Chemical and Biological Studies on Traditional Anti-.
malarial Plants from Meru and Kilifi Districts
By
KIRJRA PETER GAKIO B.Ed. (Sc.) HODS (Kenyatta)
A thesis submitted to the School of Pure and Applied Sciences for partial fulfilment
of Master of Science degree ofKenyatta University
SEPTEMBER 2004
Kirira, Peter Gakio
Chemical and
biological studies on
\ 'I~ ~Im I~ 11\II\11111111MII~ \1\II
2005/216932
IV
Declaration
Declaration by candidate
This thesis is my original work and has not been presented for any degree in another
university.
Signature .. ~ Date ~~.(!~9.f...
Kirira Peter Gakio
Declaration by Supervisors
This thesis has been submitted in partial fulfilment of Masters of Science degree in
the School of Pure and Applied Sciences, Kenyatta University with our approval as
supervisors.
Signa~·fl~~~te .~t~H~~·
Prof. Isaiah ~iege
Chemistry Department,
Kenyatta University,
Nairobi,
Kenya.
Signature .•.... Date ..~JI.~'.~.lf.
Dr. Alphonse Wanyonyi
Chemistry Department,
Kenyatta University,
Nairobi,
Kenya.
Signature. Date ",j,yOf.(
Dr. Geoffrey Rukunga
Centre for Traditional Medicine and Drug Research (CTMDR),
Kenya Medical Research Institute (KEMRI),
Nairobi,
Kenya.
vAcknowledgements
I thank Godfor enabling me do this work t.o its completion. Truly it has taken His hand.
I am greatly indebted to Prof Isaiah O. Ndiege who always worked round the clock to
ensure that this work was a success. His contribution in running NMR spectra, organising
for acquisition of MS data oversees and correction of the thesis made completion of this
work possible. Many thanks go to Dr. Goeffrey M. Rukunga for allowing me to carry out
the research at CTMDR, KEMRI. His great contributions to this work including
providing an enabling environment, advice during difficult times and sourcing for funds
will always be remembered and are greatly appreciated. I also wish to thank Dr.
Alphonse Wanyonyi for his contribution to this work.
I greatly appreciate the help I got from CTMDR staff at KEMRI; especially Dr. J. Orwa,
Dr. E. Matu, Mr. P.G. Githaiga and Mr. F. Tolo for their support; Mr. C. Muthaura and
Mr. E. Munene for assisting me to obtain samples and to freeze dry them; Ms. S. Wachira
and Ms. B. Irungu who advised me greatly in fractionation and structural elucidation; Mr.
J. Waweru who helped with in vitro assays; and Mr. S. Mwangi, Ms. T. Ndunda, Ms. M.
Wanjohi and Ms. C. Kimani. Last but not the least I wish to thank colleagues at KEMRI
including Mr. M. Rono and Ms. E. Kigondu who assisted in many ways in the laboratory.
I also wish to thank the staff of Malaria Department at KEMRI through the head Dr. S.
Omar for allowing me to use their culture laboratory. I am also grateful to the director
Institute of Primate Research (IPR) through Dr. D. Yole and Mr. F. Nyundo for allowing
me to do scintillation counting at the institute. .
I also thank my colleagues at Kenyatta University including Mr. J. Kobia and Ms. C.
Ochieng' whose moral support and presence helped me in many ways, especially during
the course work. Special appreciations also go to Mr. E. Maina of Chemistry Department,
Kenyatta University for providing columns used during isolation of the compounds and
to the whole department for assisting me in many ways throughout the entire period.
Lastly I wish to appreciate the advice I received from my senior colleagues in the
research group, Mr. M. Omolo and Mr. H Malebo, on spectral analysis.
I also wish to recognise Prof RT. Majinda of the University of Botswana and University
of Nairobi through Prof LO. Ndiege for doing spectral analysis of the compounds
isolated during this study. I also wish to thank Mr. Wanyama, ICIPE for helping me to
obtain ElMS data.
Finally, I wish to salute the family of Mr. and Mrs. S.M. Mwangi for their love and
support and to sincerely appreciate the help and support my parents Mr. and Mrs. D.K
Mwangi for providing finances during the entire course, their patience and continued
support in many ways, you made this possible. Heart felt gratitude also go to Mr. G.
Ngondi whose many skills and advice will always be remembered. I also wish to
appreciate Ms. A Kande, a dear friend who lifted my spirits during difficult periods and
who was always there for me.
May Almighty God bless you all.
VI
Dedication
This thesis is dedicated to my father D.K. Mwangi my mother J. Wanjiru and Mr &
Mrs Solomon Maina Mwangi.
•
..
VII
LIST OF ABBREVIATIONS
amu Atomic Mass Unit
CC Column Chromatography
CDCh Deuterated Chloroform
CHCh Chloroform
C6H14 Hexane
CI Chemical ionisation
COSY Correlation Spectroscopy
CQ Chloroquine
CTMDR Centre for Traditional Medicine and Drug Research
DDT Dichlorodiphenyltrichloroethane
DEET N,N- Diethyl-m-toluamide
DEPT Distortionless Enhancement by Polarisation Transfer
ElMS Electron Impact Mass Spectroscopy
EtOAc Ethyl acetate
HETCOR Heteronuclear Correlation Spectroscopy
HPLC High Performance Liquid Chromatography
HMBC Heteronuclear Multiple Bond Correlation
HMQC Heteronuclear Multiple Quantum Correlation
Hz Hertz
IR Infrared
KEMRI Kenya Medical Research Institute
m.p Melting point
MQ Mefloquine
MS Mass Spectroscopy
NMR Nuclear Magnetic Resonance
NOE Nuclear Overhauser Enhancement
ppm parts per million
ptlc preparative thin layer chromatography
R, Retention factor
USA United States of America
tic Thin layer chromatography
UV Ultra Violet
l3C NMR Carbon 13 Nuclear Magnetic Resonance
IHNMR Proton Nuclear Magnetic Resonance
11
Table of Contents
TABLE OF CONTENTS ii
DECLARATION iv
ACKNOWLEDGEMENTS v
DEDICATION vi
LIST OF ABBREVIATIONS vii
ABSTRACT .:.: viii
CHAPTER 1: INTRODUCTION 1
1.1 Background 1
1.2 Malaria vector 2
1.3 Malaria parasite 2
1.4 Control strategies 4
1.4.1 Vaccine development. .4
1.4.2 Vector controL............................... .. 5
1.4.3 Chemotherapy 8
1.4.4 Combination of drugs 15
1.4.5 Triple therapy 17
1.5 Hypothesis 18
1.6 Objectives 18
1.6 Justification 18
CHAPTER 2: DRUG DISCOVERy ~ 20
2.1 Introduction 20
2.2 Brine shrimp lethality bioassay 22
2.3 Pharmacological models 22
2.3.1 In vitro model 23
2.3.2 In vivo model.. 26
2.4.3 In situ modeL...... . 26
CHAPTER 3: ANTI-PLASMODIAL PLANTS AND ACTIVE PRINCIPLES 27
3.1 Introduction 27
3.2 Anti-plasmodial compounds from nature......................................................................................... .. 27
3.2.1 Phenols......................................................................................................................................... . 27
3.2.2 Chalcones . 28
3.2.3 Flavonoids 29
3.2.4 Naphthoquinones 29
3.2.5 Anthraquinones and xanthonones 30
3.2.6 Terpenes and related compounds 30
3.2.7 Alkaloids 34
3.3 Anti-malarial plants 37
3.3.1 Carissa edulis (Forsk.) Vahl (Apocynaceae) 37
3.3.2 Neoboutonia macrocalyx Pax (Euphorbiaceae) 38
3.3.3 Acacia nilotica (L.) Del. (Leguminosae) 39
3.3.4 Strychnos heningsii Gilg. (Loganiaceae) 40
3.3.5 Azadirachta indica A. Juss (Meliaceae) 41
3.3.6 Myrica salicifolia A. Rich. (Myricaceae) 42
3.3.7 Fagaropsis angolensis (Engl.) Dale (Rutaceae) 42
3.3.8 Zanthoxylum usambarensis (EngI.) Kokwaro (Rutaceae) 43
3.3.9 Harrissonia abyssinica Oliv (Simaroubaceae) 44
3.3.10 Withania somnifera (L.) Dunal (Solanaceae) 45
111
CHAPTER 4: BIO-ASSAYS 47
4.1 Bioassay of crude extracts 47
4.1.1 Brine shrimp toxicity test 47
4.1.2 Anti-plasmodial screening 48
4.1.3 Summary 51
4.2 Bio-assay of mixtures and isolated compounds 52
4.2.1 Brine shrimp toxicity test 52
4.2.2 In vitro anti-plasmodial screening 53
4.2.3 Conclusion 55
CHAPTER 5: STRUCTURAL ELUCIDATION 56
5.1 Structural elucidation of compounds isolated from Neoboutonia macrocalyx 56
5.1.1 Stigmasterol (PI) (126) 56
5.1.2P2a 58
5.1.3 6,7-Epoxy-4,5,9-trihydroxy-13-hexadecanoate-20-dodecanoate-l-tiglien-I-3-one (P2b) (140) 58
5.1.4 P3 64
5.1.5 6-7 -Epoxy-4,5,9,20-tetrahydroxy-13-tetradecanoate-1-tiglien-3-one (P4) (141) 64
5.1.6 4 ,9-Dihydroxy- 20-hexadecanoate-13 -dodecanoate-1 ,6-tigliadien -3-one (P5) (142) 70
5.1.7 Methy13-heptaeicosanoyloxyoleanoate (P6) (143) 76
5.1.8 Montanin-20-palmitate (P7) (79) 81
5.1.9 12-Deoxyphorbol-13-pentadecanoate (P8) (145) 86
5.2 Structural elucidation of compounds isolated from Fagaropsis angolensis 92
5.2.1 KI 92
5.2.2 FASD 2 93
5.2.3 FASD 3 93
5.2.4 FASD 4 93
5.2.5 FASM 1 93
5.2.6 FASM 2 93
5.2.7 FASM 3 93
5.2.8 FASM 4 94
5.2.9 FASC 1 94
CHAPTER 6: CONCLUSION AND RECOMMENDATIONS 94
6.1 Conclusion 94
6.2 Recommendations 97
CHAPTER 7: EXPERIMENTAL 99
7.1 Materials, reagents and equipment... 99
7.1.1 Reagents 99
7.1.2 Equipments for the in vitro culture 99
7.1.3 Disposable plastics and glassware . '99
7.1.4 Recycled glassware 100
7.1 .5 Sterilizing materials................................................................................................................................. 100
7.2 Plant Material : 100
7.2.1 Sampling 100
7.3 General procedures 100
7.3.1 Extraction 100
7.3.2 Brine shrimp assay 101
7.3.3 In vitro anti-plasmodial assay : 101
7.3.4 Culture preparations 102
7.4 Isolation of compounds from Neoboutonia macrocalyx : 110
7.5 Isolation of compounds from Fagaropsis angolensis 114
REFERENCES 120
APPENDICES 140
Vlll
Abstract
More than half of the world's population live in areas where they are at risk of malaria
infection. Africa contributes 90% of> 2 million deaths and> 500 million clinical cases
annually. Malaria accounts for 10% of the continental disease burden. Pregnant women
and children under 5 years are the main risk groups in the endemic areas. Malaria control
is becoming more difficult due to the spread of resistance of anopheline mosquitoes and
the parasites to insecticides and drugs, respectively. The absence of an effective
commercial anti-malarial vaccine that offers full protection despite several decades of
intensive research is a dangerous situation. Emergence of resistant malaria parasites has
caused a crisis in the use of anti-malarial drugs for prophylaxis and therapy.
Chemotherapy being the primary strategy of malaria control in the developing world,
new anti-malarial compounds with different modes of action are urgently required.
Plants are used widely in traditional health systems to treat a variety of diseases. Ethno-
botanical information has previously provided potent anti-malarial compounds like
quinine, artemisinin, cryptolepine and nitidine that led to the development of synthetic
drugs such as chloroquine and artemether. It is therefore necessary to evaluate the
traditional anti-malarial plants with the aim of incorporating them into the national health
care systems or discovering new potent anti-plasmodial molecules which may be
developed into drugs or used as templates for the development of new more potent
synthetic analogues.
In the search for new anti-malarial principles, we carried out bio-evaluation of 10 plants
used to treat malaria in Meru and Kilifi Districts. Preliminary activity and cytotoxicity
studies of extracts were carried out using brine shrimp tests. Only Neoboutonia
macroca/yx, Azadirachta indica, Fagaropsis ango/ensis and Harrisonia abyssinica
showed toxicity (LDso 41.69 ± 0.9, 101.26 ± 3.7, 173.48 ± 0.6 and 234.71 ± 11.5 11g/ml ,
respectively) for aqueous extracts. However, methanol extracts from N macroca/yx, F.
angolensis, A. indica, Zanthoxylum usambarense, Strychnos heningsii, Carissa edulis,
Withania somnifera and H. abyssinica (LDso 21.04 ± 1.8, 57.09 ± 1.4, 61.43 ± 2.9, 97.66
± 3.6,101.22 ± 3.2,186.71 ± 6.9, 207.27 ± 0.7 and 217.34 ± 7.2 ug/ml, respectively)
exhibited toxicity against brine shrimp nauplii. Extracts were screened against CQ-
susceptible and CQ-resistant strain of Plasmodium falciparum (NF54 & ENT30,
respectively). The order of anti-plasmodial activity was as follows: F. angolensis (ICso
10.65 ± 1.23), Z. usambarense (ICso 14.33 ± 4.22), S. heningsii (ICso 73.39 ± 9.75),
Myrica sa/icifolia (ICso85.97 ± 5.48), H. abyssinica (ICso 89.74 ± 8.12), N macroca/yx
(ICso92.85 ± 7.65), C. edulis (ICso> 250), A. indica (ICso> 250), Acacia nilotica (ICso>
250) and W. somnifera (ICso> 250 ug/ml) for aqueous extracts against ENT30 and: F.
angolensis (ICso 5.04 ± 0.68), Z. usambarense (ICso 5.54 ± 1.70), M salicifolia (ICso
55.89 ± 2.00), A. nilotica (ICso 73.59 ± 2.87), N macrocalyx (ICso 78.44 ±2.89), H.
abyssinica (ICso 79.50 ± 3.31), W. somnifera (ICso 145.86 ± 2.23), S. heningsii (ICso
190.0 ± 16.85), C. edulis (ICso> 250), A. indica (ICso> 250 ug/ml) for methanol
extracts against the same isolate. Two plants, F. ango/ensis and Z. usambarense, showed
good in vitro anti-plasmodial activity (ICso 5.0-11 ug/ml) against CQ-resistant strain.
IX
For NF54, the order was: Z. usambarense (ICso 5.25 ± 0.27), F. angolensis (ICso6.13 ±
1.15), M salicifolia (ICso66.84 ± 2.88), S. heningsii (ICso67.16 ± 8.78), N. macrocalyx
(ICso84.56 ± 8.93), H. abyssinica (ICso 86.56 ± 3.21), C. edulis (ICso 148.53 ± 12.65),
A. nilotica (ICso 153.79 ± 15.79), A. indica (ICso> 250), W. somnifera (ICso > 250
ug/ml) for the aqueous extracts and: Z. usambarense (ICso 3.20 ± 0.45), F. angolensis
(ICso4.68 ± 0.09), M salicifolia (ICso51.07 ± 1.70), A. nilotica (ICso70.33 ± 1.89), H.
abyssinica (ICso72.66 ± 1.39), N. macrocalyx (ICso78.40 ± 4.68), W. somnifera. (ICso
125.59 ± 1.30), S. heningsii (ICso 157.91 ± 10.03), C. edulis (ICso> 250), A. indica
(ICso > 250 ug/ml) for the methanol extracts. Two plants, F. angolensis and Z.
usambarense, showed good in vitro anti-plasmodial activity (ICso3-6 ug/ml) against CQ-
sensitive strain.
Extracts of F. angolensis and N. macrocalyx were subjected to bioassay guided
fractionation to avail 9 compounds. 6,7-Epoxy-4,5,9-trihydroxy-13-hexadecanoate-20-
dodecanoate-l-tiglien-1-3-one (140), 6,7-epoxy-4,5,9,20-tetrahydroxy-13-tetradecanoate-
I-tiglien-3-one (141), 4,9-dihydroxy-20-hexadecanoate-13-dodecanoate-l,6-tigliadien-3-
one (142), 12-deoxyphorbol-13-pentadecanoate (145) and methyl 3-
heptaeicosanoyloxyoleanoate (143) are being reported for the first time while
stigmasterol (126) and montanin-20-palmitate (79) are known compounds. A further two
compounds, P3 and KI, were also isolated but not characterized.
The order of in vitro anti-plasmodial activity of some of the isolated compounds is: 4,9-
dihydroxy-20-hexadecanoate-13-dodecanoate-l ,6-tigliadien-3-one and montanin-20-
palmitate mixture (ICso 241.39±4.73), P3 (ICso 241.63±18.24), 4,9-dihydroxy-20-
hexadecanoate-13-dodecanoate-1,6-tigliadien-3-one (>250), stigmasterol (ICso >250) and
KI (ICso >250 ug/ml) against ENT30. Similarly, only P3 had mild anti-plasmodial
activity (ICso 237.47±11.23 ug/ml) against NF54. P3 had high cytotoxicity levels (LDso
18.36 ug/ml) as compared to the other compounds (LDso > 100 ug/ml).
1CHAPTER 1
INTRODUCTION
1.1 Background
Malaria is a parasitic protozoan disease, caused by minute protozoa m the genus
Plasmodium, is transmitted by the infected female anopheline mosquito. It infects human
and insect hosts alternatively. The name was derived from the Italian word mal-aria or
"bad air" and is also known as Roman fever. The disease probably originated in Africa
(along with mankind) as evident in the discovery of fossils of mosquitoes up to 30
million years old. From Africa, the disease accompanied human migration to the
Mediterranean shores, India and South East Asia (http://www-
microb.msb.le.ac.uk/224/BradleylHistory.html).
Malaria is a serious health care problem in the tropics and sub-tropics and has far
reaching medical, social and economic consequences (Nchinda, 1998). Globally, it
claims 2-3 million lives, and accounts for> 500 million clinical cases, annually. These
estimates have been increasing steadily over the last three decades (WHO, 2000;
Krogstand, 1996). It is estimated to cause 9 and 2.3% of disease burden in Africa and the
world, respectively. It is ranked third after pneumococcal acute respiratory disease
(3.5%) and tuberculosis (TB) (2.8%) among the infectious disease threats in Africa
(Nchinda, 1998; WHO, 1996). The high risk groups include those in whom immunity
has not yet developed (travellers, immigrants and children) and those in whom immunity
has diminished (pregnant women, immuno-compromised subjects and people from
endemic areas who have ceased to be routinely exposed to re-infection) (WHO, 1998;
Trigg and Kondrachine, 1998). To date, the pattern of immune response fully predictive
of protection has not been identified or validated. Acquired immunity wanes rapidly in
the absence of parasite exposure, and protection has been similarly short-lived in those
few sub-unit vaccine trials that have demonstrated measurable efficacy (Bradley et aI.,
1987). However, such vaccines might be useful for travellers.
Malaria in Africa account for approximately 90% of the global cases (WHO, 1996).
Annually, 25.3 million cases of malaria are reported in eastern Africa; of which 8.2
2million cases out of30 million Kenyans as compared to 8.6,5.3,2.0 and 1.2 million cases
for Tanzania, Uganda, Burundi and Rwanda, respectively (Jean-Marie, 2002). It is also
responsible for the greatest number of consultations (30% of new cases) in medical
centers within the public health service in Kenya. Consequently, it is most common
reason for hospital admission (22,000 cases, annually) in public hospitals. Each year,
malaria kills 26,000 children below five years
(http://www.msf.org/countries/page.cfm?articleid). More than 90% of malaria in Kenya
is caused by Plasmodium falciparum parasite transmitted by female Anopheles gambiae
mosquito (Khaemba et aI., 1994).
1.2 Malaria vector
The female anopheline mosquito is responsible for the transmission of human malaria.
Out of the 380 species of anopheline mosquitoes, only 60 can transmit malaria
(Kakkilaya, 2002). Their distribution varies from one region to another. The female
anopheline mosquito lays 30-150 eggs every 2-3 days. They exhibit the most regular
cycles of blood feeding and egg laying. When a mosquito feeds on blood from an
infected individual, it sucks the gametocytes (the sexual forms of the parasites). The
gametocytes continue the sexual phase of their lifecycle and the sporozoites invade the
salivary glands of the infected mosquito. When the infected female mosquito bites
human host for a blood meal, which it needs to nourish its eggs, it inoculates the
sporozoites into human blood stream, thus spreading the infection (Kakkilaya, 2002).
