UNIVERSITY OF CAPE COAST PHYTOCHEMISTRY, ANTI … MALCOLM... · True Alkaloids 40 Protoalkaloids ... 12 Examples of True Alkaloids 41 13 Examples of Protoalkaloids 42 14 Examples

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


ii

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


iii

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.


iv

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.


v

DEDICATION

To my lovely wife, Naomi Arthur Fynn (Mrs) and children, Nhyiraba,

Nyameyie, Judalyn and Jedida


vi

TABLE OF CONTENTS

Page

DECLARATION ii

ABSTRACT iii

ACKNOWLEDGEMENTS iv

DEDICATION v

TABLE OF CONTENTS vi

LIST OF TABLES xiii

LIST OF FIGURES xiv

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


vii

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 36

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 42

Nomenclature of Alkaloids 43

Pharmacological Uses of Alkaloids 44

Distribution of Alkaloids 44

The Family Euphorbiaceae 46

The Family Apocynaceae 47

The Family Asteraceae 48


viii

The Family Loganiaceae 49

The Papaveraceae Family 50

The Family Rutaceae 51

The Family Solanaceae 53

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


ix

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

Total Antioxidant Activity by the Phosphomolybdenum Method

106

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


x

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

Column chromatographic separation 0f the crude alkaloid extract

125

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


xi

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 168


xii

APPENDIX A: 1H-NMR of M1 in MeOD at 500 MHz

APPENDIX B: Integrated 1H-NMR of M1 in MeOD at 500 MHz APPENDIX C: 13C-NMR of M1 in MeOD at 500 MHz

APPENDIX D: Expanded 13C-NMR of M1 in MeOD at 500 MHz

APPENDIX E: Mass spectrum of M1 APPENDIX F: Elemental analysis of M1

APPENDIX G: 1H-NMR of M5 in MeOD at 500 MHz APPENDIX H: Integrated 1H-NMR of M5 in MeOD at 500 MHz APPENDIX I: 13C-NMR of M5 in MeOD at 500 MHz

APPENDIX J: Expanded 13C-NMR of M5 in MeOD at 500 MHz APPENDIX K: Mass spectrum of M5

CURRICULUM VITAE

LIST OF PUBLICATIONS

197

198

199

200

201

202

203

204

205

206

207


xiii

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


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


xiv

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


xv

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

38

Thermolysis of Tryptophan (1) to Form Tryptamine (2)

By-products of the thermolysis of tryptophan to form tryptamine

85

86

39 Pathways for the Generation of the Various Mediators of

Inflammation

99

40 Pathway for the Detoxification of Reactive Oxygen species by


xvi

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

47

Fragmentation Pattern of Compound M1

The structure of compound M5

136

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,

Diclofenac

144

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;


xvii

***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

Diclofenac on Carrageenan-induced Foot Edema in Rats

150

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

56 Plot of Percent Inhibition Against Concentration of Extracts and Isolated Compounds 159

57 DPPH Absorption Spectra of Extracts and Isolated Compounds 159


xviii

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


xix

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


1

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)


2

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).

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

INFLAMATION

Cancer

Cardiovascular

Alzheimer’s diseases

Pulmonary diseases

Arthritis

Autoimmune diseases

Neurological diseases

Diabetes


3

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.


4

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).


5

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


6

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).


7

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


8

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


9

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


10

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.


11

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.


12

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


13

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.


14

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


15

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


16

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


17

(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


18

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-


19

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


20

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


21

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).

OCOOCH3

OH

O

O

O

OO H

OHH

O

OH

O

RO

OH

HH

OH

H

O

OiBu

iBu :

Saudinolide

Cromiargyne

O

4-Deoxyphorbol diesters

AcO

R2O

O

OAcOR1

H

HO

BzO

OBz : R1: Ac

R2 : AC

Jatrophane polyesters

Figure 3: Examples of diterpenoids isolated from the family Euphorbiaceae


22

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.

