaloids tabtoxin and a deep blue coloured pyocyanine which have relatively powerful biological activity (Aniszewski, 2007).
H O N CH3 O N O H N N CH H H3C 3 N H C 3 Pyocyanine Echinuline
Figure 34: Alkaloids of Fungi and bacteria
Animals
The kingdom animalia contains different classes of alkaloids, especially in millipedes, salamanders, toads, frogs, fish and mammals. They occur particularly in the genera saxidomus, Salamandra, Phyllobates, Dendrobates,
69
Digitized by UCC, Library
Castor, Moschus, Solenopsis, Odontomaschus, Glomeris and Polyzonium. Many alkaloids have been recently isolated from the sponges (Gallimore et al., 2005).
For instance, ptilomycalin A and its analogues have been isolated from
Ptilocaulis spiculifer, Hemimycale spp., Crambe spp, Monanchora arbuscula,
Monanchora ungiculata as well as from some starfishes such as Fromia monilis and Celerina heffernani. From the Caribbean sponge Monanchora unguifera the guanidine alkaloids- batzelladine J, ptilomycalin A, ptilocaulin and isoptilocaulin have been recently isolated. Many of these alkaloids display ichthyotoxicity, and antibacterial, antifungal and antiviral activities
(Aniszewski, 2007). Antiviral activity has been exhibited against Herpes
Simplex virus (HSV-1) and also in inhibiting the HIV virus and cytotoxicity against murine leukaemia cell lines (L1210) and human colon carcinoma cells
(HCT-16). From two Thorectidae sponges-Thorectandra and Smenospongia, six new brominated indole alkaloids have been isolated (Segraves and Crews,
2005). These alkaloids have a wide range of biological activities and are good therapeutic agents (Aniszewski, 2007). The skin of amphibians contains alkaloids especially indole alkaloids. Costa et al (2005) have isolated bufetenin from Anura species. This alkaloid is a component of chemical defence system in these species. Bufetenin acts as a potential hallucinogenic factor showing similar activity to LSD upon interaction with the 5HT2 human receptor (Costa et al., 2005). Toads belonging to the genus Melanophryniscus contain toxic alkaloids in their skin (Mebs et al., 2005). And alkaloids of the pumiliotoxin
70
Digitized by UCC, Library
(PTX) group and indolizidines have been isolated from Melanophryniscus montevidensis.
Defensive substances such as alarm and trail pheromones secreted by certain arthropods have alkaloid-like structure, e.g. from the venom of the fire ant Solenopsis invicta Forel, several 2,6-dialkylpiperidines have been isolated
(Bhat et al., 2007). In general, arthropod natural products are only produced in trace amounts in specialized exocrine glands (Bhat et al., 2007)
The ovaries and liver of the puffer fish (swellfish, Japanese fugu, Spheroides rubripes, S. vermicularis) contain tetradotoxin, one of the most toxic low molecular weight poisons known (Bhat et al., 2007). This alkaloid has also been isolated from goby fish Gobius criniger, the Californian newt Taricha torosa and the skin of frog belonging to the genus Atelopus. The lady bird
(Coccinellidae) and other beetles also contain alkaloids such as adaline, coccinelline, podamine, epilachnene, myrrhine, propeleine, propyleine and stenusine. Conversely, some moths (e.g. Utethesia ornatrix) depend on alkaloids for defence. Utethesia ornatrix sequesters pyrrolizidine alkaloids as a larva from the food plants such as Crotalaria (Campo et al., 2001). Some poisonous frogs (Mantella) digest alkaloids in their food. The strawberry poison frog (Dendrobates pumilio) contains dendrobatid alkaloids that are considered to be sequestered through the consumption of alkaloid-containing arthropods distributed in the habitat (Takada et al., 2005). Some species of ants
(Anochetum grandidieri and Tetramorium electrum), containing pyrrolizidine alkaloids, have been found in the stomachs of Mantella frogs (Clark et al.,
71
Digitized by UCC, Library
2005). It is now known that over 800 biologically active alkaloids have been isolated from the amphibian skin (Daly et al., 2005). All these alkaloids seem to be derived from dietary sources except samandarines and pseudophrynamines.
It has been found out that beetles are sources for batrachotoxins and coccinelline-like tricyclics and ants and mites for pumiliotixins Also, ants are sources for decahydroquinolines, izidines, pyrrolidines and piperidines (Daly et al., 2005). Several brominated indole alkaloids such as deformylflustramine and flustramine have been isolated from the North Sea Bryozoan (Flustra foliacea)
(Peters et al., 2004). Deformylflustramine A and B have been known to have affinities in the lower micromolar range with the neuronal nicotinic acetylcholine receptor (nAChR). It has been reported that erythrian alkaloids (β- erythroidine and dihydro-β-erythroidines) with neuromuscular transition blocking activity resembling the effects of curare are present in the milk of goats (Capra) which grazed the leaves of Erythrinia poeppigiana (Soto-
Hernandez and Jackson, 1993). The spectrum of alkaloids in mammals ranges from isoquinoline derivatives, via β-carbolines, through to thiazolidines, arising from vitamin B6, chloral and glyoxylic acid (Bringmann et al., 1991). And that the formation of endogenous alkaloids occurs naturally in man and mammals
(Bringmann et al., 1991). A few alkaloids have been isolated from mammals, for example muscopyridine from the scent of gland of musk deer, Moschus moschiferus. Similarly, bufetenin has also been isolated from human urine.
However, recent reports confirm the presence of numerous β-carboline alkaloids-pinoline, norharman, harman, harmine, β-CCE, hydro-β-carbolines in
72
Digitized by UCC, Library
various tissues and fluids of mammals (Cao et al., 2007). Other well known mammalian alkaloids are salsolinol, norlaudanosoline (THP), dideoxynorlaudanosoline 1-carboxylic acid and spinaceamines. New isoquinoline alkaloids have been identified in mammals (Brossi, 1991;
Rommelspracher et al., 1991).
Alkaloids in nature are a part of production and consumer (feeding) chains.
They contribute to species growth, pleasure, pathology and they play a role in the processes of agressivity and defence by the species.
N N N N N N H3CO H H H CH3 CH3 Norharman Harman Harmine
NH N N N HO N H CH3 H3CO N H H CH3 CH3 MTHBC Harmaline Harmol Figure 35: Alkaloids of Animals
Tests for Alkaloids
Alkaloids are detected by using group of reactions typical of a whole group of alkaloids and specific reactions for an individual alkaloid due to their chemical properties, structure and the presence of functional groups
(Melentyeva and Antonova, 1988).
The group reactions are based on the ability of the alkaloids to yield simple or complex salts with various acids, heavy metal salts, complex iodides and other substances. The detection reactions are either precipitation or colour reactions.
Some of the precipitation reactions include the following:
73
Digitized by UCC, Library
1. A solution of iodine in potassium iodide (Bouchardat’s, Wagner’s or Lugol’s
reagent)
This reagent gives a brown precipitate with acidified aqueous solutions of
alkaloid salts. These reagents only differ in the concentration of the iodine and
potassium iodide.
2. A solution of mercury iodide in potassium iodide (Mayer’s reagent)
With most acidified or neutral alkaloid solutions, it yields white or slightly
yellowish precipitates. This reagent precipitates almost all the alkaloids except
caffeine and colchicine.
3. A solution of bismuth iodide in potassium iodide (Dragendorff’s reagent)
The reagent gives orange-red or reddish-brown amorphous and barely
crystalline precipitates with solutions of alkaloid sulphates and chlorides.
Dragendorff reagent was developed for detecting alkaloids, heterocyclic
nitrogen compounds and quaternary amines (Wagner et al., 1984). At least six
different Dragendorff reagents are known each containing potassium iodide.
4. Phosphomolybdic acid (Sonnenschein’s reagent)
This reagent is one of the most sensitive for alkaloids. It gives yellowish
amorphous precipitates that change to blue and green colour with time due to
the reduction of molybdic acid.
5. Phosphotungstic acid (Scheibler’s reagent)
This reagent forms amorphous white precipitates with almost all the alkaloids.
6. Tannic acid solution
74
Digitized by UCC, Library
This reagent contains a freshly prepared 10% aqueous tannic acid solution with
a 10% alcohol solution. The reagent forms white or yellow precipitates with
alkaloid salts in a neutral and weakly acidic medium.
7. 1% aqueous picric acid solution (Hager’s reagent)
The solution precipitates picrates with almost all the alkaloids except caffeine,
colchicine, coniine, morphine and theobromine. However, caffeine, a purine
derivative, does not precipitate like most alkaloids. It is usually detected by
mixing with a very small amount of potassium chlorate and a drop of
hydrochloric acid, evaporating to dryness and exposing the residue to ammonia
vapour. A purple colour is produced with caffeine and other purine derivatives
(Murexide test).
In addition to precipitation reactions, colour reactions can be used to test
for alkaloids. Colour reactions are based on the chemical reaction of water
removal, or on the oxidation of the alkaloids, or their condensation with
aldehydes. All these reactions proceed in the presence of concentrated sulphuric
acid absorbing water and are based on the features of the chemical structure of
the alkaloids and their functional groups. The most common reagents for these
coloured reactions are pure concentrated sulphuric acid, concentrated nitric acid,
and a mixture of these acids (Erdman’s reagent), a mixture of concentrated
sulphuric acid and molybdenum trioxide (Froehde’s reagent) and a mixture of
formaldehyde and concentrated sulphuric acid (Marchi’s reagent). For some
alkaloids, these reactions can be specific, while for others they can fail to be
characteristic. For example, a reaction with Marchi’s reagent is specific for
75
Digitized by UCC, Library
morphine, codeine and papaverine, while this reaction is not specific for other alkaloids (Melentyeva and Antonova, 1988). Care must be taken in the application of these alkaloidal tests, as the reagents also give precipitates with proteins. So one can use acidic water-alkaline-extraction method to remove the proteins and test for alkaloids.
Extraction and Isolation of Alkaloids
Extraction methods vary with the scale and purpose of the operation, and with the raw material. Alkaloids are mostly alkaline and exist in organic salts form as citrate, oxalate, tartrate, succinate, etc. Few exist in inorganic salt form, such as berberine or morphine (as morphine sulphate) and in free form such as amide alkaloids. Alkaloids in the free or salt form can be extracted with inorganic acidic water in order to replace organic acids with inorganic acid salt and increase its solubility. Both the free and salt alkaloids are soluble in alcohol and so heated alcohol under reflux extraction or ultrasonic alcohol extraction can be used. Most of the free alkaloids are lipophilic and can be extracted with organic solvents such as chloroform, benzene, ether, etc. Most alkaloids obtained by extraction are mixtures according to the class of alkaloids, basicity, solubility differences and the functional groups present. The following methods can be used in alkaloid extraction.
Acidic-water Extraction
This method is used to extract alkaloids which exist in the salt form where the organic acid salt is replaced with inorganic acid salt, thereby increasing the solubility of water. The method usually uses 0.1%-1% sulphuric
76
Digitized by UCC, Library
acid, hydrochloric acid, acetic acid or tartaric acid solution, by dipping, maceration, percolation and sometimes refluxing (if the sample is less starchy) extraction (Yubin et al., 2014). The method is relatively simple; however, there is the wastage of solvents, difficulty in solvent recovery and has more water- soluble impurities. The alkaloids can be purified using cationic exchange resin.
Aqueous-alcohol Extraction
Both free and salt alkaloids are soluble in alcohol and alcohol reflux, cold maceration, percolation, etc can be used in extracting them. With this method, different alkaline salts can be obtained and in addition water-soluble impurities are less. However, more fat-soluble impurities are extracted. Total alkaloids can be obtained by recovering the alcohol, adding dilute acidified water, basifying and extracting with suitable lipophilic organic solvent.
Organic Solvent Extraction
Most free alkaloids are lipophilic and chloroform, benzene, ether and methylene chloride can be used to extract them either by impregnating, refluxing or continuous refluxing extraction. To make the alkaloids free and also increase the solvent penetrating the plant tissue, a small amount of alkaline wetting is recommended (Yubin et al., 2014).
With this method water-soluble impurities are less and the fat-soluble impurities can be removed by acidic extraction. In addition, volatile alkaloids such as ephedrine can be obtained by steam distillation while sublimated alkaloids such as caffeine can be extracted using sublimation method.
77
Digitized by UCC, Library
Beta-carboline Alkaloids
Beta-carboline alkaloids are a large group of natural and synthetic indole alkaloids with different degrees of aromaticity. Some of these alkaloids are widely distributed in nature, including various plants, foodstuffs, marine creatures, insects, mammalians as well as human tissues and body fluids (Cao et al., 2007). These compounds are of great interest due to their diverse biological activities. Particularly, these compounds have been shown to intercalate into
DNA, to inhibit CDK, topisomerase, and monoamine oxidase, and to interact with benzodiazepine receptors and 5-hydroxy serotonin receptors. These chemicals also show a broad spectrum of pharmacological properties including sedative, anxiolytic, hypnotic, anticonvulsant, antitumor, antiviral, antiparasitic as well as antimicrobial activities (Cao et al., 2007). The prevalence of β- carboline alkaloids is associated with the ease of forming the β-carboline core from tryptamine in the intramolecular Mannich reaction. Simple (non- isoprenoid) β-carboline derivatives include harmine, harmaline, harmane and a slightly more complex structure of canthin-6-one.
Nomenclature of Beta-carboline Alkaloids
The beta-carboline alkaloids are a large group of natural and synthetic indole alkaloids that possess a common tricyclic pyrido [3.4-b] indole ring structure (Cao et al., 2007). These compounds are classified according to the saturation of their nitrogen-containing six-membered ring. Unsaturated members are named as fully aromatic β-carbolines (βCs), whereas the partially or completely saturated ones are known as dihydro-β-carbolines (DHβCs) and
78
Digitized by UCC, Library
tetrahydro-β-carbolines (THβCs), respectively. These tricyclic compounds usually contain several substituents both in the pyrido ring and/or the indole ring. The photophysical properties of β-carboline alkaloids are strongly affected by the presence of two different nitrogen atoms in the tricyclic system, the pyridinic and the pyrrolic nitrogens. The pyridinic nitrogen is more basic than the pyrrolic one, while its basicity increases upon excitation (Carmona et al.,
2000) and is affected by the substituents presence in the structure (Hidalgo et al., 1990). Depending upon pH and solvent, β-carbolines can exist in four forms
(Varela et al., 2001): the cation, the neutral form, a zwitterion (or an alternative quinine-type canonical form), and an anion.
Distribution of Beta-carboline Alkaloids
The plants that are rich in β-Carboline alkaloids include harmal
(Peganum harmala) which contains harmane, harmine and harmaline and the
Calabar bean (Physosstigma venenosum) containing physosstigma. Peganum harmala is medicinal plant which is used traditionally as an emmenagogue and abortifacient in the Middle East and North Africa (Mahmoudian et al., 2002).
The extracts of Peganum harmala have been traditionally used for hundreds of years to treat the alimentary tract cancers and malaria in Northwest China (Chen et al., 2005).
The Indian tribes in the south-western Amazon basin use plants containing β-
Carboline alkaloids as hallucinogenic drinks “ayahuasca” or snuffs. From the past decades, numerous simple and complex β-carboline alkaloids containing saturated or unsaturated tricyclic ring systems have been isolated from various
79
Digitized by UCC, Library
plants as the major bioactive constituents. Reports up to 2003 on the isolation and characterization of simple β-carboline alkaloids including harman and norharman have been documented (Pfau and Skog, 2004). Increasing evidence shows that β-carboline alkaloids and related derivatives widely occur in nature, especially in various tissues and body fluids of humans. And human beings are sufficiently exposed to various β-carboline alkaloids, which are both present in plants used for the preparation of hallucinogenic drinks and medicinal drugs, and in tobacco smoke and well-cooked food (Cao et al., 2007). Additionally, it has been found that humans can endogenously form various β-carboline alkaloids, such as norharman and harman.
There have been many reports of the presence of simple and complex β- carboline alkaloids in extracts from the leaves, barks and roots of a variety of plants.
Additionally, numerous simple or complex β-carboline alkaloids have been isolated and characterized from various marine invertebrates including hydroids Aglao-phenia, bryozoans -Cribricellina,Caten-icella (Prinsep et al.,
1991; Harwood et al., 2003), soft corals Lignopsis, tunicates- Eudistoma,
Didemnum, Lissoclinum, Ritterella, Pseudodis-toma (Schuup et al., 2003) and various sponges. Marine ascidians belonging to the genus Eudistoma (family
Polycitoridae) are another rich source of biologically active β-carboline derivatives. Examples of such β-carboline alkaloids include eudistomins A-T
(Rinehart et al., 1984; Kobayashi et al., 1984), eudistomidins A-F (Kobayashi et al., 1986, 1990), eudis-talbins A and B (Buckholtz et al., 1980), eudistomin U
80
Digitized by UCC, Library
and isoeudistomin U (Badre, et al., 1994), eudistomin V (Davis, et al., 1998) and two new trypargine derivatives (Cao, et al., 2007).
It has been established that the simple β-carboline alkaloids, such as tetrahydro-
β-carboline-3-carboxylic acid and 1-methyl-tetrahydro-β-carboline-3-carboxylic acid, are easily formed from tryptophan or tryptamine and formaldehyde or pyruvate or acetate precursors by Pictet-Splengler reaction in foods and berverages. Quite recently, it had been proven that various tetrahydro-β- carboline and β-carboline alkaloids in variable but appreciable levels are present in foods, alcoholic and non-alcoholic beverages, and fruit and fruit-derived products.
The presence of β-carboline and its analogues in many ingested foodstuffs strongly proved that diet is an important exogenous source of these compounds in mammals and humans. The ingestion of these compounds could be partially responsible for their further endogenous presence in various mammals' tissues, organs and physiological fluids besides certain endogenous formation by putative biosynthesis pathway (Myers, 1989; Herraiz, et al., 1993).
Since the isolation and characterization of endogenous pinoline (6-methoxy- tetrahydro-β-carboline) from an extract of pineal gland tissue by Farrel and
Mclsaac, many researchers have focused on the detection and identification of
β-carboline alkaloids in mammals (Cao et al., 2007). Present reports confirm the presence of numerous β-carboline alkaloids - norharman, harman, harmine, β-
CCE, harmaline, harmalan and several different tetra-hydro-β-carboline in various tissues and fluids of a variety of mammals.
81
Digitized by UCC, Library
Biosynthesis of Beta-carboline Alkaloids
In the formation of simple β-carboline alkaloids, such as tetrahydro-β- carboline-3-carboxylic acid, 1-methyl-1-tetrahydro-β-carboline-3-carboxylic acid, harmine and harmaline, pyruvic acid acts as the keto acid precursor in the
Pictet-Splengler reaction in foods and beverages involving the use of tryptophan or tryptamine. It is a cyclisation reaction involving indoleamines and acetaldehyde to give simple tetrahydro-β-carboline alkaloids. Oxidation of these simple β-carboline alkaloids gives the β-carbolines.
R2 R R O R 2 1 2 R1 R R1 NH2 1 CH H N N N N H 1 N CH H CH H R1 R1
R2 R2 NH R1= H, CH3 R1 O R N 1 R2 = H, COOH, COOC2H5 N R1 N R R2 = H, OH H H 1
Figure 36: Biosynthesis of simple beta-carboline alkaloids
Synthesis of Beta-carboline Alkaloids
N-alkylated tryptamines have complex psychoactive properties. Routes for their synthesis from the Internet websites involve the thermolytic decarboxylation of tryptophan to tryptamine as a precursor to these compounds.
High boiling solvents and ketone catalysts are employed to facilitate the decarboxylation process. However, there may be the formation of tetrahydro-β- carboline (THBC) derivatives which may result from reaction with both the solvent and the ketone catalysts (Brandt et al., 2006). This underlines the problems associated with illicitly manufactured drugs and precursors that may
82
Digitized by UCC, Library
contain significant levels of impurities of which nothing is known of their toxicities. The possible interaction of the contaminants and the principal product in the human body may affect the efficacy of the drug and may put the user at mortal risk.
Tryptophan (1) (Trp) and its analogues are readily available and are used as starting materials for the synthesis of the corresponding tryptamine (2) precursor via thermal decarboxylation. The chemically based conversion of Trp is by far the simplest way to the synthesis of tryptamine and is done by refluxing in a high boiling solvent with some modifications in achieving success. For example, Hashimoto et al., (1986) used cyclohexanol as solvent and observed an increased reaction times and a higher yield of amine product with 2-cyclohexen-1-one as impurity. Other researchers used diphenylmethane and diphenyl ether. Alternatively, there is also a two-step catalytic decarboxylation by reacting tryptophan with copper acetate or zinc acetate with the formation of metal chelate compounds that are then decarboxylated to produce tryptamine hydrochloride, with indole as a by-product.
Other used L-tryptophan in refluxing tetralin with a catalytic amount of various carbonyl compounds. This method has been modified where Trp was decarboxylated in cyclohexanol: one method used tetralin that contains its peroxide, another used tetralone followed by tetralin. A quantitative decarboxylation of Trp in acetophenone at 1300C, using organic peroxides as catalysts has also been reported (Brandt et al., 2006). A study of various hydroxy- and methoxy-aromatic ketones as the decarboxylation media
83
Digitized by UCC, Library
concluded that of tryptophan and other α-amino acids proceed via the formation of stable Schiff base intermediates-imines (Brandt et al., 2006). Some of these intermediates after acidic or basic hydrolysis undergo transamination to a degree depending on the ketone used with yields of 60-100 % tryptamine. An interesting approach uses carvone (5-isoprenyl-2-mehtyl-cyclohex-2-enone) in spearmint (Mentha spicata) oil as the ketone catalyst and either xylene or white spirit as the refluxing solvent. It has been suggested that dill (Anethum graveolens), caraway (Carum carvi) which contains carvone or pennyroyal
(Mentha pulegium) which contains D-pulegone, (5R)-methyl-2-isopropylidene- cyclohexanone) essential oils could also employed as catalysts. Oil of turpentine
(the steam-volatile oil from rosin, exudates of pine trees) can also be used as solvent. What is of synthetic interest is the range of side products that may be present as trace constituents in the final products which may act as indicators to the synthetic route. This problem has been rectified (Brandt et al., 2006) by the analytical characterization of the synthetic route to tryptamine via decarboxylation of Trp in the presence of ketone catalysts, with an emphasis on the identification of possible by-products. It is a two-stage synthesis from tryptophan to tryptamine and its subsequent methylation to N,N- dimethyltryptamine using methyl iodide and benzyltriethylammonium chloride/NaOH phase transfer catalyst (the so called Breadth of Hope
Synthesis).
According to this method, decarboxylation of tryptophan was achieved by the suspension of tryptophan in a high boiling-point solvent under a nitrogen
84
Digitized by UCC, Library
blanket. The mixture was heated at reflux and stirred vigorously until a clear reaction mixture was observed. TLC analysis of the product mixture indicated that tryptophan was no longer present. Quantitative estimation of the final product mixture was performed using a standard addition technique and the calculated yields were in agreement with that obtained from flash chromatography in the isolation of tryptophan.