1.3 Malaria parasite
Malaria is an infectious disease caused by single-celled protozoan parasites of the genus
Plasmodium. About 120 species of Plasmodia have been identified but only four are
capable of infecting humans; P. ovale, P. vivax, P. malariae and P. falciparum,
subsequently causing a mild form of benign tertian malaria, benign tertian malaria,
quartan malaria and malignant subtertian malaria, respectively. The rest attack a variety
of animal hosts. P. vivax is the most wide-spread species extending throughout the
tropics, sub-tropics and the temperate areas, where it predominates and is rarely found in
Africa (WHO, 1997). P. falciparum is the most virulent species and predominates in the
3tropical and sub-tropical regions of Africa and South East Asia while P. malariae has the
same range but much less virulent (Ridley, 1997). P. ovale occurs in Central West Africa
and sporadically in the West Pacific region (powells, 1989). The mortality rate of P.
falciparum malaria remains high (10-50%) in spite of the advances made in medicine
within the last four decades (Warrel, 1988). The acute form causes fevers and serious
neurological complications such as cerebral malaria. Chronic infection of the disease
causes severe anaemia while pre-natal infection results in low birth weight (Greenwood
et a/., 1992).
The life cycle of Plasmodium parasite consists of two phases; the schizogonic (asexual)
in the vertebrate host and sporogonic (sexual) phase in the anopheline mosquito (Jensen,
1983). A mosquito feeding on blood during sporogony takes up the male and female
gametocytes which fuse to form a zygote that penetrates the stomach of the mosquito to
form an oocyst (Gamham, 1966). Within the oocyst, large numbers of sporozites
develop and pass through the body cavity with some entering the salivary glands.
Sporozites are inoculated into a new host through the infected saliva when a female
anopheline mosquito takes blood. The sporozites introduced by infected anopheline
mosquito into the vertebrate host disappear from the blood stream within half an hour and
enter parenchyma cells of the liver, where they develop into large schizonts. The
schizonts rapture after an incubation period of 1-2 weeks, releasing the merozoites into
the blood stream. A merozoite enters an erythrocyte and develops successfully into a
ring, trophozoite and schizont which rapture to release other merozoites, each of which
enters a new erythrocyte to perpetuate the erythrocytic cycle. The asexual cycle takes
about two days (36-48 hours) for P. falciparum, 72 hours for P. malariae and 48 hours
for P. vivax and P. ovale. Some merozoites in the red blood cells (RBC) differentiate
into male and female gametocytes. Female anopheline mosquitoes pick up malaria
parasites when they feed on the blood of infected humans. The duration of the extrinsic
incubation period in the mosquito varies with the temperature and Plasmodium species.
Below temperatures of 16 and 18°C P. vivax and P. falciparum, respectively, cannot
complete their developmental cycle (Wernsdorfer and Macgregor, 1988).
41.4 Control strategies
Control of malaria is generally achieved through chemotherapy and vector control.
Besides, attempts to develop vaccines for the prevention of malaria are actively being
pursued.
1.4.1 Vaccine development
Although the malaria parasite was discovered more than 120 years ago, it is only during
the past three decades that serious attempts on vaccine development have been made.
Effective vaccines for malaria could interrupt the life cycle of the parasite at different
stages in the human host. There are a number of stages of the parasite life cycle that are
being targeted for the development of vaccines. To date, more than 15 vaccine trials
have either been completed or are in progress, and many more are planned (Good, 2001).
Important candidate antigens include the surface proteins (SP) of the asexual merozoite
stage, the form that invades the host erythrocyte (Cowman, 2002). In the Republic of
Tanzania, a double-blind phase ill trial of the Colombian SPf66 peptide vaccine
demonstrated that it reduced the numbers of first malarial fevers in children by one third
(WHO, 1996). Peptide-based vaccines have successfully been used though they face the
challenge of toxic adjuvants, which are critical for immunogenicity of the synthetic
peptides (Ben Mohamed et al., 2002).
Other candidate trial vaccmes includes nucleic acids (Doolan and Hoffman, 2002),
asexual stage (Kumar et aI., 2002) and pre-erythrocytic stage (Ballou et al., 2002). RTS,
S is a novel pre-erythrocytic malaria vaccine based on the circumsporozoite surface
protein (CSP) of P. falciparum linked to hepatitis B surface antigen (HBs) and combined
with a novel adjuvant system (S/AS2) (Doherty et a/., 1999). A randomised trial of the
efficacy ofRTS,S/AS2 against natural P.falciparum infection in semi-immune adult men
in Gambia showed efficacy of 71% during the first 9 weeks of follow-up (Bojang et a/.,
2001). It is still undergoing trials in several countries. Scientists from several
organizations worldwide are currently working together to develop a multistage,
multigene DNA-based vaccine against P. falciparum malaria. This collaborative vaccine
5development effort is named "multi-stage DNA-based malaria vaccine operation"
(Kumar et aI., 2002).
Many factors make vaccine development difficult and challenging. Firstly, the size and
genetic complexity of the parasite mean that each infection presents thousands of
antigens to the human immune system. To date, at least 40 promising antigens have been
identified. Understanding these can be useful for vaccine development. Secondly, the
parasite changes through several life stages even while in the human host, presenting a
different subset of molecules for the immune system to combat at each stage. Thirdly,
the parasite has evolved a series of strategies enabling it to confuse, hide and misdirect
the human immune system. Finally, it is possible to have multiple malaria infections of
not only different species but also of various strains at the same time (Hoffman et a/.,
1996).
The recent sequencing of P. falciparum and An. gambiae genomes and development of
methods to manipulate the genes has provided an insight into the possible construction of
gain-of-function and loss-of-function mutants to analyse the role of parasite proteins.
This has provided new information on the role of merozoite antigens in erythrocyte
invasion and also allows new approaches to address their potential as vaccine candidates
(Cowman et al., 2002). In the absence of an operational vaccine either due to cost
functions, malaria control strategies need to be employed. Vaccines may be a tool for
malaria control in the developed world. However, in the third world, vector control may
be more appropriate than vaccines. It is also easier to control mosquito populations
within a given geographical area than giving vaccines for protection or administration of
drugs to individual persons.
1.4.2 Vector control
Vector control involves the use of methods targeted for controlling mosquito population
at larval or adult stages of their life cycle. Vector control has turned out to be an
effective method for malaria control (htp://www.liv.ac.uk/lstm/malaria/Techvector.html).
6Insecticides have been used in adult vector control. These are natural or synthetic
chemical compounds that will kill a given target insect. Insecticides especially
organochlorines like dichlorodiphenyltrichloroethane (DDT) have been used to control
mosquitoes. By 1964, malaria was eradicated from parts of India by use ofDDT house
spraying (Sharma and Mehrotra, 1986). However, its efficacy has reduced due to the
resistance developed by many insects (Kumar, 1984). DDT is non-biodegradable hence
a major environmental problem. It is also toxic to non-target organisms including man
(Charles et a/., 1995). Other synthetic insecticides such as organophosphate
(methylparathion, malathion, parathion, diazinon and fenthion) (Kumar, 1984),
organocarbamates (Baygone and 1-naphthyl-N-methycabarmate) (Kirk and Othmer,
1981) and synthetic pyrethroids such as permethrin; allethrin and cyclethrin have been
also used as effective insecticides (Metcalf et aI., 1962). However, they suffer from
resistance developed by mosquitoes against them and toxicity to non-target organisms
like DDT (Kirk and Othmer, 1981).
Larval population management has also been employed for control of malaria. In the
U.S.A, oil sprays on water has been used to control mosquito larvae (Wigglesworth,
1976). However, concerns about fate of other non-target organisms have discouraged
this practice. Inorganic larvicides like Paris green have also been used but the high heavy
copper content and its toxicity to other aquatic organisms have limited their practice
(Metcalf et al., 1962). The synthetic insecticides (organochlorines, organophosphates,
organocarbamates and pyrethroids) have all been used in larval control (Kirk and
Othmer, 1981). However, their persistence in environment, toxicity to non-target
organism and the increase in resistance developed by target mosquito have discouraged
their widespread use (Kumar, 1984; Kirk and Othmer, 1981). One of the earliest
researchers of the use of plants extracts against mosquito larvae found that plant alkaloids
like nicotine, anabasine, methylanabasine and lupinine extracted from Russian weed,
Anabasis apylla, killed larvae of Culex pipens Linn, C. territans Walker and C.
quinquefasciatus Say (Campbell et aI., 1933). Other bio-organic larvicides include
rotenone, azadaractin and unsaturated amides (Jacobson, 1989).
7Repellants have also been used to reduce host vector contact. These are substances that
protect man, animals, plants or products from insect attacks by making food or living
conditions unattractive or offensive. Synthetic repellants such as dimethyl phthalate and
2-ethyl-1,3-hexanediol have not provided a big impact in controlling the rate of mosquito
bites and transmission of malaria parasites since most of these repellants are highly
volatile and thus provides only short lived protection against the vector (Kirk and
Othmer, 1981). N,N-Diethyl-m-toluamide (DEET) is currently the most used synthetic
repellent (Beroza, 1970). However, concerns have been raised about its safety (Leach et
al., 1988; Qui et al., 1998). Other promising structurally similar synthetic chemical
repellents include n-propyl-N,N-diethylsuccinimate and O-chloro-N,N-diethylbenzamide
(Metcalf et a/., 1962). The discovery of p-menthane-3,8-diol as an effective mosquito
repellent from Eucalyptus citriodora (Curtis et a/., 1987; Barasa et a/., 2002) has
increased the choice of topical repellents. This compound is rapidly replacing DEET
from the market and is sold as Mosiguard® (Govere et a/., 2000). However, repellents
push vectors from a protected to unprotected host giving rise to ethical issues. These
issues are currently a subject of open and controversial debate.
There has been increasing interest in the use of insecticide treated nets (ITNs) for malaria
control. Bed nets are widely used against nuisance mosquitoes in China, Thailand, Latin
America, Papua New Guinea and Africa (Curtis, 1995). Since most Anopheles species
are active at night, it has been assumed that nets should reduce the chances of contracting
malaria (Lindsay and Gibson, 1988). In Gambia, Guinea Bissau and elsewhere,
introduction of ITNs in the communities remarkedly reduced parasite prevalence and
malaria cases (Marbiah et al., 1998). However, some mosquitoes bite before bed time.
Besides, some of the pyrethroids used in bed-net treatment may cause allergic reactions
when they come into contact with human skin (Wagner, 1994). Many people in rural
areas may not have beds. Nets may also be unaffordable to the rural communities.
Improper use of ITNs, non compliance and the unaffordable re-treatment regime has
reduced their impact on malaria transmission (Lines and Addington, 2001). Other
supplementary methods like chemotherapy still need to be investigated and new agents
discovered.
81.4.3 Chemotherapy
This involves use of chemical agents to fight the parasites once they are in the body. In
most cases, anti-malarial drugs are targeted against the asexual erythrocytic stage of the
parasite. The parasites degrades haemoglobin in its acidic vacuole, producing free heme
able to react with molecular oxygen and generate reactive oxygen species as toxic by
products (Francis et al., 1997). The most common pathway for detoxification of heme
moieties is polymerisation as malaria pigment (Slater et a/., 1991; Pagola et a/., 2000).
Majority of anti-malarial drugs act by disturbing the polymerisation (and the
detoxification by any other way) of heme, thus killing the parasite with its own metabolic
waste (Egan and Marques, 1999). The main chemical classes of active schizontocides are
4-aminoquinolines, aryl alcohols including quinoline alcohols and anti-folate compounds
which inhibit the synthesis of parasitic pyrimidines (Robert et a/., 2001). The newest
class of anti-malarials is based on the natural endoperoxide, artemisinin and its
hemisynthetic derivatives and synthetic analogs. Some anti-biotics are also used,
generally in combination with quinoline alcohols (pukrittayakamee et a/., 2000). Few
compounds are active against gametocytes and the intra-hepatic stages of the parasite.
The available commercial anti-malarial drugs can be classified into 5 groups based on
their chemical structural relationships.
1.4.3.1 8-Aminoquinolines
This is the only class of gametocytocides. Very few of these have been developed into
effective anti-malarials but they are useful in chemoprophylaxis.
1.4.3.1.1 Primaquine (1) has been widely used for the treatment of the hypnozoites (liver
reservoirs) responsible for the relapsing forms of P. vivax and P. ovale (Robert et a/.,
2001). However, primaquine (1) was recently reconsidered for malaria
chemoprophylaxis to eliminate P. falciparum at the early stage of infection, when
parasites develop in the liver, thus preventing the clinical symptoms (Basco et a/., 1999).
Despite its good oral absorption, the drug has a short half-life and needs to be
administered daily. Serious toxicity can be a major problem in patients with glucose-6-
9phosphate dehydrogenase deficiency (Robert et aI., 2001). Primaquine interferes with the
mitochondrial function of Plasmodium parasites (Robert et aI., 2001).
1.4.3.1.2 Tafenoquine (2) is a primaquine analog with a longer elimination half-life (14
days compared to 4 hours for primaquine) (peters et aI., 1993). It has a larger therapeutic
index than primaquine. This drug may be useful for chemoprophylaxis of P. falciparum
and for prevention of relapses of vivax malaria (Lell et aI., 2000).
1.4.3.2 4-Aminoquinolines
The main anti-malarials are the 4-aminoquinolines because they have been proven to be
the most successful class of compounds for the treatment and prophylaxis of malaria
(Robert et aI., 2001). They are easily synthesized, cheap and generally well tolerated.
The compounds as well as quinoline alcohols are active against the intra-erythrocytic
stages of the parasite. They are able to accumulate to high concentrations within the acid
food vacuole of Plasmodium and kill the parasite (O'Neill et aI., 1998). Several of these
are available in the market.
1.4.3.2.1 Chloroquine (CQ) (3) was introduced in 1944 and soon became the mainstay
of therapy and prevention, since the drug is cheap, non-toxic, and active against all
strains of malaria parasites. In 1994, CQ was the third most widely consumed drug in the
world after aspirin and paracetamol (Foster, 1994). The precise mode of action of the 4-
aminoquinoline-based anti-malarial drugs and the mechanism of parasite resistance are
still not completely understood. Chloroquine resistance was observed in South East Asia
and South America at the end of the 1950s and in Africa in the late 1970s (Trape et aI.,
1998; Legarde et aI., 1998). Resistant parasites accumulate CQ less avidly than sensitive
ones. Resistance can be reversed in vitro using drugs such as verapamil known to reverse
drug resistance in tumour cells (Bray and Ward, 1998). In spite of its reduced efficacy,
chloroquine is still the most used anti-malarial drug in most parts of Africa, due to cost
10
and the widespread prevalence of partial immunity among symptomatic children older
than five and adults. Moreover, tumour necrosis factor (TNF), a cytokine responsible for
some cerebral damages which is produced by immune system during the malaria crisis,
has been proven to have a synergistic effect with chloroquine, thus enhancing the effect
of the drug (Kwiatkowiski and Bates, 1995).
1.4.3.2.2 Amodiaquine (4) is chemically related, but more effective than CQ in clearing
parasitemia in cases of uncomplicated malaria, even against some chloroquine-resistant
strains (O'Neill et aI., 1998; Ringwald et al., 1996). However, drug resistance and
potential hepatic toxicity limit its use. Amodiaquine (4) has been shown to bind to heme
and inhibit heme polymerization in vitro, with better efficiency than CQ (Foley and
Tilley, 1998).
R
~CIA)l)
3 R =HNCH(CH3l(CH2bN(C2H5l2
CH;1N(C2H5l2
4 R~HN-o-OH
1.4.3.3 Quinoline methanols
1.4.3.3.1 Quinine (5), the active ingredient of cinchona bark, was introduced into Europe
from South America in the 17th century. It had the longest period of effective use, but
there is now a decrease of the clinical response of P. falciparum in some areas
(Pukrittayakamee et al., 1994; Zalis et a!., 1998). Nevertheless, it remains an essential
anti-malarial drug for sevete falciparum malaria and intravenous infusion is, in this case,
the preferred route. Quinine interacts weakly with heme, but has been shown to inhibit
heme polymerization in vitro (Robert et a!., 2001). The mechanism of resistance to
quinine is unknown, but a similar one to mefloquine has been suggested (Foley and
Tilley, 1998).
1.4.3.3.2 Mefloquine (6) (Lariam®) is structurally related to quinine, and its long half-
life (14-21 days) has probably contributed to the rapid development of resistance
(Wongsrichanalai et a!., 2002). For this reason, mefloquine should be used in
combination with other anti-malarial agents (Robert et a!., 2001).
11
5 6
1.4.3.4 Other aryl methanols
1.4.3.4.1 Halofantrine (Halfan®) (7) is effective against chloroquine-resistant malaria
(Ter Kuile et al., 1993). However, cardiotoxicity has limited its use as a therapeutic
agent (Nosten et a/., 1993). Mefloquine usage appears to lead to selection of parasites
resistant also to halofantrine (Wongsrichanalai et al., 1992). Furthermore, it is an
expensive drug.
7
1.4.3.5 Acridines
1.4.3.5.1 Pyronaridine (8), an acridine derivative, is a synthetic drug widely used in
China and may be useful for multi-resistant falciparum malaria (Elueze et al., 1996;
Ringwald et al., 1999). The current oral formulation is reported to be effective and well
tolerated in China. However, its oral bio-availability is low, contributing to an
unacceptably high cost of the treatment. It is likely that drug resistance would emerge
rapidly if pyronaridine is used as monotherapy (Robert et al., 2001).
8
As reported above, resistance to a lot of anti-malarial drugs has been observed in clinical
isolates, but resistance to mefloquine, quinine, and halofantrine appears to be inversely
12
correlated with resistance to chloroquine and amodiaquine, suggesting that the
development of a high level of resistance to chloroquine makes the parasite more
sensitive to the aryl methanols (Ward et a/., 1995).
1.4.3.6Folate antagonists
These compounds inhibit the synthesis of pyrimidines in the parasite, and consequently
DNA (Sirotnak, 1984). There are two groups of antifolates: (i) the dihydrofolate
reductase (DHFR) inhibitors, like pyrimethamine (9) and proguanil (10); and (ii) the
dihydropteroate synthase (DHPS) inhibitors including sulfones and sulphonamides like
sulfadoxine (11) and dapsone (12), respectively (Stanley et al., 1991). Due to the
observed marked synergistic effect, a drug in the first group is usually used in
combination with another in the second one (Cowman, 2001). Unfortunately, resistance
to this group of anti-malarials is widespread in Asia, India, and Africa (Cowman, 2001;
Ogutu et aI., 2000).
C'-oR R=NHC?,NHC?,NHCH(CH3)2
NH NH
9 10
11 12
1.4.3.6.2 Dapsone (12) is the parent of the sulfone drugs, and the major therapeutic agent
in this group for the treatment of leprosy (Stanley et al., 1991). It is also administered to
treat dermatitis, herpetiformis and malaria, and is used in combination with radiotherapy
in the treatment of gynecologic neoplasms (Stanley et al., 1991). Dapsone is also sold for
use as an accelerator in epoxy resins cross linking (Amato et a/., 2002).
13
1.4.3.7Other anti-malarial drugs
1.4.3.7.1 Atovaquone (13), a hydroxynapthoquinone, became available in 1992 and is
successful against uncomplicated P. falciparum malaria. It should be taken with food, to
improve absorption (Delmont, 2002). ~nCI
Wo..00-1....,1 I~ OHo
13
1.4.3.7.3 Lumefantrine (14) (beflumetol) was first registered in China as an anti-
malarial drug in the 1987. Pre-clinical trials showed that it had an EDso value of 1.02
mg/kg per day against P. berghei in mice. Little is known about its cross resistance with
other drugs (WHO, 1990). Studies of314 patients of Yunnan Province and Hainan island
in China gave cure rates> 96% with fever subsidence in 38-41 hours and parasite
clearance in 62-67 hours (WHO, 1990).
14
1.4.3.7.4 Promethazine
Promethazine is an affordable anti-histamine that acts through competition for H-l
receptor sites, on effector cells, with histamine. This H-l antagonist is also used as
adjunct therapy in the treatment of malaria in English-speaking West African countries.
The drug is given as an anti-emetic with chloroquine to prevent or alleviate chloroquine-
associated pruritus. Commonly, a dose of 5.0-10 mg is given simultaneously, or just
prior to administration of chloroquine in children withfalciparnm malaria in Nigeria. In
adults, daily doses of 25 mg are well tolerated (Oduola et al., 1998).
14
1.4.3.8Artemisinin derivatives
Artemisinin derivatives are the fastest active anti-malarial drugs (Meshnick et aI., 1996).
Four compounds have been used, the parent one, artemisinin (15), extracted from
Artemisia annua (Jain et aI., 1996) and other three derivatives that are actually more
active than artemisinin itself (Meshnick et aI., 1996; Cumming et aI., 1998). They
include the water-soluble hemisuccinate, artesunate (16); the oil-soluble, artemether (17);
and arteether (18). All of them are readily metabolized to the biologically active
metabolite, dihydroartemisinin.