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

H H

H

OAc

OH

RO

HO

Boeticol Kamaladiol-3-acetate

Cis/ trans Securinegin

R= p-coumaroyl


23

(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


24

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


25

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).

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),

N

H

O

O

H

Virosecurinine

NHH3CO

H3CO

H

Crotonosine

N

OCH3

O

Onosmin A

N

OH

H

O

H

Onosmin B

NH

O

HN

O

O O

Aurentiamide acetate

O

NH

CH3

O

OH

N-(2-hydroxy-1-phenylpropyl) benzamide


26

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.


27

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).

Figure 6: Examples of flavonoids isolated from the family Euphorbiaceae

O O

O

OH

OH

HO

OH

OHOH

HO

OH

OH

OH

OH OH

HO

O

OH

OH

OO

Kaempferol Quercetin

Myricetin

O

O

Flavone

O

O

O

OOH

HO

OH

HO

OH

OH

Chrysin Catechin


28

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


29

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).

Figure 7: Examples of tannins isolated from the family Euphorbiaceae.

O

O

O

O

OH

HO

HO OH

Ellagic acid

OH

O

OH

HO

HO

Gallic acid

O

OH

OH

OH

OH

HO

OH

Gallocatechin

CO

CHOMe

O

O

O

OH

HO

OH

OH

OHOH

HO

HO

Corilagin O

O

HO

HO OHOH

OH

O

O O

OO

HOH2C

Furosin


30

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).


31

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

O O

Coumarin

O O

O O O O

O O

O O

O O

HO

H3CO

HO

H3CO

H3CO

Umbelliferone

ScoparoneScopoletin

OHHO

Daphnethin

OPsoralen

O

Seselin


32

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).


33

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

NO

O O

OH

OH

OH

OH

R3

OCH3

R1

R2

R4

HN

CN

O

O

O

OH

OH

HOOH

H3CONO

O

O

HO

OH

OH

OH

OCH3

CH3

R1= CH3; R2= OH; R3= H; R4= CN- AcalyphinR1= CH3; R2= H; R3= OH; R4= CN-EpiacalyphinR1= H; R2= OH; R3= H; R4= CN- NoracalyphinR1= H; R2= H; R3= OH; R4= CN-EpinoracalyphinR1= CH3; R2= OH; R3= H; R4= CONH2- Acalypin amide

ar-Acalyphidone CH3

Seco-Acalyphin

N

O

OO

CH3

O

HOOH

OH

OCH3

CONH2

H

Epiacalyphin amide ycloside


34

and esterified triterpenes alcohol fractions and a new acyclic triterpenes alcohol

named peplusol (Salatino and Negri, 2007).

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

CO O C

OHOH

HO

HO

O

OO

OHO

C O

HO

OH

OH

O

C O

OH

OH

HO

OH

1246 TGG

R

R

OH

Peplusol

R=


35

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).

OH

MeO

HO

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.


36

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.


37

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.


38

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.


39

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


40

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


41

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

N

C2H5

R1R1H

R1

R2

Vinorelbine

NH

N

OOCH3

H3COOC

H3CO

H

HH

Reserpine

CO

OCH3

OCH3

OCH3

H

R2

NH

NOH

N

N

C2H5

OCOCH3COOCH3HOR

H3COOC

1. R= CH3

Vinblastine

2. R= CHO

Vincristine

N

N

H3CO

OH

Quinine

Figure 12: Examples of true alkaloids.


42

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


43

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.

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)

O

NH

Solasodine

NCH3

CH3

CH3

HO

CH3

HH

H

Solanidine

O

N

H

HTomatine

CHOH

NHCH3H3C

Ephedrine


44

(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


45

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


46

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.


47

NH

N

O O

H2NR2

R1

A, R1=H, R2=OCH3B,R1=OCH3 R2=H

NH

N

NR2

R1

C,R1=OCH3 R2=H

D,R1=H R2=OCH3

NH

N

NH3CO

E

NH

N

HN

H3CO

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


48

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


49

7-0-angelyl-9-0-sarracinylretronicine (Aniszewski, 2007). Cheng and Roeder

(1986) have isolated two pyrrolizidine alkaloids (senkirkine and doronine) from

Emilia sonchifolia.