Figure 37: Thermolysis of tryptophan (1) to form tryptamine (2)
Accordingly, 1,1-disubstituted 1,2,3,4-tetrahydro-β-carbolines (THBC) were synthesized as follows: the reference materials for confirming the identification of the THBC by-products were prepared by a modified Pictet-
Spengler procedure (Kuo et al., 2004). Tryptamine (300 mg, 1.87 mmol) was added to a solution of 30 mL toluene and 2 mL trifluoroacetic acid. The appropriate ketone (28 mmol) was added and the mixture stirred at 600C overnight. The reaction mixture was concentrated under reduced pressure and the crude residue made alkaline with 10% (w/w) aq. Sodium hydroxide. The free basic compounds were extracted three times with 40 mL chloroform and
85
Digitized by UCC, Library
washed twice with water. The chloroform layer was evaporated under reduced pressure and subjected to flash chromatography using chloroform-methanol- ammonia (0.88 s.g.) 9:1:0.1 as eluent. The corresponding THBCs were isolated as oils and dried under vacuum over P2O5 where some of the products solidified. THBC derivatives 6 and 7 were synthesized simultaneously using pulegone as the ketone catalyst with heating at 600C for 3 days.
Figure 38: By-products of the thermolysis of tryptophan to form tryptamine
The 1,1-disubstituted-tetrahydro-β-carbolines 3-8 were identified as the major by-products during the decarboxylation particularly when cyclohexanol was used as the solvent. N-Benzyllidene-tryptamine was formed during decarboxylation in diphenylmethane, possibly in the presence of benzaldehyde contamination of the solvent.
Pharmacological Uses of Beta-carboline Alkaloids
Many researchers have focused on the effects of β-Carboline alkaloids on the central nervous system (CNS), such as their affinity with benzodiazepine
86
Digitized by UCC, Library
receptors (BZRs), 5-HT2A and 5-HT2C (Cao et al., 2007). However, recent attention has been shifted to their potent antitumor, antiviral, antimicrobial and antiparasitic activities. The individual β-carboline alkaloids have been shown to bind to different targets leading to various pharmacological activities. Both harmine and harmaline have been shown to be hallucinogenic in humans.
Harmine has been shown to be inactive after oral (up to 960 mg) and subcutaneous (up to 70 mg) administration, but induced some subjective effects at 35-45 mg (Scoltin et al., 1970) and hallucinogenic effects at 150-200 mg via intravenous administration (Naranjo et al., 1967).
Also, harmaline produced subjective effects in humans at a dose which is half of what is required for harmine and its hallucinogenic effect was above 1 mg/Kg.
It has been observed that these hallucinogens produce their psychoactive effects, at least in part, via interaction with 5-HT2 serotonin receptors in the brain. It has been debated as to whether β-carboline alkaloids elicit hallucinogenic actions in a manner consistent with classical hallucinogens because many previous investigations demonstrated the modest interaction of β- carboline alkaloids with 5-HT receptors. It is possible that the 6-methoxyl moiety contributes to the hallucinogenic effects of these compounds. What is more, the higher saturation in the tricyclic rings makes higher hallucinogenic effects.
It is worth noting that harman and related β-carboline alkaloids play a role in the process of substance abuse and dependence. The benzodiazepine receptors of the mammalian central nervous system are able to mediate the
87
Digitized by UCC, Library
anxiolytic, anticonvulsant, sedative/hypotic action and myorelaxant of diazepam
(Cao, et al., 2007). During the past two decades, a wide variety of non-benzo- diazepine molecules have been found to bind with high affinity to the benzodiazepine receptors especially β-carboline alkaloids. Many of these com- pounds have now been found to be benzodiazepine receptor inverse agonist or antagonist (Cao, et al., 2007). For instance, 3-(ethoxy-carbonyl)-β-carboline (β-
CCE) and 3-(methoxycarbonyl)-β-carboline (β-CCM) were inverse agonists in many animal behaviour models. The alkaloid also improved performance in various learning and memory tests in animals when given prior to training (Cao et al., 2007). The same alkaloid is able to exert stress-like effects including the inhibition of locomotor exploration in post-weanling rats.
In contrast, pinoline showed no affinity for the benzodiazepine receptors and had no convulsive activity. Rather, it demonstrated an anticonvulsive, anxiogenic and antidepressant effects in some animal models. Hence, the mechanism of action of pinoline is attributable to its neuropharmacological effect and not its interaction with benzodiazepine receptors.
β-carboline alkaloids have also demonstrated promising antitumor activities during the last decades. Ishida et al., (1999) reported that harmine and β- carboline analogues exhibited significant activities against several human tumor cell lines including three drug-resistant KB sublines with various resistance mechanisms, and a-(4-nitrobenzylidine)-harmine had a broad cytotoxicity spectrum against 1A9, KB, SaOS-2.A549, SK-MEL-2, U-87-MG and MCF-7 cells with ED50 values ranging from 0.3 to 1.2μg/mL. Structure activity
88
Digitized by UCC, Library
relationship analysis suggest that (1) introducing alkoxy substituents at C-7 leads to enhanced cytotoxic activities, (2) the length of C-7 alkoxy chain affects both cytotoxicity and cell line specificity, (3) N9-alkylated β-carboline derivatives exhibit strong cytotoxic effect, (4) C-6 brominated β-carboline derivatives show selective cytotoxic activities, (5) N2-alkylated β-carboline derivatives display specific cytotoxic activities and that (6) the 3,4-dihydro-p- carboline derivatives are inactive. It has been reported that 3-substituted β- carboline derivatives showed cytotoxic activities against human tumor cell lines including HL-60, KB, Hela and BGC (Cao et al., 2007). Bis-3,4-dihydro-β- carbolines and bis-β-carbolines have been synthesized and have been found to be cytotoxic to L-1210 cells with micro-molar IC50, (Cao et al, 2007).
Numerous β-carboline derivatives with substituents at different positions have been synthesized and evaluated for their antitumor activities in vitro and in vivo
(Cao et al., 2004, 2005). Most of the synthesized compounds showed significant cytotoxic activities in vitro against a panel of human tumor cell lines including non-small cell lung carcinoma (PLA-801), liver carcinoma (HepG2 and Bel-7402), gastric carcinoma (BGC-823), cervical carcinoma (HeLa) and colon carcinoma (Lovo). Structure activity relationship analysis indicates that
(1) the β-carboline structure is an important basis for the design and synthesis of new antitumor drugs, (2) appropriate substituents at position-1. 3 and 9 of β- carboline ring might play a crucial role in determining their enhanced antitumor activities, (3) the antitumor potencies of β-carboline derivatives are enhanced by the introduction of benzyl substituent into the position-2, (4) the acute toxicity
89
Digitized by UCC, Library
of β-carboline derivatives reduced dramatically by the introduction of an appropriate substituent into the position-3 and 9 and (5) the β-carboline derivatives have the potential to be used as antitumor drug leads.
Moreover, β-carboline amino acid ester conjugates also exhibit potent cytotoxic activities against human tumor cell lines including cervical carcinoma (Hela), human breast cancer (MCF-7) and liver carcinoma (HepG2). The Lys/Arg conjugates have the highest activities against human cervical carcinoma cells.
Many marine species contain β-carboline alkaloids with potent antitumor properties. For instance, eudistomin exhibited potent cytotoxic activities in vitro against murine P-388 cells with IC50 value of 0.01μg/mL and the antitumor assay in vivo gave a T/C of 137% at 100 mg/kg, and a further antitumor activity in vivo against L1210, A549 and HCT-8 cell lines. It has been reported that 6-hydroxymanz-amine A and 3,4-dihydromanzamine A were cyto- toxic against L1210 (IC50 1.5 and 4.8 μg/mL. respectively) and KB cells (IC50
2.5 and 0.61μg/mL, respectively in vitro. Accordingly, manzamine A, 8- hydroxymanzamine A and 8-methoxymanzamine A showed significant cytotoxicities against KB (IC50 0.05, 0.30 and 0.33 μg/mL respectively and
Lovo (IC50 0.15 0.26 and 0.1 μg/mL. respectively) cell lines. However, only manzamine A exhibited cytotoxicity in the P-388 assay with 1C50 0.07 μg/mL
(Ichiba et al., 1994). A new manzamine dimer,-neo-Kauluamine exhibited cytotoxicity with an IC50 1.0 μg/mL against human lung and colon carcinoma cells (El Sayed et al., 2001), while Kauluamine was inactive in anticancer as- says (Ohtani et al., 1995). Simple β-carboline alkaloids isolated from marine
90
Digitized by UCC, Library
bryozoan Cribricellina cribraria, differed markedly in their degree of biological activity in the P-388 cytotoxicity assay (Prinsep et al., 1991). Also, l-Vinyl-8- hydroxy-β-carboline had IC5o value of 100 g/mL against P-388, whereas other
1-alky substituted derivatives such as harman and 1-ethyl-p-carboline were weakly cytotoxic. These results suggested that the vinyl group might be important for P-388 cytotoxicity.
Apart from harmine and harman, the cantin-6-one alkaloids isolated from Eurycoma longifolia exhibit cytotoxic activities against a panel of human cancer cell types including breast, colon, fibrosarcoma, lung, melanoma,
KB.KB-V1 and murine lymphocytic leukaemia P-388 (Li et al., 1993). The β- carboline alkaloids are also potent antiviral agents. Rinehart et al., (1984) have reported of the antiviral activities of eudistomins C, E, K and L against herpes simplex virus-1 (HSV-1), in vitro, were in the range in the range of 25-250 ng/12.5 mm disc. The eudistomins D, H, I, N and Q were also found to exhibit modest activities against HSV-1 (Kobayashi et al., 1984). Also, high activities for eudistomin K sulfoxide and the indole unsubstituted derivative eudistomin K against both HSV-1 and polio vaccine type-1 virus have been reported (Lake et al., 1988, 1989). The alkaloids of the bryozoan Cribricellina cribraria, also displayed modest antiviral activities against HSV-1 and poliovirus grown on the
BSC cell line (Prinsep et al., 1991). Harman and its derivatives inhibit HIV replication in H9 lymphocyte cells, and 9-n-butyl-harmine showed potent activities with EC50 and therapeutic index values of 0.037 μM and 210 re- spectively (Ishida et al., 2001).
91
Digitized by UCC, Library
From the structure activity relationship, significant antiviral activity is depended upon both natural stereochemistry at both C (1) and C (13b) and the presence of the C (1) -NH2 substituent. Recently, manzamine A,8-hydroxymanzamine A and 6-deoxy-manzamine X were also found to possess anti-HIV activities against human peripheral blood mononuclear (PBM) cells with median effective concentrations (EC50) 0.59, 4.2 and 1.6 μM respectively (Cao et al., 2007).
Recent reports indicate β-carboline alkaloids have potent antimicrobial activities. The eudistomins H, I, O and P exhibited modest antimicrobial activities against Saccharomyces cerevisiae and the eudistomins D, I, N, O, P and Q showed moderate activities against Bacillus subtilis, a gram-positive bacterium. In another studies, alkaloids from the bryozoan Cribricellina cribraria are active against two Gram-negative bacteria, Pseudomonas aeruginosa and Escherichia coli), A gram-positive bacterium (Bacillus subtilis) and three fungi-Candida albicans, Trichophyton mentagrophytes and
Cladisporum resinae (Prinsep et al., 1991).. During the last decades, the antiparasitic activities of β-carbolines have attracted increasing attention.
Harmaline exhibited significant antiparasitic activities against Leishmania mexicana amazonensis both in vitro and in vivo (Evans et al., 1987) and also showed antileishmanial activity toward the intracellular amastigote form of
Leishmania. Recently, a series of 1-amino substituted β-carbolines were synthesized and screened against the parasites T. cruzi (Tulahuen C4 strain), P. falciparum (Kl strain), L. donovani (MHOM-ET-67/L84 strain) and T.b. rhodesiense (STIB 900 strain) by the World Health Organization (WHO)
92
Digitized by UCC, Library
(Boursereau et al., 2004), all compounds were observed to exhibit significant antiparasitic activities.
The structure activity relationships studies showed that the presence of a carbomethoxy at position-3 and an aryl substituent at position-1 in β-carboline nucleus effectively enhanced the antifilarial activities particularly against A. viteae. Manzamine A and its hydroxy derivatives, (-)-8-hydroxymanzamine A, were found to be active against the asexual erythrocytic stages of Plasmodium beighei. Interestingly, three 50 µM/kg i. p. dose of ent-8-hydroxymanzamine A were found to be curative and totally cleared the parasite, and two oral doses
(100μM/kg) provided a remarkable reduction of parasitemia.
The antimalarial activities of manzamines against malaria parasite
Plasmodium falciparum (Rao et al., 2003; Winkler et al., 2006) and Leishmania donovani (Rao et al., 2003), the causative agent for visceral leishmaniasis have been reported. Moreover, 3-carboline derivatives isolated from Eurycoma longifolia were found to be effective antimalarial against three Plasmodium falciparum clones, W2, D6 and TM91C235 (Kuo et al., 2003). There have been few publications on the antithrombotic activities of β-carboline derivatives.
Tang et al., (1999, 2001) first reported that perlolyrine and its analogues exhibited potent anti-aggregation activities in vitro and antithrombotic activities in vivo. Conclusively the proposed biosynthesis pathways of those "endogenous alkaloids'" in human body fluids and tissues have attracted much concern because of their possible influence on the central nervous function. However, it
93
Digitized by UCC, Library
has been debated whether substantial amounts of them are derived from diet or physiologically (Salmela et al., 1993).
Invariably, the β-carbolines have extensive biochemical activities and multiple pharmacological effects. Individual compounds might selectively interact with specific targets so as to lead to a variety of pharmacological actions in vitro and in vivo. Therefore, the β-carboline alkaloids might be a particularly promising lead compounds for discovering and developing novel clinical drugs. However, it is also worthy to note that certain β-carbolines are very dangerous. Harman and norharman are comutagens or precursors of mutagens; TaClo, TaBro and N- methylated β-carboline derivatives are potent endogenous neurotoxins; and N- nitroso derivatives of β-carboline and APNH derivatives are endogenous mutagens and carcinogens. Moreover, humans are continuously exposed to endogenous and exogenous β-carboline alkaloids. Therefore, further studies in vivo with respect to possible actions on human health are urgently required.
INFLAMMATION
Inflammation is the body’s response to disturbed homeostasis caused by infection, injury or trauma resulting in systemic and local effects. An inflammatory reaction serves to establish a physical barrier against the spread of infection and to promote healing of any damaged tissue (Hansson, 2005). It is a protective response that involves immune cells, blood vessels and molecular mediators purposely to eliminate the initial cause of injury, clear out worn necrotic cells and tissues damaged from the original damaged and also to initiate tissue repair. There are other instances where immune responses are mounted
94
Digitized by UCC, Library
inappropriately due to exposure to ultraviolet light, chemicals, innocuous foreign particles (pollen) or even tissues of the body itself (auto immunity).
In the absence of inflammation, wounds and infections would never heal and progressive destruction of the tissue would compromise the survival of the organism. However, inflammation which runs unchecked can also lead to a host of diseases, such as hay fever, atherosclerosis, and rheumatoid arthritis. An inflammatory reaction may be triggered by infection (invasion and multiplication within tissues by various bacteria, fungi, viruses and protozoa, which in many instances, cause damage by release of toxins that directly destroy host cells), trauma, thermal injury, chemical injury, and immunologically mediated injury. It is characterized by excessive heat, swelling, pain, and redness. It is a common factor in arthritic diseases or osteoarthritis. Inflammation can be categorized into two folds, that is, acute and chronic inflammation. Acute inflammation is the rapid response to an injurious agent that serves to deliver mediators of host defence leukocytes and plasma proteins to the site of injury. Acute inflammation has five cardinal signs: dolor (pain), calor (heat), rubor (redness), tumor
(swelling) and functionalaesa (loss of function). The redness and heat are due to the increased blood flow to the affected area, swelling is due to the accumulation of fluid, pain is due to the release of chemicals that stimulate nerve endings and loss of function is due to a combination of factors. These signs are evident when acute inflammation occurs on the surface of the body where as in internal organs several of these signs are not present. Pain only occurs when there are sensitive nerve endings at the inflamed area. For example, in the acute inflammation of the
95
Digitized by UCC, Library
lung (pneumonia), pain will only be felt if the inflammation affects the parietal pleurae since the pain-nerve endings are located there. The characteristic heat of inflammation occurs when there is entry of large amount of blood to the inflamed area at body core temperature onto the normally cooler area.
It has three major components: vasodilation, vascular leakage, edema and leukocyte emigration (mostly polymorphonuclear cells). When a host encounters an injurious agent, such as an infectious microbe or dead cells, phagocytes that reside in all tissues try to get rid of these agents. At the same time, phagocytes and other host cells react to the presence of the foreign or abnormal substance by liberating cytokines, lipid messengers, and the various other mediators of inflammation. Some of these mediators act on endothelial cells in the vicinity and promote the efflux of plasma and the recruitment of circulating leukocytes to the site where the offending agent is located. The recruited leukocytes are activated by the injurious agent and by locally produced mediators, and the activated leukocytes try to remove the offending agent by phagocytosis (Amponsah, 2012).
As the injurious agent is eliminated and anti-inflammatory mechanisms become active, the process subsides and the host returns to a normal state of health. The acute inflammatory response is enhanced by chemical mediators such as kinin system, vasoactive amines, arachidonic metabolites, complementary cascade and coagulation cascade. If the injurious agent cannot be quickly eliminated, the result may be chronic inflammation. Chronic inflammation is a pathological condition characterised by recurrent active inflammation, tissue destruction, and attempts at repair. It is not characterised by the classic signs of acute inflammation listed
96
Digitized by UCC, Library
above (Amponsah, 2012). The immune response is enhanced as a result of lymphocytes, plasma cells and macrophages. Phagocytosis in chronic inflammation is of two types namely; immune and non-immune phagocytosis.
This is because it is dependent on the inciting agent (antigenic or non-antigenic).
Necrosis occurs afterward and is followed by repair of damaged tissues through new blood cell formation, fibroblastic proliferation and collagen deposition
(fibrosis). Chronic inflammation, also known as low level inflammation has been implicated in a host of degenerative diseases such as heart disease, cancer, chronic lower respiratory disease, stroke, Alzheimer’s disease, diabetes and nephrit which contributes considerably to mortality (Amponsah, 2012). Chronic inflammation can be triggered by cellular stress and dysfunction such as that caused by excessive consumption of calories, elevated blood sugar levels and oxidative stress. It is now clear that the destructive capacity of chronic inflammation is unprecedented among physiological processes (Amponsah,
2012). Recent research has identified age-associated aberration of mitochondrial function as a principal activator of chronic inflammation. Specifically mitochondrial dysfunction brings about chronic inflammation firstly through the accumulation of free radicals which induces mitochondrial membrane permeability. Secondary, molecular components normally contained within the mitochondria leaks into the cytoplasm. Thirdly, cytoplasmic pattern recognition receptors (cPRRs) which detects and initiates an immune response against intracellular pathogens, recognizes the leaked mitochondrial molecules as potential threats. Finally, upon detection of the potential threats, cPRRs’s form a
97
Digitized by UCC, Library
complex called the inflammasone that activates the inflammatory cytokine interleukin-1β, which then recruits components of the immune system to destroy the “infected” cell. There are other inducers of chronic inflammation such as circulating sugars which end up forming advanced glycated end products with lipids and proteins. Also, pro-inflammatory instigators such as uric acid crystals, oxidized lipoproteins, homocysteine etc. together promote a perpetual low level chronic inflammatory state called para-inflammation (Amponsah, 2012).
Inflammatory Pathway
The acute inflammatory response occurs in three distinct phases. The first phase is caused by an increased vascular permeability resulting in exudation of fluids from the blood into the interstitial space; the second phase involves the infiltrations of leukocytes from the blood into the tissue while the third phase involves granuloma formation and tissue repair (Amponsah, 2012).
Mediators of inflammation originate either from plasma (e.g. complement proteins kinins) or from cells. The production of active mediators is triggered by microbial products or by host proteins (kinins) and coagulation systems that are themselves activated by microbes and damaged tissues. Generally the mediators of inflammation (figure 38) are histamine, prostaglandins (PGs), leukotrienes
(LTB4), nitric oxide (NO), platelet-activation factor (PAF), bradykinin, serotonin, lipoxins, cytokines, and growth factors (Armah et al., 2015)
98
Digitized by UCC, Library
Figure 39: Pathways for the generation of the various mediators of inflammation.
Experimental Models of Inflammation
Paw oedema, sponge implantation and air pouch granulomas are among the models that are used in inflammation studies. These models employ a variety of agents like formalin, Freunds adjuvant, carrageenan, monosodium urate crystals and zymosan (Singh, 2000). Others include vasoactive agents (e.g. platelet activating factor and histamine), weakened bacteria such as E. coli, chemotactic factors (e.g. N-formyl-norleucyl-phennylalanine), injection of polymorphonuclear leucocyte, leucotriene B4 and arachidonic acid in acetone
(Issekutz and Issekutz , 1989). Injecting these agents into various parts of the body may induce acute inflammatory response.
Models of Acute Inflammation
Acute inflammatory response can be assessed by monitoring reactions such as foot volume increase produced by oedema (e.g. in the rat’s paw),
99
Digitized by UCC, Library
presence of plasma markers in the skin, measurement of inflammatory mediators in plasma exudates, local rise in the temperature of the skin, hyperaemia (an increase in vascular permeability), monocyte infiltration, polymorphonuclear leucocyte and lymphocyte accumulation (Issekutz and
Issekutz, 1989). Hyperaemia and the emigration of leucocytes are the basic manifestations of the acute inflammatory reaction (Issekutz, 1981). Among the lot, the most acceptable preliminary screening test for anti-rheumatic activity is the carrageenan - induced acute footpad oedema in laboratory animals. This model has been widely used to screen new anti-inflammatory drugs (Singh,
2000) and has been used in this current investigation with very excellent result.
Carrageenan-induced Paw Edema
This model is based on the principle of release of various inflammatory mediators by carrageenan. The carrageenan-induced edema model in rodents is based on the principle of release of various inflammatory mediators by carrageenan and is the most accepted in vitro experimental model for anti- rheumatic activities in laboratory animals (Singh et al., 2000).
Oedema formation due to carrageenan in the rat paw is a biphasic event. The initial phase is attributed to the release of histamine and serotonin. The second phase of oedema is due to the release of prostaglandins, protease and lysosome
(Amponsah, 2012). Subcutaneous injection of carrageenan into the rat paw produces inflammation resulting from plasma extravasation, increased tissue water and plasma protein exudation along with neutrophil extravasation, all due to the metabolism of arachidonic acid (Chatpaliwar et al., 2002). The first phase
100
Digitized by UCC, Library
begins immediately after injection of carrageenan and diminishes in two hours.
The second phase begins at the end of the first phase and remains through the third hour up to five hours.