7)0.0o .o
o
7)0.0o .o
OR
16 R = OCO(CH2)2COONa
17 R = Me
18 R = Et
15
Artemisinin is active at nanomolar concentrations in vitro on both CQ-sensitive or
resistant P. falciparnm strains. The treatment of several million patients with artemisinin
derivatives for acute malaria failed to detect any significant toxicity (Newton and White,
1999; Price et aI., 1999; Van Vugt et aI., 2000), even in pregnant women (McGready and
Nosten, 1999). Artemis inin and its derivatives appear to be the best alternative for the
treatment of severe malaria (Dhingra et a/., 2000), and artemether has been included in
the WHO List of Essential Drugs (Robert et a/., 2001) for the treatment of severe multi-
drug resistant malaria. In this family, the US Army through the Walter Reed Institute of
Researchers have patented a stable, water-soluble derivative, artelinic acid that is
currently being tested in animals (Li et a/., 1998).
A key advantage of these endoperoxide-containing anti-malarial agents, which have been
used for nearly two decades, is the absence of any drug resistance (White, 1998). When
several strains of P. berghei or P. yoelii were exposed to selection pressure by artemisinin
or synthetic analogs in infected mice, resistance proved very difficult to induce. A low
level of resistance has been observed, which disappeared as soon as the drug-selection
pressure was withdrawn. Similarly, with a synthetic analog of artemisinin (B07), the
15
resistance to the drug was lost when drug pressure was removed and was not regained
even when drug pressure was re-applied (peters and Robinson, 1999).
The major drawback of artemisinin derivatives is their short half-life (3-5 h). When used
in monotherapy, a treatment as long as 5 days is required for complete elimination of the
parasites. Consequently, they are preferentially used in combination with other anti-
malarial agents such as sulfadoxine-pyrimethamine (SP), benflumetol (lumefantrine) or
mefloquine to increase cure rates and to shorten the duration of therapy in order to
minimize the emergence of resistant parasites (White, 1998).
1.4.4 Combination of drugs
Resistance of P. falciparum to anti-malarials especially quinoline-based drugs such as
quinine has greatly affected chemotherapy and chemoprophylaxis of malaria. Several
attempts have, therefore, been made in developing drug combinations that could
circumvent chloroquine resistance in P. falciparum infections and hopefully restore
efficacy. The use of drugs in combination where scientifically justified, provides a
means of reducing the doses of individual drugs and also a possible way of circumventing
or delaying the induction of drug resistance (peters, 1987). Malaria chemotherapy and
prophylaxis is now targeting combination of drugs due to increased resistance of
individual conventional anti-malarials such as chloroquine. The basic tenet of
combination therapy is that the probability of resistance developing simultaneously to
two chemotherapeutic agents with independent mechanisms of action is extremely low
(one in 1012 treatments). This frequency is the product of the probabilities of the
acquisition of a resistant mutation to each drug multiplied by the number of parasites in a
typical infection (White, 1998). The anti-malarial activity of the combined drug may
either be potentiated (synergism), additive or antagonistic. An ideal combination is one
that is potentiating, well matched, has reduced toxicity and delays the emergence of
resistance to the individual components.
The combination of sulfadoxine and pyrimethamine (Fansidar®) represents one of the
most important chemotherapeutic agents currently used in treatment of chloroquine-
16
resistant malaria (Sowumni, 2002). However, more recent studies have indicated there is
evidence of increasing resistance to Fansidar® by P. falciparum in East Africa (Ogutu et
al., 2000). Combination of chloroquine and promethazine reversed chloroquine
resistance in standard P. falciparum clones and patient parasite isolates from Nigeria
(Oduola et al., 1998). The combination reduced the inhibitory concentration fifty (IC5o)
for chloroquine against the resistant parasites by 32-92%.
Malarone®, a new drug combination that was released in Australia in 1998, is a
combination of proguanil and atovaquone. It is highly effective for treatment of acute
uncomplicated malaria caused by P. falciparum resistant to first line treatment anti-
malarials although it is a very expensive drug (Kremsner et aI., 1999). A related
combination of atovaquone and tetracycline has also shown good synergism (Canfield et
al., 1995).
Maloprim®, a combination of dapsone and pyrimethamine has also been developed
though resistance to this drug is now widespread and its use in malaria chemotherapy is
no longer recommended (Canfield et al., 1995).
The most recent addition to malaria treatment in Africa is Lapdap®, a combination of
chlorproguanil and dapsone that was developed by the World Health Organization. It has
recently been approved by the UK Medicines and Healthcare Products Regulatory
Agency (MHRA) for the treatment of uncomplicated P. falciparum, the most life-
threatening malaria parasite. Lapdap® is effective against drug-resistant parasites and
has been available in several African countries since the end of 2003. Affordable new
anti-malarials are very much needed in sub-Saharan Africa, where other treatments are
failing due to increasing parasite resistance. Lapdap® is cheap to produce and has a short
half-life, which may be associated with better safety, although further assessment of this
new drug is required (Kanyok, 2003).
The effect of combination therapy is enhanced by the inclusion of an artemisinin
derivative. Artemisinin combinations decrease parasite density more rapidly than any
17
other anti-malarial drugs (White, 1997). When used alone, the short half-life of the
artemisinin derivatives minimises the period of parasite exposure to sub-therapeutic
blood levels. In combination to another drug with a longer half-life, the short half-life
and rapid parasite clearance time of artemisinin derivatives mean that fewer parasites are
exposed to the companion drug after elimination of the artemisinin component.
Furthermore, exposure occurs when blood levels of the drug close to a maximum are still
present (White, 1998). Another benefit of artemisinin combinations is the 90% reduction
in gametocyte levels in treated patients (price et aI., 1996). These characteristics
minimise the probability that a resistant mutant will survive therapy and may also reduce
overall malaria transmission rates. Combination of artemether or artesunate and
mefloquine has been used and is currently the standard treatment in areas of multi-drug
resistance in South East Asia (Robert et aI., 2001). When in association with
lumefantrine (benflumetol, a slow eliminating oral drug), artemether is as effective as
artesunate-mefloquine combination, and better tolerated. Artemether clears most of the
infection, and the lumefantrine concentration that remains at the end of the 3-5 day
treatment course is responsible for eliminating the residual parasites (Robert et aI., 2001;
Looareesuwan et aI., 1996).
In 2004, the Kenyan government changed the first-line anti-malarial therapy to
Coartem® (artemether-lumefantrine) due to the high levels of resistance to Fansidar®
(sulfadoxine-pyrimethamine) and chloroquine.
1.4.5 Triple therapy
Due to resistance of P. falciparum to single and double therapy (Mutabingwa et aI.,
2001), triple therapy is the most viable approach of malaria control. It slows down
development of resistance to the individual drugs (McIntosh and Greenwood, 1998).
Chloroquine plus sulfadoxine-pyrimethamine (SP), amodiaquine (alone or in
combination with SP) and Lapdap® a combination of chloroquine, proguanil and
dapsone (Winstanley et aI., 2002) have been used successfully and are also affordable.
Combinations of artesunate sulfadoxine-pyrimethamine and primaquine have also been
used (Chokejindachai et aI., 1999). Even with combination therapy, there seems to be no
18
hope of eliminating resistance development hence the need to find more effective anti-
plasmodial compounds for possible development into effective anti-malarial drugs.
Consequently, over 300,000 compounds isolated from plants have been tested for anti-
malarial activity by the Walter Reed Army Research Institute between 1965 and 1986
(http://www.userrpage.fu~berlin.de/~kayser/sec_2.htm).
1.5Hypothesis
There are Kenyan plants used in traditional malaria therapy in Kilifi and Meru districts
that may provide stable, isolable and identifiable compounds retaining their anti-
plasmodial activity singly or severally.
1.6 Objectives
The general objective was to validate the anti-plasmodial activity of traditional anti-
malarial plants from Meru and Kilifi and identify the biochemical principles therein.
These were to be achieved specifically through:
(i) Screening for anti-plasmodial activity of several plant extracts through in
vitro assay and cytotoxicity determination using brine shrimp nauplii;
(ii) Identification of the plants extracts with the highest anti-plasmodial
activity followed by detailed phytochemical investigations;
(iii) Isolation and purification of the anti-plasmodial compounds through
bioassay-guided fractionation of the plant extracts by chromatographic
techniques (CC, TLC, HPLC);
(iv) Identification of the active compounds using conventional spectroscopic
techniques (UV, IR, NMR, MS, Optical rotation and X-ray
crystallography);
(v) Bioassay of the pure isolated compounds for anti-plasmodial activity in
vitro.
1.6 Justification
Malaria has plagued humans throughout history and results in the death of over 2 million
people per year. Each year, there are 5 million malaria infections which lead to over
19
30,000 deaths in Kenya (Jean-Marie, 2002). The disease is endemic in the lowlands,
particularly the coastal strip and Lake Victoria basin where transmission is sufficiently
intense that both incidence and prevalence of infection reach more than 90% of the
population within 10-12 weeks after the beginning of the rainy season. Avoidance of
mosquito bites and use of vector control measures to reduce malaria transmission, such as
insecticide-treated nets (ITNS), indoor residual spraying (IRS); environmental
management to minimize potential mosquito breeding sites or make them unsuitable for
the development of mosquito larvae have been tried without much success. The re-
emergence of malaria as a public health problem is mainly due to the development of
resistance by P. falciparum to cheap highly effective drugs like chloroquine,
pyrimethamine, mefloquine, proguanil and sulphonamides. The failure to realise an
effective malaria vaccine has also contributed significantly to infection rate increase in
addition to development of resistance to insecticides.
Plant based systems continue to play an essential role in health care. It has been
estimated by the World Health Organisation that approximately 80% of the world
inhabitants rely on traditional medicines for primary health care (Arvigo and Balick,
1993; Farnsworth et al., 1985). Plants have provided a number of useful clinical anti-
malarial agents such as quinine and artemisinin, and have considerable potential as
sources of new drugs. Several natural products isolated from plants used in traditional
medicine have shown anti-plasmodial action in vitro and represent sources of potential
novel anti-malarial drugs.
20
CHAPTER 2
DRUG DISCOVERY
2.1 Introduction
The usual sequence to discover biologically active compounds from plant extracts is as
follows: Collection of ethno-botanical information, botanical identification of plants to be
investigated, preparation of extracts, pharmacological screening of bio-active
constituents, structural elucidation of bio-active compounds, structural modification and
synthesis, toxicological assessment of bio-active principles, synthetic derivatives and
clinical evaluation for therapeutic efficacy.
The desire to overcome some of the liabilities of available anti-malarial drugs makes the
discovery of new chemical entities even more challenging. It is desirable that new drugs
targeting treatment of uncomplicated malaria are efficacious against drug resistant
strains, provide cure within three days to ensure better compliance, be safe for small
children and pregnant women, have appropriate formulations and packaging for tropical
conditions, and be affordable. Other profiles include drugs that can be used for
intermittent treatment in pregnancy and in early infancy, as well as for severe and P.
vivax malaria (Solomon, 2003). There is also the prospect of combining new compounds
into combination products. The availability of the parasite, host and vector genomes has
given a boost to our search for new anti-malarial drugs. However, genomics need to link
with chemistry and high throughput screening before it can deliver drugs (Ridley, 2002;
Rosenthal, 2001). Pharmaceutical companies can support the genomics efforts by
providing chemistry and high throughput screening for the new drug targets being
identified.
In order for new chemical entities to make it to market as a safe and effective drug, they
must pass through a series of hurdles. The initial set of hurdles to overcome is passing
from the different drug discovery stages to the pre-clinical phase. A target based
discovery programme progresses from target identification to validation, hit generation
largely from high throughput screening, lead optimization and a lead drug candidate.
These steps are all important (Rosenthal, 2001). Analysis of why drugs fail in the clinic
21
shows that in 39% of cases are due to biopharmaceutical issues such as bio-availability
and formulation and 21% due to toxicity (Lipinski et aI., 1997). These issues are as
important as efficacy, which contribute 29% of failures (Solomon, 2003). Traditional
compound discovery screening involves cell-based screens to identify compounds with
potential therapeutic activities. Currently, available anti-bacterial and anti-malarial
compounds in the clinic today have benefited from semi-rational optimization
programmes based on compounds, often natural products identified by whole cell
screening. Therefore, integration of the strengths of traditional screening techniques with
target based rational approaches should be enhanced. Rodent malaria models are helpful
not only for defining the pharmacology and proof of concept efficacy studies, but also for
rapid feedback to medicinal chemists on aspects such as oral activity of compounds
(Solomon, 2003). The human malaria parasites do not infect rodents and specific rodent
malaria parasites such as P. berghei have been used. This has worked well for drugs
without specific enzyme target such as chloroquine and artemisinin. These drugs are
believed to target haem metabolism, in the food vacuoles, that are similar in rodent and
human malarial parasites. This scenario ceases to be true for enzyme targets where there
are differences in the protein sequence, structure and inhibitory specificity. In the
absence of sufficient similarity between P. berghei or other models and P. falciparum
enzymes, one has to either rely on cell culture data combined with rapid pharmacokinetic
evaluation or move to more expensive and low throughput primate models that can
sustain falciparum infection. Once a compound has shown satisfactory efficacy in
animal model, it is subjected to pre-clinical assessment which evaluates initial parameters
such as drug metabolism, pharmacokinetics, and toxicity in animals. After successfully
passing through the pre-clinical stage, the new chemical entities can progress to clinical
development. The investigational new drug (IND) will then enter phase I (safety and
tolerability in healthy volunteers); proceed to phase II (efficacy in a small number of
patients); and phase III (efficacy in large population of patients). Should the drug pass all
three clinical phases, it is submitted to regulatory authorities for approval as new drug
(Solomon, 2003).
22
A significant factor that increases costs in drug development is the high failure rate of
new chemical entities resulting in unnecessary development costs. It has been estimated
that about 1 in 5000 compounds make it from discovery to the pre-clinical stage. In
addition, 1 in 25 compounds that make it from the pre-clinical stage to IND stage will
make it to the market. Efforts are directed at a new series of in vitro assays (some being
developed) that serve as reliable indicators to minimize attrition rate of new chemical
entities. These models are helpful in reducing pre-clinical and clinical failure rates by
attempting to accurately evaluate efficacy and safety much earlier in the drug discovery
process (Solomon, 2003).
2.2 Brine shrimp lethality bioassay
The brine shrimp lethality assay is used in the preliminary determination of activities
based on cytotoxicity. It is an inexpensive, fast and general bio-assay for screening
compounds that can be done in-house. It was originally proposed by Meyer et a/. (1982)
and refined by McLaughlin et a/. (1988) and is an easy and quick way to detect general
bio-activity in plant extracts. Meyer et a!. (1982) developed fractionation monitoring of
physiologically-active plant extracts based on the assumption that bio-active compounds
are almost always toxic in high doses to zoological organisms. It is a useful tool to
monitor the isolation ofbio-active constituents.
Brine shrimp test has been previously utilised in various bio-assay systems such as
analyses of pesticide residues (Michael et a/., 1956), mytotoxins (Brown et aI., 1968),
stream pollutants (Hood et a/., 1960), anaesthetics (Robinson et aI., 1965), toxicity of oil
dispersants (Zillioux et a!., 1973), co-carcinogenic phorbol esters (Kinghorn et aI., 1967)
and anti-malarial compounds (Solis et aI., 1995).
2.3 Pharmacological models
Several models are available for the screening of plant extracts and pure compounds.
These tests require relatively small amount of samples. They are rapid, simple, reliable
and reproducible. Some of the models that have been used in the pharmacological
screening include in vitro, in situ, and in-vivo (Rasoanaivo, 1993).
23
2.3.1 In vitro model
The screening of plants for their anti-plasmodial activities was first employed by Spencer
et al. (1947) with avian malaria as the test parasites. It remained an open question
whether Spencer's result could be transferred to human malaria parasites raising the need
to develop a test method which could involve human malaria parasites, such as P.
falciparum, in the anti-malarial assays. The development of the technique for in vitro
culture of P. falciparum by Trager and Jensen (1976) led to a better understanding of the
biology of malaria parasites. One of its applications is the in vitro drug sensitivity assay
which, like in the case of anti-biotic sensitivity assay, can determine sensitivity or
resistance of the parasites to anti-malarial drugs. However, unlike anti-biotic assay, in
vitro drug sensitivity test for anti-malaria drugs is not yet standardised.
Subsequently, an in vitro anti-malarial drug screening method for P. falciparum was
developed by Desjardin et al. (1979). The method is based on the ability of the drugs to
inhibit uptake to radio-labelled nucleic acid precursor, CH]-hypoxanthine as determined
by scintillation counting. From the data, the concentration of the drug required to inhibit
the growth of 50% of the parasites (ICso) is calculated. The method is semi-automatic so
that a large number of samples can be tested simultaneously (Nkunya, 1992). The in
vitro anti-plasmodial assay continues to play an important role in screening of novel
compounds, analysis of in vitro cross resistance, effects of drug combinations,
determination of the phenotype of reference clones and strains, and epidemiological
description of drug resistance (Ringwald and Basco, 1999). There are three in vitro
models involving the use of isolated organs, parasite cultures, enzyme inhibition and
receptor binding assays.
2.3.1.1 Isolated organs
The technique measures contraction or relaxation of an organ or a portion of it removed
from a freshly killed animal and suspended in a tissue bath containing physiological
solution maintained at 37°C and chosen to nourish the tissue. Contraction, relaxation or
inhibition of contractions of the tissue caused by addition of the test extracts with or
without addition of the reference antagonist or agonist compounds as well as their
24
duration are indicative of pharmacological-activity and therefore are recorded through a
transducer. Some of the isolated organs commonly used in pharmacology are ileum and
vas deferens of guinea pig, uterus and duodenum of a rat and heart of a rabbit among
others. This technique is very useful in the investigation of the mechanism of action of
pure compounds (Rosel and Aguvel, 1975), but is rarely used as a screening method with
plant extracts.
2.3.1.2 Enzyme inhibition (EI) and receptor binding assays
It measures the inhibitory effect of plant extracts on specific enzymes involved in the
expression of a disease. The receptor binding methods evaluate the ability of a
compound or a drug to recognize a receptor selectively. EIA has been used as a tool in
the search for new drug substances, investigating the biochemical mechanisms of action
of drugs, screening of plant extracts and in the search for a new drug substances (y.leiryb
et a/., 1972). Ohkanda et al (2001) has shown potent anti-malarial activity of
peptidomimetic inhibitors of farnesyl transferase protein.
2.3.1.3Parasite cultures
External agents such as bacteria, viruses, fungi and protozoa cause infectious and
parasitic diseases. Progress in basic and applied science has made it possible to cultivate
these agents in vitro. Culture-based assays involve the evaluation of the inhibitory effect
of plant extracts on bacteria, viruses, fungi, parasite and cell growth. They are usually
cultivated at 37°C in a defined synthetic medium supplemented by some kind of serum.
The inhibition of bacteria, fungi, viruses, protozoa or cell growth by addition of test drug
or substance is indicative of biological activity. This is expressed quantitatively as
inhibitory concentration fifty (ICso), which is the dose of plant extracts that inhibit the
living material growth by 50%.
Culture methods have successfully been used in the screening of anti-malarial, anti-
fungal, anti-bacterial, anti-viral, anti-parasitic and anti-tumour drugs (Rasoanaivo, 1993).
The in vitro continuous culture of P. falciparnm was pioneered by Trager and Jensen
(1976; 1977; 1978) and Jensen (1979; 1983) and contributes to the decisive progress in
25
the anti-malarial drug research. This method enables both asexual intra-erythrocytic and
sexualforms of the parasite to be obtained and used for in vitro assays.
In vitro anti-plasmodial test is better than the in vivo test in that it is faster and P.
falciparum parasite responsible for the falciparum malaria in man is used as the test
organism unlike in vivo, which takes a longer time and uses P. berghei, which infect
mice. The in vitro anti-plasmodial assay can be used for the detection of interaction
between drugs (synergism, antagonism or additive properties) (Martin et aI., 1987). If for
example chloroquine and another substance are combined in a series of concentrations, it
is possible to evaluate the influence of the substance on the ICso of chloroquine against
sensitive or resistant strains of P. falciparum. Initially, a normal in vitro anti-plasmodial
assay is carried out to establish the ICso of chloroquine and that of the substance alone.
These results determine the doses to be used in the drug combination study. Serial
concentrations of the substance ranging from half of the ICso down to one tenth are
combined, respectively, with the same concentrations of chloroquine. The ICso of each
compound acting alone is assigned an isobolar unit of 1.0 and the ICso values of each
compound used in combination is converted to fractional isobolar equivalent as follows:
Isobolar equivalent for chloroquine = ICso (chloroquine + substance)
ICso (chloroquine alone)
Isobolar equivalent for extract = ICso (substance in combination)
ICso (substance alone)
A plot of isobar equivalents of chloroquine in combination (in the abscissa) against isobar
equivalents of the substance in combination (in the ordinate) gives an isobar graph.