O

N

O

O

O O

N

O

O

OO

N

O

O

O

OH

O

N

O

O

OO

N

O

O

O

H

Senecovernine

HOHO

Senecionine

HO

Retrorsine

HO

O O

HO

N

HOH

Senkirkine Dehydrosekirkine Retronecine

HO

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).


50

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

O

N

N

O

N

N

O

N

N

O

N

Sungucine

O

N

O

N

ON

O

N

O

H3CO

H3CO

StrychnineBrucine

N

O

N

N

O

N

O

OH

Strychnogucine A

Strychnogucine B

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


51

(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).

N

OCH3

OCH3

H3CO

H3CO

Papaverine

ON CH3

ON CH3

HO

HO

H3CO

HO

Morphine Codeine

ON

O

O

Thebaine

NO

OCH3

OCH3

O

OO

CH3

Hydrastine

N

OCH3

OCH3

O

O

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


52

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.


53

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


54

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).

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.

NCH3

CH3

CH3

HO

CH3

HH

H

Solanidine

O

NH

HTomatine

OCH2OH

H

O

NCH3

Hyoscyamine

N

NCH3

N

NH N

NH

Nicotine Myosmine Anabasine


55

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)

N COOCH3

OCOC6H5H

H

CH3N COOH

OHH

H

CH3

CocaineEcgonine

OHN CH3

Tropine

N CH3

O

CH3

Hygrine

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


56

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.

N

HOH

OH

N

HOH

OH

N

AcOH

OH

N

ROH

OH

R=2-MeBut

7-(2-Methylbutyrl)retronecine

N

HOH

OR

R=2-MeBut

9-(2-Methylbutyryl)retronecine

Retronecine

7-Acetylretronecine

Heliotridine

N

HOH

(+)-Supinidine

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


57

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.

N

OHH

Lupine

NN

OH

HLupanine

NNH

OH

H

Angustifoline

N

N

O

Anagyrine

O

O

HN

NH

NH3C

H

H3C

CH3

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


58

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).


59

N

O

N

O

H

O

O

O

O

H

Cepharantine

N

O

O

O

N

O

O

O

OCH3

AtherospermidineLiriodenine

N

H3CO

HOCH3

H3CO

H3COCorydine

N

H3CO

H3COCH3

HO

H3CO

N

H3CO

H3COH

HO

H3CO

Isocorydine Norisocorydine

H

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.


60

NO

N

O

OOO

OH

H

Berbamine

N

OO

O

O

Glaucine

N

OCH3

H3CO

O

O

CH3

Nantenine

N

O

OOCH3

OCH3

Berberine

H

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).


61

OCH3

OH

OH

N

C2H5

OCH3

H3CH2CO

OCOC6H5

OCH3

COCH3

OH

OH

N

C2H5

OCH3

H3CH2CO

OCOC6H5

OCH3

OH

OH

OH

N

C2H5

OCH3

H2CH3CO

Aconine Aconitine

OH

OH

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).


62

NCH3

H

H

CH3

HO

O

O

Jervin

O

HN CH3

H

H

H3C

H

CH3

HO

H3C

H

H

H

CyclopamineN

O

HO OCOOCH3

OH OCOCHCH3

OHOHCH3

CH3

OCH3

H

COOCH3 CH3

Protoveratrine

HO

CH3

H

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


63

(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.

N

N N

NH

CH3

CH3O

O

N

N N

NH3C

CH3

CH3O

O

N

N N

NH

CH3

HO

O

Theobromine Caffeine Theophylline

NH

N

OCH3

OCH3

H3CO

H3CO

H

Emetine

NH

N

OCH3

OCH3

HO

H3CO

H

Cephaeline

Calycosidine

H3CH2C H3CH2C

Figure 28: Alkaloids of Rubiaceae


64

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


65

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).

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.