Animals (rats/chicks) are divided into groups of about five each (n=5) prior to the day of experiment. The control group receives vehicle orally, while other groups receive test and standard drugs. The left paw is marked with ink at the level of lateral malleolus; basal paw volume is measured by volume displacement method using Plethysmometer, by immersing the paw till the level of lateral malleolus. The animals are then given drug treatment. One hour after dosing (pre- emptive), the rats are challenged by a subcutaneous injection of 0.1mL of 1% solution of carrageenan into the sub-plantar side of the left hind paw. The paw volume is measured again at 1, 2, 3, 4 and 5 hours after the challenge. The increase in paw volume is calculated as percentage compared to the basal volume.
The difference of average values between treated animals and control group is calculated for each time interval and evaluated statistically. The percent Inhibition is then calculated (Armah, et al., 2015).
OXIDATIVE STRESS
Metabolic processes in the body generate highly reactive species, known as free radicals, which injure cellular molecules. Free radicals are highly reactive atomic or molecular species that contain one or more unpaired electrons in their outermost atomic or molecular orbital and are capable of free existence
(Sen et al., 2010). Free radicals react quickly with the nearest stable molecule to capture the electron they need to gain stability. The ‘injured’ molecule loses its
101
Digitized by UCC, Library
electron, becoming a free radical itself. They can damage vital cellular components like nucleic acids, cell membranes and mitochondria, resulting in subsequent cell death. As all aerobic organisms utilize oxygen during cellular respiration and normal metabolism, the generation of free radicals by biochemical cellular reactions and from the mitochondrial electron transport chain is inevitable (Abdillahi, et al., 2011). The free radicals include reactive
. . - oxygen and nitrogen species such as superoxide (O2 ¯), hydroxyl (OH ) , peroxyl
(ROO-), peroxinitrite (ONOO¯), and nitric oxide (NO·) radicals. All these are produced through oxidative processes within the mammalian body (Abdel-
Hameed, 2009). They may also be generated through environmental pollutants such as cigarette smoke, automobile exhaust fumes, radiation, air pollution and pesticides (Sen et al., 2010). To protect the cells and organ systems of the body against reactive oxygen and nitrogen species, humans have evolved a highly sophisticated and complex antioxidant protection system, that functions interactively and synergistically to neutralize free radicals. These antioxidants are capable of stabilizing or deactivating, free radicals before they attack cells
(Almeida, et al., 2011). Antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase destroy toxic peroxides. In addition to antioxidant enzymes, non-enzymatic molecules play important roles in antioxidant defence systems. These non- enzymatic molecules are of an exogenous nature and are obtained from foods. They include α-tocopherol, β- carotene, and ascorbic acid, and such micronutrient elements as zinc and selenium (Aremu, et al., 2011). Normally, there is a balance between free
102
Digitized by UCC, Library
radical generation and scavenging (Aremu, et al., 2011). Oxidative stress results from an imbalance between excessive generation of oxidant compounds and insufficient anti-oxidant defence mechanisms (Aremu, et al., 2011). When the natural antioxidant mammalian mechanism becomes inadequate, the excess of free radicals can damage both the structure and function of cell membranes in a chain reaction leading to degenerative diseases and conditions such as
Alzheimer’s disease, cataracts, acute liver toxicity, arteriosclerosis, nephritis, diabetes mellitus, rheumatism and DNA damage which can lead to carcinogenesis (Aremu, et al., 2011).
ANTIOXIDANTS
All cells in eukaryotic organisms contain powerful antioxidant enzymes.
Endogenous antioxidants made in the body are believed to be more potent in preventing free radical damage than exogenous antioxidants. The major classes of endogenous antioxidant enzymes are the superoxide dismutases, catalases and glutathione peroxidases (Almeida, et al., 2011), α-lipoic acid and coenzyme
Q10. In addition, there are numerous specialized antioxidant enzymes reacting with and, in general, detoxifying oxidant compounds.
Superoxide dismutases are present in almost all aerobic cells and in extracellular fluids (Aremu, et al., 2011). Superoxide dismutase enzymes contain metal ion cofactors that, depending on the isozyme, can be copper, zinc, manganese or iron. They catalyse the breakdown of the superoxide anion into oxygen and hydrogen peroxide as shown in figure 39. Catalases, on the other hand, are
103
Digitized by UCC, Library
enzymes that catalyse the conversion of hydrogen peroxide to water and oxygen, using either an iron or manganese cofactor (Sen et al, 2010).
Figure 40: Pathway for the detoxification of reactive oxygen species by superoxide dismutase, catalase and peroxidases.
Determination of Antioxidant Properties
The antioxidant activities of putative antioxidants have been attributed to various mechanisms, among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction and radical scavenging.
Several methods have been used to assess antioxidant activity of compounds, extracts and nutritional supplements. These include the DPPH radical scavenging, lipid peroxidation, reducing power and total antioxidant capacity assays. Because different reactive oxygen species have different reaction mechanisms, attempting to evaluate antioxidant activity using one assay in order to claim ‘‘total antioxidant activity’’ is oversimplified and inappropriate.
Therefore in this study, the DPPH free radical scavenging activity, total phenolic activity and the total antioxidant activity assays were used to assess the antioxidant activity of the extracts and isolates.
104
Digitized by UCC, Library
Total Antioxidant Capacity
The total antioxidant capacity refers to a full spectrum of antioxidant activity against various reactive oxygen/nitrogen radicals. The major advantage of this test is that it measures the antioxidant capacity of all antioxidants in a biological sample or extract and not just the antioxidant capacity of a single compound. Major antioxidant capacity assays can be roughly divided into two categories:
(1) hydrogen atom transfer (HAT) reaction based assays and
(2) single electron transfer (ET) reaction based assays (Amponsah, 2012).
These two mechanisms yield identical results, but they differ in terms of kinetics and the potential for side reactions to occur.
HAT-based procedures measure the classical ability of an antioxidant to quench free radicals by hydrogen donation (Amponsah, 2012);
X + AH → XH + A , where (AH = any H donor). HAT- based assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity, total radical trapping antioxidant parameter, and crocin bleaching assays. HAT reactions are solvent and pH independent and usually are quite rapid; typically they are completed in seconds to minutes. A disadvantage of the procedure, however, is that the presence of reducing agents, such as metals, can lead to high apparent reactivity.
ET-based methods detect the ability of a potential antioxidant to transfer one electron to reduce a species. They measure the capacity of an antioxidant to reduce an oxidant, which changes colour when reduced. The degree of colour
105
Digitized by UCC, Library
change is correlated with the sample’s antioxidant concentration (Amponsah,
2012). ET-based assays include the total phenols assay with Folin-Ciocalteau reagent, Trolox equivalence antioxidant capacity, ferric ion reducing antioxidant power, total antioxidant potential assay using a Cu (II) complex as an oxidant, phosphomolybdenum method and DPPH. ET reactions are usually slow and can require long times to reach completion, so antioxidant calculations are based on percent decrease in product rather than on kinetics. Trace compounds and metals also interfere with ET methods and can account for high variability and poor reproducibility of results (Amponsah, 2012).
DPPH Radical Scavenging Activity
The antioxidant ability of a sample can be estimated by determining the hydrogen donating ability of the sample in the presence of 2,2-diphenyl-1-picryl- hydrazyl or 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical at 517 nm.
The DPPH assay is a valid and simplest assay to evaluate scavenging activity of antioxidant, since the radical compound is stable and does not have to be generated as in other radical scavenging assays (Muller, et al., 2011).
DPPH assay method is based on the reduction of purple methanolic DPPH to a yellow coloured diphenyl picrylhydrazine and the remaining DPPH which shows a maximum absorption at 517 nm is measured (Muller, et al., 2011). The decrease in absorbance of DPPH at its absorption maxima of 517 nm is proportional to concentration of free radical scavenger added to DPPH reagent solution. Decrease in the DPPH solution absorbance indicates an increase of the
106
Digitized by UCC, Library
DPPH radical scavenging activity. The DPPH radical scavenging activity is calculated according to the following equation:
% DPPH radical scavenging activity = 1 - [Asample /Acontrol] 100
Where Asample and Acontrol are absorbances of sample and control
The concentration of sample required to scavenge 50% of DPPH is expressed as
IC50 (Muller, et al., 2011).
Total Antioxidant Activity by Phosphomolybdenum Method
It is a spectroscopic method for the quantitative determination of antioxidant activity, through the formation of phosphomolybdenum complex as described by Lallianrawna et al., (2013). The assay is based on the reduction of molybdenum, Mo (VI) to Mo (V), by the extract and subsequent formation of a green phosphate/Mo (V) complex at acidic pH which is measured at 695 nm.
Total Phenolic Activity by Folin-ciocalteau Method
The antioxidant activities of most plants have been ascribed to their phenolic constituents (Khomsug et al., 2010). In this study, the phenolic constituent of the extracts were determined using the method described by
Lallianrawna et al., (2013). This method depends on the reduction of Folin-
Ciocalteau reagent by phenols to a mixture of blue oxides which have a maximal absorption in the region of 760 nm. The reaction equation is as follows:
Folin: Mo+6 (yellow) + ѐ (from antioxidant) → Mo+5 (blue)
107
Digitized by UCC, Library
Where the oxidizing reagent is a molybdophosphotungstic heteropolyacid and comprised of 3H2O·P2O5·13WO3·5 MoO3·10H2O, in which the hypothesized active centre is Mo+6.
The method is simple and sensitive, and can be useful in characterizing and standardizing botanical samples. However, the reaction is slow and occurs at acidic pH.
108
Digitized by UCC, Library
CHAPTER THREE
MATERIALS AND METHODS
Chemicals
All organic solvents used for the research were of analytical grade and obtained from BDH Laboratory Supplies (Merck Ltd., Lutterworth, UK). The standard reference drug, Diclofenac, was purchased from Troge (Hamburg,
Germany) while all other chemicals were obtained from Sigma-Aldrich Company
Ltd, (Poole, Dorset, UK).
General Experimental Procedures
1H and 13C NMR were obtained on a JEOL 500 MHz spectrometer instrument. Chemical shifts were reported in δ (ppm) using the solvent (CDCl3 or methanol-D4), standard and coupling constants (J) were measured in hertz
(Hz). The high resolution (Q-ToF) mass spectroscopy instrument, SYNAPTG2-
Si#UGA333 (Thermo Fisher Scientific, UK), with an electrospray ionization probe was used for accurate mass measurement over the full mass range of m/z
50-2000. Nano-electrospray analyses were performed in positive ionization mode by using NanoMate to deliver samples diluted into MeOH+10%
0 NH4OAc. The temperature was set at 200 C, sheath gas flow of 2 units and capillary (ionizing) voltage at 1.4 kV. Column chromatography was performed with aluminum oxide neutral gel (grade II, 70-230 mesh) and TLC with silica gel F254. Alkaloid detection was performed using Dragendorff’s reagent,
Mayer’s reagent and 3% Ce (NH4)2SO4 in 85% H3PO4.
109
Digitized by UCC, Library
Melting points were determined using electrochemical melting point-9100 apparatus.
Collection and Authentication of Plant Sample
The root bark of Anthostema aubryanum (Euphorbiaceae) was harvested from Adukrom in the Nzema East Metropolis in the Western region of Ghana.
The plant was identified by Mr. Agyarkwa of the Department of Botany, School of Biological Sciences, College of Agriculture and Natural Sciences, University of Cape Coast, Cape Coast where a voucher specimen with reference number
(HBS/Antho/2014/R2895) has been deposited in the herbarium.
Processing of Plant Material
The root bark of A. aubryanum was air dried for three weeks. The dried material (1200 g) was coarsely milled and packed into brown paper bags and kept at the laboratory until required for use.
Phytochemical Screening of Crude Plant Extract
The root bark of A. aubryanum was screened for phytochemical constituents as per the procedures given by Harborne (1998) with modifications by Wanyama et al., (2011).
25 g of the plant sample was first defatted with petroleum ether (40/60) solvent in a Soxhlet apparatus for 3 hrs. The ether extract was concentrated to 50 mL.
Analysis of the extract for various liposoluble chemical constituents were carried out as described below.
110
Digitized by UCC, Library
Tests on the ether extract
Test for terpenoids
Ten (10 mL) of the ether extract was evaporated to dryness. The residue was dissolved in acetic anhydride (0.5 mL) and then in 0.5mL of chloroform.
The solution was transferred to a dry test tube and conc. Sulphuric acid was added to the bottom of the test tube by means of a dropping pipette. A brownish-red or violet ring was formed and the supernatant layer turned green indicating the presence of terpenoids.
Test for carotenoids
Ten (10 mL) of the ether extract was evaporated to dryness after which 2-
3 drops of concentrated sulphuric acid in chloroform were added. No intense blue colour developed showing the absence of carotenoids in the plant extract.
Test for fatty acids
Ten (10 mL) of ether extract was exhaustively extracted with aqueous sodium hydroxide solution. The aqueous alkaline layer was then acidified with conc. HCl (pH= 3-4), thereby liberating the fatty acids from their alkaline salts.
The acid solution was then shaken several times with small portions of petroleum ether in a separating funnel to extract the fatty acids. The ether layer was then evaporated to dryness. An oily residue was observed which showed the presence of fatty acids.
Test for flavonoid aglycones
Three (3 mL) of the ether extract was evaporated to dryness. The residue was dissolved in 1-2 mL of methanol. A piece of magnesium ribbon was then
111
Digitized by UCC, Library
added to the solution followed by 4-5 drops of concentrated HCl. A pink or magenta-red colour developed within 3 min indicating the presence of flavonoid aglycones.
Test for anthraquinone aglycones (emodols)
To three (3 mL) of the ether extract in a test tube was added 1 mL of 10% sodium hydroxide solution. A red colour was formed showing the presence of anthraquinone aglycones.
Test for coumarins
Three (3 mL) of the ether extract was evaporated to dryness. The residue was then dissolved in 2 mL of hot water and the solution allowed to cool to room temperature. The filtrate was divided into equal parts, one of which served as a reference. The other portion of the solution was made alkaline by adding
0.5 mL of 10% ammonia solution. An intense fluorescent colour was observed under UV light indicated the presence of coumarins and their derivatives.
Tests on the alcohol extract
The mack obtained after extracting the root bark with ether was dried and extracted three times with 95% ethanol. The alcohol extract was concentrated under reduced pressure to 50 mL. The extract was screened for phenolic compounds according to their physicochemical properties as described below.
Test for tannins
One (1 mL) of the alcohol extract was diluted with 2 mL of distilled water to which 2-3 drops of iron (III) were added. A blue-black colour showed the presence of catechol tannins.
112
Digitized by UCC, Library
Test for reducing sugar
One (1 mL) of the alcohol extract was diluted with (1-2mL) of distilled water. One (1 mL) of Fehling solutions (I and II) were added to the solution and the mixture was then heated. A brick-red precipitate was formed indicating the presence of reducing sugars.
Test for alkaloids
Fifty (50 mL) of the extract was transferred to a capsule and evaporated on a water bath. Ten (10 mL) of dilute HCl (10%) was added to the residue. The solution was basified by adding aqueous ammonia (10%) to a pH of 8-9 and then extracted with chloroform. The chloroform extract was evaporated to dryness and the residue dissolved in HCl (20 mL, 2%) and the solution divided into two portions. One portion was kept as a reference.
Precipitation reaction test for alkaloids
These tests were carried out by using Mayer’s, Dragendorff’s, Wagner’s,
Hager’s and Tannic acid reagents on the second portion of the test solution which was also divided into five portions. 2 -3 drops of the alkaloid test reagents were added to the test solutions.
The alkaloids formed coloured precipitates with the test reagents; orange-red
(Dragendorff’s), slightly yellowish (Mayer’s), brown (Wagner), yellow
(Hager’s) and white (tannic acid) which indicated the presence of alkaloids.
Colour reaction test for alkaloids
These tests were also carried out as with the precipitation tests but by using the Froehde’s, Marchi’s and the Molisch’s test reagents. The alkaloids also
113
Digitized by UCC, Library
formed different colours for the colour reaction test; blue-black (Froehde’s reagent), black-green (Marchi’s reagent) and pale yellow (Molisch’s reagent), indicating the presence of alkaloids.
Tests on the hydrolysed alcohol and aqueous extracts
To the ethanol extract (25 mL) was added HCl (15 mL, 10%) and the mixture heated under reflux for 10 minutes. During the hydrolysis of the glycosides, the solution became opalescent due to the formation of aglycones as a precipitate. The mixture was cooled and extracted three times with ether (10 mL) using a separating funnel. The ether extract (35 mL) were combined and dried over anhydrous magnesium sulphate.
Test for anthracyanoside glycosides
The ether extract (5 mL) was evaporated to dryness. The residue was then dissolved in methanol (2 mL, 50%) by heating and then magnesium ribbon added followed by 5-6 drops of conc. HCl. There was a red solution which turned blue in alkaline medium indicating the presence of anthracyanosides.
Test for polyuronide glucosides
The plant sample after the extraction with ether and alcohol was dried. It was then extracted with warm distilled water for 20 minutes. The solution was filtered and concentrated to 50 mL. Two (2 mL) of this aqueous extract was added dropwise to a test tube containing 10 mL of methanol. No violet or blue colour precipitate was observed showing the absence of polyuronide glucosides.
114
Digitized by UCC, Library
Test for glucosides
The aqueous extract (2 mL) was transferred into a petri dish and was evaporated to dryness. 2-3 drops of concentrated sulphuric acid was added and allowed to stand for 5 minutes. 3-4 drops of methanol saturated with thymol
(Molisch’s reagent) was then added. The absence of a red colour meant the absence of glucosides.
Test for saponins
Two (2 mL) of the diluted aqueous extract (1:1) with distilled water was shaken in a test tube for 20 minutes. The appearance of foam that lasted for more than 20 min indicated the presence of saponins.
Test for anthraquinone glycosides
To the alcohol extract (25 mL) was added 15 mL of 10% dilute hydrochloric acid and the mixture heated under reflux for 10 minutes. The solution became opalescent due to the formation of aglycones as precipitates during the hydrolysis of the glycosides. The mixture was cooled and then extracted three times with ether (10 mL) using a separating funnel. The ether extract (30 mL) was then dried over anhydrous magnesium sulphate. 4 mL of the extract was concentrated to 2 mL ammonia solution was then added with shaking. A red colour was observed indicating the presence of aglycones in oxidized form.
Test for flavonoid glycosides
The ether extract (5 mL) from the hydrolysed alcohol extract was evaporated to dryness. The residue was dissolved in 2 mL of 50% methanol by
115
Digitized by UCC, Library
heating and then added a small piece of magnesium ribbon followed by the addition of 5-6 drops of concentrated hydrochloric acid. A red solution was formed indicating the presence of flavonoid glycosides.
Test for cyanogenic glycosides
Fresh plant material (1.0 g) was cut into pieces and placed in a test tube with 3.0 mL of distilled water and 6 drops of chloroform, followed by briefly crushing the material with a glass rod. The test tube was stoppered with a cork containing a strip of picrate-impregnated paper hanging down from the stopper and incubated at ambient temperature for 2 h. The assay was performed in triplicate. A colour change of the picrate-impregnated paper from yellow to brown-red indicated the release of hydrogen cyanide and hence cyanogenic glycosides.
Picrate paper preparation
Strips of filter paper (5.0 X 1.5cm) were soaked in an aqueous solution of
0.05M picric acid previously neutralized with sodium bicarbonate, and filtered.
The impregnated paper was left to dry at ambient temperature.
Extraction of Plant Material
The dried and powdered root bark of Anthostema aubryanum (1.20 Kg) was moistened with NH3 (aq) (25%) and extracted by Soxhlet in 70% MeOH (2x
2.5 L) for 48 h. The combined extracts were concentrated under reduced pressure to afford a brownish crude extract (32.20 g). The crude extract (31.20 g) was dissolved in 5% acetic acid, refrigerated for 24 h and filtered. The clear acidic solution was extracted with Hexane (3x200 mL). The Hexane layer was
116
Digitized by UCC, Library
discarded and the aqueous phase basified with 10% NH3 (aq) (25%), extracted with CH2Cl2 (3x150 mL). This organic layer was dried using MgSO4 and evaporated under reduced pressure to dryness, light brownish crude (0.680 g, yield= 0.1%) were obtained. The screening of this extract using Dragendorff’s reagent, Mayer’s reagent and 3% Ce (NH4)2SO4 in 85% H3PO revealed the presence of alkaloids. The two extracts were kept in desiccators in the laboratory until needed.
Anti-inflammatory Assay of Extract
Experimental animals
Sprague Dawley rats were obtained from Noguchi Memorial Institute for
Medical Research, Accra, Ghana, and were housed in stainless steel cages (30 ×
47 × 20 cm) at a population density of 5 rats per cage. Food (Cheletin diet, from
GAFCO Tema, Ghana) and water were available ad libitum through 1-qt gravity- fed feeders and waterers. The room temperature was maintained regularly
(25±2°C) with humidity of 30-60%, and overhead incandescent illumination was maintained on 12-hour light-dark cycle. Daily maintenance was conducted during the first quarter of the light cycle. Wood shavings were used as bedding for the animals. Group sample size of 5 was used throughout the study.
Carrageenan-induced edema in rats
To evaluate folkloric claims, the effects of extracts and isolated compounds from the root bark of A. aubryanum was studied using acute in vivo carrageenan- induced hind paw oedema model of inflammation in rats (Kumar et al., 2014).
Carrageenan (10 µl of a 2% suspension in saline) was injected subplantar into the
117
Digitized by UCC, Library
right footpads of the rats. The foot volume was measured before injection and at hourly intervals for 5 hours after injection by water displacement plethysmography as described by Fereidoni et al., (2000) using an electronic Von
Frey plethysmometer (Model 2888, IITC life science inc. Ca 91367 Canada). The oedema component of inflammation was quantified by measuring the difference in foot volume before carrageenan injection and at the various time points.
Anti-inflammatory Assay of Crude Methanolic Extract
The experiment was aimed at investigating the effect of the extract and standard drug (diclofenac) on edema 1 hour after carrageenan challenge and continuing up to 5 hours. The drug was given through the intraperitoneal (i.p) route and the extracts by the oral route. The test animals received the extract (30,
100 and 300 mg/kg, p.o.), diclofenac (10, 30 and 100 mg /kg, i.p.) whereas the control animals received only the vehicle (2 mL/Kg normal saline). The foot volumes were individually normalized as percentage of change from their values at time zero and then averaged for each treatment group. The total inflammation during the entire observation period for each treatment was also calculated in arbitrary unit as the area under the curve (AUC) and compared with the untreated group (Mireku et al., 2014). The doses for the hydro-alcoholic extracts were prepared by dissolving a known weight of the extract in 2 % tragacanth mucilage.
All experimental protocols were in compliance with the National Institute of
Health guidelines for the care and use of laboratory animals and were approved by the Department of Biomedical and Forensic Science, College of Agriculture and Natural Sciences, University of Cape Coast Ethics Committee.