Interpretation of this graph could indicate synergism, antagonism or additive properties of
the drug. This technique has been successfully used to demonstrate drug-potentiating
effect in both synthetic and natural products (Martin et aI., 1987; Rastimamanga-Urverg
et al., 1992).
26
One of the major disadvantages is that the parasite in vitro method detects the anti-
plasmodial activity but does not give the information about the mechanism of action of
bio-activeconstituents.
2.3.2 In vivo model
This model utilizes intact, non-anaesthetized animal. The process involves the induction
of a specific disease, in most cases by the administration of an appropriate reference
substance or organism, to selected animals. Test drugs are given orally, intraperitonically
or intravenously. Changes of some biological parameters are recorded to evaluate the
efficacy of the test substance. The disadvantages include availability of substantial
amounts of test substances and high risk of false results. Nonetheless, it has been
successfully used in anti-malarial drug screening programs (Rasoanaivo, 1993). It is
usually the last step before the involvement of human subjects in drug testing. It avails
more toxicological and drug efficacy data and is therefore critical in the decisions to
proceed to the human subject level. Several levels may be involved here; rodent models
in particular those using P. berghei, P. yoelii, P. vinkei, P. chabaudi and infections in
mice (peters, 1980; Cox, 1988) and primates models of P. falciparum, P. vivax and P.
knowlesi infection using Owl and squirrel monkeys and or Aotus and Saimiri species
(Kocken et al., 2002; Cogswell, 1999). Most primate malaria parasites exhibit tertian
periodicity, completing one asexual cycle every 48 hours. Exceptions include P.
knowlesi which has a quotidian (24 hour) cycle, P. inui and P. brasilianum which have a
quartan (72 hour) cycle. In the rhesus macaque, P. knowlesi produced a fulminating,
often fatal infection within 13.6 days (Cogswell, 1999). In vivo model is a very
expensive undertaking.
2.4.3 In ~itumodel
This model utilizes infected anaesthetized animal. Test compounds are injected directly
into target organs. This model is not very common in screening programs (Rasoanaivo,
1993). It is rarely used as it does not mimic the usual drug administration mode in
humans.
27
CHAPTER 3
ANTI-PLASMODIAL PLANTS AND ACTIVE PRINCIPLES
3.1Introduction
The resurgence of malaria is attributed to the development of resistance by the malaria
parasite,especially P. falciparum, to the most of the available drugs and the resistance of
vector anopheline mosquitoes to insecticides (Mutabingwa et aI., 2001; Kumar, 1984).
Discovery of new drugs in this field is therefore a health priority especially if their mode
ofaction is different from those of the available drugs. Several new molecules are under
investigation. Chemotherapy is currently the primary strategy for malaria control in the
world. However, due to resistance to the commonly used anti-malarial drugs, new
compounds are required. The plant kingdom offers a territory little explored for the
presence of pharmacologically-active compounds yet heavily depended on by indigenous
traditional communities.
A number of alkaloids, terpenoids, quinoid and phenolic compounds from higher plants
have shown activity against protozoa (Phillipson and O'Neill, 1987). The majority of
these compounds have been evaluated for in vitro and in a few cases in vivo activity in
animal models. Very few of the reported anti-malarial compounds have been assessed
clinically. Several thousands of anti-plasmodial compounds from all classes of natural
products have been isolated from different plants. A review of the same is attempted
below, though far from exhaustive.
3.2 Anti-plasmodial compounds from nature
3.2.1 Phenols
Simple phenols that are widely distributed in nature have shown characteristic inhibition
of malaria parasite growth. From Hypericum calycinum (Hypericaceae), a prenylated
phloroglucinol derivative (19), inhibited P. falciparum growth in vitro with an EC50 value
of 0.88 ug/ml (Decosterd et aI., 1991). Anti-plasmodial activity of 2'-
epicycloisobrachycoumarinone epoxide (20) and its sterioisomer isolated from Vernonia
brachycalyx (Asteraceae) have been reported. Both sterioisomers show similar in vitro
activity against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum
28
with ECso values of 0.11 and 0.15 ug/ml respectively (Oketch-Rabah et al., 1997a). A
new coumarin derivative, 5,7-dimethoxy-8 -(3'-hydroxy -3'-methy I-I '-butene )-coumarin
(21) has also been isolated from Toddalia asiatica and was found to have ICso value of
16.2± 1.4 and 8.8 ± 1 .6 ug/ml against chloroquine-sensitive and resistant P. falciparum
isolates,respectively (Oketch-Rabah et al., 2000) ..
Y"0~
OH S~:~'0 : 0 0OH
19 20 21
3.2.2 Chalcones
Phlorizidin (22), from Micromelum tephrocarpum (Rutaceae), was one of the first
chalcone glycoside reported to exhibit anti-parasitic activity (Kayser et al., 1998). In
ethno-medicine M. tephrocarpum is used to treat malaria because of the bitter taste, a
property shared with quinine and other anti-malarial herbs. Phlorizidin inhibits the
induced permeability in Plasmodium infected erythrocytes to various substrates including
glucose. The most promising compound in this class of natural products is licochalcone
A (23). It was first isolated from Glycyrrhiza glabra (Fabaceae) and was the subject of
intensive preclinical studies (Zhai et al., 1995). Starting with liclochalcone A as a lead
structure, a large number of chalcones have been synthesised and structure-activity
relationships documented (Nielsen et al., 1998).
~OH~OHOH0 In
GlueO 0
OH
o
22 23
29
3.2.3Flavonoids
Flavonoids are widespread In the plant kingdom. Following the detection of anti-
plasmodial flavonoids from Artemisia annua (Asteraceae) this class of compound has
attracted renewed interest. Elford et al (1985) demonstrated that methoxylated
flavonones artemetin (24) and casticin (25) act synergistically with artemisinin against P.
falciparum in vitro. As part of a multi-disciplinary research programme on anti-
plasmodial drugs, additional Artemisia species have been screened in Thailand (Elford,
1985), and exiguaflavanone A (26) and B (27) isolated from Artemisia indica
(Asteraceae) exhibited in vitro activity against P. falciparum with ECso values of 4.6 and
7.1 ug/ml, respectively (Elford, 1985).
OR
Hz(:
26 R=H
25 R=H 27 R=CH3
o
OH 0
3.2.4 Naphthoquinones
Naphthoquinones and other related quinoid compounds are one of the major natural
product classes with significant activity against Leishmania, Trypanosoma and
Plasmodium. Many have been isolated but frequently their potential use has been limited
by low bio-availability and high toxicity (Wright and Phillipson, 1990; Sepuvelda-Boza
and Cassels, 1996; Fournet et a/., 1992; Akendengue et al., 1999). The plant derived
product hydrolapachol (2-hydroxy-l,4-napthoquinone) (28) was shown to have activity
against P. lophurae in ducks in the 1940s (Hooker, 1936; Hudson, 1984). Lately,
plumbagin (29), a cytotoxic napthoquinone has been isolated from Plumbago zeylanica
has been found to exhibit anti-plasmodial activity (ICso 178.12 and 188.8 ng/ml) against
chloroquine-sensitive (D6) and resistant (W2) isolates, respectively (Lin et aI., 2003;
Obua et al., 2002).
o
~OH0Y
o
OH 0
~ o
28 29
30
3.2.5Anthraquinones and xanthonones
This group is related to naphthoquinones in structure and biological activity. The main
chemical difference between the groups is the tricyclic aromatic system with a para-
quinoid substitution. Anthraquinones isolated from the tropical tree Morinda lucida
(Rubiaceae) were tested for anti-plasmodial activity in vitro. Digitolutein (30), rubiadin-
I-methyl ether (31) and damnacanthal (32) showed activity on chloroquine-resistant P.
falciparum isolate (ECso ;:::21.4 - 82.9 J.lM) (Sittie et a/., 1999). Other rare anthraquinones
have been identified as potential anti-plasmodial drugs. From Psychotria camponutans
(Rubiaceae), the benzoisoquinoline-5-10-dione (33) has been isolated and tested against
P.falciparum (ECsoO.84 ug/ml) (Solis eta/., 1995).
30 RJ = H; R2 = CH3; R3 = OCH3
31 RJ = OCH3; R2 = CH3; R3 = H
32 RJ = OCH3; R2 = CHO; R3 = OCH3
o$'"N. I I"" ""
o
33
Anti-plasmodial xanthones have been isolated from Garcinia cowa (Guttiferae).
Preliminary screening of five prenylated xanthones demonstrated significant activity
against P. fa/ciparum in vitro with ECso ranging between 1.5 and 3.0 ug/ml.
Cowaxanthone (34) displayed a good anti-plasmodial potential (ECso = 1.5 ug/ml)
compared to that of pyrimethamine (ECso 2.8 ug/ml) (Likhitwitayawuid et aI., 1998).
OH 0 OH
OH
34
3.2.6 Terpenes and related compounds
3.2.6.1 Monoterpenes
Monoterpenes are examples of simple anti-protozoal drugs. Espintanol (35) isolated from
Oxandra espinata (Annonaceae) was shown to exhibit an IC90 of 25-100 ug/ml against
twenty different T. cruzi strains (Kayser et aI., 1998). Piquerol A (36) from the same
plant had an ICso of 100 ug/rnl against P. falciparum (Kayser et aI., 1998).
31
OH'¢Y'
OMe
35 36
3.2.6.2 Sesquiterpenes
The anti-protozoal potential of sesquiterpenes is well established since artemisinin (15)
and its derivatives were identified as new drugs with high clinical preference. In addition
to sesquiterpene endoperoxides, other sesquiterpenes with anti-plasmodial activity have
been reported. The sesquiterpene lactone parthein (37) has an EC50 value of 1.29 ug/ml
against P. falciparum in vitro (picmun et aI., 1979). From Neuroleaena lobata
(Asteraceae), a medicinal plant used in Guatemala for the treatment of malaria infection,
activity was documented for germacranolide sesquiterpene lactones, neurolenin A (38)
and B (39) (Francois et aI., 1996). Two sesquiterpenes, 5-isopropyl-3,3,9-tri-
methylbicyclo-nona-5-en-4-o1 (40) and 9,1O-tris-epoxy-pentadec-12-1 ,2-diene (41) with
significant anti-plasmodial activities (ECso < 4 ug/ml) were isolated from red marine
algae Laurencia implicate (Rhodomelaceae) and brown algae Potriera hornemannii
respectively (Rhizophylladaceae) (Konig et aI., 1991).
a~CH2
H:zC
o CHz
o
38R=H
39 R=OAc
37
Br
Br_~o,- )11
~-V-
40 41
32
3.2.6.3 Diterpenes
Diterpenes from many species are well known for their biological activity and are among
the most widely distributed terpenes in the plant kingdom (Kayser et a/., 1998).
However, most of them combine high anti-parasitic activity with high cytotoxicity to
mammalian cells (Oketch-Rabah et a!., 1998). The macrocyclic germacrane dilactone,
16,17-dihydrobrachy-calyxolide (42), from Vernonia brachycalyx (Asteraceae) has good
anti-plasmodial activity (EC50 17 ug/ml on P. falciparum) but also inhibits the
proliferation of human lymphocytes at the same concentration indicating general toxicity
(Oketch-Rabah et a/., 1998). Other anti-plasmodial diterpenes are phytol (43) and 6-E-
geranylgeraniol-19-oic acid (44) isolated from Microglossa pyrifolia (Asteraceae). They
have been found to have high anti-plasmodial activity, IC50 8.5 ug/ml (PoW); 11.5 ug/ml
(Dd2) and IC50 12.9 ug/ml (PoW); 15.6 ug/ml (Dd2), respectively (Kohler et aI., 2002).
o
OH
~CHPH
2
42 43 44
3.2.6.4 Triterpenes
Triterpenes and saponins from plant sources are known for their biological activity, but
exhibit some toxicity to humans and other mammals. Despite the fact that triterpene
action in biological systems is well known, the first rational report on their anti-protozoal
activity was described in late 1970s (Kayser et al., 1998). Betulinic acid (45), also
known for its anti-neoplastic effect, was identified by bio-guided fractionation to be the
anti-plasmodial principle of Triphyophyllum peltatum (Dioncophyllaceae) and
Ancistrocladus heyneanus (Ancistrocladaceae). It had an EC50 value of 10.46 ug/rnl
against P. falciparum in vitro and moderate cytotoxicity (EC50 >20 ug/ml) (Bringmann et
al., 1997; Majester-Sarvonia et al., 1991). The use of saponins as drugs is limited to due
to the poor bio-availability, reduced absorption in the gastrointestinal tract and
haemolytic toxicity when given orally. Despite this fact, medicinal plants that contain
33
saponins are known. From Asparagus africanus (Liliaceae), a new steroidal saponin,
muzanzagenin (46) that exhibited anti-plasmodial activity of ECso 61 JlM against K39
isolateof P. falciparum, was isolated (Oketch-Rabah et aI., 1997b).
_ I (?yc","
HO-~~
45 46
3.2.6.5Limonoids
Limonoids are also known as bitter terpenoids (Kayser et al., 1998). One well known
plant family rich in these is Meliaceae. Azadirachta indica, the neem tree, widely used
as an anti-plasmodial plant in Asia belongs to this family. Nimbolide (47) (ECso = 0.95
ng/mI, P. falciparum Kl) was the first to be identified as the active anti-plasmodial
principle of the neem tree (Rochanakij et a/. , 1985). Subsequently, gedunin (48) was also
found to be active in vitro against P. falciparum parasites with ECso values in the range
of 0.72-1.74 ug/ml (Khalid et a/. , 1989; Badam et a/. , 1987; McKinnon et al., 1997).
a COOCH3
~
a
4-0a
47 48
3.2.6.6 Quassinoids
They are biosynthetically related to triterpenes and share the same metabolic precursors.
The anti-plasmodial activity ofbrusatol (49) and ailanthinone (50) is high (Cabral et a/. ,
1993). The most active compound in this group is simalikalactone D (51) from Simaba
guianensis (Simaroubaceae) (ECso < 0.02 ug/ml) but was found to be too toxic in vivo
(Cabral et al., 1993). Quassinoids inhibits protein synthesis (Kirby et a/. , 1989). Activity
of the compounds in this group is due to the methylene-oxygen bridge. This explains
why quassin (52) is inactive, while its synthetic derivatives chaparrinone (53) and 15-
desacaetylundulatone (54) have high anti-plasmodial activity against P. falciparum NF54
(ECso= 0.037 and 0.047 ug/ml, respectively) (Francois et aI., 1998).
34
OH
H~O HO. ~ _.
o R
o 0
OH
OH
~
O"O
o R
"" 010
49 R = O-tigJate 50 R = O-tigJate 51 R = O-tiglate
52
OH
~
HO" _-
o .
o
"" , 0 0
R
53 R=H
54 R = O-tiglate
3.2.7 Alkaloids
Alkaloids constitute one of the most important classes of natural products providing
drugs for humans since ancient times. Most alkaloids are well known because of their
toxicity or use as psychedelic drugs, for instance cocaine and morphine, but many
alkaloids have a deep impact on the treatment of parasitic infection, for example quinine
(Kayser et aI., 1998).
3.2.7.1 Naphthylisoquinoline alkaloids
This type of alkaloids shows a remarkable activity against P. falciparum in vivo and in
vitro. Extracts from the species of Triphophyllum peltatum (Dioncophyllaceae) and the
isolated compounds; dioncopeltine A (55), dioncophylline B (56) and C (57) exhibit high
anti-plasmodial activity (Francois et al., 1997). Doncopeltine A was shown to suppress
parasitaemia almost totally while dioncophylline C (57) cured infected mice completely
after oral treatment with 50 mg/kg daily for 4 days without noticeable toxic effects
(Francois et al., 1997). Recently, a novel dimeric anti-plasmodial napthylisoquinoline
alkaloid heterodimer, korundamine A (58), has been isolated from another species,
Ancistrocladus korupensis in the same family. It is one of the most potent naturally
occurring anti-plasmodial naphthylisoquinoline dimmers yet identified by in vitro
screening with an ECso of 1.1 ug/ml against P. falciparum (Hallock et aI., 1998).
35
OH
58OH
3.2.7.2 Quinolines
Up to the middle of this century, quinine (5) was used for the treatment of malaria, and
with the widespread development of chloroquine-resistant strains of Plasmodium
falciparum it has become important again (Kayser et aI., 1998). Quinine was the lead
structure in the discovery of synthetic derivatives (like chloroquine (3) and mefloquine
(6)) that have higher anti-malarial activity. Other natural quinoline derivatives like 2-n-
propylquinoline (59), chimanine B (60) and 2-n-pentylquinoline (61) have been shown to
exhibit activities ofECso = 25-50 ug/ml against parasites causing cutaneous leishmaniasis
(Kayseret aI., 1998).
59 R, = C3H7• R2 = H
60 R, = CH=CHCH3. R2 = H
61 R, = C5H". R2 = H
3.2.7.3 Bisbenzylisoquinolines
A number of different bisbenzylisoquinolines with anti-protozoal activity have been
identified. In vitro anti-plasmodial activity of most bisbenzylisoquinolines is < 1.0 ug,
close to the ICso of chloroquine (ICso - 0.2 ug/ml). For instance, pycnamine (62) from
Trichilia sp. was found to have ICso value of 0.15 ug/ml (Kayser et aI., 1998). However,
monomeric benzylisoquinolines do not have potential anti-plasmodial activity (Kayser et
al., 1998). Some aporhinoids, like isoguattouredigine (63) from Guatteria foliosa have
been tested for anti-plasmodial activity (Kayser et aI., 1998).
36
63
3.2.7.4 Indoles
Indoles comprise a group of alkaloids with varied biological activity (Kayser et a/.,
1998). The indole sub-structure is widely distributed in the plant kingdom. Some indoles
are reported to possess anti-protozoal activity. For instance, cryptolepine (64) and related
indole-quinolines isolated from Cryptolepis sanguinolenta were active in vitro against
(ECso= 27-41 ng/ml) against P. falciparum (yV2, D6 and Kl) though they were found to
be mildly active in vivo (10.8-19.4% through suppression of P. yoelii at 100 mg/kg/day)
(Kayser et a/., 1998).
64
3.2.7.4 Phenanthridine and benzophenanthridine
These alkaloids are mostly found within three plant families only; Papaveraceae,
Fumariaceae and Rutaceae (Krane et aI., 1984). Some examples of benzophenanthridine
alkaloids obtained from plant sources are fagaronine (65), nitidine (66), sanguinarine (67)
and chelirubine (68). Anti-malarial activity of nitidine has been reported (IC5o= 9 - 108
ng/ml) against P. falciparum (Gakunju et aI., 1995).
MeO
OH
OMe MeO
MeO MeO
65 66
67 R=H
68 R =-OMe
Traditional medicinal practices have previously provided potent anti-malarial compounds
like quinine and artemisinin or lead compounds for synthetic anti-parasitic drugs. It is
37
therefore important to test for anti-plasmodial activities of medicinal plants that herbalists
use to cure malaria. In this study, anti-plasmodial activities of 10 plants used by
herbalists in Kilifi District (endemic zone) and Meru District (epidemic zone) to treat
malariawere evaluated with laboratory adapted isolates of P. falciparum in vitro.
3.3Anti-malarial plants
Previous survey done by the Centre for Traditional Medicine and Drug Research
(CTMDR) at KEMRI identified 10 plant species as potential candidates for activity tests
against P. falciparum. These included Strychnos heningsii, Zanthoxylum usambarensis,
Carissa edulis, Withania somnifera, Fagaropsis angolensis, Neoboutonia macrocalyx,
Harrissonia abyssinica, Azadirachta indica, Myrica salicifolia and Acacia nilotica. They
are used singly or in combination depending on the illness (Kokwaro, 1993; Rukunga,
Personal communication). These plants were identified after interviewing traditional
healers in Meru and Kilifi District and selected on the basis of their success against
malaria. Previous ethno-botanical, pharmacological and chemical investigations of the
plants are summarized below.
3.3.1 Carissa edulis (Forsk.) Vahl (Apocynaceae)
Warm root decoction of C. edulis (Apocynaceae) locally referred to as Kamuria (Meru) is
taken for indigestion, lower abdominal pains and for treatment of malaria. An infusion of
roots together with other medicinal plants is used for treating chest pains (Kokwaro,
1993). It exhibits anti-diabetic activity (El-Fiky et aI., 1996). Previous chemical
investigations revealed presence of alkaloids (Omino and Kokwaro, 1993) and 2-
hydroxyacetophenone (Bentley et aI., 1984). The methanolic extract of Carissa edulis
contains about 5% sesquiterpenes (Achenbach et aI., 1985). Besides carissone (69),
cryptomeridiol (70) and ~-eudesmol (71), three hitherto unknown sesquiterpenes of the
eudesmane-type (72, 73) and a novel germacrane derivative (74) have been isolated from
this plant (Achenbach et aI., 1985).