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

N

HO

O

O

Lycorine

N

O

O

OCH3

Haemanthamine

HN

O

CH3

OH

H3CO

Galanthamine

OHH

OH

NH

NH

Elaeagine


66

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).

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

NHO

H

Nitramine

NHN

O

Nitraramine

N

NH

Nitrarine

NH

NH

Komavine

NH

N

Acetylkomavine

NH

NH3CO

CH3

Harmaline

NH

NHO

CH3NH

N

CH3

HO

Harmol Harmolol

O


67

sedative, anxiolytic, hypnotic anticonvulsant as well as antimicrobial activities

(Cao et al., 2007)

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.).

NH

NH2HONH

NHO

NH

N

Serotonin Psilocin

POH

OO

HO

Psilocybin

CH3

CH3

CH3

CH3


68

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,


69

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).

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,

N

N

HN

H3C CH3

O

O CH3

H

Echinuline

N

N

H3C

O

Pyocyanine

H


70

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


71

(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.,


72

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


73

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.

NH

N

Norharman

NH

N

Harman

NH

N

CH3H3CO

Harmine

NH

NH

CH3

MTHBC

CH3

NH

N

CH3

H3CO

Harmaline

NH

N

CH3

HO

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:


74

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


75

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


76

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


77

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.


78

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


79

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


80

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


81

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.


82

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.

NH

NH2

R2R1

CH1

R1

O

NH

N

R2R1

CH

R1

NH

N

R2R1

CH

R1

NH

R1 NH

R2

R1 NH

R1 N

R2

R1

H

O R1= H, CH3

R2 = H, COOH, COOC2H5

R2 = H, OH

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


83

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


84

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


85

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


86

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


87

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


88

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


89

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


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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


91

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).


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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)


93

(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


94

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


95

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


96

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


97

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


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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)


99

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),


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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


101

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


102

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


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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


http://en.wikipedia.org/wiki/Copper

http://en.wikipedia.org/wiki/Manganese

http://en.wikipedia.org/wiki/Iron

http://en.wikipedia.org/wiki/Catalase

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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.


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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


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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


107

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)


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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.


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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%

NH4OAc. The temperature was set at 2000C, 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.


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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.


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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


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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.


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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


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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.


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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


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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


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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


118

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.


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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


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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


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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.

100AUC

AUCAUCedemaoofinhibition%

control

treatmentcontrol

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.

Y= a+(b-a)

�1+10(LogEC50-X)�


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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


123

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.


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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.


125

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-


126

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.

Figure 41: Schematic representation of the isolation of alkaloids

Fraction I (190 mg) Fraction II (230 mg) Fraction III (250 mg)

100%

DCM

DCM-EtOAc

50:25 v/v

DCM-EtOAc

50:25 v/v

DCM-EtOAc

50:25 v/v

MeOH 100%

CC, alumina (70 – 230 mesh)

NH3(aq)

DCM

Organic layer

Crude alkaloid (0.68g)

Marc Extract (31.20 g)

Organic layer Aqueous layer

Root Bark

NH3(aq) MeOH (70%)

HOAc

DCM

Aqueous layer


127

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.

M1

M5


128

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.

PTLC, alumina

Toluene: EtOAC 75:25 v/v

Crystallization, EtOAC

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

Fraction I (190 mg)

M1 (160mg)


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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

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.

Sub-Fraction II1 (120mg) Sub-Fraction II2 (100mg)

M3 (90mg)

Fraction II (230 mg)

M2 (80mg)

Crystallization, EtOH Crystallization, EtOH

PTLC, alumina

Toluene: EtOAc75:25 v/v


130

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.

Sub -fraction IIIa (100 mg Sub -fraction IIIb (140

M5 (120 mg)

)

M4 (70 mg)

Fraction III (250

mg)

Crystallization EtOH

PTLC, alumina

Toluene: EtOAC 75: 25

v/v

Crystallization EtOH


131

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).


132

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


133

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


134

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)

N

N

O

A

B C

D

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

spectrum. In addition to the methoxy group, the 1H –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


135

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


136

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.