118
Digitized by UCC, Library
Anti-inflammatory Assay of Crude Alkaloid Extract
The crude alkaloid extract was tested for anti-inflammatory activities using the method stated above.
Antioxidant Assay of Extracts
Total phenolic content assay
The total phenolic content (TPC) of crude methanol extract was determined using the modified Folin-Ciocalteau method (Lallianrawna, et al.,
2013). In this method, I mL of the extract solution (1.0 mg/mL) in distilled water was introduced into a test tube followed by 1 mL of Folin-Ciocalteau reagent and
I mL of 2.0% sodium carbonate. The content of the test tube was mixed thoroughly and the reaction mixture was allowed to stand for 2 h with shaking at
250C in an incubator. The mixture was then centrifuged at 3000 rpm for 10 minutes before measuring the absorbance of the resulting complexes at 760 nm using UV-VIS spectrophotometer (Cecil CE 7200 spectrophotometer, Cecil instrument limited, Milton Technical Centre, England). Quantification of total phenolic was based on a vitamin E standard curve generated by preparing 0-100
μg L-1of vitamin E. The TPC were expressed as milligrams of vitamin E equivalents (VEE)/g extract.
Total antioxidant capacity assay
The assay is based on the reduction of molybdenum, Mo +6 to Mo +5, by the extracts and subsequent formation of a green phosphate-molybdate (Mo +5) complex at acidic pH (Lallianrawna, et al., 2013). Test tubes containing l mL each of the extracts (0.25-2 mg/mL) and 3 mL of the reagent solution (0.6 M
119
Digitized by UCC, Library
sulphuric acid, 28 mM disodium phosphate and 4 mM ammonium molybdate) were incubated at 95 oC for 90 minutes. After the mixture had cooled to room temperature, the absorbance of the solution was measured at 695 nm. Four concentrations of Vitamin E (0.025, 0.05, 0.1 and 0.2 mg/mL) was used to construct a calibration curve. A blank solution was prepared by adding every other solution but without extract or standard drug. The antioxidant capacity was expressed as mg of Vitamin E equivalent (VEE)/g of extract. This procedure was used for all the extracts and the isolates.
In vitro qualitative DPPH test
The qualitative test for antioxidant activity was performed using the rapid
DPPH radical scavenging assay (Muller, et al., 2011). 10 µl of the crude methanolic extract was applied on silica gel plates 60 F254 (Merck, 0.25 mm thick) and allowed to dry completely. The plate was then sprayed with a solution of 2% DPPH in methanol. A pale yellow to white spot over a purple background indicated a radical scavenging activity of the particular extract/isolate.
Quantitative Antioxidant Assays of Extracts
For the DPPH assay, the antioxidant activity of the crude methanol extract was assessed in terms of the hydrogen donating or radical scavenging abilities of the extract using the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method of Muller et al., (2011). Aliquots of the crude extract (0.25-2.0 mg/mL) and vitamin E (standard) (0.04-1.28 mg/mL) were mixed with 100 mM
Tris-HCl buffer (800 μL, pH= 7.4). Then 1 mL of freshly prepared 500 µM
DPPH in methanol was added to the mixture and allowed to stand for 30 min at
120
Digitized by UCC, Library
room temperature in the dark. The mixture was shaken vigorously and the absorbance was measured at 517 nm with a spectrophotometer, (Cecil CE 7200 spectrophotometer, Cecil instrument Ltd, All samples were analyzed in triplicate.
Pure methanol was used as a blank. The actual decrease in absorption induced by the test sample was compared with the positive control, vitamin E. The amount of remaining DPPH against the sample concentration was plotted to obtain the amount of antioxidant (μg) necessary to decrease free radicals by 50% (IC50). A smaller IC 50 value corresponds to a higher antioxidant activity (Muller, et al.,
2011).
Statistical Analysis of Data
The raw scores for right foot volumes were individually normalized as percentage of change from their values at time zero then averaged for each treatment group. Total foot volume for each treatment was calculated in arbitrary unit as the area under the curve (AUC). To determine the percentage inhibition for each treatment, the following equation was used.
AUC AUC inhibition% edemaoof control treatment 100 AUC control
Differences in AUCs were analyzed by one way analysis of variance followed by
Student-Newman-Keuls’ post hoc t test. Doses and concentrations responsible for 50 % of the maximal effect (EC50 and IC50) for each drug/extract were determined using an iterative computer least squares method, with the following nonlinear regression (three-parameter logistic) equation.
a+(b-a) Y= 1+10(LogEC50-X)
121
Digitized by UCC, Library
Where, X is the logarithm of dose and Y is the response. Y starts at a (the bottom) and goes to b (the top) with a sigmoid shape. The fitted midpoints
(ED50/IC50 values) of the curves were compared statistically using F test
(Armah, et al., 2015). Graph Pad Prism for Windows version 5.0 (Graph Pad
Software, San Diego, CA, USA) was used for all statistical analyses. P < 0.05 was considered statistically significant (Amponsah, 2012).
Fractionation of Alkaloid Extract
All doses of the dichloromethane/ alkaloid extract administered through the same (oral) route displayed either comparable or better anti-inflammatory activity as the standard drug- diclofenac.
Therefore the alkaloid extract was fractionated using column and preparative thin layer chromatography coupled with spectroscopic analysis to isolate and characterize the major anti-inflammatory constituents present in the root bark of
Anthostema aubryanum (Baill).
Chromatographic materials
One type of stationary phase material was used for the column chromatographic technique: Aluminum oxide neutral gel (70-230) mesh (ASTM,
Merck Germany). Aluminum pre-coated silica gel plates 60 F254 (0.25 mm thick) were used for the analytical thin layer chromatography (TLC).
Detection for analytical thin layer chromatography
The zones on TLC plates corresponding to separated compounds were detected under UV light 254 nm and 365 nm and also by spraying with
122
Digitized by UCC, Library
Dragendorff’s and Ehrlich’s reagents followed by heating at 105 °C for 5-10 minutes.
Column Chromatography
The wet method was used in packing the column with aluminum oxide neutral gel (70-230 mesh). A column with a diameter of 0.20 cm and height 3.20 cm was filled to one-third with dichloromethane and the aluminum oxide neutral gel was gently packed on the glass column. The extract was dissolved in a minimum amount of solvent and adsorbed onto a quantity of aluminum oxide neutral gel. It was then allowed to dry completely and then placed on top of the already packed column. The mobile phase (solvent or mixture of solvents) was then placed on top of the packed column to separate the extract into different fractions and the eluent collected into glass beakers.
Preparative-layer chromatography
The method of Waksmundzka-Hajnos et al (2006) was used.
Chromatography was performed on 20 cm x 20 cm glass plates precoated with
0.25 mm layers of aluminum oxide neutral gel 60 F254 (Merck). Samples were applied by the use of a Desaga (Heidelberg, Germany) AS 30 automatic applicator or were applied to the edge of the layer by use of capillary tubes.
Plates were developed face-down to a distance of 10 cm, in a horizontal Teflon
DS chamber (Chromdes, Lublin, Poland) after conditioning for 15 min with dichloromethane. After development, the mobile phase was evaporated to dryness and layers were scraped into glass beakers.
123
Digitized by UCC, Library
Development of thin layer chromatogram
The technique of thin layer chromatography-TLC (Bobbit, 1964) was used to resolve the crude extract into its components, develop the best solvent system for isolation and to check the purity of the isolated compounds. It was a qualitative measure involving the use of solvents of different polarities and ratios to obtain a suitable solvent system. The TLC plates were of dimensions 5 cm x 20 cm and precoated with silica gel 60 F254 with 0.2 mm layer thickness (Merck).
The plates were activated by heating them in the oven at 1100C for about 5 minutes before being used.
The one way ascending technique was used. The chamber was developed for at least an hour before developing the plates to ensure homogeneity of the atmosphere (to achieve equilibrium between the gaseous phase and the liquid phase). Samples of mixtures/extracts to be analysed by TLC were dissolved in an organic solvent and were applied on the TLC plates as spots with the aid of capillary tubes at one end of the plate in a straight line about 2 cm above the edge and 1.5 cm away from the margins. The spots were dried and the plates placed inside a chromatographic tank containing the mobile phase. The mobile phase ran along the TLC plate in an ascending manner due to capillary action, carrying with it the components of the extract. When the solvent reached a reasonable height the operation was stopped and the solvent front marked. After development, the plates were air-dried for about 5 minutes. The separated compounds were identified by observing them under ultra violet light for fluorescence; spots were also developed in iodine tank followed by spraying with Dragendorff’s reagent.
124
Digitized by UCC, Library
Isolation of Compounds from the Crude Alkaloid Extract
Column chromatographic separation of the crude alkaloid extract
Neutral aluminum oxide gel (40g, 70-230 mesh ASTM) was wet packed into a glass column (3.2cm × 0.2 cm). The crude alkaloid extract (0.68 g) was dissolved in a minimum amount of methanol and mixed with 5 g of alumina gel, allowed to dry to attain the same consistency as the alumina gel that was used, and spread gently on top of the packed column. A wad of glass wool was placed on top of the packed column in order not to disturb the surface of the packing.
The elution was done with a mixture of CH2Cl2-EtOAc then EtOAc-MeOH and
MeOH following a gradient of polarity.
Elution with a mixture of CH2Cl2 - EtOAc (50:25 v/v) led to fraction I which was colourless (190 mg). Elution with CH2Cl2 - EtOAc (50:50 v/v) gave a brownish fraction II (230 mg). The fraction III was obtained with a mixture of
EtOAc – MeOH (50:25 v/v) to MeOH (100%). This fraction was light yellow
(250 mg). Each fraction collected was tested for alkaloids by the use of
Dragendorff’s reagent and confirmed by using Ehrlich’s reagent. The fraction I was purified by preparative TLC on aluminum oxide neutral gel and crystallized in EtOAc to give 160 mg of compound M1 (Rf 0.7 in toluene-
EtOAc 50:50 v/v) which was a yellowish needle-like crystals. Fraction II was purified by preparative TLC on aluminum oxide gel neutral. The elution with toluene-EtOAc (75:25 v/v) gave sub-fractions II1 and II2 which were respectively crystallized in absolute ethanol and yielded the compounds: M2 (80 mg, Rf 0.40 in toluene-EtOAc 50:50 v/v) and M3 (90 mg, Rf 0.50 in toluene-
125
Digitized by UCC, Library
EtOAc 50:50 v/v). These two compounds were brownish and off-white
amorphous powder respectively. The compound M4 was a reddish-brown
powder and M5 was light yellowish crystals.
Root Bark
NH3(aq) MeOH (70%)
Marc Extract (31.20 g)
HOAc DCM
Organic layer Aqueous layer
NH (aq) 3 DCM
Organic layer Aqueous layer
Crude alkaloid (0.68g)
CC, alumina (70 – 230 mesh) DCM-EtOAc 100% DCM-EtOAc DCM-EtOAc 50:25 v/v DCM 50:25 v/v 50:25 v/v MeOH 100%
Fraction I (190 mg) Fraction II (230 mg) Fraction III (250 mg)
Figure 41: Schematic representation of the isolation of alkaloids
126
Digitized by UCC, Library
M1
M5
Figure 42: TLC analysis of crude alkaloid extract
Further purification of fraction III by preparative TLC on aluminum oxide gel neutral with toluene-EtOAc (75:25 v/v) gave sub-fractions IIIa and
IIIb. These sub-fractions were crystallized in absolute ethanol to give compounds: M4 (70 mg, Rf 0.45 in toluene-EtOAc 50:50 v/v) and M5 (120 mg,
Rf 0.60 in toluene-EtOAc 50:50 v/v) respectively.
127
Digitized by UCC, Library
Isolation of Compound M1
This compound was isolated from Fraction I (190 mg) by preparative- layer chromatography as described above. After development the mobile phase was evaporated to dryness and plates were scraped under UV lamp using sharp knife. The scraped material was dissolved in dichloromethane and after evaporating the mobile phase was washed several times with petroleum ether
(40/60) and crystallized in ethyl acetate to give compound M1 (160 mg, Rf 0.7 in toluene-EtOAc 50:50 v/v) as a yellowish needle-like crystals with a characteristic odour.
Fraction I (190 mg)
PTLC, alumina
Toluene: EtOAC 75:25 v/v Crystallization, EtOAC
M1 (160mg)
Figure 43: Schematic representation of the isolation of M1
Isolation of Compounds M2 and M3
These compounds were isolated from Fraction II (230 mg) by preparative-layer chromatography as described above. The scraped material was dissolved in dichloromethane to give sub-fractions II1 and II2. The sub-fraction
II1 was washed several times with hexane and crystallized in absolute ethanol to yield compound M2 (80 mg, Rf 0.40 in toluene-EtOAc 50:50 v/v) which was a brownish amorphous powder. The sub-fraction II2 was also washed several times with hexane and crystallized in absolute ethanol to give an off-white
128
Digitized by UCC, Library
amorphous compound M3 (90 mg, Rf 0.50 in toluene-EtOAc 50:50 v/v). The figure below illustrates the isolation procedure for compounds M2 and M3
Fraction II (230 mg)
PTLC, alumina
Toluene: EtOAc75:25 v/v
Sub-Fraction II1 (120mg) Sub-Fraction II2 (100mg)
Crystallization, EtOH Crystallization, EtOH
M2 (80mg) M3 (90mg)
Figure 44: Schematic representation of the isolation of M2 and M 3
Isolation of Compounds M4 and M5
The same procedure was followed as above in isolating compounds M4 and
M5 from fraction III (250 mg). Two sub-fractions IIIa and IIIb were obtained which were washed several times with hexane and crystallized in absolute ethanol to yield compounds: M4 (70 mg, Rf 0.45 in toluene-EtOAc 50:50v/v) and M5 (120 mg, Rf
0.60 in toluene-EtOAc 50:50 v/v). The compound M4 was a reddish-brown powder and M5 was light-yellowish crystals.
129
Digitized by UCC, Library
Fraction III (250 mg) PTLC, alumina
Toluene: EtOA 75: 25 C v/v
Sub - fraction IIIa (100 mg Sub -fraction IIIb (140 Crystallization EtOH Crystallization EtOH
M4 (70 mg) M5 (120 mg) ) Figure 45: Schematic representation of the isolation of M4 and M5
After the extraction and the isolation process, only compounds M1 and M5
were found to be pure enough, based on their TLC analysis, for spectroscopic
analyses. They were therefore sent to Greenwich University, UK for spectral
analyses.
Anti-inflammatory Activity of Isolated Compounds
The isolated compounds M1 and M5 were pure and were therefore tested
for their anti-inflammatory potential using the method described above. However,
the doses for the isolated compounds and the standard drug- diclofenac were 3, 10
and 30 mg/Kg body weight.
In vitro DPPH radical scavenging activity of isolated compounds
The free radical scavenging activity of the isolated compounds M1 and M5
was determined using the method stated above.
130
Digitized by UCC, Library
CHAPTER FOUR
RESULTS AND DISCUSSION
INTRODUCTION
The preliminary phytochemical analyses have revealed that the crude extract of Anthostema aubryanum is characterized by the presence of alkaloids, terpenoids, flavonoids, coumarins, anthraquinones, fatty acids, reducing sugars, cyanogenic glycosides, tannins and saponins. Carotenoids and glucosides were not detected or were absent. The presence mainly of alkaloids, flavonoids, steroids and terpenoids may largely contribute to the observed pharmacological activity because more chemicals belonging to these phytochemicals present in other medicinal plants had previously been reported to exhibit such pharmacological activity (Agnihotri, et al., 2010). It has been established that flavonoids are the major anti-inflammatory agents. Some of them act as phospholipase inhibitors and some have been demonstrated as TNF-α inhibitors in different inflammatory conditions (Agnihotri, et al., 2010). Flavonoids inhibit human neutrophil elastase (HNE) and the matrix metalloproteinases (MMP-2).
Biochemical investigations have also shown that flavonoids can inhibit both cyclooxygenase and lipoxygenase pathways of the arachidonic metabolism depending upon their chemical structures (Agnihotri et al., 2010).
131
Digitized by UCC, Library
Table 1: Phytochemical Analysis of A. aubryanum
Constituents Observation
Alkaloids +
Terpenoids +
Flavonoid aglycones +
Coumarins +
Anthraquinone aglycones +
Fatty acids +
Reducing sugars +
Tannins +
Anthraquinone glycosides +
Flavonoid glycosides +
Saponins +
Carotenoids -
Glucosides -
Cyanogenic glycosides +
(+) = Present, (-) = Absent Source: Laboratory data (2015)
Quercetin is a bioflavonoid compound that blocks the release of histamine and other anti-inflammatory enzymes. Although human studies with arthritic patients are lacking at this time, anecdotal evidence is strong for this application, as is experimental research investigation. There are no well-known side effects or drug-nutrient interactions for quercetin (Agnihotri, et al., 2010).
Alkaloids in asserted skeletal type based on pyridine ring system have been
132
Digitized by UCC, Library
presented with striking anti-inflammatory activity, e.g. Berberine from Berberis is a traditional remedy against rheumatism (Agnihotri, et al., 2010).
Terpenoids significantly inhibit the development of chronic joint swelling. In
Western medicine, the treatment often involves topical application of corticosteroids which are symptomatically effective but have inherent disadvantages. Terpenoids may affect different mechanism relevant to inflammations arising in response to varied etiological factors (Changa et al.,
2008). Phytol, the aliphatic diterpene found in F. thonningii has anti- inflammatory effects and has been reported as a potential therapeutic agent for the treatment of rheumatoid arthritis and possibly other chronic inflammatory diseases such as asthma (Dangarembizi et al., 2013). Hence the use of A. aubryanum against inflammation, wounds and infectious diseases may be rationalized by the presence of these compounds in the plant.
This is the first phytochemical report on the constituents of Anthostema aubryanum.
Characterization and Identification of Isolated Compounds
Comprehensive chromatographic analysis coupled with spectroscopic study has led to the isolation, characterization and identification of two of the major anti-inflammatory alkaloids as 5-methoxy-canthin-6-one [1] and canthin-
6-one [2].
Identification of M1 as 5-methoxy-canthin-6-one [1]
M1 was obtained as yellow needles with a characteristic odour and bright yellow-green fluorescence at 360 nm. The bright yellow-green UV fluorescence
133
Digitized by UCC, Library
at 360 nm suggested it to be a canthinone alkaloid and this was confirmed by phytochemical analysis (Zapesochnaya et al., 1991).
It gave a positive test with Dragendorff’s reagent on analysis by TLC suggesting it to be an alkaloid. It was soluble in chloroform, m.p 223-2340C; (nm, log ε):
EtOH- 269 sh. (4.31), 277(4.41), 297 sh. (3.93), 308 sh.(3.90), 355 sh.(4.01),
376(4.09).
Elemental analysis: Found: C, 72.03; H, 3.92; N, 11.08. C15H10N2O2 requires C.
71.99; H, 4.03, N, 11.19 %; δH (500MHz, MeOD, J/Hz) 8.02 (1H,, H-1, J=5.0),
8.68 (1H,d,H-2, J=5.0), 7.27 (1H,s, H-4, J=10), 8.24 (1H,d, H-8, J=7.7), 7.59
(1H,t, H-9, J=7.7), 7.73 (1H,t, H-10, J=7.7), 8.58 (1H,d, H-11, J=7.7), 4.06 (s,
3H). HR-MS (m/z) 251.0898 [M+H] - (calc. for C15H10N2O2) (Appendix 1C).
It has a DBE of 12, indicating an ABCD aromatic system (O’Donnell &
Gibbons, 2007)
A B C N N D
O
The 1H and 13C-NMR data (table 2, Appendix 1A-B) were similar to those of compound 2; however, ring D was seen to be a single substituted aromatic system and a deshielded methoxy singlet was present at δ 4.06 in the 1H-NMR
1 spectrum. In addition to the methoxy group, the H –NMR for M1 displayed seven signals in the aromatic region (δ7.27-8.68). Two proton doublets at δH
8.02 and 8.68 (J=5.0Hz) are characteristic of pyridine protons and assignable to
134
Digitized by UCC, Library
H-1 and H-2 respectively, and a pair of doublets occurring at δH 8.24 and δH
8.58 (J=7.7Hz) are typical of indole protons and are assignable to H-8 and H-11 respectively. The pair of triplets at δH 7.59 and δH 7.73 (J=7.7) further confirmed the presence of indole protons and are assignable to H-9 and H-10 respectively.
Further ortho coupled aromatic system was evident: a cis double-bond positioned next to aromatic nitrogen at δH 8.68 and δH 8.02 (J=5.0). In addition to seven methine carbons, the 13C-NMR spectrum revealed the presence of seven aromatic quaternary carbons (δ 124.29-146.78) and one deshielded signal consistent with a carbonyl carbon (δ158.40). Ring A is an aromatic system by correlations. The triplet at δ 7.59 (H-9; J=7.7Hz) correlated to the triplet at δ
7.73 (H-10; J=7.7 Hz) which in turn coupled to the doublet at δ 8.58 (H-11;
J=7.7 Hz).
Ring C was consistent with ortho coupled pair of hydrogens (H-1, δ 8.02 and H-
2, δ 8.68 J=5.0 Hz) positioned on a pyridine ring. H-1 is correlated to the quaternary carbon C-15 (δ 131.33), completing the assignment of ring B, while
H-2 is also correlated to the C-14 quaternary (δ 131.89). The correlation between H-1 and H-11 concluded the β-carboline skeleton of the canthin-6-one structure.
The resonance at δ152.0 could be unambiguously assigned to C-5 by irradiating the 5-methoxyl protons at δ 4.06. The complex multiplet of C-5 can be converted to a clean doublet due to the coupling to the H-4 proton. The doublets of C-14 at δ 131.89 and C-15 at δ 131.33 could each be analyzed in terms of three-bond coupling of 8.02 Hz. The assignment can be confirmed by irradiating
135
Digitized by UCC, Library
the H-4 proton at δ 7.27, where the signal at 131.89 will be reduced from a doublet to a singlet. The triplet at 128.85 (3JCH = 11.0Hz) with no one- or two- bond coupling is easily assigned to C-16 which is at a higher field due to the para-position effect of the 5-methoxyl substituent. The double doublet of C-6 at
δ 158.40 could be analyzed in terms of two-bond coupling of 2.2 Hz between C-
6 and H-4 and three-bond coupling of 11.0 Hz between C-6 and H-4. This is the first report of the occurrence of this compound from the root bark of
Anthostema aubryanum and hence Euphorbiaceae.