69 70 71
38
74
3.3.2Neoboutonia macrocalyx Pax (Euphorbiaceae)
The stem of N. macrocalyx (Euphorbiaceae) commonly known as Mutuntuki (Meru) is
used to treat headache and fever problems. To the best of our knowledge, there has been
no previous chemical investigation on this plant. However, from the dichloromethane
extract of the leaves of Neoboutonia melleri two new tigliane diterpenoids, mellerin A
(75) and B (76) along with three known sterols (24R)-24-ethylcholesta-3~,5a,6~-triol
(77) and 7f3-hydroxysitosterol (78), respectively, have been isolated (Zhao et a/., 1998).
75
"M
no ~,m5JJ .
OH
77
76
HO
78
Lately, from the stem bark of N. glabrescens, two daphnane diterpenoids,
montanin-20-palmitate (glabrescin) (79) and montanin (80), neoboutonin (81) a
degraded diterpenoid with a novel skeleton) and neoglabrescins A (82) and B (83)
(two rhamnofolane derivatives), have been isolated (Tchinda et a/., 2003).
OH
HO
81
39
~
~ 0
Ho-
~ .OH
OH
HO HO
82 83
Other compounds isolated from the same family include the tigliane diterpenoids 4-
deoxyphorbol ester (84) from Euphorbia obtusifolia (Marco et a/., 1999), langduin A
(85), 12-deoxyphorbol-13-hexadecanoate (86) and prostratin (87) from E. fischeriana
(Ma et al., 1997), baliospermin (88) from Baliospermum montanum (Harbone and
Herbert, 1993)
O
~~·i,B.)y '.iB = isobutyrate
CH20H 85
84
87 R = Ac
~
' ,OR
s-: .HO \
°HO ,-:;
88
and the daphnane diterpenoids huratoxin (89) and 12p-acetoxy huratoxin (90) from
Wikstroemia retusa (Yaga et aI., 1993).
89 R =H
90 R =OAc
3.3.3Acacia nilotica (L.) Del. (Leguminosae)
Bark juice of A. nilotica (Leguminosae), sub-family Mimosoideae, locally known as
Mkufu (Gogo) and Munga (Giriama) is used for the treatment of sore throat and coughs.
40
The leaves are boiled in tea or coffee as a treatment for chest pains or pneumonia while
boiledroots are used for indigestion or stomach troubles. Bark and roots are used as an
aphrodisiac, roots for gonorrhea and treatment of chest diseases. A decoction of the bark
is also given to children with fever (malaria), drunk to aid digestion, impotence or as a
powerful stimulant. It exhibits anti-mutagenic, chemopreventive (Kaur et aI., 2002),
strong anti-oxidant (Saleem et aI., 2001), anti-bacterial (Kambizi and Afolayan, 2001),
anti-hypertensive, anti-spasmodic (Gilani et aI., 1999), anti-plasmodial (El-Tahir et aI.,
1999a), anti-inflammatory (Dafallah and Al-Mustafa, 1996), anti-microbial (Sotohy et
aI., 1995) and molluscidal (Kela et aI., 1989) properties. Previous chemical
investigations revealed presence of proanthocyanidins, phenols (Dube et aI., 2001),
polyphenols and tannins (Sotohy et aI., 1995). There has been no report of the isolation
of the anti-plasmodial compounds.
3.3.4Strychnos heningsii Gilg. (Loganiaceae)
Root decoction of S. heningsii (Loganiaceae) locally referred to as Muchambi (Meru) is
used for treatment of malaria, chest pains and internal injuries. Fresh roots are also used
(chewed) as antidote for snake bite (Kokwaro, 1993). It exhibits anti-diarrhoeal activity
(Shoba and Thomas, 2001) and anti-alcoholic effect (Sukul et al., 2001). Previous
chemical investigations revealed the presence of brucine (91) and strychnine (92) (Gu et
al., 1997) in addition to other alkaloids like strychnobrasiline (93), strychnocarpine (94),
strychnopivotine (95) and strychnosilidine (96) (Kuehne and Xuf, 1997). Strychnine
isolated from Strychnos icaja has shown anti-malarial activity (philippe et al., 2003).
41
~
NMe
. II
:7
~ I f"! 0
Ac
91 Rl = R2= MeO
92 Rl = R2= H
93 94
Ir;"hnCrY,
Ac
MeO
MeO
95 96
3.3.5 Azadirachta indica A. Juss (Meliaceae)
A. indica (Meliaceae) commonly known as Mwarubaine (Giriama) has been associated
with many medicinal uses. Neem leaves are traditionally being used as curative against
malaria, several fungal and bacterial diseases (Parida et aI., 2002). It is used by several
communities in Asia for the treatment of malaria (parida et aI., 2002). It has anti-
plasmodial, anti-inflammatory, anti-pyretic, hypoglycaemic, anti-microbial and anti-
cancer properties and pesticidal activities (Murthy et al., 1978; Patel and Trivedi, 1962;
Khan and Wassilew, 1987; Udeinya, 1993; Schmutterer, 1995; Kusumran eta/., 1998; EI
Tahil et aI., 1999; Sai Ram et aI., 2000). Previous chemical investigations revealed
presence of several tetracyclic triterpenoids including 6a-O-acetyl-7-deacetylnimoci-nol
(97) and meliacinol (98) (Siddiqui et aI., 2000). Nimbolide (47) (EC50 = 0.95 ng/ml, P.
falciparum KI) was the first to be identified as the active anti-malarial principle of the
neem tree (Rochanakij et aI., 1985). Subsequently, gedunin (48) was also found to be
active in vitro againstP.falciparum parasites with EC50 values in the range of 0.72-1.74
ug/ml (Khalid et aI., 1989; Badam et al., 1987; McKinnon et aI., 1997). Low activity of
extracts in in vitro anti-plasmodium test frequently reported could be due to presence of
prodrugs (Gakunju et aI., 1995). It could also be due to low concentration of active
principle due to change-in climate and soil composition.
42
c
o~~:'
, I
• OAc ,'--0
97 98
3.3.6Myrica salicifolia A. Rich. (Myricaceae)
The root of M salicifolia (Myricaceae) commonly known as Muthaa (Giriama) is used as
a slow acting medicine in stomach problems and headache (symptoms associated with
malaria). The bark is chewed for tooth ache problems whereas powdered young leaves
are used to treat skin infections (Kokwaro, 1993). Pharmacological and chemical
investigations of this plant are yet to be reported.
3.3.7Fagaropsis angolensis (Engl.) Dale (Rutaceae)
The entire plant of F. angolensis (Rutaceae) locally known as Murumu or Mukuria
Mpungu (Meru) is used for treatment of malaria (Khalid and Waterman, 1985). Previous
chemical investigations revealed presence of indole alkaloid (99), canthin-6-one (100)
(Bettarini et aI., 1993), nitidine (66), 6-hydroxymethylnitidine (101) (Khalid and
Waterman, 1985) and rutaevin (102) (Khalid et aI., 1986). Pycnanthus angolensis from
the same family has shown anti-hyperglycemic (Fort et aI., 2000) and anti-malarial
activity (Ancolio et aI., 2002). Fractions containing nitidine (66) have shown in vitro
anti-malarial activity (ICso 9-108 ng/ml) against P. falciparum for a range of
chloroquine-susceptible and resistant strains (Gakunju et aI., 1995).
MeO'09~b o=BI ~ ~ 7 ~ ~ N /;
~ N N-_ o OMe
99 100
Qa
~o
Mea >a
Mea a a
OH
a
101 102
43
3.3.8Zanthoxylum usambarensis (Engl.) Kokwaro (Rutaceae)
Thebark, fruits and roots of Z. usambarensis (Rutaceae) locally referred to as Mugucuwa
(Meru)are used as a cough mixture. A decoction of the bark is drunk to treat rheumatism
while fruit infusion with milk is used as a remedy for fever, sore throat, tonsolitis and
chest pains. It is also used in concotion of other plants like Prunus sp. to treat malaria
(Kokwaro, 1993). It exhibits anti-bacterial and anti-inflammatory activities (Matu and
Van Staden, 2003). Bio-assay-guided fractionation of the dichloromethane extracts of
the roots and the bark of Z. usambarense led to the isolation for the first time of two
physiologically active compounds; canthin-6-one (100) (fungicide) and pellitorine (103)
(insecticide) together with (+)-sesamin (104), (+)-piperitol-3,3-dimethylallyl ether (105),
norchelerythrine (106) and oxychelerythrine (107) (He et a/., 2002).
0:0I >80--'>-- °:0,°~\< ••...
103 104 105
106 107
Other alkaloidal compounds isolated are (-)-usambarine (108), (-)-cis-N-methylcanadine
(109), boccolline (110), chelerythrine (111), (+)-N-methylplatydesmine (112), (-)-
edulinine (113), (-)-oblongine (114), (+)-tembetarine (115), (+)-magnoflorine (116),
nitidine (66), (Kato et aI., 1996) and the coumarin derivatives toddaculin (117), 0-
methylcedrelopsin (118), toddalolactone (119), pimpinellin (120) and (121) phellopterin
(Kokwaro et aI., 1983). The anti-plasmodial activity of this plant may be due to the
presence ofnitidine (66) which has been shown to exhibit anti-malarial activity (Gakunju
et aI., 1995).
44
0
0 >> 00108 R = Me OMe
OMe
OMe 109 R=H OMe
110 111OR
0
OMeo.»-; "-'::// ~..-< 0 OH MeO
I //
112 113
~~
",ag1 + /MeO HO // N, 1 // + N/HO -,OH HO "-':: HO "-'::1// 1//
OH MeO MeO
114 115 116
117 Rl = OMe R2 = CH2CHCMe2 R3 = OMe R4 = H
118 Rl = H R2 = OMe R3 = OMe R4 = CH2CHCMe2
119 Rl = OMe R2 = CH2CH(OH)C(OH)Me2 R3 = OMe R4 = H
OMeMeo&Ol "'"
:::". 0 0
~
120 121
3.3.9Harrissonia abyssinica Oliv (Simaroubaceae)
The root decoction of H. abyssinica (Simaroubaceae) commonly known as Mutagata
(Meru) is used as a remedy for fever, insomnia, nausea, vomiting (symptoms associated
with malaria), bubonic plague, swelling of the testicles and tuberculosis. Leaf extract
alone or together with roots is used for snake bite treatment. Extract of the whole plant is
used as a remedy for stomach ache and abdominal pains (Kokwaro, 1993). Previous
45
chemical and biological investigation has led to isolation of harrison in (122) and pedonin
(123). Harrisonin has been shown to be the anti-plasmodial principle in this plant (Kubo
et aI., 1976; Rajab et aI., 1997).
o
122 123
3.3.10 Withania somnifera (L.) Dunal (Solanaceae)
Root sap of W. somnifera (Solanaceae) locally known as Mugumbao (Meru) is used for
treating stomach-ache, especially gastric ulcer, while the root decoction is good for
treatment of colds in children, skin rashes, excess bile, labour pains, gonorrhoea and
general ill health. It is also used as an anti-malarial remedy. The roots may also be
prepared by drying and grinding into a fine powder. A tea spoonful of this powder is
taken in a cup of tea or with honey once or twice a day. It has no pronounced taste but
has a slight smell. Heated leaves are applied to various parts of the body as pain killers
(Kokwaro, 1993). It has anti-tumour (Davis and Kuttan, 2002; Prakash et a/., 2001),
anti-oxidant (Bhattachayra et al., 2002), anti-clastogenic (Ghoshal et a/., 2001),
neuroprotective (Jain et a/., 2001), nootropic-like (Dhuley, 2001) and anti-stress (Singh et
aI., 2001) properties. Previous chemical investigations revealed presence of coumarins
(scopoletin (124) and aesculetin), ~-amyrin (125), stigmasterol (126), sitosterol (127)
(Abou-Douh, 2002), calystegine (128) (Bekkouche et aI., 2001) and withanolides C-G,L-
Q (129-139) (Cai et aI., 1994).
Meo~
HO~O/J,,,o
H
124 125 126
HO
127
129
132
135
138
46
HO 130
H
)~OH
~ OH
128
131
134
-, -,
OH
137
CH20H
139
Confirmation of the anti-malarial activity of the above 10 plants and identification of the
bio-active principles therein is required to ascertain their efficacy.
o
133
o 0
136
o
47
CHAPTER 4
BIO-ASSAYS
4.1 Bioassay of crude extracts
4.1.1 Brine shrimp toxicity test
4.1.1.1 Aqueous extracts
The crude water extracts were screened for toxicity on brine shrimp nauplii and the lethal
dose that kills 50% of the organism (LDso) obtained (Table 1).
Table 1: Lethal dose (LDso) of aqueous plant extracts against brine shrimp nauplii
Plant species Family LD50llg/ml
Carissa edulis Apocynaceae 260.34±1.5
Neoboutonia macrocalyx Euphobiaceae 41.69±0.9
Acacia nilotica Leguminosae 368.11±6. 7
Strychnos heningsii Loganiaceae 293.93±5.0
Azadirachta indica Meliaceae I01.26±3.7
Myrica salicifolia Myricaeae 328.22±10.9
Fagaropsis angolensis Rutaceae 173.48±0.6
Zanthoxylum usambarense Rutaceae 260.90±1.1
Harrisonia abyssinica Simaroubaceae 234.71±11.5
Withania somnifera Solanaceae 301.44±7.2
Emetine hydrochloride 19.7±1.2 ug/ml used as a positive control
A crude is considered active up to a concentration of 240 ug/rnl (Meyer et a/., 1982) and
therefore N macrocalyx, A. indica, F angolensis and H. abyssinica were considered
active with increasing activity in that order (LDso 41.69±0.9 > 101.26±3.7 > 173.48±0.6
> 234.71±11.5). The other plant extracts had no significant toxic effects. Hence the
order of decreasing activity was N macrocalyx > A. indica > F angolensis > H.
abyssinica > C. edulis > Z. usambarense > S. heningsii > W. somnifera >M. salicifolia >
A. nilotica.
4.1.1.2 Methanol extracts
Like aqueous extract, N macrocalyx showed the highest toxicity (LDso 21.04 ± 1.8
ug/ml). F angolensis, A. indica, Z. usambarense, S. heningsii, C. edulis, W. somnifera
and H. abyssinica showed moderate toxicity with activity decreasing in that order (LDso
= 57.09 ± 1.4, 61.43 ± 2.9,97.66 ± 3.6,101.22 ± 3.2,186.71 ± 6.9,207.27 ± 0.7 and
48
217.34 ± 7.2 ug/rnl, respectively) (Table 2). A. nilotica and M salicifolia showed no
cytotoxicity (ICso > 240 ug/ml). Hence the order of decreasing toxicity activity was N.
macrocalyx > F angolensis > A. indica > Z. usambarense > S. heningsii > C. edulis >
W.somnifera > H. abyssinica >A. nilotica >M salicifolia.
Table 2: Lethal dose (LD50)of plant methanol extracts against brine shrimp nauplii
Plant species Family LDso ug/ml
Carissa edulis Apocynaceae 186.71±6.9
Neoboutonia macrocalyx Euphobiaceae 21.04±1.8
Acacia nilotica Leguminosae 267.31±3.1
Strychnos heningsii Loganiaceae 101.22±3.2
Azadirachta indica Meliaceae 61.43±2.9
Myrica salicifolia Myricaeae 328.22±1O.9
Fagaropsis angolensis Rutaceae 57.09±1.4
Zanthoxylum usambarense Rutaceae 97.66±3.6
Harrisonia abyssinica Simaroubaceae 217.34±7.2
Withania somnifera Solanaceae 207.27±0.7
Emetine hydrochloride 21.1±O.9 ug/ml used as a positive control
The aqueous and methanol plant extracts were further subjected to in vitro anti-
plasmodial screening.
4.1.2 Anti-plasmodial screening
The crude water and methanolic extracts were screened for in vitro anti-plasmodial
activity against two P. falciparum isolates (NF54 and ENT30, CQ-susceptible and
resistant, respectively). The mean inhibitory concentration fifties (IC50) for the extracts
are sumrnarised in tables 3 and 4. Chloroquine (CQ) diphosphate was used as the
standard drug.
4.1.2.1 Aqueous extracts
F angolensis and Z. usambarense showed good activity (ICso 10.65 ± 1.23 and 14.33 ±
4.22 ug/ml, respectively) against the chloroquine-resistant isolate (ENT 30). S. heningsii,
M salicifolia, H. abyssinica and N. macrocalyx showed mild activity (IC5o73.39 ± 9.75,
85.97 ± 5.48, 89.74 ± 8.12 and 92.85 ± 7.65 ug/ml, respectively) against the same isolate.
However, C. edulis, A. indica, A. nilotica and W. somnifera had no activity (ICso > 250
49
ug/ml). The order of decreasing in vitro anti-plasmodial activity of aqueous extracts was
established as F. angolensis > Z. usambarense > S. heningsii > M. salicifolia > H.
abyssinica > N. macrocalyx > C. edulis > A. indica > A. nilotica > W. somnifera
against the CQ resistant strain (ENT 30) (Table 3).
Table 3: In vitro anti-plasmodial activity (IC5o) of aqueous plant extracts on two P.
falciparum strains
IC50±S.D (ug/ml)
Plant Family ENT 30 NF 54
Carissa edulis Apocynaceae >250 148.53±12.65
Neoboutonia macrocalyx Euphobiaceae 92.85±7.65 84.56±8.98
Acacia nilotica Leguminosae >250 153.79±15.79
Strychnos heningsii Loganiaceae 73.39±9.75 67.l6±8.70
Azadirachta indica Meliaceae >250 >250
Myrica salicifolia Myricaeae 85.97±5.48 66.84±2.88
Fagaropsis angolensis Rutaceae 1O.65±1.23 6.l3±1.l5
Zanthoxylum usambarense Rutaceae 14.33±4.42 5.25±0.27
Harrisonia abyssinica Simaroubaceae 89.74±8.l2 86.56±3.21
Withania somnifera Solanaceae >250 >250
Chloquine IC50 =0.03±O.005, 0.067±O.016 ug/ml for NF 54 and ENT 30, respectively acted as the positive
control
Similarly, Z. usambarense and F. angolensis showed good activity (IC5o 5.25 ± 0.27 and
6.13 ± 1.15 ug/ml, respectively) against the chloroquine-sensitive isolate (NF 54). M.
salicifolia, S. heningsii, N. macrocalyx and H. abyssinica showed mild activity of (IC5o
66.84 ± 2.88, 67.16 ± 8.78, 84.56 ± 8.93 and 86.56 ± 3.21 ug/ml, respectively) against
the same strain. C. edulis and A. nilotica showed low activity (IC5o 148.53 ± 12.65 and
153.79 ± 15.79 ug/ml, respectively) while A. indica and W. somnifera had no activity
(IC5o > 250 ug/ml). The order of decreasing in vitro anti-plasmodial activity of aqueous
extracts was shown to be Z. usambarense > F. angolensis > M salicifolia > S. heningsii
>N. macrocalyx > H. abyssinica > C. edulis > A. nilotica > A. indica> W. somnifera
against the CQ sensitive strain (NF 54) (Table 3).
4.1.2.2 Methanol extracts
Like the aqueous extract, F. angolensis and Z. usambarense showed good activity (IC5o
5.04 ± 0.68 and 5.54 ± 1.70 ug/ml, respectively) against the chloroquine-resistant isolate
(ENT 30). M. salicifolia, A. nilotica, N. macrocalyx and H. abyssinica showed mild
50
activity (IC5o 55.89 ± 2.00, 73.59 ± 2.87, 78.44 ±2.89 and 79.50 ± 3.31 ug/ml,
respectively) against the same isolate. W. somnifera and S. heningsii showed low activity
(IC5o 145.86 ± 2.23 and 190.0 ± 16.85 ug/ml, respectively) while C. edulis had no
activity (IC5o> 250 ug/ml). The order of decreasing anti-plasmodial activity of methanol
extracts was found to be F. angolensis > Z. usambarense > M salicifolia > A. nilotica >
N macrocalyx > H. abyssinica > W. somnifera > S. heningsii > C. edulis > A. indica
against the CQ resistant strain (ENT 30) (Table 4).