NN

O

OCH3

1

2

4

5

6

8

9

10 11

12

13

14

15

16

Figure 46: Fragmentation pattern of compound M1

N

N

O

OCH3

N

N

O

O-

N

N

O

N

N

-CH3

m/z 251 m/z 235

-15

-CO

-28

m/z 207

m/z 179

-CO-28

N

NH

m/z 153

-C2H2

-26N

m/z 125

-HCN

-27

+ +

+

++

+


137

Table 2: 1H-NMR and 13C-NMR spectral data and 1H-13C long-range


Carbon Position

Type δH δC 2J 3J

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)


138

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


139

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.

Figure 47: The structure of compound M5

NN

O

1

2

4

5

6

8

9

10 11

12

13

14

15

16


140

Table 3: 13C-NMR chemical shifts (ppm) of canthin-6-one and compound M5

Carbon position

*Canthin-6-one Compound M5

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)


141

Table 4: 1H-NMR and 13C-NMR spectral data and 1H-13C long-range


Carbon Position

Type δH δC 2J 3J

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



142

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


143

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



144

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.


145

Methanol crude

0 2 4 6 80

20

40

60

80

10030mg/kg

100mg/kg

300mg/kg

Control

Time/hrs

% I

ncre

ase i

n f

oo

t vo

lum

e

crude auc

contr

ol30 10

030

0

0

100

200

300

400

******

***

Crude extract of A. aubryanum (mg/kg BDW)To

tal fo

ot

oe

de

ma

(c

alc

ula

ted

as

AU

C)

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.


146

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.

0 2 4 6 80

20

40

60

80

10030mg/kg

100mg/kg

300mg/kg

Control

Time/hrs

% I

ncre

ase i

n f

oo

t vo

lum

e


147

Anti-inflammatory activity of the alkaloid extract

All doses of the dichloromethane/alkaloid extract administered through


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


148


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



149

M1

0 2 4 6 80

20

40

60

80

10010mg/kg

30mg/kg

100mg/kg

Control

Time/hrs

% I

ncre

ase i

n f

oo

t vo

lum

e

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.


150

M5

0 2 4 6 80

20

40

60

80

10010kg/mg

30kg/mg

100kg/mg

Control

Time/hrs

% I

ncre

ase i

n f

oo

t vo

lum

e

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


151

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.


152

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 20 40 600.0

0.2

0.4

0.6

0.8

r2 = 0.9632

conc.

Ab

sorb

ance

(n

m)

Figure 54: Absorbance against concentration of vitamin E used in the calibration curve.


153

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


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


154

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


155

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


156

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


Many medicinal plants possessing antioxidant activities have been

shown to possess protective effects on the erythrocyte membrane from


157

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).


158

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


Figure 55: Concentration response curves for standard drug, extracts and isolated compounds.


159

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

0

20

40

60

80

100

120

0 100 200 300 400 500 600

% in

hib

itio

n

Concentration(µg/ml)

M1 CA M5 CRUDE VIT E


160

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).


161

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.


162

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.


163

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).


164

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


165

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


166

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


167

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.


168

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APPENDICES

Appendix A: 1H-NMR of M1 in MeOD at 500 MHz


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Appendix B: Integrated 1H-NMR of M1 in MeOD at 500 MHz


199

Appendix C: 13C-NMR of M1 in MeOD at 500 MHz


200

Appendix D: Expanded 13C-NMR of M1 in MeOD at 500 MHz


201

Appendix E: Mass spectrum of M1


202

Appendix F: Elemental analysis of M1


203

Appendix G: 1H-NMR of M5 in MeOD at 500 MHz


204

Appendix H: Integrated 1H-NMR of M5 in MeOD at 500 MHz


205

Appendix I: 13C-NMR of M5 in MeOD at 500 MHz


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Appendix J: Expanded 13C-NMR of M5 in MeOD at 500 MHz


207

Appendix K: Mass spectrum of M5


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