10 11 1
9 12 14 2 8 13 15 N N 16
4 6 5 O
OCH3
+ + + N N -CO -CH3 N N N -15 -28 N O O - OCH3 O O m/z 207 m/z 251 m/z 235
-28 -CO + -HCN + + -C2H2
-27 NH N N -26 N N m/z 125 m/z 153 m/z 179
Figure 46: Fragmentation pattern of compound M1
136
Digitized by UCC, Library
Table 2: 1H-NMR and 13C-NMR spectral data and 1H-13C long-range
correlations of M1 in MeOD at 500 MHz
Carbon Type δH δC 2J 3J Position 1 CH 8.02 d (5.0) 115.53 C-1, H-2
2 CH 8.68 d (5.00 146.78 C-2, H-1
4 CH 7.27 s (10.0) 140.42
5 C - 152.00 C-5, OCH3
6 C - 158.40 C-6, H-4
8 CH 8.24 d (7.7) 118.07 C-8, H-9
C-8, H-10
9 CH 7.59 t (7.7) 129.84 C-9, H-11
10 CH 7.73 t (7.7) 127.33 C-10, H-8
11 CH 8.58 d (7.7) 126.58 C-11, H-10
C-11, H-9
12 C - 124.29 C-12, H-10
C-12, H-8
13 C - 137.70 C-13, H-9
C-13, H-11
14 C - 131.89 C-14, H-2
15 C - 131.33 C-15, H-4
16 C - 128.85 C-16, H-2
C-16, H-5
-OCH3 4.06 s 59.80 C-5
Source: Laboratory data (2015)
137
Digitized by UCC, Library
Identification of M5 as canthin-6-one [2]
M5 was obtained as light yellow needles with a light blue fluorescence at
360 nm. It gave a positive test with Dragendorff’s reagent on analysis by TLC. It was soluble in chloroform; m.p 156-1570C, (Lit. 155-156, Zapesochnaya, et al.,
1991); (nm, log ε): EtOH-251(4.10), 259(4.12), 268(4.07), 300(3.92), 347(3.94),
362(4.15), 380(4.13).
Elemental analysis: Found: C, 76.32; H, 3.63; N 12.78. C14H8N2O requires C,
76.35; H, 3.66; N, 12.72%; δH (500MHz, MeOD, J/Hz) 8.0 (1H,d, H-
1,J=5.0Hz), 8.72 (1H,d, H-2, J=5.0), 8.11 (1H,d, H-4, J=10.0), 6.93 (1H,d,
J=10.0) 8.47 (1H,d, H-8, J=10.0), 7.68 (1H,t, H-9, J=8.5), 7.52 (1H,t, H-10,
J=8.5), 8.18 (1H,d, H-11,J=8.5). HR-MS (m/z) 221.0755 [M+H]- (calc. for
C14H8N2O)
It has a DBE of 12 which completes an ABCD aromatic ring system.
The spectral data (1H-NMR, 13C-NMR, table 4, appendix 2A-B) of compound 2 revealed that this compound was canthin-6-one, previously isolated from
Ailanthus altissima (Simaroubaceae) by Koike and Ohmoto (1985). The NMR spectra (table 4; appendix 2A-B) show that it is unsubstituted as shown by the characteristic doublets of the H-4 and H-5 protons with the constant J= 10 Hz, and also by the doublets of a pair of vicinal protons (H-1 and H-2). In addition to the signals of these protons of rings C and D (H-1, H-2, H-4 and H-5), its spectra also contain the signals of four aromatic protons (H-8, H-9, H-10 and H-
11) which are characteristic of an unsubstituted canthinone. In the long-range selective proton decoupling, irradiation of either H-1 proton at δ 8.0 or the H-4
138
Digitized by UCC, Library
proton at 8.1 reduced the triplet signal at δ 133.21 to a doublet, revealing three- bond couplings among C-15, H-1 and H-4. Irradiation of either the H-2 proton at δ 8.7 or the H-5 at δ 6.9 reduced the triplet signal at δ 136.92 to a doublet, revealing three-bond couplings among C-16, H-2 and H-5. At the same time, irradiation of the H-1 or H-2 proton reduced the double doublet at signal δ
132.09 to a doublet, revealing two-bond coupling between C-14 and H-1 of 3.7
Hz and three-bond coupling between C-14 and H-2 of 8.1 Hz. Irradiation of the
H-4 or H-5 proton reduced the double doublet signal at δ 160.96 to a doublet, showing two-bond coupling between C-6 and H-4 of 2.2 Hz and three-bond coupling between C-6 and H-5 of 11.0 Hz. On the basis of the above evidence and comparison with the published data, the structure of M5 was established as canthin-6-one. Canthin-6-one alkaloids occur plentifully in many plants of
Simaroubaceae and Rutaceae (Koike and Ohmoto, 1985). However, to the best of our knowledge, this is the first report of its occurrence in A. aubryanum and hence Euphorbiaceae.
10 11 1
9 12 14 2 8 13 15 N N 16 4 6 5 O
Figure 47: The structure of compound M5
139
Digitized by UCC, Library
13 Table 3: C-NMR chemical shifts (ppm) of canthin-6-one and compound M5
Carbon *Canthin-6-one Compound M5 position 1 115.37 117.85
2 144.84 146.88
4 138.57 140.69
5 127.98 127.11
6 158.21 160.96
8 116.29 118.13
9 129.84 129.92
10 124.69 124.31
11 121.61 120.00
12 123.33 125.65
13 138.24 140.29
14 128.99 133.21
15 130.91 132.13
16 135.23 136.92
Source: Laboratory data (2015), *(Koike and Ohmoto, 1985)
140
Digitized by UCC, Library
Table 4: 1H-NMR and 13C-NMR spectral data and 1H-13C long-range
correlations of M5 in MeOD at 500 MHz
Carbon Type δH δC 2J 3J Position 1 CH 8.00 d (5.0) 117.85 C-1, H-2
2 CH 8.72 d (5.0) 146.88 C-2, H-1
4 CH 8.11 d (10.0) 140.69
5 CH 6.9 3d (10.0) 127.11
6 C - 160.96 C-6, H-4
8 CH 8.47 d (8.5) 118.13 C-8, H-10
9 CH 7.68 t (8.5) 129.92 C-9, H-11
10 CH 7.52 t (8.5) 124.31 C-10, H-8
11 CH 8.18 d (8.5) 120.00 C-11, H10 C-11, H-9
12 C - 125.65 C-12,H-10, C-12, H- 8
13 C - 140.29 C-13, H-9, C-13, H-11
14 C - 132.09 C-14, H-1 C-14, H-2
15 C - 133.21 C-15, H-1,C-15, H-4
16 C - 136.92 C-16, H-2, C-16, H-5
Source: Laboratory data (2015)
141
Digitized by UCC, Library
BIOASSAYS
Anti-inflammatory activity of root bark extract
The stem and root bark of Anthostema aubryanum are routinely employed in traditional medicine to treat a variety of disease conditions including inflammatory pain, wounds, boil and edema. Many compounds with numerous pharmacological activities have been isolated from Euphorbiaceae but little is known about the pharmacology of the root bark of Anthostema aubryanum.
In our experimental conditions, we first used a positive control diclofenac which showed a time-dependent anti-inflammatory effect at all hours (figure 48). The
AUC calculation showed that the three tested doses (10, 30 and 100 mg/Kg
BDW) of diclofenac suppressed the carrageenan-induced edema under the experimental condition by 36.16 ± 2.4, 48.94 ± 2.2 and 59.20 ± 2.6 respectively.
From figure 46, it can be seen that oral administration of the methanolic extract of the root bark of A. aubryanum similarly suppressed the carrageenan-induced inflammation in a dose-and time-dependent manner. The extract was given orally to the rats at 30 mg/kg, 100 mg/kg and 300 mg/kg (weight of concentrated solution), 1 hour before induction of oedema with carrageenan. Diclofenac (10-
100 mg/kg, i.p) was used as reference drug. Induction of acute inflammation in control rats resulted in a prominent increase in paw thickness, which began 1 hour after intraplantar injection of carrageenan and reached a peak of inflammation after 2 hours (figure 48) and slowly declined for the next 3 hours. The extract caused significant (P < 0.000 1) dose-dependent inhibition of the carrageenan - induced inflammation in the six weeks old rats, the effect of which began 2 hours after carrageenan injection (figure 48). Diclofenac (10-100 mg /kg, i.p) showed
142
Digitized by UCC, Library
significant (P < 0.001) effect on the time course curve and dose dependently reduced the total oedema (figure 54). Values are means ± S.E.M. (n=5). ***P <
0.0001; ***P < 0.001; ***P < 0.01 compared to vehicle-treated group (One-way
ANOVA followed by Newman-Keul’s post hoc test). Dose response curves for the inhibition of foot oedema are shown in figure 52. The anti-edematogenic activity was quantified using the ED50. This is the dose required to reduce the inflammation by 50%. The stronger the anti-inflammatory actions of the drug, the lesser the quantity needed to inhibit the edema by 50%. Diclofenac showed the highest anti-inflammatory activity, followed by the crude extract (Table 5).
Table 5: Effect of crude extracts and standard drug on carrageenan-induced edema
Extracts/Drug ED50 (mg/Kg) ±SEM
Total Crude 5.29 ± 0.02
Alkaloidal crude 13.84 ± 0.01
Diclofenac 1.99 ± 0.01
Source: Laboratory data (2015)
143
Digitized by UCC, Library
Figure 48: Time-course edema development following carrageenan injection into rat paws and dose (mg/Kg)-dependent anti-inflammatory effect of the standard positive control, diclofenac.
144
Digitized by UCC, Library
Methanol crude 100 30mg/kg 80 100mg/kg 300mg/kg 60 Control
40
20 Increase in foot volume Increase
% 0 0 2 4 6 8 Time/hrs
crude auc 400 *** 300 *** *** 200
100
0 l 0 0 0 o 3 0 0 tr 1 3 n o c otal foot oedema (calculated asAUC) oedema (calculated foot otal
T Crude extract of A. aubryanum (mg/kg BDW)
Figure 49: Effect of the methanol root bark extract (30-300 mg/kg oral), on time course curve (a) and the total edema response (expressed as AUC, b) for 5 hours, in carrageenan - induced paw oedema in rats. ***P < 0.0001;*** P < 0.001; ***P < 0.01 compared to vehicle-treated group.
145
Digitized by UCC, Library
100 30mg/kg 80 100mg/kg 300mg/kg
60 Control
40
20
Increase in foot volume Increase
% 0 0 2 4 6 8 Time/hrs
Figure 50: Effect of the alkaloidal extract (30-300 mg/kg oral), on time course curve (a) and the total oedema response (expressed as AUC, b) for 5 hours, in carrageenan - induced paw oedema in rats.***P < 0.0001; *** P < 0.001;***P < 0.01 compared to vehicle-treated group.
146
Digitized by UCC, Library
Anti-inflammatory activity of the alkaloid extract
All doses of the dichloromethane/alkaloid extract administered through the same (oral) route displayed either comparable or better anti-inflammatory activity as the diclofenac. Results of the anti-inflammatory activity of the crude alkaloid extract (Table 5; figure 49), shows that oral administration of the alkaloid extract similarly suppressed the carrageenan-induced inflammation in a dose-and time-dependent manner. The crude extract exhibited potent anti-inflammatory activity than the alkaloid extract possibly due to synergism. Thus the present study has shown that the root bark of A. aubryanum possesses potent anti- inflammatory activity and therefore justifies its use in folkloric medicine in treating and managing inflammatory conditions.
Anti-Inflammatory activity of the isolated compounds
Oral administration of 5-methoxy-canthin-6-one [M1] (3-100 mg/kg) showed a dose -dependent inhibition of oedema in the six weeks-old rats (figure
52). It recorded a maximum inhibition of 27.08 ± 3.12% at 30 mg/kg and an ED50 value of 60.84 ± 0.01 mg/kg. Also, canthin-6-one [M5], showed a dose-dependent inhibition of oedema in the rat model (figure 53) with maximum inhibition of
17.9% at 30 mg/kg and an ED50 of 96.64 ± 0.01 mg/kg. The overall anti- inflammatory activity of the isolated compounds during the entire observation period was also assessed through the AUC analysis with due comparison with the positive control, diclofenac. All doses (3-100 mg/Kg) of M1 and M5 and diclofenac displayed significant (p 0.0001) edema reduction when compared with the untreated group. Interestingly, all doses of M1-M5 administered through
147
Digitized by UCC, Library
the same (oral) route displayed either comparable or better anti-inflammatory activity as diclofenac. The 5-methoxycanthin-6-one [M1] exhibited a higher anti- inflammatory activity than its unsubstituted analogue [M5] (Table 8). The observed activity of M1 is due to the presence of the methoxy group which makes it less polar/lipophobic or more lipophilic to be able to cross the membranes or the blood brain barriers. While the presence of other minor constituents with a similar pharmacological effect cannot be ruled out, the isolated compounds as major constituents of the root bark of A. aubryanum are likely to play a major role for the reported medicinal uses of the plant. The dose response curves (figure 53), show the highest activity for diclofenac, crude extract, alkaloid extract, 5- methoxycanthin-6-one and canthin-6-one. This is shown by the sigmoid nature of the curves, the more sigmoid the curve, the higher the activity.
Table 6: Effect of M1 and M5 on carrageenan-induced edema
Alkaloid/Drug ED50 mg/Kg ± SEM 5-methoxy-canthin-6-one 60.84 ± 0.01 Canthin-6-one 96.64 ± 0.01 Diclofenac 1.99 ± 0.01 Source: Laboratory data (2015)
148
Digitized by UCC, Library
M1 100 10mg/kg 80 30mg/kg 100mg/kg 60 Control
40
20 Increase in foot volume Increase
% 0 0 2 4 6 8 Time/hrs
Figure 51: Effect of 5-methoxy-canthin-6-one (3-30 mg/kg; i.p) on time course curve (a) and the total oedema response (expressed as AUC, b) in carrageenan - induced paw edema in rats. ***P < 0.0001; *** P < 0.001; ***P < 0.01 compared to vehicle-treated group.
149
Digitized by UCC, Library
M5 100 10kg/mg 80 30kg/mg 100kg/mg 60 Control
40
20 Increase in foot volume Increase
% 0 0 2 4 6 8 Time/hrs
Figure 52: Effect of canthin-6-one (3-30 mg/kg; i.p) on time course curve (a) and the total oedema response (expressed as AUC, b) in carrageenan – induced paw edema in rats. ***P < 0.0001, ***P < 0.001, ***P < 0.01 compared to vehicle-treated group
150
Digitized by UCC, Library
Figure 53: Dose response curves for standard drug, extracts and isolated compounds on carrageenan - induced foot edema in rats.
Antioxidant Activity of Extracts
Antioxidant activity of crude extracts and isolated compounds
The qualitative DPPH test showed the two extracts and the isolated compounds bleaching the purple DPPH radical, thus giving pale spots over a purple background. This indicates that they contain some antioxidant constituents.
The DPPH assay is a valid and simplest assay to evaluate scavenging activity of antioxidant, since the radical compound is stable and does not have to be generated as in other radical scavenging assays. Antioxidants scavenge the DPPH radical by donating a proton. Different authors use different initial radical concentrations and different reaction times. The extract showed a concentration dependent DPPH radical scavenging activity. The decrease in the absorbance of
DPPH was due to phytoconstituents in the plant extracts acting as antioxidants by hydrogen donation.
151
Digitized by UCC, Library
Quantitative antioxidant assay of extracts
Three methods were used to determine quantitatively the antioxidant activity of both the crude and the alkaloid extracts. They are the total phenolic content, total antioxidant capacity and DPPH radical scavenging assays.
Total phenolic content
The total phenolic content of the extracts was determined using the Folin- ciocalteau reagent and vitamin E as standard. The total phenolic content was expressed as mg of vitamin E equivalents (VEE) per g of extract. Table 7 shows the total phenolic contents of the crude methanolic (CE) and alkaloid (AC) extracts. The crude methanolic extract had the highest phenolic content.
Table 7: Total phenolic content of root extracts
Extracts (1.5mg/mL) Mean (mg VEE/g) ± SEM CE 74.53±0.00
AE 59.54±0.00
Source: Laboratory work (2015)
0.8 r2 = 0.9632 0.6
0.4
bsorbance (nm) bsorbance 0.2 A
0.0 0 20 40 60 conc.
Figure 54: Absorbance against concentration of vitamin E used in the calibration curve.
152
Digitized by UCC, Library
Total antioxidant capacity
In the total antioxidant capacity assay, vitamin E was used as standard.
The antioxidant activity was expressed as mg of vitamin E equivalent (VEE) per g of extract. All the extracts showed increase in antioxidant activity with increase in concentration. The total crude extract showed the highest total antioxidant capacity (Table 8).
Table 8: Total antioxidant capacity of root extracts
Extracts (1.5mg/mL) Mean (mg VEE/g) ± SEM CE 95.57±2.31
AE 72.44±0.01
Source: Laboratory data (2015)
The relationship between the antioxidant capacity and total phenolic content analysis was highly significant (r2 = 0.86)
A high total phenolic content value is often correlated with high antioxidant activity, though not all plant extracts exhibit the same pattern due to their different antioxidant mechanisms (Mazlan, et al., 2013) and also Folin-ciocalteau reagent not being specific to just phenolic contents but to any other substances that could also be oxidized by the reagent (Khomsug et al., 2010).
Phenolic compounds are widely distributed in plants and have gained much attention due to their antioxidant activities and free radical scavenging abilities which have beneficial implications for human health (Mazlan, et al., 2013).
The phenolic compounds may contribute directly toward the observed high antioxidant activity through different mechanisms exerted by different phenolic
153
Digitized by UCC, Library
compounds or through synergistic effects with other non phenolic compounds
(Mazlan, et al., 2013).
It has been established that compounds with high antioxidant activities may also contribute toward the inhibition of tyrosinase, cholinesterase (AChE) and nitric oxide (NO) production in cells. Inflammatory conditions may enhance the production of reactive oxygen/nitrogen species (ROS/NOS), which leads to oxidative stress that can damage important organic substrates. Antioxidants can scavenge free radicals and protect organisms from ROS/NOS-induced damage, leading to a reduction in inflammation (Abdillahi et al., 2011; Almeida et al.,
2011). Antioxidants can also prevent major degenerative diseases and aging and might have protective effects toward Alzheimer’s disease (Aremu et al., 2011).
The inhibition of cholinesterase is suggested to be quite useful in the treatment of
Alzheimer’s disease and other diseases including senile dementia, ataxia and
Parkinson’s disease. Alzheimer’s disease is the result of a deficiency in the cholinergic system due to the rapid hydrolysis of acetylcholine. Hence, nerve impulse transmission is terminated at the cholinergic synapses. By suppressing cholinesterase, cholinergic neurotransmission can be restored (Mazlan, et al.,
2013). Tacrine is one of the synthetic drugs used for treating the symptoms of cognitive dysfunction or memory loss associated with Alzheimer’s disease.
However, adverse effects have been reported for these synthetic drugs, including gastrointestinal disturbances and suppression of bioavailability. Oxidative – related processes coupled with tyrosinase activity can also trigger melanogenesis, which causes skin pigmentation (Abdillahi et al., 2011). There are no reports of
154
Digitized by UCC, Library
the cholinesterase inhibition properties of any Anthostema species. However,
Anthostema species are expected to have cholinesterase (AChE) inhibition properties because it has been reported that plants belonging to the Euphorbiaceae family have AChE inhibitory potential (Mazlan, et al., 2013).
Thus, the high levels of antioxidant activity found in the plant extract may also result in a higher inhibition of tyrosinase and cholinesterase activities as well as nitric oxide production. Antioxidant activity of plant extract is not limited to phenolic compounds. Activity may also be due to the presence of other antioxidant secondary metabolites, such as flavonoids, volatile oils, carotenoids and vitamins. Flavonoids are good antioxidants which scavenge and reduce free radical formation (Grassi et al., 2010). The C-glucosylflavonoids (orientin, vitexin and isovitexin) which have been isolated from many medicinal plants such as Ficus thonningii, pigeon pea, linseed oil and in rooibos tea possess antioxidant properties (Dangarembizi, et al., 2013). Orientin possesses free radical scavenging activity based on its ene-diol functionality i.e. its dihydroxy substituents in the B ring and the double bond characteristic of the C ring
(Dangarembizi, et al., 2013). Vitexin and isovitexin also possess antioxidant though to a lesser extent than orientin due to the lack of OH substituent. In addition to flavonoid, stilbenes also exhibit antioxidant activity. Resveratrol and its methylated derivatives, trans-3,3’, 5,5-tetrahydroxy-4-methoxystilbene, possess antioxidative effects against oxidative stress induced by reactive nitrogen species and reactive oxygen species (Dangarembizi, et al., 2013). Resveratrol and its derivatives have also been shown to reduce peroxynitrite which is one of the
155
Digitized by UCC, Library
most potent reactive nitrogen species (Olas et al., 2008). High levels of peroxynitrite are generated in inflammatory based disease conditions
(Dangarembizi, et al., 2013). There is also the possibility of synergistic interactions between flavonoids and stilbenes.
DPPH radical scavenging activity of extracts of A. aubryanum
The results of the free radical scavenging potential of the total and alkaloid extracts of A. aubryanum using DPPH free radical scavenging method are shown in the table below. The reference drug, vitamin E (0.003-0.03 mg/mL) and the extracts (0.5-1.5 mg/mL) exhibited concentration-dependent free radical scavenging activity (table 9). The concentration that provided 50% radical scavenging (IC50) of the crude extract was determined as 8.84±0.02 compared to the vitamin E standard of 8.61±0.00.
The order of decreasing activity (as defined by IC50 in mg/mL) was found to be: vitamin E total crude alkaloid crude. The results indicate that the root bark of A. aubryanum possess potent antioxidant activity.
Table 9: DPPH scavenging activity of extracts of A. aubryanum root bark
Extracts IC50 (μg/mL) ± SEM
Total Crude 8.84 ± 0.01
Alkaloidal crude 23.12 ±0.01
Vitamin E 8.61 ± 0.01
Source: Laboratory data (2015)
Many medicinal plants possessing antioxidant activities have been shown to possess protective effects on the erythrocyte membrane from
156
Digitized by UCC, Library
acetaminophen-induced membrane peroxidation (Ahur et al., 2010). The antihaemolytic and haematinic potential of medicinal plants is possibly due to its antagonistic activity against the depletion of glutathione and hence prevention of inflammation. Thus the present study has shown that the root bark of A. aubryanum possess significant antioxidant properties and may contribute to the retardation of the inflammatory process. This is because inflammatory tissue injuries are mediated by reactive oxygen metabolites from phagocytic leukocytes (e.g. neutrophils, monocytes, macrophages and eosinophils) that invade the tissues and cause injury to essential cellular components (Amponsah,
2012).
Antioxidant Activity of Isolated Compounds
Quantitative DPPH radical scavenging test
5-methoxycanthin-6-one [1] canthin-6-one [2] showed various degrees of antioxidant properties, with 5-methoxycanthin-6-one being the most active
(Table 10). Vitamin E (VE) was used as the standard antioxidant drug. The order of decreasing activity as indicated by the IC50 is VE > 5-methoxycanthin-
6-one > canthin-6-one. From the concentration response curves for the standard drug, extracts and isolated compounds (figure 53), the more sigmoid the curve, the higher the activity. The standard drug and the crude extract show the highest activity followed by the alkaloid extract, 5-methoxycanthin-6-one and canthin-
6-one. These results are further confirmed by the percent inhibition curve
(figure 56) and the DPPH absorption spectra (figure 57).