Table 4: In vitro anti-plasmodial activity (ICso) of methanol extracts on two P. falciparum
strains
ICsO±S.D(ug/ml)
Plant species Family ENT 30 NF 54
Carissa edulis Apocynaceae >250 >250
Neoboutonia macrocalyx Euphobiaceae 78.44±2.89 78.40±6.84
Acacia nilotica Leguminosae 73.59±2.87 70.33±l.89
Strychnos heningsii Loganiaceae 190.0±16.85 157.91±10.03
Azadirachta indica Meliaceae >250 >250
Myrica salicifolia Myricaeae 55.89±2.00 51.07±1.70
Fagaropsis angolensis Rutaceae 5.04±0.68 4.68±0.09
Zanthoxylum usambarense Rutaceae 5.54±l.70 3.20±0.45
Harrisonia abyssinica Simaroubaceae 79.50±3.31 72.66±l.39
Withania somnifera Solanaceae 145.86±2.23 125.59±l.30
Choloquine IC50 =O.03±0.005, 0.067±O.016 ug/ml for NF 54 and ENT 30, respectively acted as the positive
control
Similarly, Z. usambarense and F. angolensis showed good activity (ICso3.20 ± 0.45 and
4.68 ± 0.09 ug/ml, respectively) against the chloroquine-sensitive isolate (NF 54). M.
salicifolia, A. nilotica, H. abyssinica and N macrocalyx showed mild activity (ICso 51.07
± 1.70, 70.33 ± 1.89, 72.66 ± 1.39and 78.40 ± 4.68 ug/ml, respectively) against the same
strain. W. somnifera and S. heningsii had low activity (ICso 125.59 ± l.30 and 157.91±
10.03 ug/ml, respectively) while C. edulis had no activity (ICso> 250 ug/ml). The order
of decreasing anti-plasmodial activity was demonstrated to be Z. usambarense > F.
angolensis > M salicifolia > A. nilotica > H. abyssinica > N macrocalyx > W.
somnifera. > S. heningsii > C. edulis > A. indica against the CQ-sensitive strain (NF
54)(Table 4).
51
4.1.3 Summary
Generally, the aqueous extracts had lower in vitro anti-plasmodial activity and toxicity
against brine shrimp nauplii than the methanol ones.
N.macrocalyx had the highest toxic effect against brine shrimp nauplii.
F. angolensis and Z. usambarense methanol extracts had good anti-plasmodial activity.
H. abyssin ica, S. heningsii, M salicifolia and N macrocalyx aqueous extracts had mild
activity (ICso 50-100 ug/ml) decreasing in that order. C. edulis and A. nilotica extracts
had low activity on NF 54 isolate and no effect on ENT 30.
Water extracts of S. heningsii had better activity (ICso 65-75 ug/ml) than the methanol
one (ICso 150-200 ug/ml).
The water extracts of W. somnifera and A. indica did not exhibit any anti-plasmodial
activity (ICso > 250Ilg/ml).
F angolensis and Z. usambarense (Rutaceae) had higher activity (ICso< 6 ug/ml) in
methanol than water (ICso 5-15 ug/ml).
M salicifolia, A. nilotica, N macrocalyx and H. abyssinica methanol extracts had
moderate activity (IC50 50-100 ug/ml) decreasing in that order whereas S. heningsii and
W. somnifera had low activity (ICso 100-200 ug/rnl).
A. nilotica methanol extract had much better activity (ICso 70-75 ug/ml) than the water
one (ICso >150 ug/ml).
The methanol extract of C. edulis showed no anti-plasmodial activity (IC50> 250llg/ml).
52
For all the extracts, it was observed that there was relatively higher activity against the
chloroquine-sensitive isolate (NF 54) than the resistant strain (ENT 30). However, this is
not always the case (Muregi et al., 2001; Wanyoike et aI., 2000).
On the basis of the high in vitro anti-plasmodial activity, least phytochemical and
pharmacological reports, F. angolensis (Rutaceae) was selected for bio-assay guided
chromatographic fractionation. N macrocalyx (Euphorbiaceae) was also selected on the
basis of moderate in vitro anti-plasmodial and high brine shrimp toxicity and absence of
chemical information on this plant despite its widespread use in both areas as an anti-
malarial plant.
4.2 Bio-assay of mixtures and isolated compounds
4.2.1 Brine shrimp toxicity test
Some of the extracts, pure and semi-purified compounds were tested for toxicity against
brine shrimp nauplii. PI, P2b, P4, P6, P7 and P, were not assayed since they were obtained
in small amounts. The results are summarised in table 5.
Table 5: Brine shrimp toxicity assay of derivatives from F. angolensis and N macrocalyx
Species Compound/extract LDso (ug/ml)
F angolensis Hexane 199.78 ± 4.6
" Dichloromethane 126.23 ± 1.6
" Ethyl acetate 27.22 ± 3.2
" Methanol 30.01 ± 4.4
" Kl 124.13 ± 0.9
N macrocalyx Hexane 136.559 ± 2.1
" Dichloromethane 106.00 ± 3.2
" Ethyl acetate 22.78 ± 0.7
" Methanol 38.02 ± 0.3
" P3 18.36 ± 2.2
" P5&7 138.82 ± 8.7
" P5" 113.15 ± 5.6
Emetine hydrochloride 19.0±O.7 ug/ml used as positive control
A pure compound is considered active if it shows cytotoxicity (LDso) at concentration of
up to 10 ug/ml (Meyer et al., 1982). Consequently, ethyl acetate (LD50= 27.22 ug/ml)
and methanol (LDso = 30.01 ug/ml) extracts of F. angolensis as well as ethyl acetate
53
(LDso = 22.78 ug/ml), methanol (LDso = 38.02 ug/ml) and P3 (LDso = 18.36 ug/ml) from
N. macrocalyx, exhibit mild toxicity.
4.2.2 In vitro anti-plasmodial screening
The pure and semi-purified compounds isolated from N. macrocalyx and F angolensis
were screened for in vitro anti-plasmodial activity against two P. falciparum isolates
(NF54 & ENT30). PI, P2b, P4, P6, P7 and Ps were not assayed since they were obtained in
small amounts. The mean inhibitory concentration fifties (ICso) obtained for the extracts
are summarised in the tables 20-21. Chloroquine diphosphate was used as the standard
drug.
The petroleum ether extract showed no in vitro anti-plasmodial activity (ICso > 250
ug/ml) against the same isolate. Similarly, stigmasterol (PI) isolated from the same
fraction exhibited low in vitro anti-plasmodial activity. Ethyl acetate and
dichloromethane extracts of N. macrocalyx showed very low anti-plasmodial activity
(ICso 205.83 ± 23.16 and 226.33 ± 15.77 ug/ml, respectively) against the chloroquine-
resistant isolate (ENT 30). The compound P3 isolated from the ethyl acetate extract
exhibited even lower activity (ICso 241.63 ± 18.24). A mixture of Ps & P7 also showed
low activity (ICso 241.39±4.73 ug/ml). The order of decreasing anti-plasmodial activity
of N. macrocalyx extracts against the chloroquine-resistant isolate (ENT 30) was
established as: ethyl acetate> dichloromethane > methanol & hexane, while that of the
pure and semi-purified compounds was mixture Ps & P7= P3 > P, & stigmasterol.
The petroleum ether extract of F angolensis had no actrvity (ICso > 250 ug/rnl).
Similarly, the compound KI isolated from this extract showed no activity. The
dichloromethane extract had mild activity (ICso 37.34 ± 2.80 ug/ml). On the other hand,
methanol and ethyl acetate extracts had moderate anti-plasmodial activity (ICso 5.298 ±
0.44 and 9.238 ± 0.21 ug/ml, respectively) against the chloroquine-resistant isolate (ENT
30). The order of decreasing anti-plasmodial activity ofF angolensis extracts against the
chloroquine-resistant isolate (ENT 30) was established as: methanol> ethyl acetate>
dichloromethane> hexane. The results are summarised in the table 6.
54
Table 6: In vitro anti-plasmodial activity (ICso) of derivatives F angolensis and N
macrocalyx against P. falciparum (ENT30).
Plant species Extract/compound ICsO±S.D(ug/ml)
N. macrocalyx Petroleum ether >250
" Dichloromethane 226.52±15.77
" Ethyl acetate 205.83±23.16
" Methanol >250
" Stigmasterol >250
" P3 241.63±18.24
" Mixture PS&P7 241.39±4.73
" Ps >250
Fangolensis Petroleum ether >250
" Dichloromethane 37.34±2.80
" Ethyl acetate 9.238±O.21
" Methanol 5.298±0.44
" Ki >250
Chloroquine Ie50 = O.071±O.025 ug/rnl acted as the positive control
The petroleum ether extract of N macrocalyx showed no activity (ICso > 250 ug/ml)
against the chloroquine-sensitive isolate (NF 54). Both stigmasterol (Pi) and the mixture
OfPS&P7 isolated from this fraction showed no activity (ICso > 250 ug/ml). Ethyl acetate
and dichloromethane extracts of N macrocalyx exhibited very low activity (ICso200.33 ±
17.16 and 221.03 ± 9.34 ug/ml, respectively). The compound P3 isolated from the ethyl'\
acetate extract exhibited even lower activity (ICso 237.47 ± 11.23). The order of
decreasing anti-plasmodial activity of N macrocalyx extracts against the chloroquine-
sensitive isolate (NF54) was established as: ethyl acetate> dichloromethane > methanol
and hexane, while that of the pure and semi-purified compounds was P3 > mixture PS&P7,
stigmasterol and Ps.
The petroleum ether extract of F angolensis had no activity (ICso> 250 ug/ml) against
the chloroquine-sensitive isolate (NF54). The compound (KI) isolated from this extract
also showed no activity. The dichloromethane extract had moderate activity (ICso 35.22
± 7.55 ug/ml). On the other hand, methanol and ethyl acetate extracts of exhibited high
anti-plasmodial activity (ICso 5.66 ± 2.77 and 8.70 ± 7.55 ug/ml, respectively). The order
of decreasing anti-plasmodial activity of F angolensis extracts against the chloroquine-
55
sensitive isolate (NF54) was established as: methanol> ethyl acetate> dichloromethane
> hexane. The results are summarised in the table 7.
Table 7: In vitro anti-plasmodial activity (IC5o) of derivatives from F. angolensis and N
macrocalyx against P. falciparum (NF54).
Plant species Extract/compound IC5o±S.D (Ilg/ml)
N. macrocalyx Petroleum ether > 250
" Dichloromethane 221±9.34
" Ethyl acetate 200.33±17.16
" Methanol extract > 250
" Stigmasterol (PI) > 250
" P3 237.47±11.23
" Mixture ofP5&P7 > 250
" P5 > 250
F angolensis Petroleum ether > 250
" Dichloromethane 35.22±7.55
" Ethyl acetate 8.70±7.55
" Methanol 5.666±2.77
" KI > 250
Chloroquine ICso =O.027±O.037 ug/ml acted as the positive control
4.2.3 Conclusion
All the compounds isolated from F. angolensis except KI could not be tested for anti-
plasmodial assay due to solubility problems. KI though not yet identified does not exhibit
any anti-plasmodial activity. Nonetheless, nitidine which has shown to exhibit anti-
plasmodial properties (Gakunju et a!., 1995) has been isolated previously from this plant.
However, in our present study we did not isolate nitidine.
Of the pure compounds isolated from N macrocalyx and tested (PI, P3 & P5) or the
mixture of P5 & P7, none showed reasonable in vitro anti-plasmodial activity to account
for its use as traditional anti-malarial remedy. However, the other isolated compounds
PZb, P4, P6, P7 and Ps were isolated in small quantities inadequate for bio-assasy.
Consequently, these other compounds may be responsible for the anti-malarial activity.
Alternatively, it may be possible that these compounds exhibit activity in combination
with each other but not singly.
56
CHAPTER 5
STRUCTURAL ELUCIDATION
5.1 Structural elucidation of compounds isolated from Neoboutonia macrocalyx
5.1.1 Stigmasterol (PI) (126)
This compound was isolated as white needles (70 mg, Rf 0.55, Si02, 10:5 n-CJIw
EtOAc) and melting point 166-168 "C. Its IR spectrum showed characteristic bands at
3424 (hydroxyl) and 1688 cm-l (double bonds).
IHNMR is summarized in table 8.
Table 8: IH NMR (200 MHz) data for stigmasterol (PI) (126) in CDCh
Proton Chemical shift (D) Multiplicity J (Hz) Integral
3 3.53 m IH
6 5.35 br, d 4.4 IH
18 l.01 s 3H
19 l.66 s 3H
21 1.15 d 5.6 3H
22&23 5.09-5.13 m 2H
26&27 0.83 d 3.0 6H
29 0.68 s 3H
Others 0.84-2.29 m 26H
Methyl signals were observed at D 0.68 (s, 3H, H-29), 0.83 (d, J = 3.0 Hz, 6H, H-26&27),
1.01 (s, 3H, H-18), 1.15 (d, J = 5.6, 3H, H-21) and 1.66 (s, 3H, H-19). The signal at D
5.35 (br d, IH) was assigned to an olefinic proton whereas that at 3.53 (m, 1H) suggested
the presence of an a-proton typical of sterols hydroxylated at C-3. The multiplet at D
5.09-5.12 (2H) suggested presence of two olefinic protons. The rest of the signals were
in a complex mass ofmultiplets spread between D 0.84-2.29.
The l3C NMR revealed 29 signals (Table 9).
57
Table 9: BC NMR (50 MHz), DEPT and HETCOR data for stigmasterol (PI) (126) in
CDCh
Carbon Chemical DEPT HETCOR Carbon Chemical DEPT HETCOR
shift (8) shift (8)
I 37.2 CH2 16 28.2 CH2
2 31.6 CH2 17 56.0 CH
3 71.8 CH 3.53 (m, H-3) 18 11.8 CH3 1.01 (s, H-18)
4 42.2 CH2 19 18.8 CH3 1.66 (s, H-19)
5 140.7 C 20 36.1 CH
6 121.7 CH 5.35 (d, H-6) 21 19.8 CH3 1.15 (d, H-21)
7 33.8 CH2 22 138.1 CH 5.11 (m, H-22)
8 31.8 CH 23 129.2 CH 5.11 (m, H-23)
9 50.0 CH 24 55.9 CH
10 36.5 C 25 29.0 CH
11 21.0 CH2 26 19.4 CH3 0.86 (d, H-26)
12 39.7 CH2 27 18.9 CH3 0.86 (d, H-27)
13 45.7 C 28 24.3 CH2
14 56.7 CH 29 12.0 CH3 0.68 (s, H-29)
15 23.0 CH2
Six methyl signals appeared at 0 11.8, 12.0, 18.8, 18.9, 19.4 and 19.8, as revealed by
DEPT analysis. DEPT analysis also revealed 9 methylene carbon signals at 0 21.0,23.0,
24.3, 28.2, 31.6, 33.8, 37.2, 39.7, and 42.2. Similarly, 11 methine carbon resonances
were observed at 0 29.0,31.8,36.1,50.0,55.9,56.0,56.7, 71.8, 121.7, 129.2 and 138.1.
The remaining three signals at 0 36.5, 45.7 and 140.7 were due to quaternary carbons.
From the NMR analysis, stigmasterol (126) was proposed.
29
21
18
27
12
2616
HO
4
126
58
The structure was further confirmed through determination of melting point and IR.
Stigmasterol is found in many higher plants such as wheat germ (Triticum) and sweetcom
(Zea mays) where it plays an important role in the membranes of plant cells (Harbone and
Baxter, 1993). The spectral data obtained was in agreement with those reported by
Budavari (1996).
5.1.2 P2a
This compound was an impurity in P2b. It wa~ isolated as a colourless gum (8.2 mg, R,
0.57, Si02, 3:1 n-CJI14: EtOAc. IH NMR and 13C NMR spectra showed that P2awas the
same as P7.
5.1.3 6,7 -Epoxy-4,5,9-trihydroxy-13-hexadecanoate-20-dodecanoate-1-tiglien-1-3-
one (P2b) (140)
This compound was isolated as light orange oil (17.7 mg, Rf0.45, Si02 gel, 3:1 n-CJI12-
EtOAc). Its positive ion mode CI mass spectrum gave quasimolecular ion adducts at m/z
829 [M+C2H5t and 801 [M+Ht which indicated that its molecular weight was 800 amu.
Its IR spectrum showed characteristic bands at 3389 (-OH), 1689 (-C=C-C=O) and 1624
cm-1 (-C=C-).
IH NMR spectral data is summarised in table 10.
The IH NMR spectrum of P2b (Table 10) was characterised by two methyl triplets at 8
0.89 (J = 6.3 Hz, 3H, Me-12") and 0.85 (J = 7.0 Hz, 3H, Me-16'), one methyl doublet at
0.93 (J = 6.7 Hz, 3H, H-18), three methyl singlets at 1.78 (3H, H-19), 1.19 (3H, H-16)
and 1.09 (3H, H-17) and a signal for a deshielded olefinic proton at 7.69 (s, IH, H-l).
The broad singlets at 8 3.73 (IH) and 3.56 (lH) were attributed to free hydroxyl groups.
In addition, two oxygenated methylene signals were observed at 84.80 (d, J = 12.0 Hz,
IH, H-20a) and 3.82 (d, J = 12.0 Hz, IH, H-20b). The doublet at 03.14 (J = 4.6 Hz, IH,
H-7) was assigned to an epoxy proton. In addition to the methylene proton signals
observed for H-12 at 02.03 (dd, J = 8.2, 15.5 Hz, IH, H-12a) and 1.57 (dd, J = 8.2, 15.5
Hz, IH, H-12b), integration revealed 38 other other methylene protons overlapping at 8
59
1.26 (br, m) and 1.60 (m) which were assigned to the CH2 of the ester moieties in the side
chains (4'-12', 4"-15"). The signals at 8 2.34 (J = 7.6 Hz, t, 2H) and 2.30 (J = 7.5 Hz, t,
2H) were assigned to H-2' and H-2", respectively. The IH NMR signals in rings A and C
of PZb were similar to those of mellerin A (75) (Zhao et aI., 1998) and 12-deoxyphorbol-
13-hexadecanoate (86) (Ma et aI., 1997) and were therefore assigned accordingly. On the
other hand, the IH NMR signals in the ring B were similar to those of montanin (80)
(Tchinda et a/., 2003) and the protons were assigned accordingly.
Table 10: IH NMR (600 MHz) and COSY data OfP2b (140) in CDCb
Proton Chemical Multiplicity J (Hz) Integral COSY
shift (0)
1 7.69 s 1H
5 4.28 s IH
7 3.14 d 4.6 IH
8 2.82 dd 4.6, 7.6 IH
10 3.90 br, s 1H
11 1.84 m 1H
12a 2.03 dd 7.3, 15.5 IH
12b 1.57 dd 9.0, 15.5 1H
14 1.12 d 7.6 1H
16 1.19 s 3H
17 1.09 s 3H
18 0.93 d 6.7 3H
19 1.78 s 3H
20a 4.80 d 12.0 1H
20b 3.82 d 12.0 1H
2' 2.34 t 7.6 2H
2" 2.30 t 7.5 2H
3',3" 1.61 m 4H
4'-15',4"-11" 1.26 br, m 40H
16' 0.85 t 7.0 3H
12" 0.89 t 6.3 3H
OH 3.56 s 1H
5-0H 3.73 s 1H H-5
5-0H
H-8
H-7, H-14
H-18, H-12a, H-12b
H-11, H-12b
H-11, H-12a
H-8
H-11
H-20b
H-20a
H-3'
H-3"
H-2', H-2",H-4', H-4"
**
*
* Complete connectivities not determined due to overlap of IHNMR signals in this range.
The l3C NMR spectrum of Pzs exhibited 48 carbon signals as 6 methyls, 26 methylenes, 7
methines and 9 quartenary carbons (Table 11).
Among the 47 carbon signals in PZb was a carbonyl groups (8 209.8, C-3), two ester
moieties (8 175.3, C-l'; 173.4, C-l") and two olefinic carbons (8163.6, C-l; 134.3, C-2).
60
The seven signals between 0 75.3-59.7 were assigned to the oxygenated carbons (C-4, C-
5, C-6, C-7, C-9, C-13, and C-20). Signals from the gem-dimethylcyclopropane moiety
were observed at 0 64.2 (C-13) and 24.0 (C-15) (both quartenary) and 31.9 (methine) as
reported previously (Marco et al., 1999). The 17 carbon signals at 0 29.1 - 29.7 (CH2, 4'-
13',4"-10") were assigned to the methylenes in the substituent ester moieties. The other
part of the molecule contained twenty carbons and was suggested to be a tigliane
diterpenoid on the basis of literature spectra data (Tchinda et aI., 2003; Zhao et aI., 1998;
Ma et aI., 1997).
The structure was therefore proposed to be a di-substituted tigliane diterpenoid (140).