157
Digitized by UCC, Library
Table 10: DPPH scavenging activity of M1 and M5
Compounds IC50 µg/mL ± SEM
5-methoxy-canthin-6-one 27.62 ± 0.01
Canthin-6-one 33.60 ± 0.01
Vitamin E 8.61 ± 0.01
Source: Laboratory data (2015)
Figure 55: Concentration response curves for standard drug, extracts and isolated compounds.
158
Digitized by UCC, Library
120 M1 CA M5 CRUDE VIT E
100
80
60 % inhibition % 40
20
0 0 100 200 300 400 500 600 Concentration(µg/ml) Figure 56: Plot of percent inhibition against concentration of extracts and isolated compounds.
Figure 57: DPPH absorption spectra of extracts and isolated compounds.
Compounds that have scavenging activities toward free radicals have been found to be beneficial in inflammatory diseases. The antioxidant activity
159
Digitized by UCC, Library
of the root bark reported in this study support its traditional use for wound healing. This is because in acute and chronic wounds, oxidants cause cell damage and thus inhibits wound healing (Thang et al., 2001). The administration of antioxidants or free radical scavengers is reportedly helpful, notably to limit the delayed sequel of thermal trauma and to enhance the healing process (Thang et al., 2001).
160
Digitized by UCC, Library
CHAPTER FIVE
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
INTRODUCTION
The present study was aimed at investigating the root bark of A. aubryanum (Baill., family, Euphorbiaceae) for phytochemical constituents and pharmacological activity using the acute carrageenan-induced foot edema model in six weeks old rats and to isolate the compounds which may be responsible for this activity. Also since free radicals and reactive oxygen species are implicated in inflammatory diseases, the antioxidant potential of extracts and isolated compounds were investigated in in vitro experimental models.
Summary
In African folk medicine, the stem and root bark of Anthostema aubryanum (Baill) are used as an effective remedy against several inflammatory ailments including rheumatism and renal inflammations.
The preliminary phytochemical analyses have revealed that methanolic extract of Anthostema aubryanum is characterized by the presence of alkaloids, steroids, flavonoids, coumarins, fatty acids, reducing sugars, cyanogenic glycosides, tannins, anthraquinones and saponins. Carotenoids and glucosides were not detected. These classes of compounds are known to have established biochemical activities and multiple pharmacological effects and hence the use of this plant in ethnomedicine may be rationalized by the presence of these compounds in this plant.
161
Digitized by UCC, Library
Oral administration of both the total crude and crude alkaloid extracts of
A. aubryanum resulted in suppression of the carrageenan-induced inflammation in a dose- and time-dependent manner and thus, presumably, inhibited the synthesis and release of prostaglandins as well as kinins responsible for the inflammation. A low ED50, indicating high anti-inflammatory activity, was recorded for the total methanol root bark extract (ED50 5.294 ± 0.02 mg/kg
BDW). The crude alkaloid extract of the root bark also exhibited dose- dependent reduction in foot volume but with comparatively lower activities than the total methanol extract (ED50 = 13.84 ± 0.01 mg/kg BDW) due to synergism.
The antioxidant activity of Anthostema aubryanum (Baill) was evaluated by the DPPH assay. The concentration that provided 50% radical scavenging
(IC50) was determined as 8.84±0.01 which was equivalent to the vitamin E standard of 8.61±0.0. Two alkaloids, 5-methoxy-canthin-6-one [1] and canthin-
6-one [2] were isolated from the root bark. The time course study clearly shows that all the two major compounds isolated from A. aubryanum displayed anti- inflammatory activity in a dose dependent manner with ED50 values of 60.84 ±
0.01 and 96.64 ± 0.01mg/kg body weight respectively.
All the isolated compounds showed concentration-dependent DPPH scavenging effect with respective IC50 values of 27.62 ± 0.01 and 33.60 ±
0.01µg/mL. The anti-inflammatory and antioxidant activities of the compounds were much lower than those of their respective extract from which they were isolated due to synergism with other secondary metabolites.
162
Digitized by UCC, Library
Conclusions
The pharmacodynamic basis supporting the use of A. aubryanum extracts in ethnomedicinal systems has been established and pharmacological studies have demonstrated the anti-inflammatory and antioxidant effects of the plant extracts and isolated compounds. The remarkable therapeutic effects exhibited by A. aubryanum are a result of array of phytochemicals presents in the plant. The antioxidant potency of the crude extract was found to be equal to that of vitamin E.
Comprehensive chromatographic analysis coupled with spectroscopic study on the root bark have resulted in the identification of two major alkaloids
[1-2] that displayed anti-inflammatory and antioxidant activities comparable with the positive controls diclofenac and vitamin E respectively. The 5- methoxycanthin-6-one alkaloid, however, exhibited higher activities than the canthin-6-one alkaloid due to the presence of the methoxy group which makes it less polar/lipophobic or more lipophilic and is able to cross the membranes or the blood brain barrier to elicit the observed pharmacological activity. Although the synergistic effects of other minor constituents with similar pharmacological effects are possible, canthin-6-one and 5-methoxycanthin-6-one as major constituents of the root bark of A. aubryanum are likely to play major role in the reported ethnomedicinal uses of the plant. The canthin-6-one alkaloid [2] has been isolated from Simaroubaceae and Rutaceae (Koike and Ohmoto, 1985;
Cebrian-Torrejon et al., 2011).
163
Digitized by UCC, Library
The canthin-6-one alkaloid has been shown to inhibit cytotoxic activities against a panel of human cancer cell types including breast, colon, fibrosarcoma, lung, melanoma, KB, KB-V1 and murine lymphocytic leukaemia P-388 (Cao, et al.,
2007). Moreover, 1-methoxy-canthin-6-one inhibited the growth of a panel of human tumor cell lines, including epiderimoid carcinoma of the nasopharynx
(KB), lung carcinoma (A-549), ileocecal carcinoma (HCT-8), renal cancer (CAK-
1), breast cancer (MCF-7) and melanoma (SK-MEL-2), with IC50 value in the range of 2.5-20 μg/mL. Also, canthin-6-one and 1-methoxycanthin-6-one exhibited aspirin, indomethacin, phenylbutazone and reserpine induced gastric and duodenal antiulcer (10 mg/Kg) activity in rats’ model.
Canthin-6-one exhibited a broad spectrum of antifungal activity against
Aspergillus fumigatus, A. niger, A. terreus, Candida albicans, C. tropicalis, C. glabrata, Cryptococcus neoformans, Geotrichum candidum, Saccharomyces cerevisiae, Trichosporon beigelii, T. cutaneum and T. mentagrophytes var. interdigitale with minimum inhibitory concentration values between 5.30 and
46μmol/L (Thouvenel et al., 2003).
Canthin-6-one also possesses a broad spectrum of leishmanicidal activity.
Canthin-6-one exhibited a strong trypanocidal activity in vivo in the mouse model of acute and chronic infection and due to its very low toxicity, it is possible that long-term oral treatment with this natural product could prove advantageous compared to the current chemotherapy of Chagas disease.
It has also been reported that canthin-6-one exhibited antiplasmodial activity with
IC50 on chloroquine/mefloquine resistant and sensitive strains of Plasmodium
164
Digitized by UCC, Library
farciparum of 2.0-5.3 and 5.1-10.4μg/mL respectively (Cebrian-Torrejon et al.,
2011).
The remarkable anti-inflammatory activity of the isolated alkaloids supports the assertion that alkaloids in asserted skeletal type based on pyridine ring system possess striking anti-inflammatory activity (Agnihotri, et al., 2010).
The multiple pharmacological effects of these β-carboline alkaloids go to prove that individual compounds might selectively interact with specific targets so as to lead to a variety of pharmacological actions in vitro and in vivo. Thus various substituents at different positions of β-carboline ring system might play a crucial role in determining their multiple pharmacological functions (Cao et al., 2007).
Therefore, the β-carboline alkaloids might be a particularly promising lead compounds for discovering and developing novel clinical drugs.
In view of the present findings and above mentioned numerous pharmacological and biochemical activities of these compounds, the ethnomedicinal uses of the
Anthostema aubryanum for inflammatory conditions, wound healing, pain suppression and as antimicrobial agent appears to be justified. To the best of our knowledge, this is the first report on the isolation of this group of alkaloids from
Anthostema aubryanum (Baill) and the family Euphorbiaceae as well as their pharmacological activity.
Recommendations
From the research results obtained, it is recommended that further elucidation of the molecular mechanisms underlying the activity of these chemicals is also critical to evaluate the possibility of using the extracts for future
165
Digitized by UCC, Library
drug development. Research could also target the effect of A. aubryanum on the nervous system, the endocrine system as well as its interaction with the immune system in fighting diseases.
The investigations of the anti-inflammatory activity of the canthinone alkaloids should be continued to determine the in vivo activities and to evaluate their toxicity.
Also structural modifications of both alkaloids, to obtain a more potent anti- inflammatory and antioxidant compounds, should be considered in future collaborative research.
Toxicity studies of the root bark extract and on newly isolated alkaloids should be considered in future work. This is due to the fact that certain β-carboline alkaloids are very dangerous. For instance, harman and norharman are comutagens or precursors of mutagens; 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (Taclo) and its analogue Tabro, and N-methylated β-carboline derivatives are potent endogenous neurotoxins; and N-nitroso derivatives of β-carboline and aminophenylnorharman (APNH) derivatives are also endogenous mutagens and carcinogens (Cao, et al. 2007). Interestingly, humans are continuously exposed to endogenous and exogenous β-carboline alkaloids. There is therefore the need to study their biological and pharmacological activities to reduce their potential risk and to develop new drugs. Moreover, further studies in vivo with respect to possible actions on human health are urgently required.
Considering the pharmacological activities shown in the present study, the root bark should be investigated for wound healing activity in future research. A
166
Digitized by UCC, Library
topical formulation could be made for both deep and superficial wounds after the toxicity profile of the extract has been established.
Suggestions for Further Research
Beta-carboline alkaloids are of great interest due to their diverse biological activities. Particularly, these compounds have been shown to intercalate into
DNA, to inhibit CDK, topisomerase and monoamine oxidase, and to interact with benzodiazepine receptors and 5-hydroxy serotonin receptors. Therefore, further research should consider the biochemical activities of the isolated alkaloids.
Also, further research should consider the biochemical and pharmacological activities of flavonoids and terpenoids present in the plant and to isolate and characterize the compounds responsible for these activities. These phytoconstituents have potent biochemical and pharmacological activities.
Last but not the least, combine therapy has been used for centuries in Africa ethnomedicine, therefore, work on the biochemical and pharmacological activities of the crude extract should be consider in further research and standardize it to augment or replace the currently available therapeutics which have several adverse effects.
167
Digitized by UCC, Library REFERENCES
Abbiw, T. (1990). Study of Tropical Shrubs and Plants. Journal of Biogeography,
591-602.
Abdillahi, H.S., Finnie, J.F., & Van Staden, J. (2011). Anti-inflammatory, antioxidant,
anti-tyrosinase and phenolic contents of four Podocarpus species used in
traditional medicine in South Africa. Journal of Ethnopharmacology, 136 (3):
496-503.
Abe, I., Takahashi, Y., Morita, H., & Noguchi, H. (2001). Benzalacetone synthase: A
novel polyketide synthase that plays a crucial role in the biosynthesis of
phenylbutanones in Rheum palmatum. Eur. J. Biochem. 268:335-9.
Abdel-Hameed, E.S. (2009). Total phenolic contact and free radical scavenging
activity of certain Egyptian Ficus species leaf samples. Food chemistry,
144:1271-1277.
Abreu, P.M., Martins, E.S., Kayser, O., Bindseil, K.U., & Siens, K. (1999).
Antimicrobial, antitumor and antileishmania screening of medicinal plants
from Guinea-Bissau. Phytomedicine, 6: 187-192.
Abou-Donia, A.H., Amer, M.E, Darwish, F.A., Kassem, F.F., Hammoda, H.M.,
Abdel-Kader, M.S., Zhou, B.N., & Kingston, D.G.I (2002). Two new
alkaloids of the crinane series from Pancratium sickenbergi. Planta Medica,
68:379-381.
Agnihotri, S, Wakode, S. and Agnihotri, A. (2010). An overview of an anti-
inflammatory properties and chemo-profiles of plants used in traditional
medicine. Indian Journal of Natural Products and Resources, 3: 150-167.
Ahur, V.M., Madubunyi, I., Adinkola, A.Y. and Udem, S.C. (2010): The effect of ethyl
acetate extract of Ficus thonningii (Blume) leaves on erythrocyte osmotic
168
Digitized by UCC, Library fragility and haematological parameters in acetaminophen-treated rats.
Com.Clin. Pathol, 10: 1107-1112.
Al-Douri, N. A. (2000). A Survey of medicinal plants and their traditional uses in
Iraq. Pharmaceutical Biology, 38:74-79.
Almeida, I.M.C., Barreira, J.C.M., Oliveira, B.P.P., & Ferreira, I.C. (2011). Dietary
antioxidant supplements: benefits of their combined use. Food and Chemical
Toxicology, 49 (120): 3232-3237.
Amaral, A. C. F., & Barnes. R. A (1997). Alkaloids of Croton celtidifolius. Planta
Med. 63: 485-490.
Amponsah, I.K. (2012). Chemical constituents, anti-inflammatory, antioxidant and
Antimicrobial activities of the stem bark and leaves of Ficus exasperate
(Vahl). (Kwame Nkrumah University of Science and Technology, Kumasi).
Retrieved from https://ir.knust.edu.gh/xmlui/handle/12345678914002.pdf.
Aniszewski, T. (1994). The biological basis of quinolizidine alkaloids. Sciences of
Legumes, 1:1-24.
Aniszewski, T. (1995). Grain legumes, evaluation and genetic resource (2nd ed.).
Cambridge University Press.
Aniszewski, T. (2005). Elements of Applied Botany. Socrates / Erasmus Programme.
Warsaw University, [In Polish]. Joensuu; Joensuu University Press.
Aniszewski, T. (2007). Alkaloids-Secrets of life, alkaloid chemistry, biological
significance, applications and ecological roles (1st ed.). Amsterdam, Elsevier.
Armah, F.A., Annan, K., Mensah, A.Y., Amponsah, I.K., Tocher, D.A., &
Habtemariam, S. (2015). Erythroivorensin: A novel anti-inflammatory
diterpene from the root-bark of Erythrophleum ivorense (A Chev.).
Fitoterapia, 105: 37-42.
169
Digitized by UCC, Library Araujo-Junior, V. T., da Silva, M. S., da Cunha, E. V. L., Agra, M. D., da Silva, R.
N., Barbosa, J. M., & Braz-Filho, R. (2004). Alkaloids and diterpenes from
Croton moritibensis. Pharm. Biol. 42: 62-67.
Aremu, A.O., Amoo, S.O., Ndhlala, A.R., Finnie, J.F., & Van Staden, J. (2011).
Antioxidant activity, acetylcholinesterase inhibition, iridoid content and
mutagenic evaluation of Leucosidea sericea. Food and Chemical Toxicology,
49 (5): 1122-1128.
Attioua, B. K., Harisolo, R., Boti, J. B., Adiko, V.A. Tonziba, F. Z., & Djakoure L. A.
(2012). Isolation and identification of alkaloids from Croton lobatus. Internal
Journal of Pharmaceutical Sciences Review and Research, 13(2): 1-4.
Athikomkulchai, S., Prawat, H., Thasana, N., Ruangrungsi, N., & Ruchirawat,
S.(2006).COX-1.COX-2 inhibitors and antifungal agents from Croton
hutchinsonianus. Chem. Pharm. Bull. 54: 262-4.
Badre, A., Boulanger, A., Abou-Mansour, E., Banaigs, B., Combaut, G. &
Francisco, C. (1994). Eudistomin U and Isoeudistomin U, New
Alkaloids from the Caribbean Ascidian Lissoclinum fragile. J. Nat.
Prod. 57: 528-533.
Bandara, B. M. R.; Wimalasiri, W. R. & Bandara, K. A. N. P. (1987). Isolation
and insecticidal activity of (-)-hardwickiic acid from Croton aromaticus.
Plants Med. 6: 575-578.
Bannister, J., Banister, W., & Rotilio, G. (1981). Aspects of the structure, function and
application of superoxide dismutase. CRC critical Reviews in Biochemistry
and Molecular biology, 22 (2): 111-180.
Bastida, J., Salles, M., Codina, C., Viladomat, F., & Luz, J.L. (1996). Alkaloids from
Behriatenuiflora Greene. Planta Medica, 64:679-680.
170
Digitized by UCC, Library Barbosa, P. R., Fascio, M., Martins, D., & Roque, N. F. (2004). Benzoyl-
methylpolyols from Croton species (Euphorbiaceae). Arkivoc, 6: 95-
102.
Barnes, R. A. & Soeiro, O. M, (1981). The alkaloids of Croton salutaris.
Phytochemistry, 20: 543-44.
Barku, V.Y.A (2015). Antioxidant and wound healing properties of some
selected plants from Kpando traditional area. Isolation of flavonoids
from Anogeissus leicarpus (DC) and Perr (Combretaceae). (University
of Cape Coast). Retrieved from
https://erl.ucc.edu.gh/jspui/bitstream/123456789/2594/BARKU%202015.pdf.
Berkada, B (1978). Preliminary report on warfarin for the treatment of herpes simplex.
J. Irish Coll. Phys. Surg. 22: 56-62.
Bhat, S. V., Nagasampagi, B. A., & Sivakumar, M. (2007). Chemistry of Natural
Products (3rd ed.). New Delhi: Narosa publishing house.
Blundell, M. (1987). Collins guide to the wild flowers of East Africa. London: Collins
Publishers.
Bobbit, J. (1964). Thin Layer Chromatography. Reinhold, New York.
Boursereau, Y., & Coldham, I (2004). Synthesis and biological studies of 1-
amino betacarbolines, Bioorg. Med. Chem. 14: 5841-4.
Bracca, A., Bader, A., Siciliano, T., Morelli, I., & Tommasi, N. D. (2003). New
pyrrolizidine alkaloids and glycosides from Anchusa strigosa. Planta Medica,
69: 835-841.
Brandt, S.D. Mansell, D., Freeman, S., Fleet, I.A., & Alders, J.F. (2006). Analytical
characterization of the routes by thermolytic decarboxylation from tryptophan
171
Digitized by UCC, Library to tryptamine using ketone catalysts, resulting in tetrahydro-β-carboline
formation. Journal of Pharmaceutical and Biomedical Analysis, 41: 872-882
Bringmann, G., Feineis, D., Friedrich, H., & Hille, A. (1991). Endogenous alkaloids
in man-synthesis analytics, in vivo identification, and medicinal importance.
Planta Medica, 57: 573-584.
Brossi, A. (1991). Mammalian-alkaloids: Conversion of tetrahydroisoquinoline-
carboxylic acids derived from Dopamine. Planta Medica, 57: 593-5100.
Bruno, E. (2012). Research in Clinical Phytopharmacology to Develop Health Care in
Developing Countries: State of the art and perspectives. Phytopharmacology,
12: 1-57.
Buckholtz N. S (1980). Neurobiology of tetrahydro-beta-carbolines. Life Sci.,
27: 893-897.
Camacho, M. R. (2000). Oxoaporphine alkaloids and quinones from Stephania dinklage
and evaluation of their antiprotozoal activities. Planta Medica, 66: 478-486.
Cao, R., Peng, W., Wang, Z., & Xu, A. (2007). β-Carboline Alkaloids: Biochemical and
Pharmacological Functions, Current Medicinal Chemistry, 14: 479-500.
Catalan, C. A. N.; Heluani, C. S.; Kotowicz, C; Gedris, T, E. & Herz, V. (2003).
A linear sesquiterpene, two squalene derivatives and two peptide
derivatives from Croton hieronymi. Phytochemistry, 64: 625-9.
Cebrian-Torrejon, G. Spellman, K., Karine, L., & Erwan, P. (2011). The
antiplasmodium effects of a traditional South American remedy: Zanthoxylum
Chiloperone Var. angustifolium against Chloroquine resistant and chloroquine
sensitive strains of Plasmodium farciparum, Revista Brasileira De
Farmacognosia, 21 (4): 652-61.
172
Digitized by UCC, Library Cebrian–Torrejon, G. Kablan, L., Ferreira, M.E., Dela Cruz, D.K., Domenech, A, and
De Bilbao, N.V. (2015). Harvesting Canthinones: Identification of the optimal
seasonal point of harvest of Zanthoxylum chiloperone leaves as a source of 5-
methoxycanthin-6-one, Natural Product Research, 29 (21): 1-5.
Changa, C., Wena, Z., Wangc, S. and Duha C. (2008). New anti-inflammatory steroids
from clavularia viridis. Formosan Soft Coral, Steroids, 73: 562-567.
Charris, J., Dominguez, J., De La Rosa, C., & Caro, C. (2000). (-)-Amuronine from the
leaves of Croton havens L. (Euphorbiaceae), Biochem. Syst. Ecol. 28: 795-98,
Chatpaliwar, V.A. Johrapukar, A.A., Wanjari M.M., Chakraborty, R.R., & Kharkar,
V.T. (2002). Anti-inflammatory activity of Martynia diandra glox. Indian
Drugs, 39: 543-545.
Chen, J.J., Chang, Y.L., Teng, C.M., & Chen, I.S. (2000). A New Aristolactam
Alkaloid and Anti-Platelet Aggregation Constituents from Piper taiwanense.
Planta Med, 66: 251-256.
Chen, K. S., Chang, Y. L., Teng, C. M., Chen, C. F., & Wu, Y. C. (2000).
Furoquinolines with antiplatelet aggregation activity from leaves of Melicope
confuse. Planta Medica, 66: 80-81.
Chen, Y. W., Li, D. G., Wu, J. X., Chen, Y. W., & Lu, H. M. (2005). Tetrandrine
inhibits activation of rat hepatic stellate cells stimulated by transforming
growth factor- beta in vitro via up-regulation of Smad 7. Journal of
Ethnopharmacology, 100: 299-305.
Cheng, D., & Roder, E (1986). Pyrrolizidine alkaloids of Emelia sonchifolia. Planta
Medica, 24: 484-486.
173
Digitized by UCC, Library Chi, Y., Jong, H., Sonk, Charg, H., Kang, S. & Kim H. (2001). Effects of naturally
occurring prenylated flavonoids on enzymes metabolizing arachidonic acid:
cyclooxygenases and lipooxygenases. Biochemical Pharmal, 62: 1185-1191.
Clark, V.C., Raxworthy, C.S., Rakotomalala, V., Sierwald, P., & Fisher, B.L. (2005).
Convergent evolution of chemical defence in poison frogs and arthropod prey
between Madagascar and the Neotropics. Proceedings from the National
Academy of Sciences of the United States of America, 102: 11617-11622.
Contreras-Guzman, E.S., & Strong, F.C. (1982). Determination of tocopherols (vitamin
E) by reduction of cupric ion. Journal of the Association of Official Analytical
Chemists, 65: 1215-1222.
Costa, T.O., Morales, K. A., Brito, J.P., Gordo, M., Pinto A.C., & Bloch, C. Jr. (2005).
Occurrence of bufetonin in the Osteocephalus genus (Anura: Hylidae).
Toxicon, 46: 371-375.
Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical Microbiology
reviews, 52: 564-582.
Critchfield, J. W., Butera, S. T., & Folks, T.M. (1996). Inhibition of HIV activation in
latently infected cells by flavonoid compounds. AIDS Res. Hum. Retroviruses,
12: 39-42.
Daly, J.W., Spande, T.F., & Garrafo, H.M. (2005). Alkaloids from Amphibian skin: A
tabulation of over eight-hundred compounds. Journal of Natural Products,
68:1556-1575.
Dalsgaard, P.W. Blunt, J.W., Munro, M.H.G., Frisvald, J.C., & Christophersen, C.
(2005). Communesin G and H, new alkaloids from the Psychotolerant fungus
Penicillium rivulum. Journal of Natural Products, 68: 258-261.
174
Digitized by UCC, Library Dangarembizi, R., Kennedy, H., Erlwanger, Moyo, D., & Chivandi, E. (2013).
Phytochemistry, pharmacology and ethnomedicinal uses of Ficus thonningii
(Blume Moraceae): A review. Afr. J. Tradit. Complementary. Altern. Med. 12:
203-212.
Davis, R.H. (1991). Cyanogens. In D’Mello, J.P.F., Duffus, C.M., & Duffus, J.H. (Eds),
toxic substances in crop plants (pp. 152-171). Cambridge, England: The Royal
Society of Chemistry Publications.
De Heluani, C. S., Catalan, C. A., N., Hernandez, L. R., Burgueno-Tapia, E., &
Joseph-Nathan, P (2000). Three new diterpenoids based on the novel
sarcopetalane skeleton from Croton sarcopetalus. J. Nat. Prod. 63: 222-
225.
Deodhar, S.D., Sethi, R. & Srimal, R.C. (1980). Preliminary Studies on anti-rheumatic
activity of curcumin. Indian J. Med. Res. 71: 632-634.
Dewick, P. M. (2002). Medicinal Natural Products. A biosynthetic approach. (2nd ed.).
Chickester-New York: John Wiley and Sons Ltd.
Duka, T. (1991). In New Concepts in Anxiety. In Briley, M., & File, S. (Eds),
controlling anxiety (pp. 440-457). CRC Press: Boca Raton, FL.
Dupont, C., Couillerot, E., Gillet, R., Caron, C., Zeches-Hanrot, M., Rion, R. F.,
&Trentesanx, C. (2005). The Benzophenanthridine alkaloid fagaronine,
induces erythroleukemic cell differentiation by gene activation. Planta
Medica, 71: 489-494.
Eisenreich, W. J., Hofner, G., & Bracher, F. (2003). Alkaloids from Croton
flavens L and their affinities to GABAA-Receptors. Nat. Prod. Res. 17: 437-
441.
175
Digitized by UCC, Library El Sayed, K.A., Kelly, M., Kara, U. A. K., Ang, K. K. H., Katsuyama, 1., Dunbar,
D. C., Khan, A. A., & Hamann, M. T. (2001). New manzamine alkaloids
with potent activity against infectious diseases. J. Am. Chem. Soc, 123:
1804-1808.
Endress, M. E., Sennblad, B., Nilsson, S., Civeyrel, L., Chase M. W., Huysmans, S.,
Grafsrom, E., & Bremer, B. (1996). A phylogenetic analysis of Apocynaceae
Str. and some related taxa in Gentianales: A multidisciplinary approach.
Opera Botanical Belgica, 7: 59-102.
Erdemgil, F. Z., Telezhenetskaya, M. V., Baser, K. H. C., & Kirimer, N. (2000).
Alkaloids of Thalictrum orientale growing in Turkey. Chemistry of Natural
Compounds, 3(2): 223-224.
Evans, A. T., & Croft, S. L (1987). Antileishmanial activity of harmaline and other
tryptamine derivatives. Phytother. Res., 1: 25-27.
Evidente, A., Cicala, M.R., Randazzo, G., Riccio, R., Calabrese, G., Liso, R., &Arrigori,
O. (1983). Lycorine structure-activity relationships. Phytochemistry, 22: 2193-
2196.
Farsam, H., Yassa, N., Sarkhail, P. & Shafiee, A. (2000). New Pyrrolizidine Alkaloids
from Heliotropium crassifolium. Planta Med., 66: 389-394.
Fereidoni, M., Ahmadiani, A. & Samnanian, S. (2000). An accurate and simple method
for measurement of paw oedema. Journal of Pharmacology and Toxicological
Methods, 43:11-14.
Frederlich, M., De Pauw, M.C., & Llabres, G. (2004). New antimalarial and cytotoxic
sungucine derivatives from Strychnos icaja roots. Planta Medica, 66: 262-269.
Forgo, P., & Hohmann, J. (2005). Leucoverine and acetylcoverine alkaloids from
Leucojum verunum. Journal of Natural Product, 68: 1588-1591.
176
Digitized by UCC, Library Gallimore, W.A., Kelly, M., & Scheuer, P.J. (2005). Alkaloids from the Sponge
Monanchora unguifera. Journal of Natural products, 68: 139-1399.
Geissman, T.A. (1963). Flavonoid compounds, tannins, lignins and related compounds.
In M. Florkin and E. H Stotz (Ed.), Pyrrole pigments, isoprenoid compounds
and phenolic plant constituents, volume 9 (pp. 348-359). Elsevier, New York,
N. Y.
Giang, P. M.; Son, P. T; Hamada, Y., & Otsuka, H (2005). Cytotoxic diterpenoids
from Vietnamese medicinal plant Croton tonkinensis Gagnep. Chem.
Pharm. Bull. 53: 296-300.
Gonzalez-Vazquez, R., Diaz, B. K., Aguilar, M. 1., Diego, N., & Lotina-
Hennsen, B. (2006). Pachypodol from Croton ciliatoglanduliferus Ort as
water-splitting enzyme inhibitor on thylakoids, J Agric, Food Chem. 54:
1217-21.
Goren, A. C., & Yue, S. M. (2005). Hasubanan type alkaloids from Stephania longa.
Journal of Natural Products, 68: 1201-1207.
Govaerts, R., Frodin, D.G. & Radcliff-Smith, A. (2000). World checklist and
bibliography of Euphorbiaceae (and Pandaceae). Kew Publishing, Royal
Botanic Gardens, Kew.
Grassi, D., Desideri, D., & Ferri, C. (2010). Flavonoids antioxidants against
atherosclerosis. Nutrients, 2: 889-902.
Guerrero, M. F., Puebla, P., Carron, R., Martin, M. L., & Roman, L. S. (2002).
Assessment of the antihypertensive and vasodilator effects of ethanolic
extracts of some Colombian medicinal plants. J. Pharm. Pharmacol.54:
1373-1378.
177
Digitized by UCC, Library Guo, Z.B., & Fu J. G. (2005). Progress of cardiovascular Pharmacologic study on
berbamine. [Abstract in English]. Chinese journal of Integrated Traditional
and Western Medicine, 25:765-768.
Hamburger, M., & Hostettman, K. (1991). Bioactivity in plants, the link between
phytochemistry and medicine. Phytochemistry, 30 (12): 3864-3874.
Hampel, C. A., & Hawley, G. G. (1976). Glossary of Chemical terms. New York:
Nostrand Reinhold Publishing Company.
Hansson, G.K. (2005). Inflammation, atherosclerosis and coronary artery disease. New
England Journal of Medicine, 23: 1685-1695.
Hao, H., & Qicheng, F. (1986). Chemical study on alkaloids from Corgalis bulleyana.
Planta Medica, 12: 193-198.
Harborne J.B. (2008). Phytochemical Methods: A guide to modern techniques of plants
analysis. Chapman and Hall, London.
Haslam, E. (1996). National polyphenols (vegetable tannins) as drugs possible modes of
action. J. Nat. Prod. 59: 205-215.
Hawthorne, W. & Jongkind, C. (2006). Forest: A guide to the forest trees, shrubs and
lianes from Senegal to Ghana. Kew Publishing, Royal Botanic Gardens, Kew.
He, J., Lion, U., Sattler, I., Gollmick, F.A., Grabley, S, Cai, J., Meiners, M., Schunke,
H., Schaumann, K., Deschert Y., & Krohn, M. (2005). Diastereomeric
quinolinone alkaloids from the marine-derived fungus Penicillium janczewskii.
Journal of Natural products, 68:1397-1399.
Hegnauer, K. (1989). Chemotaxonomie der pflanzerr (8th ed.). Basel, Birkhauser-Verlag
Publishers.
Hernandez. J., & Delgado, G. (1992). Terpenoids from aerial parts of Croton draco.
Fitoterapia. 63: 377-378.
178
Digitized by UCC, Library Hidalgo, J., Balon, M., Carmona, C., Munoz, M., Pappalardo, Rafael R., Marcos, E.,
& Sanchez, J. (1990). AM1 Study of a (β-carboline set). Part II: pyrrole-N
deprotonated species. Phytochemistry, 35: 1025-1032.
Hoelzel S. C. S. M., Vieira, E. R., Giacomelli, S. R. Dalcol M, Zanatta, N., & Morel,
A. F. (2005). An unusual quinolinone alkaloid from Waltheria douradinha.
Phytochemistry, 66: 1163-1167.
Hostettman, K. (2005). Methylpyrole tropane alkaloids from the bark of Erythroxylum
vacciniifolium. Journal of National Products, 68: 1153-1158.
Houghton, P.J. (1995). The role of plants in traditional medicine and current therapy.
Journal of Alternative and Complementary Medicine, 1:131-143.
Hoult, J. R, S., & Paya, M. (1996). Pharmacological biochemical actions of simple
coumarins, natural products with therapeutic potential. Gen. Pharmacology,
27: 713-722.
Ichiba, I., Corgiat, J. M., Scheuer, P. J., & Borges, M. K (1994). 8-Hydroxymanzamine
A, a beta-carbolines alkaloid from a sponge, Pachypellina sp. Journal of
Natural Products, 57: 168-170.
Ishida, J., Wang, H.K., Bastow, K.F. Hu C.Q., & Lee K. H. (1999). Antitumor agents
201.Cytotoxicity of harmine and ß-carboline analogues. Bioorg. Med. Chem.
Lett. 9: 3319-24.
Ishida, J., Wang, H. K., Oyama, M., Cosentino, M. L., Hu, C. Q., & Lee, K. H.
(2001). Anti-AIDS agents, 46. Anti-HIV activity of harman, an anti-HIV
principle from Symplocos setchuensis, and its derivatives. J. Nat. Prod., 64:
958-962.
Issekutz, C.A., & Issekutz, T.B. (1989). Quantization of blood cell accumulation and
vascular responses in inflammatory reactions. J. Nat. Prod, 6 (4): 235-238.
179
Digitized by UCC, Library Issekutz, A.C. (1981). Vascular responses during neutrophic acute inflammation, their
relationship to in vivo neutrophil emigration. Laboratory investigation, 45 (5):
435-441.
Itokawa, H., Ichihara, Y., Mochizuki, M., Enomori, T., Morita, H., Shirota, 0.,
Inamatsu, M., & Takeya, K. (1991). A cytotoxic substance from Sangre de
Grado. Chem. Pharm. Bull. 39: 1041-2.
Jakubke, H. D. Jeschkeit, H., & Eagleson, M. (1994). Concise Encyclopaedia
Chemistry. Berlin, New York, Walter de Gruyter.
Joubert, E., & Ferreira, D. (1996). Antioxidants of rooibos tea, a possible exploration
for its health promoting properties. The South African Journal of Food Science
and Nutrition, 8: 79-83.
Judd, W. S., Campbell, C. S., Kellogg, E. A., & Stevens, P. C. (1999). Plant
Systematics. A phylogenetic approach. Flora Europaea, volumes, 1-5.
Cambridge University press.
Jussieu, A. H. L. (1824). De Euphorbiaceanum Generibus Medicisque earumdem
Viribus tentamen. Tabulis aeneis 18 illustratum 56.
Keating, G. J., & O’ Kennedy, R. (1997). The Chemistry and Occurrence of Coumarins.
In R. O’ Kennedy & R. D. Thornes (Ed.), Coumarins: biology, applications
and mode of action. (pp. 348-355) John Wiley and Sons. Inc. New York.
Keawpradub, N., Eno-Amooquaye, E., Burke, P. S., & Houghton, P. S. (1999).
Cytotoxic activity of indole alkaloid from Alstonia macrophylla. Planta
Medica, 65: 311-315.
Khomsug, P., Thongjaroenbuangam, W., Pakdeenarong, N., Suttajit, M., &
Chantiratikul, P. (2010). Antioxidative activities and phenolic content of
180
Digitized by UCC, Library extracts from Okra (Abelmoschus esculentus L.). Research Journal of
Biological Sciences, 5 (4): 310-313.
Kobayashi, J., Harbour, G .C., Gilmore J., & Reinhart, K.L., Jr (1984). Eudistomins A,
D, G, H, I, J, M, N, 0, P, and Q, bromo, hydroxy, pyrrolyl and iminoazepino
β-carbolines from the antiviral Caribbean tunicate Eudistoma olivaceum. J.
Am. Chem. Soc. 106: 1526-28.
Kobayashi, J., Nakamura, H., Ohizumi, Y., & Hirata, Y. (1986). A novel
antagonist of serotonergic receptors, hymenidin, isolated from the Okinawan
marine sponge Hymeniacidon sp. Tetrahedron Lett. 27: 1191-7.
Kobayashi, J., Chong. J., Ohta. T., Nozoe. S., Ohizumi, Y., & Sasaki, T. (1990).
Eudistomidins B, C, and D: Novel antileukemic alkaloids from the
Okinawan marine tunicate Eudistoma glaucus. J. Org. Chem.55: 3666-
3670.
Kobayashi, J., Tsuda, M., & Rawasaki, N. (1994). 6-Hydroxymanzamine A and
3,4-dihydromanzamine A, new alkaloids from the Okinawan marine sponge
Amphimedon Sp. J. Nat. Prod., 57: 1737-1745.
Kobayashi, M., Chen, Y.J., Aoki, S., In, Y., Ishida. T., & Kitagawa, I. (1995).
Four new β-carboline alkaloids isolated from two Okinawan marine sponges
of Xestospongia sp. and Haliclona sp. Tetrahedron, 51: 3727-3743.
Koike, K. & Ohmoto, T. (1985). Carbon- 13 Nuclear Magnetic Resonance study of
Canthin-6-one Alkaloids. Chem. Pharm. Bull. 33 (12): 5219-5244.
Kostova, I., Iossifova, T., Rostan, L., Vogler, B., Kraus, W., & Navas, H. (1999).
Chemical and biological studies on Croton panamensis latex (Dragon's
Blood). Pharm. Pharmacol. Lett. 9: 34-42.
181
Digitized by UCC, Library Krebs, H. C., & Ramiarantsoa, H (1996). Clerodane diterpenes and other
constituents of Croton hovarum. Phytochemistry, 41: 561-567.
Krebs, H. C., & Ramiarantsoa, H (1997). Clerodane diterpenes of Croton hovarum.
Phytochemistry, 45: 379-386.
Kuo, P. C., Shi, L.S., Damu, A. G., Su. C. -R., Huang, C. H., Ke, C-H., Wu, J.
B., Lin, A. J., Bastow, K. F., Lee, K. F., & Wu, T. S. (2003). Cytotoxic
and antimalarial beta-carboline alkaloids from the roots of Eurycoma
longifolia. J. Nat. Prod., 66: 1324-1331.
Lake, R. J., Blunt, J. W., & Munro. M. H. G. (1989). Eudistomins from the New
Zealand ascidian Ritterella sigillinoides. Aust. J. Chem. 42: 1201-1206.
Lake, R.J., Brennan, M.M., Blunt, J.W., Munro, M.H.G., & Pannell, L.K. (1988).
Eudistomin K sulfoxide -an antiviral sulfoxide from the New Zealand ascidian
Ritterella sigillinoides. Tetrahedron Lett. 29: 2255-2256.
Lallianrawna, S., Muthukumaran, R., Raite, V., Gurusubramanian, K., & Kumar, S.N.
(2013). Determination of total phenolic content, total flavonoid content and
total antioxidant capacity of Ageratina adenophora. Science Vision, 148-156.
Lansiaux, A., Bailly, C., Houssier, C., Colson, P., Pauw-Gillet, M, C, de Frederich et
al., (2002). Apoptosis of H-60 Leukaemia cells induced by the bisindole
alkaloids sungucine and isosungucine from Strychnos icaja. Planta Medica,
35: 73-74.
Lewis, J.R. (2000). Amaryllidaceae, muscarine, imidazole, oxazole, thiazole and
peptide alkaloids and other miscellaneous alkaloids. Natural Product Reports,
8 (17): 52-54.
182
Digitized by UCC, Library Li G.Y., Li, B.G. Yin, H.Y., Liu, G.Y., & Zhang, G.L. (2005). Sesterterpenoids,
terretonins A-D and an alkaloid, Asterrelenin, from Aspergillus terreus.
Journal of Natural Products, 68:1243-1246.
Li, H.Y., Koike K., & Ohmoto T (1993). New alkaloids, picrasidines W, X and Y, from
Picrasma quassioides and X-ray crystallographic analysis of picrasidine Q.
Chem Pharm Bull, 41:1807-1811.
Lin, C.R., Amaya, F., Barret, L., Wang, H., Takada, J., Samad, T.A., & Woolf, C.J.
(2006). Prostaglandin E2 receptor EP4 contributes to inflammatory pain
hypersensitivity. Journal of Pharmacology and Experimental Therapeutics.
319:1096-1103.
Ma, W.G., Fukushi, Y., & Tahara, S. (1999). Fungitoxic alkaloids from Hokkaido
corydalis species. Fitoterapia, 70: 258-261.
Macabeo, A. P., Krohn, K., Gehle, D., Read, R. W., Brophy, J. S., Cordell, G. A. et
al. (2005). Indole alkaloids from the leaves of Philippine Alstonia scholaris.
Phytochemistry, 66: 1158-1162.
Maciel, M. A. M., Pinto, A. G., Arruda, A. C., Pamplona, S. G. S. R., Vanderlinde, F.
A., Lapa, A. J., Echevarria, A., Grynberg, N. E., Colus, I. M. S., Farias, R. A.
F., Costa, A. M. L., & Rao, V. S. N. (2000). Ethnopharmacology,
phytochemistry and pharmacology; a successful combination in the study of
Croton cajucara, J. Ethnopharmacol. 70: 41-55.
Magalhaes, J. C., Criddle, D. N., Tavares, R. A., Melo, E, M., Mota, T. L., & Leal-
Cardoso, J. H. (1998). Intestinal myorelaxant and anti-spasmodic effects of
the essential oil of Croton nepetaefolius, and its constituents cineole, methyl-
eugenol and terpineol. Phytother. Res. 12: 172-77.
183
Digitized by UCC, Library Mahmoudian, M., Jalilpour, H., & Salehian, P. (2002). Toxicity of Peganum harmala:
Review and a case report. Iranian, J. Pharmacol. Ther.1: 1-4.
Mazlan, N.A., Mediani, Ahmed, Abas, F., Ahmad, S., Shaari, K., Khamis, S., & Lajis,
N. H. (2013). Antioxidant, antityrosinase, anticholinesterase and nitric oxide
inhibition activities of three Malaysian Macaranga species. The Scientific
World Journal, 45: 312-320.
Mebs, D., Pogoda, W., Maneyro, R., & Kwet, A. (2005). Studies on the poisonous
skin secretion of individual red bellied toads, Melanophryniscus montevidensis
(Anura, Bufonidae) from Uruguay. Toxicon, 46: 641-650.
Melentyeva, G., & Antonova, L (1988). Pharmaceutical Chemistry. (2nd ed). Mir
Publishers, Moscow.
Miller, J.R. (2003). Graph Pad in Prism version 4.0 step-by step examples. San Diego,
CA, Graph Pad Software Inc.
Milo, B., Riseo, F., Vila, R., Iglesias, J., & Canigueral, S. C. (2002).
Characterization of a fucoarabinogalactan, the main polysaccharide from
the gum exudate of Croton urucurana. J. Nat. Prod. 65: 1143-6.
Minh, P.T. H.., Ngoc, P.1.1., Quang, D. N., Hashimoto, T., Takaoka, S., &
Asakawa, Y. (2003). A novel ent-kaurane diterpenoid from the Croton
tonkinensis Gagnep. Chem. Pharm. Bull. 51: 590-595.
Mireku, E.A, Mensah A.Y., Mensah M.L.K., & Habtemariam, S. (2014a). Anti-
inflammatory properties of the stem- bark of Anopyxis kalineanaand its major
constituent, methyl angolensate. Phytother, Res, 28: 1855-1860.
Mireku, E.A., Mensah, A.Y., Mensah, M.L.K., Ekuadzi, E & Dickson, R.A. (2014b).
Antimicrobial and Antioxidant Activities of the Stem Bark of Cussonia
bancoensis. Journal of Medical and Biomedical Sciences, 3 (2): 7-13.
184
Digitized by UCC, Library Muller, L., Frohlich, K., & Bohm, V. (2011). Comparative antioxidant of carotenoids
measured by Ferric reducing antioxidant power (FRAP), ABTS bleaching
assay (α TEAC), DPPH assay and peroxyl radical scavenging assay. Food
Chemistry, 129 (1): 139-148.
Murillo, R. M., Jakupovic, J., Rivera. J., & Castro, V. H. (2001). Diterpenes and
other constituents from Croton draco. Rev. Biol. Trop.49: 259-64.
Murray, M. (1995). The Healing Power of Herbs. Prima Publishing, Rocklin, CA.
Muganza, M.D., Fruth, B.I., Nzunzu Lami, J., Mesia, G.K., Kambu,O.K., & Tona, G.L.
(2012). In vitro antiprotozoal and cytotoxic activity of 33 ethnopharmacologically
selected medicinal plants from Democratic Republic of Congo. J.
Ethnopharmacol, 141: 301-308.
Muqishima, T., Tsuda, M., Kasai, Y., Ishiyama, H., Fukushi, E., & Kawabata, J.,
(2005). Absolute stereochemistry of citrinadas A and B from marine-derived
fungus. The Journal of Organic Chemistry, 70: 9430-9435.
Myers, R.D., (1989). Isoquinolines, beta-carbolines and alcohol drinking: involvement
of opioid and dopaminergic mechanisms. Experienta, 45: 436-443.
Nakaoji, K., Nayeshiro, H., Tanahashi, T., Su, Y., & Nagakura, N. (1997).
Bisbenzylisoquinoline alkaloids from Stephania cepharantha and their effects
on proliferation of cultured cells from the Murine Hair apparatus. Planta
Medica, 63: 425-428.
Namba, T., Morita, O., Huang, S. L., Goshima, K., Hattori, M., & Kakiuchi, N. (1988).