16' l'
CH3(CH2)13C02\
\
\13
16
I
,,' 17r--~--Y
19
°
1" 12"
CH2OCO(CHvlOCH3
20
140
61
Table 11: l3C NMR (150 MHz), HMBC and HMQC data for PZb (140) in CDCh
Carbon Chemical shift (0) DEPT HMQC HMBC (HtoC)
1 163.6 CH 7.69 (s,H-l) 3,4,9,19
2 134.3 C
3 209.8 C
4 72.5 C
5 69.6 CH 4.28 (s, H-5) 3,7, 10,20
6 60.7 C
7 65.7 CH 3.14 (d, H-7) 5,9,14,20
8 36.2 CH 2.82 (dd, H-8) 6, 10, 11, 13, 15
9 75.3 C
10 49.8 CH 3.90 (s, H-lO) 2,3,5,8,11
11 38.1 CH 1.84 (m, H-ll) 8, 10, 13
12 31.9 CH2 2.03 (dd, H-12a) 9, 14, 15, 18
1.57 (dd, H-12b) 9,14,15,18
13 64.2 C
14 31.9 CH 1.12 (d, H-I4) 7,9,12,16,17
15 24.0 C
16 22.8 CH3 1.19 (s,H-16) 13,14,17
17 15.8 CH3 1.09 (s, H-17) 13,14, 16
18 19.0 CH3 0.93 (d,H-I8) 9,12
19 9.8 CH3 1.78 (s,H-I9) 1,3
20 65.7 CH2 4.80 (d, H-20b) 5,6,7,1"
3.82 (d, H-20a) 5,6,7, I"
I' 175.3 C
2' 34.4 CH2 2.34 (t, H-2') 4'
3' 24.8 CH2 1.61 (m, H-3') 1',5'
4' 29.1 CH2 1.28 (m, H-4') 2',6'
5' 29.3 CH2 1.26 (m, H-5') 3',7'
6' 29.4 CH2 1.26 (m, H-6') 4',8'
7' 29.6* CH2 1.26 (m, H-7') 5',9'
8' 29.6* CH2 1.26 (m, H-8') 6',10'
9' 29.6* CH2 1.26 (m, H-9') 7', II'
10' 29.6* CH2 1.26 (m, H-I0') 8',12'
11' 29.6* CH2 1.26 (m, H-l1') 9',13'
12' 29.6* CH2 1.26 (m,H-12') 10',14'
]3' 29.6* CHz 1.26 (m, H-I3') II', 15'
14' 32.0 CH2 1.26 (rn, H-14') 12'
15' 22.7 CH2 1.26 (m, H-15') 13'
16' 14.1 CH3 0.85 (t, H-15') 14'
1" 173.4 C
2" 34.2 CH2 2.30 (t, H-2") 20,4"
3" 24.9 CH2 1.61 (m, H-3") 1",5"
4" 29.1 CH2 1.26 (m, H-4") 2",6"
5" 29.2 CH2 1.26 (m, H-5") 3",7"
6" 29.3 CH2 1.26 (m, H-6") 4",8"
7" 29.5 CH2 1.26 (m, H-7") 5",9"
8" 29.6* CH2 1.26 (m, H-8") 6",10"
9" 29.6* CH2 1.26 (m, H-9") 7", II"
10" 29.7 CH2 1.26 (m, H-lO") 8",12"
11" 22.7 CH2 1.26 (m, H-II ") 9"
12" 14.1 CH3 0.89 (t, H-12") 10"
*Assigmnents interchangeable
62
TheCI spectrum was characterised by three regions. At the higher region, the molecular
ionpeak was observed at m/z 801 at 100% relative abundance. At the lower region, two
peakswere observed at m/z 327 and 309 amu with relative abundance of 21 and 22%
respectively. This was lower than the expected di-substituted fragment at 360 amu. The
peakat m/z 309 [M*+Ht can be explained by inductive cleavage that leads to loss of
threehydroxyl groups and M* is therefore the mass of dehydrated fragment. The mass at
mlz 327 is due to addition of reactive species (CH5+) to the dehydrated fragment. The
other region showed a peak at m/z 537 with relative abundance of 6%. This was
attributed to the loss of hexadecanoic acid (CH3(CH2)14C02H) from the molecule and the
low relative abundance (6%) suggested that the molecule was very unstable. The
subsequent loss of the dodecanoic acid (CH3(CH2)IOC02H)moiety was confirmed by the
presence of the peak at m/z 309 in the positive ion mode APCI mass spectrum. This was
further confirmed by EI mass spectrum fragments at m/z 223 and 185 which could be
assigned to pentadecanoyl and lauroyl ion fragments, respectively (Scheme 1).
Consequently, the molecular formula was deduced as C4gHgo09.
The proposed structure was further supported by the EI mass fragmentation pattern in
scheme 1.
Scheme 1: Proposed fragmentation pattern of'P», (140) in EIMS
+
140
o
OH CH2'}'OCO(CH2l1oCH3o OH ~
mlz 544
j .~C(CH2110CH3·30H
mlz 239
+
+ O=C(CH21lOCH3
mlz 185
o
m/z 293
63
Theproton assignments were further confirmed by COSY experiments (Fig. 1).
Fig1: Correlations observed in IH_IH COSY OfP2b (140)
, n(\
HO 'H CH2OCOCH2CH2CH2(CH2hCH3'-.f
140
The connectivities of the methyl, methylene, methine and quartenary carbons was
determined on the basis of IH_l3C long range correlation signals in the HMBC spectrum
(Table 11, Fig. 2).
Fig 2: Correlations observed in the HMBC spectrum of P2b (140)r-;
CH3( CH2)11 CH2CH2CH2CO
64
COSY, HMBC and HMQC spectra confirmed the existence of two side chain ester
moieties (hexadecanoyl at C-13 and dodecanoyl at C-20). The IUPAC name was
deduced as 6-7-epoxy-4,5 ,9-trihydroxy-13 -hexadecanoate-20-dodecanoate-1-tiglien-3-
one (140). The compound is being reported for the first time. It has a very close
semblanceto baliospermin (88) (Euphorbiaceae) previously isolated from the roots of
Baliospermum montanum and found to have cytotoxic and anti-cancer activity (Harbone
andHerbert, 1993). It only differs with three methylene carbons at the C-13 ester moiety
andthe 12 carbon ester moiety at C-20.
5.1.4 P3
This compound was isolated as orange crystals (61.7 mg, Rf 0.4, Si02, 2:1 CHCb:
MeOH). The IR spectrum showed characteristic bands at Umax 3152, 1714 and 1639 cm-l.
TheUV spectrum of P, exhibited a maximum absorption peak at A.nax 210, 362 and 415
nm, characteristic of compounds with conjugated double bonds. Structural elucidation is
III process.
5.1.5 6-7-Epoxy-4,5,9,20-tetrahydroxy-13-tetradecanoate-l-tiglien-3-one (P4) (141)
This compound was isolated as orange oil (17.8 mg, R, 0.24, Si02, 2:1 CHCh: EtOAc).
The positive ion mode CI mass spectrum gave quasimolecular ion adducts at m/z 619
[M+CzHsf and a quasimolecular ion peak at m/z 591 [M+H] + which indicated its
molecular weight to be 590. The IR spectrum showed characteristic bands at Umax 3397 (-
OH), 1684 (-C=C-C=O) and 1623 cm-1(-C=C-).
The IH NMR spectrum is summarized in table 12.
65
Table 12: IHNMR (600 MHz) and COSy data OfP4(141) in CDCb
Proton Chemical shift (0) Multiplicity J (Hz) Integral COSY
I 7.71 s 1H
5 4.26 s IH
7 3.26 d 4.4 IH
8 2.82 dd 4.4, 7.5 IH
10 3.91 s IH
11 l.84 m IH
l2a 2.06 dd 7.3, 15.3 IH
l2b l.56 dd 9.3,15.3 IH
14 l.13 d 7.5 IH
16 1.19 s 3H
17 l.07 s 3H
18 0.92 d 6.6 3H
19 l.77 s 3H
W 3.~ s lli
2' 2.30 t 7.5 3H
3' l.61 m 2H
4' - 13' l.26 br, m 20H
14' 0.88 t 7.0 3H
OH 4.92 s 1H
OH 3.97 br, s IH
OH 3.57 s IH
H-8
H-14, H-7
H-18, H-12b, H-12a
H-ll, H-12b
H-ll, H-12a
H-8
H-ll
H-3'
H-2'
*
H-13
* Complete connectivities not determined due to overlap of IH NMR signals in this range.
The IH NMR spectrum of P, revealed 5 methyl groups at 8 0.88 (t, J = 7.0 Hz, 3H, H-
14'); 1.77 (s, 3H, H-19); 0.92 (d, J = 6.6 Hz, 3H, H-18); l.19 (s, 3H, H-16) and l.07 (s,
3H, H-17). A signal for a deshielded olefinic proton was observed at 87.71 (s, IH, H-1).
The broad singlets at 8 4.92 (lH), 3.97 (IH) and 3.57 (IH) were attributed to free
hydroxyl groups. One oxygenated methylene signal observed at 8 3.83 (s, 2H, H-20)
suggested that the two protons were chemically equivalent. This suggested that the ester
moiety substituent was at C-13, not C-20. The IH NMR signals of rings A and C of Pa
were similar to those of mellerin A (75) (Zhao et al., 1998) and 12-deoxyphorbol-13-
hexadecanoate (86) (Ma et al., 1997) and were therefore assigned accordingly. On the
other hand, the IH NMR signals of ring B were similar to those of montanin (80)
(Tchinda et al., 2003) and the protons were assigned accordingly. The signals of a and B-
methylene protons of the ester moiety were at 82.30 (t, J = 7.5 Hz, 2H, H-2') and l.61
(m, 2H, H-3'), respectively. The rest of the methylene groups in the ester group were
66
observed as broad multiplet at 1.26 (24H, H-4'-H-13'). These are in agreement with
reported literature values (Tchinda et a/., 2003; Zhao et aI., 1998; Ma et aI., 1997).
The BC NMR spectra ofP 4 revealed 34 carbon signals as five methyls, fourteen
methylenes, seven methines and eight quartenary carbons (Table 13).
Among the 34 carbon signals in P4 was a ketone carbonyl (8 210.0), an ester carbonyl (8
175.4) and two olefinic carbons (8 163.9 and 134.2). The seven signals observed
between 0 61.9 - 78.1 were assigned to the carbons containing oxygen functions (C-4, C-
5, C-6, C-7, C-9, C-13 and C-20). Signals from the gem-dimethylcyclopropane moiety
were visible at 8 64.1 and 23.9 (both quartenary) and 8 31.8 (methine) as previously
reported (Marco et al., 1999).
On basis of IH and I3C NMR the diterpenoid 141 was proposed.
19
16
I
," 17r--;--y
o
141
67
Table 13: J3CNMR (150 MHz), HMBC and HMQC data for P4 (141) in CDCb
Carbon Chemical shift CD) DEPT HMQC HMBC (H to C)
1 163.9 CH 7.71 (s,H-l) 3,4,9,19
2 134.2 C
3 210.0 C
4 72.6 C
5 71.6 CH
6 61.9 C
7 65.7 CH
8 36.3 CH
9 75.4 C
10 49.7 CH
II 38.3 CH
12 31.9 CH2
4.26 (s, H-S) 3,7, 10,20
3.26 (d, H-7)
2.82 (d, H-8)
5,9,14,20
6, 10, 11, 13, 15
3.91 (s,H-lO)
1.84 (m, H-ll)
2.05 (dd, H-12a)
1.56 (dd, H-12b)
2,3,5,8,11
8,10,13
9, 14, 15, 18
9,14,15,18
13 64.1 C
14 31.8 CH 1.13 (d,H-14) 7,9,12,16,17
15 23.9 C
16 22.8 CH3 1.19 (s,H-16) 13,14, 17
17 15.7 CH3 107 (s,H-17) 13,14, 16
18 19.0 CH3 0.92 (d,H-18) 9,12
19 9.7 CH3 1.77 (s,H-19) 1,3
20 64.8 CH2 3.83 (s, H-20) 5, 7
]' 175.4 C
2' 34.4 CH2 2.30 (t, H-2') 4'
3' 24.8 CH2 1.61 (m, H-3') 1',5'
4' 29.1 CH2 1.26 (m, H-4') 2',6'
5' 29.2 CH2 1.26 (m, H-5') 3',7'
6 29.3 CH2 1.26 (m, H-6') 4',8'
7 29.4 CH2 1.26 (m, H-7') 5',9'
8 29.6* CH2 1.26 (m, H-8') 6',10'
9 29.6* CH2 1.26 (m, H-9') 7', 11'
10 29.6* CH2 1.26 (m, H-lO') 8',12'
11 29.7 CH2 1.26 (m, H-l1 ') 9',13'
12' 32.0 CH2 1.26 (m, H-12') 10',14'
13' 22.7 CH2 1.26 (m, H-13') 11'
]4' 14.1 CH3 0.88 (t, H-14') 12'
* Assignments interchangeable
The mass fragments at m/z 363 [M-CH3(CH2)12C02H + H] + and 327 [M-
CH3(CH2)12C02H - H20 + H] + in the positive ion mode CI mass spectrum were due to
the tetradecanoic acid skeleton. This was further confirmed by EI mass spectrum
fragments at m/z 211 that could be assigned to myristoyl ion fragment (Scheme 2). The
other part of the molecule contained twenty carbons suggesting a diterpene skeleton
(Tchinda et aI., 2003; Zhao et aI., 1998; Ma et aI., 1997). The molecular ion was
observed at m/z 590 and the formula of'P, deduced to be C3~5408.
68
The structure was further supported by the mass fragmentation pattern as detailed in
scheme 2.
Scheme 2: Proposed fragmentation pattern of P, (141) in ElMS
02C( CH2)12CH3-c,
m/z 211
141 m/z362
J
+
m/z 255 m/z 109
l-40H
i=Oo CH20H
m/z 189
The proton assignments were further confirmed by COSY experiment (Fig. 3).
69
Fig3: Correlations observed in IH)H COSY spectrum of P4 (141)
141
The connectivities of the methyl, methylene, methine and quartenary carbons was
determinedon the basis of IH_13Clong range correlation signals in the HMBC spectrum
(Table13, Fig. 4 ).
Fig.4: Correlations observed in HMBC spectrum of P, (141)
141
COSY,HMBC and HMQC spectra indicated the existence of a tetradecanyl ester moiety
and confirmed that compound 141 IS 6-7-epoxy-4, 5,9,20-tetrahydroxy-13-
tetradecanoate-l-tiglien-3-one (141). This compound is being reported for the first time.
70
Thiscompound has a very close semblance to baliospermin (88) and only differs with
twomethylene carbons in the ester moiety. Compound 88 was isolated from the roots of
Baliospermum montanum (Euphorbiaceae), and has cytotoxic and anti-cancer activity
(Harboneand Herbert, 1993)
5.1.6 4,9-Dihydl'oxy-20-hexadecanoate-13-dodecanoate-l,6-tigliadien-3-one (P5)
(142)
The compound was isolated as colourless oil (51.3 mg, R, 0.70, Si02, 3:1 n-CJI14:
EtOAc). The IR spectrum revealed peaks at 'Umax3379 (-OR), 1707 (-C=C-CO-) and
1612em" (-C=C-). The UV spectrum of'P, exhibited a maximum absorption peak at Amax
325nm, characteristic of compounds with conjugated double bonds.
IHNMR spectra data is summarized in table 14.
Table14: IH NMR (600 MHz) and COSY data ofPs (142) in CDCh
Proton Chemical shift Multiplicity J (Hz) Integral COSY
(8)
7.61 s IH
5a 2.51 d 12.0 IH
5b 2.38 d 12.0 IH
7 5.72 d 4.4 IH
8 3.00 dd 4.4,6.6 IH
10 3.29 s IH
11 1.96 m IH
12a 2.06 dd 7.1,14.8 IH
12b 1.56 dd 11.3, 14.8 IH
14 0.81 d 6.6 IH
16 1.19 s 3H
17 1.07 s 3H
18 0.91 d 6.0 3H
19 1.76 s 3H
20a 4.48 d 12.4 IH
20b 4.45 d 12.4 IH
2', 2.30 t 7.5 2H H-3'
2" 2.29 t 7.6 2H H-3"
3',3" 1.61 m 4H H -2', H-4', H-2", H-4"
4'-15',4"-11" 1.26 br, m 40H *
16' 0.87 t 7.0 3H H-15'
12" 0.89 t 7.1 3H H-ll"
H-5b
H-5a
H-8
H-7, H-14
H-12a, H-12b, H-18
H-ll, H-12b
H-ll, H-12a
H-8
H-ll
* Complete connectivities not determined due to overlap of IH NMR signals in this range.
71
TheIH NMR spectrum of'P, revealed 6 methyl signals at 0 0.87 (t, J = 7.0 Hz, 3H, H-
16');0.89 (t, J = 7.1, 3H, H-12"); 0.91 (d, J = 6.0, 3H, H-18); 1.78 (d, J = 1.6 Hz, 3H, H-
19); 1.19 (s, 3H, H-16) and 1.07 (s, 3H, H-17), two olefinic signals at 07.61 (s, 1H, H-1)
andS.72 (d, J = 4.2 Hz, 1H, H-7) and two oxygenated methylene signals at 0 4.48 (d, J =
12.6Hz, 1H, H-20a); 4.4S (d, J = 12.2 Hz, 1H, H-20b). It has been reported that in all
tigliane diterpenoids isolated in nature, H-8 is ~, 9-0H and H-lO are a. (Ma et aI., 1997).
On comparing the IH NMR data of Ps with the literature, close similarity was observed
with 12-deoxyphorbol-13-hexadecanoate (86) (Ma et aI., 1997; Tchinda et al., 2003)
except for additional signals at 0 0.88 (t, J = 7.0, 9H, H-18, H-1S', H-12"), 1.26 (br, m,
40H, H-4', H-S', H-6', H-7', H-8', H-9', H-10', H-11', H-12', H-13', H-14', H-1S', H-16', H-
4", H-S", H-6", H-7", H-8", H-9", H-10", H-11 "), 1.61 (m, 4H, H-3', H-3"), 2.30 (t, J =
7.SHz, 2H, H-2') and 2.29 (t, J = 7.6 Hz, 2H, H-2"). These observations suggested that
Ps is a di ester of 12-deoxyphorbol-13-hexadecanoate (86) and that the orientation ofH-8,
9-0H and H-I0 were ~, a. and u, respectively (Ma et aI., 1997). Previous IH NMR work
showed that ~o of the two protons (H-Sa and H-Sb) in the presence of 4a.-OH is larger
than that with 4~-OH (Gschwendt and Hecker, 1969). In IH NMR spectrum of Pc, two
non-equivalent methylene protons appeared as two doublets at 0 2.S1 (J = 12.0 Hz, IH,
H-Sa) and 2.38 (J = 12.0 Hz, IH, H-Sb) with ~o= 0.13, which confirmed presence of 4~-
OR. Therefore, Ps was deduced to have 4~-OH, 8~-H, 9a.-OH, 10a.-H, lla.-Me, 13a.-OR
and 14a.-R. The signals of the ester moieties observed at 2.30 (t, J = 7.S Hz, 2H, H-2'),
2.29 (t, J = 7.6 Hz, 2H, H-2") and the broad signal at 1.26 (br m, 38H, H-4', H-S', H-6', H-
7', H-8', H-9', H-IO', H-ll', H-12', H-13', H-14', H-lS', H-16', H-4", H-S", H-6", H-7", H-
8", H-9", H-IO", H-ll ") are in agreement with literature values (Tchinda et aI., 2003;
Zhao et aI., 1998; Ma et aI., 1997).
The l3C NMR spectra of P, revealed 48 carbon signals consisting of 6 methyls, 27
methylenes, 6 methines and 9 quartenary carbons (Table IS).
On further study, several characteristic carbon signals were determined including the
ketonic carbonyl (0209.1),2 ester carbonyls (0 17S.9, 173.6),2 trisubstituted double
72
bonds (0 161.5, d, C-1; 135.0, s, C-6; 133.8, d, C-7; 132.8, s, C-2), 3 oxygenated
quartenary carbons (0 75.9, C-9; 73.7, C-4; 63.3, C-13) and 1 oxygenated methylene (0
69.5, C-20). The other signals were assigned to tigliane diterpenoid skeleton.
Comparison of the spectra data of P, with those of tigliane diterpenoids 75, 84, 86 and
87, the cyclopropane ring characteristic of these diterpenoids was observed at C-13 and
C-14. The other part of the molecule contained twenty carbons. The 13C NMR data of the
diterpenic skeleton of Ps were also found to be similar to those of the recently reported
mellerin A (75) and 12-deoxyphorbol-13-hexadecanoate (86) (Zhao et a/., 1998; Ma et
al., 1997). The diterpenoid 142 was proposed.