Studies on cardio-active crude drugs. Effects of coumarins on cultured
myocardial cells. Planta Med. 54: 277-282.
Naranjo, C. (1967). In Ethnopharmacologic search for Psychoactive Drug. US
Government Printing Office, Washington DC.
185
Digitized by UCC, Library Nazrullaev, S.S., Bessonova, I. A., & Akhmedkhodzhaeva, K. S. (2001). Estrogenic
activity as a function of Chemical structure in Haplophyllum quinoline
alkaloids. Chemistry of Natural Compounds, 37 (6): 551-555.
Ngadjui, B.T., Abegaz. B. M.., Keumedjio, F., Folefoc, G. N., & Kapche, G.W.F.
(2002). Diterpenoids from the stem bark of Croton zambesicus.
Phytochemistry, 60: 345-348.
Njoronge, G. N., & Bussman, R. W. (2006). Herbal usage and informant consensus in
ethnoveterinary management of cattle diseases among Kikuyus (Central
Kenya). J. Ethnopharmacol, 108: 332-339.
Njoronge, G.N. & Kibunga J. W. (2007). Herbal medicine acceptance, sources and
utilization for diarrhoea management in a cosmopolitan urban (Thika, Kenya).
Afr. J. Ecol. 45: 65-70.
O’Donnell, G., & Gibbons, S. (2007). Antibacterial activity of two canthin-6-one
alkaloids from Allium neapolitanum. Phytotherapy research, 213-218.
Ohtani, I. 1., Ichiba, T., Isobe, M., Kelley-Borges, M., & Scheuer, P. J. (1995). New
manzamine alkaloids with potent activity against infectious diseases. J. Am.
Chem, Soc. 117: 10743-10746.
Olas, B., Wachowicz, B., Nowak, P., Stochmal, A., Oleszek, W., Glowacki, I., & Bald,
E. (2008). Comparative studies of the antioxidant effects of a naturally
occurring resveratrol analogue trans 3,3,5,5–tetrahydroxy-4-methoxystilbene
and resveratrol against oxidation and nitration of biomolecules in blood
platelets. Cell Biol Toxicol. 24: 331-340.
Orallo, F, (2004). Acute cardiovascular effects of (+) - nantenine, an alkaloid isolated
from platycapnos spicata, in anaesthetised normotensive rats. Planta Medica,
70: 117-126.
186
Digitized by UCC, Library Otshudi, A. L., Vercruysse A., & Foriers A. (2000). Contribution to the ethnobotanical,
phytochemical and pharmacological studies of traditionally used medicinal
plants in the treatment of dysentery and diarrhoea in Lomela area, Democratic
Republic of Congo (DRC). Journal of Ethnopharmacology, 71: 411-423.
Otshudi, A.L., Apers, S., Pieters, L., Claeys, M., Pannecouque, C., Clerg, E. de, Van
Zeebroeck, A., Lauwers, S., Frederich, M., & Foriers, A. (2005). Biologically
active bisbenzylisoquinoline alkaloids from the root bark of Epinetrum
villosum. Journal of Ethnopharmacology, 102: 89-94.
Palmeira, S. F., Conserva, L. M., & Silveira, E. R. (2005). Two clerodanes and
flavonoids from Croton brasiliensis. J. Braz. Chem. Soc. 16: 1420-24.
Pan, L., Zhang, X. F., Deng, Y., Wang, H., & Deng L. S (2010). Chemical constituents
investigation of Daphne tangutica. Fitoterapia, 81: 38-41.
Pelser, P. B., Vos de, H., Theuring, C., Beuerle, T., Vrieling, K., & Hartmann, T.
(2005). Frequent gain and loss of pyrrolizidine alkaloids in the evolution of
Senecio section Jacobaea (Asteraceae). Phytochemistry, 66: 1285-1295.
Perret, S., Whitfield, P. J., Sanderson, L., & Bartlett, A. (1995). The plant molluscicide
Milletia Thoningii (Leguminoseae) as a topical antischistosomal agents. J.
Ethnopharmacol. 47: 49-54.
Peters, L. Wright, A.D. Kehraus, S., Gundisch, D., Tilotta, M.C., & Koning G.M.
(2004a). Prenylated indole alkaloids from Flustra foliacea with subtype
specific binding on NAChRs. Planta Medica, 70: 883-886.
Peters, L., Wright, A.D., Krick, A., & Koning G.M. (2004b). Variation of brominated
indoles and terpenoids within single and different colours of the marine
bryozoan Flustra foliacea. Journal of chemical Ecology, 30: 1165-1181.
187
Digitized by UCC, Library Pfau, W. & Skog, K (2004). Exposure to beta-carbolines norharman and harman.
Phytochemistry, l3 (802) 115-26.
Phipps, S.M., Sharaf, M.H., & Butterweck, V. (2007). Assessing antioxidant activity in
Botanicals and other dietary supplements. Pharmacopeia Forum, 33 (4):
810-814.
Potterat, O., Puder, C., Bolek, W., Wagner, K., Ke, C., Ye, Y., & Gillardan, F. (2005).
Clauzine Z, new carbazole alkaloids from Clausena excavata with inhibitory
activity on CDK5. Die Pharmacia, 60: 637-639.
Poulton, J. E. (1989). Toxic compounds in plant foodstuffs: cyanogens. In JE Kinsella &
WG Soucie (Eds), food protein (pp. 381-401). The American Oil Chemists
society, Campaign, IL: Irwin
Poulton J E. (1990). Cyanogenesis in Plants. Plant Physiol, 94: 401-405.
Pieters, L., De Bruyne, T., Van Pod, B., Vingerhoets, R., Totte, J. & Van Den
Benghe, D. (1995). In vivo wound healing activity of Dragon's Blood (Croton
spp.), a traditional South American drug, and its constituents. Phytomedicine,
2: 17-21.
Prinsep, M. R., Blunt, J. W., & Munro, M. H. G. (1991). New cytotoxic beta-carboline
alkaloids from the marine bryozoan, Cribricellina cribraria. J. Nat. Prod, 54:
1068-76.
Przybylak, J.K., Ciesiolka, D., Wysocka, W., Garcia- Lopez, P. M. Ruiz -Lopez, M. A.
Wysocki, W., & Gulewicz, K. (2005). Alkaloid profiles of Mexican wild lupin
and an effect of alkaloid preparation from Lupinus exaltatus seeds on growth
and yield of paprika (Capsicum annuum L.). Industrial Crops and Products,
21 (1): 1-7.
188
Digitized by UCC, Library Puebla, P., Correa, S. X., Guerrero, R., & Feliciano, A. S. (2005). Phenylbutanoid
derivatives from Croton schiedeanus. Biochem. Syst. Ecol. 33: 849-4.
Purseglove, J.W. (1979). Tropical crops. Dicotyledons. London: Longman.
Rakotoson, J. H., Fabre, N., Jacquemond, I., Hannedouche, S. Fourasie, I., & Moulis, C.
(1998). Alkaloids from Galipea officinalis. Planta Medica, 64: 762-763.
Ramawat, K. G., Doss, S., & Mathura, M. (2009). The chemical diversity of bioactive
molecules and therapeutic potential of medicinal plants. In K. G. Ramawat
(Eds.), Herbal drugs ethnomedicine to modern medicine, (pp. 7-31). Springer -
Verlag Berlin Heidelberg.
Rao, L. V., Santarsiero, B. D., Mesecar, A. D., Schinazi, R. F., Tekwani, B. I., &
Hamann, M. T. (2003). New manzamine alkaloids with activity against
infectious and tropical parasitic diseases from an Indonesian sponge. J. Nat.
Prod. 66: 823-828.
Rates, S.M.K., (2001). Plants as source of drugs. Toxicon, 39: 603-613.
Rinehart. K. L., Jr., Kobayashi, J., Harbour, G. C., Hughes, R. G., Jr., Mizask, S. A., &
Scahill, T. A. (1984). Eudistomins C E K and L, potent antiviral compounds
containing a novel oxathiazepine ring from the Caribbean tunicate Eudistoma
olivaceum, J. Am Chem. Soc. 106: 1524-26.
Risco, E., Ghia, P., Vila. R., Iglesias. 1., Alvarez, E., & Canigueral, S. (2003).
Immunomodulatory activity and chemical characterisation of sangre de drago
(dragon's blood) from Croton lechleri. Planta Med. 69: 785-792.
Rizk, A. F. K., & El- Missiry M. M. (1987). In naturally occurring phorbol esters. In F.
J. Evans (Ed). Diterpenes of Euphorbiaceae. (pp. 107-138). Boston, MA:
McGraw-Hill.
189
Digitized by UCC, Library Roengsumran, S., Musikul, K., Petsom, A., Vilaivan, T., Sangvanich, P., Pompakakul,
S., Puthong, S., Chaichantipyuth, C., .Jaiboon. N., & Chaichit, N. (2002).
Croblongifolin, a new anticancer clerodane from Croton oblongifolius. Planta
Med. 68: 274-7.
Rommelspracher, H., May, T., & Susilo, R. (1991a). β-carbolines and
tetrahydroisoquinolines: Detection and function in mammals. Planta Medica,
57: 585-592.
Rommelspacher, H., Schmiditd, L. G., & May, T. (1991b). Plasma norharman
(beta-carboline) levels are elevated in chronic alcoholics. Alcohol Clin Exp.
Res. 15: 553-558.
Roth, A., Kuballa, B., Bounthanh, C., Cabalion P., Sevenet, T., Beck, J.P., & Anton, R.
(1986). Cytotoxic activity of polyindole alkaloids of Psychotria forsteriana
(Rubiaceae). Planta Medica, 32: 450-453.
Said, O., Khalil, K., Fulder, S., & Azaizeh, H. (2002). Ethnopharmacological Survey of
medicinal herbs in Israel, the Golan Heights and the West Bank region.
Journal of Ethnopharmacology, 83: 251-256.
Salmela, K. S., Roine, R. P., Koivisto, T., Hook-Nikanne, J. Kosunen.T.U., &
Salaspuro, M. (1993). Characteristics of Helicobacter pylori alcohol
dehydrogenase. Gastroenterology, 105: 325-30.
Salatino, F., & Negri G. (2007). Traditional uses, Chemistry and Pharmacology of
Croton Species (Euphorbiaceae). J. Braz. Chem. Soc, 18 (1): 11-33.
Scalbert, A. (1991). Antimicrobial properties of tannins. Phytochemistry, 30: 3875-
3883.
190
Digitized by UCC, Library Schuup, P., Poehner, T., Edrada, R., Ebel, R., Berg, A., Wray, V., & Proksch, P.
(2003). Eudistomins W and X, two new beta-carbolines from the Micronesian
tunicate Eudistoma sp. Tetrahedron, 5 (9): 29-32.
Scoltin, T. A., Di Stefano, V., & Au, W. Y. W. J. (1970). Human pharmacology of
ayahuasca. Pharmacol. Exp. Ther. 173: 26-32.
Schwarz, A., Felippe, E. C., Bernardi, M. M., & Spinosa, H. S. (2005). Impaired
female sexual behaviour of rat offspring exposed to Solanum lycocarpum
unripe fruits during gestation and lactation: Lack of hormonal and fertility
alterations. Pharmacology, Biochemistry, and Behaviour, 81: 928-934.
Seigler, D.S. (1994). Annals of the Missouri Botanical Garden. New York, Port
Chester : Melbourne.
Sen, S., Chakraborty, R., Sridhar, C., Reddy, Y.S.R., & Biplab D. (2010). Free
radicals, antioxidants and Phytomedicines: Current status and future Prospect.
International Journal of Pharmaceutical sciences Review and Research, 3 (1):
91-100.
Sheen, W. S., Tsai, I L., Teng, C. M., Ko, F. N., & Chen I. S. (1996).
Indolopyridoquinazoline alkaloids with antiplatelet aggregation activity from
Zanthoxylum integrifolium. Planta Medica, 62: 175-176.
Singh, E., Sharma, E.S., Pareck, A., Dwivedi, J., Yadav, S., Sharma S. (2011).
Phytochemistry, Traditional uses and cancer chemopreventive activity of
Amla (Phyllantus emblica): The Sustainer. S. App. Pharm, Sci. 6: 179-183.
Singh, H., Kumar, S., Dewan, S., & Kumar, V.L. (2000). Inflammation induced by
latex of Calotropis procera- a new model to evaluate anti-inflammatory drugs.
Journal of Pharmacological and Toxicological Methods, 43: 219-224.
191
Digitized by UCC, Library Soto-Hernandez, M., & Jackson, A. H. (1993). Studies of alkaloids in foliage of
Erythrinia berteroana and E. poeppigiana: detection of β-erythroidine in
goats’ milk. Phytochemical Analysis, 4: 97-99.
Staerk, D., Lemmich, E., Christensen, J., Kharazmi, A., Olsen, C. E., & Jaroszewski, J.
W. (2000). Leishmanicidal, antiplasmodial and cytotoxic activity of indole
alkaloids from Corynanthe pachyceras. Planta Medica, 66: 531-536.
Struwe, L., Albert, V. A., & Bremen, B. (1994). Cladistics and family level
classification of the Gentianales. Cladistics, 10: 175-206.
Stuart. K. L., & Graham, L (1973). Alkaloid biosynthesis in Croton linearis.
Phytochemistry, 25: 71-75.
Suarez, A. I., Suleyma, B., Delle Monache, F., Compagnone, R. S., & Arvelo. F.
(2004). Three new Glutarimide Alkaloids from Croton cuneatus. Nat. Prod.
Res. 18: 421-426.
Suladze, T.S., & Vachnadze, V.Y. (2002). Alkaloids of Veratrum lobelianum growing
in Georgia. Chemistry of Natural Compounds, 38 (5): 470-478.
Sultankhodzaev, M.N. Tashkhodzhaev, B., Averkiev, B.B., & Antipin, M., Yu, K.
(2002). Secokaraconitine, a new diterpene alkaloid from Aconitum
karacolicum. Chemistry of Natural Compounds, 38 (1): 78-82.
Takada, W., Sakata, T., Shimano, S., Enami, Y., Mori, N., Nishida, T., & Kuvahara, Y.
(2005). Scheloribatidmites as the source of pumiliotixins in dendrobatid frogs.
Journal of Chemical Ecology, 31: 2403-2415.
Tan, C. H., Wang, B. D., Jiang, S. H., & Zhu D. Y. (2002). New lycopodium
alkaloids from Huperzia serrata. Planta Medica, 68: 186-188.
192
Digitized by UCC, Library Tandon, V., Yadav, A.K., & Das, B. (2011). Phytochemicals as cure of worm infections
in traditional medicine systems, emerging trends in Zoology, Narendra
Publishing House.
Thang, P.H., Patrick, S., Teik, L.S. & Yung, C.S. (2001). Antioxidant effects of the
extracts from the leaves of Chromolaena odorata on human dermal fibroblast
and epidermal Keratinocytes against hydrogen peroxide and hypoxanthine-
xanthine oxidase induced damage, Burns, 27: 319-327.
Thanikaimani, G. (1986). Evolution of Menispermaceae. Canadian Journal of Botany,
64: 3130-3133.
Thastrup, O., Knudsen, J. B., Lemmich, J., & Winther, K. (1985). Inhibition of human
platelet aggregation by dihydropyrano – and dihydrofurano coumarins, a new
class of cAMP phosphodiesterase inhibitors. Biochem. Pharmacol, 34: 2137-
2140.
Thongtan, J., Kittakoop, P., Ruangrungsi, N., Saenboonrueng, J., & Thebtaranonth. Y
(2003). New antimycobacterial and antimalarial 8,9-secokaurane diterpenes
from Croton kongensis. J. Nat. Prod. 66: 868-70.
Thouvenel, C., Gantier, J.C., Duret, P., Fourneau, C., Hocquemiller, R. (2003).
Antifungal Compounds from Zanthoxylum chiloperone Var. angustifolium.
Phytotherapy Research, 17: 678-680.
Tsacheva, I., Rostan, J., Iossifova, T., Vogler, B., Odjakova, M., Navas, H., Kostova, I.,
Kojoharova, M., & Kraus, W. (2004). Complement inhibiting properties of
dragon's blood from Croton draco. Z. Naturforsch, C: J. Biosci., 59 (78): 528-
32.
Tsuchiya, H. M., Sato, T., Miyazaki, S., Fujiwura, S., Tanigaki, M., Ohyama, M.,
Tanaka, T.Z., & Linuma, M. (1996) Comparative Study on the antibacterial
193
Digitized by UCC, Library activity of phytochemical flavanones against methicillin–resistant staphylococcus
aureus. J. Ethnopharmacol, 50: 27-34.
Tulyaganov, T. S., Nazarov, O. M., Levkovich, M. G., & Abdullaev, N. D. (2001a).
Alkaloids of the Nitraria genus. Komavine and acetylkomavine. Chemistry of
Natural Compounds, 37 (1): 61-62.
Tulyaganov, T. S., & Allaberdiev, F. K. (2001b). Alkaloids of Nitraria sibirica.
Dihydroschoberine and nitrabirine N-Oxide. Chemistry of Natural Compounds,
37(6) 556-558.
Unver, N., Gozler, T., Walch, N., Gozler, B and Hesse, M (1999). Two novel
dinitrogenous alkaloids from Galanthus plicatus subsp. Byzantinus
(Amaryllidaceae). Phytochemistry, 50: 1255-1261.
Unver, N., Kaya, G.I., Werner, C., Verpoorte, R., & Gozler, B. (2003). Galanthindole:
A new indole alkaloid from Galanthus plicatus sp. Byzantinus (Amaryllidaceae).
Planta Medica, 69: 869-871.
Vani, T., Rajani, M., Sarker, S., & Shishoo, C.J. (1997). Antioxidant properties of the
Ayurvedic formulation triphala and its constituent. International Journal of
Pharmacognosy, 35: 313-317.
Vaisberg, A. J., Milla, M., Planas, M. D., Cordova, .1. L., Deagusti, E. R., Ferreyra,
R., Mustiga, M. D., Carlin, L., & Hammond, G. B. (1989). Taspine is the
cicatrizant principle in Sangre de Grado extracted from Croton lechleri.
Planta Med. 55: 140-145.
Varchi, G., Battaglia, A., Samori, C., Baldelli, E., Danieli, B., Fontana, G., Guerrini,
A., & Bombardelli, E. (2005). Synthesis of deserpine from reserpine. Journal
of Natural Products, 68: 1629-1631.
194
Digitized by UCC, Library Varela, A. P.; Burrows, H. D.; Douglas, P., & Miguel, M. da G..I (2001). Electronic
spectroscopy of the P-carboline derivatives nitronorharmanes, nitroharmanes,
nitroharmines and chloroharmines in homogeneous media and in solid matrix,
Photochem. Photobiol., 146: 29-35.
Vavreckova, C., Gawlik, I., & Muller, K. (1996a). Benzophenanthridine alkaloids of
Chelidonium majus. II Potent inhibitory action against the growth of human
Keratinocytes. Planta Medica, 62: 491-494.
Vavreckova, C., Gawlik, I., & Muller, K. (1996b). Benzophenanthridine alkaloids of
Chelidonium majus. I. Inhibition of 5-and 12-lipoxygenase by a non-redox
mechanism. Planta Medica, 62: 397-401.
Volken, M.C. (1999). Biological and phytochemical investigations of Euphorbiaceae
from Papua New Guinea. (Swiss Federal Institute of Technology, Zurich).
Retrieved from https://e-collection.library.ethz.ch/eserv/eth:23324/eth-23324-
02.pdf.
Vongchareonsathit, A., & De-Eknamkul, W (1998): Rapid TLC-densitometric analysis
of plaunotol from Croton sublyratus leaves. Planta Med. 64: 279-80.
Wagner, H. Bladt, S., & Zyainski, E.M. (1984). Plant drug analysis. A Thin Layer
Chromatography Atlas. Berlin-Heidelberg, Myer Publishers.
Waksmundzka-Hajnos, M., Wawrzynowicz, T., Hajnos, M.L., & Jozwiak (2006).
Preparative Layer Chromatography. Chromatographic Science Series, vol.95,
Taylor and Francis, New York.
Walt, J.M., & Breyer-Brandwijk.M.G., (1962). Medicinal and poisonous plants of
Southern and Eastern Africa. E. and S. Livingstone: Edinburgh.
Wanyama, P.A.G., Kiremire, B.T., Murumu, J.E.S., & Kamoga, O. (2011). Textile
Dyeing and Phytochemical characterization of crude plant extracts derived
195
Digitized by UCC, Library from selected dye-yielding plants in Uganda. Internal journal of Natural
Products Research, 1 (2): 26-31.
Weniger, B., Italiano, L. Beck, J.P., Bastida, J., Bergonon, S., Codina, C., Lobstein, A.,
& Anton, R. (1995). Cytotoxic activity of Amaryllidaceae alkaloids. Planta
Medica, 61: 77-79.
Wu, S.J., Tsai, J.Y., Chang, S.P., Lin, D.L., Wang, S.S., Huang, S.N. and Ng, L.T.
(2006). Supercritical Carbon dioxide extract exhibits enhanced antioxidant and
anti-inflammatory activities of Physalis peruviana. Journal of Ethnopharmacology,
108: 407-413.
Wungsintaweekul, J., & De-Eknamkul, W (2005). Biosynthesis of plaunotol in Croton
stellatopilosus proceeds via the deoxyxylulose phosphate pathway. Tetrahedron
Lett. 46 (21): 25-28.
Yubin, J.I., Miao, Y., Bing, W., & Yao, Z. (2014).The extraction, separation and
purification of alkaloids in the natural medicine. Journal of Chemical and
Pharmaceutical Research, 6 (1): 338-345.
Zapesochnaya, G.G., Pervykh, L.N., & Kurkin, V.A. (1991). A study of the herb Aerva
lanata. Chemistry of Natural Compounds, 27 (3): 336-340.
196
Digitized by UCC, Library APPENDICES
1 Appendix A: H-NMR of M1 in MeOD at 500 MHz
197
Digitized by UCC, Library
1 Appendix B: Integrated H-NMR of M1 in MeOD at 500 MHz
198
Digitized by UCC, Library 13 Appendix C: C-NMR of M1 in MeOD at 500 MHz
199
Digitized by UCC, Library 13 Appendix D: Expanded C-NMR of M1 in MeOD at 500 MHz
200
Digitized by UCC, Library Appendix E: Mass spectrum of M1
201
Digitized by UCC, Library
Appendix F: Elemental analysis of M1
202
Digitized by UCC, Library 1 Appendix G: H-NMR of M5 in MeOD at 500 MHz
203
Digitized by UCC, Library
1 Appendix H: Integrated H-NMR of M5 in MeOD at 500 MHz
204
Digitized by UCC, Library 13 Appendix I: C-NMR of M5 in MeOD at 500 MHz
205
Digitized by UCC, Library 13 Appendix J: Expanded C-NMR of M5 in MeOD at 500 MHz
206
Digitized by UCC, Library Appendix K: Mass spectrum of M5
207
Digitized by UCC, Library