16' I'
CH3CH2(CHV12CH2C92
\ I
~_\-\-\ _13 "',/ 17
IS
16
19 1" 1211
o
142
73
Table 15: 13CNMR (150 MHz), HMBC and HMQC data for Ps (142) in CDCh
Carbon Chemical shift Co) DEPT HMQC HMBC (Hto C)
1 161.5 CH 7.61 (s,H-l) 3,4,9,19
2 132.8 C
3 209.1 C
4 73.7 C
5 39.0 CH2 2.51 (d, H-5a) 3,7,10,20
2.38 (d, H-Sb) 3,7,10,20
6 135.0 C
7 133.8 CH 5.72 (d,H-7) 5,9,14,20
8 39.5 CH 3.00 (dd, H-8) 6,10,11,13, 15
9 75.9 C
10 55.8 CH 3.29 (s,H-I0) 2,3,5,8,11
11 36.4 CH 1.98 (m, H-ll) 8,10,13
12 31.8 CH2 2.06 (dd, H-12a) 9,14,15,18
1.56 (dd, H-12b) 9,14,15,18
13 63.3 C
14 32.5 CH 0.81 (d, H-14) 7,9,12,16,17
15 22.7 C
16 23.2 CH3 1.19 (s,H-16) 13,14,17
17 15.3 CH3 1.07 (s,H-17) 13,14, 16
18 18.5 CH3 0.91 (d, H-18) 9,11,12
19 10.1 CH3 1.78 (s,H-19) 1,3
20 69.5 CH2 4.48 (d, H-20a) 5,7,1"
4.45 (d,H-20b) 5,7,1"
I' 175.9 C
2' 34.6 CH2 2.30 (t, H-2') 4'
3' 24.8 CH2 1.61 (m, H-3') 1',5'
4' 29.1 CH2 1.26 (m, H-4') 2',6'
5' 29.2 CH2 1.26 (m, H-5') 3',7'
6' 29.2 CH2 1.26 (m, H-6') 4',8'
7' 29.3 CH2 1.26 (m, H-7') 5',9'
8' 29.4 CH2 1.26 (m, H-8') 6',10'
9' 29.6 CH2 1.26 (m, H-9') 7', 11'
10' 29.6 CH2 1.26 (m, H-I0') 8',12'
11' 29.6 CH2 1.26 (m, H-l1 ') 9',13'
12' 29.6 CH2 1.26 (m, H-12') 10', 14'
13' 29.6 CH2 1.26 (m, H-13') 11',15'
14' 31.9 CH2 1.26 (m,H-13') 12'
15' 22.8 CH2 1.26 (m, H-14') 13'
16' 14.1 CH3 0.87 (t, H-16') 14'
I" 173.6 C
2" 34.3 CH2 2.29 (t, H-2") 20,4"
3" 24.9 CH2 1.61 (m,H-3") 1",5"
4" 29.2 CH2 1.26 (m, H-4") 2",6"
5" 29.2 CH2 1.26 (m, H-5") 3",7"
6" 29.3 CH2 1.26 (m, H-6") 4",8"
7" 29.4 CH2 1.26 (m, H-7") 5",9"
8" 29.6 CH2 1.26 (m, H-8") 6", 10"
9" 29.6 CH2 1.26 (m, H-9") 7", 11"
10" 29.7 CH3 1.26 (m, H-l 0") 8",12"
11" 22.7 CHz 1.26 (m, H-l1 ") 9"
12" 14.1 CH3 0.89 (t, H-12") 10"
74
CImass spectrum revealed two major peaks at m/z 495 and 295 with relative abundance
of 30 and 100%, respectively. The signal at m/z 295 was characteristic of loss of two
hydroxyl groups suggesting inductive cleavage of the two free hydroxyl groups on the
molecule to form a stable secondary molecular ion fragment as the base peak (100%
relative abundance). The observation strongly suggests that the isolated molecule was
unstable in the CI conditions. The peak observed at m/z 495 suggested the loss of a
dodecanoic acid (CH3(CH2)lOC02H) from a dehydrated molecule. This implied that the
other ester moiety must have been larger than dodecanoyl group since substituents with
larger mass are cleaved preferentially during a-cleavage of molecules. Since the
molecular ion signal was not observed, it was not possible to determine unambiguously
the size of the ester moiety at this stage. During this study, similar tigliane diterpenoids
were isolated containing a 6,7- epoxide moiety. Since epoxides are of high reactivity and
undergo acid catalysed reactions with extreme ease, it is likely that chloroform used
during isolation of these compounds provided the ideal conditions and could have led to
acid catalysed ring opening of the epoxide to form this compound. P2b isolated during
this study had an epoxide at C-6 and C-7. Structural elucidation of this compound
showed that it also had a dodecanyl ester moiety. Since the two compounds were isolated
from the same extract, it is therefore possible that this compound is a reduction product of
P2b. Therefore, the second ester moiety was deduced as hexadecanoyl (CH3(CH2)14C02).
This was further supported by the fragments at m/z 239 and 185 in the EI mass spectrum
(Scheme 3) which were assigned to palmitoyl and lauroyl ion fragments, respectively.
The molecular formula of P, was therefore deduced as C4sHso07.
The proposed structure was further supported by mass fragmentation pattern as detailed
in scheme 3.
75
Scheme 3: Proposed fragmentation pattern of P, (142) in EIMS
+
+o =C(CHV14CH:!
mlz 239
o
142
m/z 526
-20H
++ 0 =C(CH2)lOCH3
m/z 185
o
mlz 294
The proton assignments of the suggested compound were further confirmed by COSY
experiment (Fig. 5).
Fig 5: Correlation signals observed in IH_IH COSY spectrum OfP5 (142)
142
On the basis ofHMBC (long range IH_13C) correlation, the ester derivatives were
assigned to C-13 and C-20 (Table 15).
76
Fig.6: Correlation observed in HMBC spectrum of P, (142)r-;
CH3(CH2)lOCH2CH2CH2CO~~
H,
142
COSY, 13C NMR, HMQC and HMBC spectra of P, indicated the presence of two ester
moieties at C-13 and C-20 and confirmed the proposed structure. The ruP AC name was
deduced as 4,9-dihydroxy-20-pentadecanoate-13 -dodecanoate-l ,6-tigliadien-3-one (142).
This compound is being reported for the first time.
5.1.7 Methyl3-heptaeicosanoyloxyoleanoate (P6) (143)
This compound was isolated as light orange oil (27 mg, Rr 0.40, Si02, 20: 1 n-
C6HI4:EtOAc). The lR spectrum showed characteristic bands at Umax 3054 (-OR), 1684 (-
C=C-) and 1264 (-C-O) em-I.
IHNMR is summarized in table 16.
77
Table16: IH NMR (600 MHz) and COSY data ofP6(143) in CDCh
Proton Chemical Multiplicity J (Hz) Integral
shift (0)
COSY
3 4.50 dd 3.3, 9.6 1H
12 5.28 t 3.4 1H
23 0.87 d 1.9 3H
24 0.90 s 3H
25 0.88 s 3H
26 0.73 s 3H
27 1.13 s 3H
29 0.93 s 3H
30 0.94 s 3H
2' 2.28 t 7.4 2H
3' 1.60 m 2H
4'-26' 1.26 br, m 46H
27' 0.86 t 5.6 3H
OCH3 3.63 s 3H
Others 1.17-1.99 m 20H
H-2a,H-2b
H-lla, H-llb
H-5
H-3'
H-2', H-4'
**
* Complete connectivities not determined due to overlap of IH NMR signals in this range.
IH NMR spectrum of P, revealed 9 methyl groups at 00.87 (d, J = 1.90 Hz, 3H, H-23);
0.90 (s, 3H, H-24); 0.88 (s, 3H, H-25); 0.73 (s, 3H, H-26); 1.13 (s, 3H, H-27); 0.93 (s,
3H, H-29); 0.94 (s, 3H, H-30); 0.86 (t, J = 5.6 Hz, 3H, H-27'); 3.63 (s, 3H, OCH3). An
olefinic proton at 0 5.28 (t, J = 3.4 Hz, 1H, H-12) was observed. In addition to several
methylene protons at 0 1.60 (m, H-3'); 2.28 (t, J = 7.4 Hz, H-2'); 1.17-1.99 (m, 20H)
integration revealed 46 other methylene protons overlapping at 0 1.26 which were
assigned to the substituent ester group (H-4'-H-26'). The observed IH NMR data of P,
was close to that of oleanolic acid (144) (Charlwood and Banthorpe, 1991).
HO
144
78
The l3C NMR spectra of P6 revealed 58 carbon signals consisting of 9 methyls, 34
methylenes, 6 methines and 9 quartenary carbons (Table 17).
Twocarbonyls were observed at 0 178.3 and 173.7. In addition, two olefinic signals were
also observed at 0 122.3 (d) and 143.8 (s). It was suggested that P6 contained an ester
groupattached at C-3 (0 173.7).
Since the IH NMR and l3C NMR data of the skeleton of P6 were identical to those of
oleanolic acid (144), P6 was proposed to be a diester of oleanolic acid, methyl 3-
heptaeicosanoyloxyoleanoate (143).
27' l'
H3C(H2Ch5C02
23
30, •.20 29
21
277
6
24
143
79
Table 17: l3C NMR (150 MHz), HMBC and HMQC data for P6 (143) in CDCh
Carbon Chemical shift (8) DEPT HMQC HMBC (H to C)
1 38.1 CH2 3,5,9,25
2 27.7 CH2 4, 10
3 80.6 CH 4.50 (dd, H-3) 1,5,23,24
4 37.7 C
5 55.3 CH
6 18.2 CH2
7 32.6 CH2
8 39.3 C
9 47.6 CH
10 37.0 C
11 22.7 CH2
12 122.3 CH
13 143.8 C
14 41.7 C
15 29.2 CH2
16 23.1 CH2
17 46.7 C
18 41.3 CH
19 45.9 CH2
20 30.7 C
21 33.9 CH2
22 32.4 CH2
23 28.1 CH3
24 15.3 CH3
25 16.7 CH3
26 16.8 CH3
27 25.9 CH3
28 178.3 C
29 33.1 CH3 0.93 (s, H -29)
30 23.6 CH3 0.94 (s, H-30)
I' 173.7 C
2' 34.9 CH2
3' 25.2 CH2
4' 23.6 CH2
5' 29.2 CH2
6' 29.3 CH2
7' 28.4 CH2
8' 29.6 CH2
9' 29.6 CH2
10' 29.6 CH2
11' 29.6 CHz
12' 29.6 CH2
13' 29.6 CH2
14' 29.6 CH2
15'-23' 29.6 (9) CH2
24' 29.7 CH2
25' 31.9 CHz
26' 23.4 CHz
27' 14.1 CH3
OCH3 51.5 CH3
1,3,7,9
4,8,9,10
5,9,14,26
1,5,7,12,14,25,26
5.28 (t, H-12)
8,10,13
9,14,18
8,13,17,27
14,18,22,28
12,14, 16,20,22,28
13,17,21,29,30
0.87 (d, H-23)
0.90 (s, H-24)
0.88 (s, H-25)
0.73 (s, H-26)
l.l3 (s, H-27)
17,19,29,30
16,18,20,28
3,5,24
3,5,23
1,5,9
7,9,14
8,13,15
19,21,30
19,21,30
2.28 (t, H-2') 4'
1.60 (m,H-3') 1',5'
1.26 (m, H-4') 2',6'
1.26 (m, H-5') 3', 7'
1.26 (m, H-6') 4', 8'
l.26 (m, H-T) 5',9'
1.26 (m, H-8') 6', 10'
1.26 (m, H-9') 7', 11'
1.26 (m, H-IO') 8',12'
1.26 (m, H-ll') 9', 13'
1.26 (m, H-12') 10', 14'
1.26 (m,H-13') 11',15'
1.26 (m,H-14') 12',16'
1.26 (m, H-15'-H-23') -
1.26 (m, H-24') 26'
1.26 (m, H-25') 27'
l.26 (m, H-26') 24'
0.86 (t, H-27') 25'
3.64 (s, OCH3) 28
80
C1mass spectrum gave quasimolecular ion peak at m/z 859 [M-H2t which indicated its
molecular weight to be 860. This was further confirmed by the mass fragments at m/z
453 [M-CH3(CH2)2SC02H + 4Ht and 429 [M-CH3(CH2)14C02H-H20 + 2Ht in the
positive ion mode C1 mass spectrum. The mass fragment observed in the E1 mass
spectrum at m/z 453 (Scheme 4) confirmed the loss of heptaeicosanoic acid. The
fragment of m/z 262 could be explained by retro-Diels-Alder of the daughter ion. The
other part of the molecule contained 31 carbons. The molecular formula was deduced as
CSJII0004.
The structure was confirmed through rigorous 2D NMR experiments (COSY, HMQC and
HMBC) and supported by the mass fragmentation pattern as detailed in scheme 4.
Scheme 4: Proposed fragmentation pattern of P, (143) in ElMS
+
+ 0= C(CH V2SCH3
m/z 393
143 m/z 453
/
+ ~co,o"
m/z 262m/z 191
m/z 203
This compound is being reported for the first time. However, long chain (>C20) fatty
acids exists in nature especially in blood plasma (Moser et al., 2002). No reference of a
plantnatural product having such long fatty acid substituent could be found.
81
5.1.8Montanin-20-palmitate (P7) (79)
This compound was isolated as colourless oil (34.7 mg, R, 0.59, Si02, 3:1 11-
C6H14:EtOAc). The IR spectrum showed characteristic bands at 'Umax 3522 (-OH), 1735
(C=O) and 1693 cm-1 (-C=C-C=O). The UV spectrum of P» revealed maxima absorption
bands at Amax 230, 248 and 334 nm, characteristic of compounds with conjugated double
bonds.
IHNMR spectral data is summarised in table 18.
Table 18: 'a NMR (600 MHz) and COSY data OfP7 (79) in CDCb
Proton Chemical Multiplicity J (Hz) Integral
shift (0)
COSY
1 7.60 s 1H
5 4.26 s 1H
7 3.33 d 4.8 1H
8 2.91 d 4.8, 2.3 1H
10 3.78 s 1H
11 2.46 m 1H
12a 2.21 dd 8.7, 14.5 1H
12b 1.62 dd 4.4, 14.5 1H
14 4.34 d 2.3 1H
16a 5.02 s 1H
16b 4.90 s IH
17 1.78 s 3H
18 1.16 d 7.1 3H
19 1.80 s 3H
20a 4.77 d 11.9 1H H-20b
20b 3.83 d 11.9 IH H-20a
2' 1.94 t 4.5 2H 3
3',3" 1.60 m 4H 2',2",4',4"
4'-11',4"-15" 1.26 br,s 40H *
12' 0.87 t 7.0 3H H-l1'
2" 2.33 t 7.5 2H 3"
16" 0.99 t 7.1 3H H-15"
5-0H 3.72 br, s IH H-5
5-0H
H-8
H-7, H-14
H-12a, H-12b, H-18
H-ll, H-12b
H-ll, H-12a
H-8
H-ll
* Complete connectivities not determined due to overlap of IH NMR signals in this range
IH NMR spectrum OfP7 revealed 5 methyl groups at 0 0.87 (t, J = 7.0 Hz, 3H, H-12');
0.89 (t, J = 7.1 Hz, H-16"); 1.16 (d, J = 7.1 Hz, 3H, H-18); 1.80 (s, 3H, H-19); 1.78 (s,
3H, H-17), several methylene groups at 05.02 (s, H-16a); 4.90 (s, H-16b); 2.21 (dd, J =
82
7.6, 14.5 Hz, 1H, H-12a); 1.62 (dd, J = 7.6, 14.5 Hz, 1H, H-12b); 4.77 (d, J = 11.9 Hz,
lH, H-20a); 3.83 (d, J = 11.9 Hz, 1H, H-20b); 2.33 (t, J = 7.5 Hz, 2H, H-2"); 1.94 (t, J =
4.5 Hz, 2H, H-2') and a deshielded olefinic proton at 0 7.60 (s, H-1). In addition to the
observed methylene proton signals, integration revealed 44 other methylene protons
overlapping between 0 1.26 and 1.60 that were assigned to the substituent ester moieties
(H-3'-H-11', H-3"-H-15"). A deshielded olefinic proton was observed at 0 7.60 (s, H-1).
Thesignals at 01.60 (m, 4H) were assigned to H-3' and H-3" respectively. The 1HNMR
spectra data of P7 was identical to that of montanin-20-palmitate (79) (Tchinda et aI.,
2003).
The l3C NMR spectra of P7 revealed 48 carbon signals consisting of 5 methyls, 27
methylenes,7 methines and 9 quartenary carbons (Table 19).
Two carbonyls were observed at 0 209.6 and 173.4. In addition, four olefinic signals
wereobserved at 0 161.0 (d), 136.5 (s), 146.4 (s) and 111.1 (t). It was suggested that P7
contains an artha-ester (0 119.4) and an ester (0 173.4) group attached at C-20. The eight
signals between 084.0 and 59.2 were assigned to the oxygenated carbons at C-13, C-14,
C-9, C-4, C-5, C-20, C-7 and C-6.
The IH NMR, l3C NMR and IR spectral data for P7 were identical to literature values
henceP7 was identified as montanin-20-palmitate (79).
16
79
83
Table 19: 13C NMR (150 MHz), HMBC and HMQC data for P7 (79) in CDCb
Carbon Chemical shift (0) DEPT HMQC HMBC (HtoC)
1 16l.1 CH 7.60 (s,H-l) 3,4,9, 19
2 136.5 C
3 209.6 C
4 72.2 C
5 70.0 CH 4.26 (s, H-5) 3,7,10,20
6 59.2 C
7 64.1 CH 3.33 (d,H-7) 5,9, 14,20
8 36.5 CH 2.91 Cd, H-8) 6,10,11,13
9 78.7 C
10 48.0 CH 3.78 (s, H-lO) 2,3,5,8, 11
11 34.7 CH 2.46 (m, H-l1) 8,10,13
12 36.4 CH2 2.21 (dd, H-12a) 9,14,15,18
1.62 (dd, H-12b) 9,14,15,18
13 84.0 C
14 81.7 CH 4.34 (d, H-14) 7,9,12,15
15 146.4 C
16 11l.1 CH2 5.02 (s, H-16a) 13,17
4.90 (s,H-16b) 13,17
17 19.0 CH3 1.78 (s, H-17) 13,16
18 20.3 CH3 1.16 (d,H-18) 9,12
19 9.9 CH3 1.80 (s, H-19) 1,3
20 65.7 CH2 4.77 (d, H-20a) 5,7,1"
3.83 (d, H-20b) 5,7,1"
I' 119.4 C
2' 34.8 CH2 1.94 (t, H-2') 4'
3' 24.9 CH2 1.60 (m, H-3') 1',5'
4' 29.1 CH2 1.26 (m, H-4') 2',6'
5' 29.4 CH2 1.26 (m, H-5') 3',7'
6' 29.6 CH2 1.26 (m, H-6') 4',8'
7' 29.6 CH2 1.26 (m, H-7') 5',9'
8' 29.6 CH2 1.26 (m, H-8') 6',10'
9' 29.7 CH2 1.26 (m, H-9') 7', II'
10' 31.9 CH2 1.26 (m, H-I0') 8',12'
II' 22.9 CH2 1.26 (m, H-II') 9',13'
12' 14.1 CH3 0.87 (t, H-12') 10'
I" 173.4 C
2" 34.1 CH2 2.33 (t, H-2") 20,4"
3" 23.4 CH2 1.60 (m, H-3") 1",5"
4" 29.3 CH3 1.26 (m, H-4") 2",6"
5" 29.5 CH2 1.26 (m, H-5") 3",7"
6" 29.6 CH2 1.26 (m, H-6") 4",8"
7" 29.6 CH2 1.26 (m, H-7") 5",9"
8" 29.6 CH2 1.26 (m, H-8") 6",10"
9" 29.6 CH3 1.26 (m, H-9") 7", 11"
10" 29.6 CH2 1.26 (m, H-I0") 8",12"
II" 29.6 CH2 1.26 (m, H-ll ") 9",13"
12" 29.6 CH2 1.26 (m, H-12") 10",14"
13" 29.7 CH2 1.26 (m, H-13") 11", IS"
14" 31.9 CHz 1.26 (m, H-14") 12",16"
15" 22.7 CH2 1.26 (m, H-15") 13"
16" 14.1 CH3 0.89 (t, H-16") 14"
84
The positive ion mode CI mass spectrum gave quasimolecular ion peak at m/z 799
[M+Ht which indicated its molecular mass to be 798. This was further confirmed by the
mass fragments at m/z 543 [M-CH3(CH2h4C02l! + Ht, 525 [M-CH3(CH2)t4C02l!-H20
+ Ht (preferential a-cleavage of the heavier substituent), 343 [M-CH3(CH2)lOC02lI +
Ht and 325 [M-CH3(CH2)10C02H-H20 + Ht (subsequent a-cleavage of the relatively
lighter substituent) in the positive ion mode CI mass spectrum. The mass fragments
observed in the EI spectrum at m/z 239 and 185 (Scheme 5) could be assigned to
palmitoyl and lauroyl ion fragments, respectively. The molecular formula was deduced
asC48H7909.
The fragmentation pattern of'P» was similar to that ofmontanin-20-palmitate (79).
Scheme 5: Proposed fragmentations of P, (79) in EIMS
+
+ O=oC(CH:z)14CH3
OH
m/z 239
o OH
mlz 543
79
j
+
+ 0 =oC(CH2)gCH3
m/z 185
o OH
m/z 324
The structure was confirmed through rigorous 2D NMR experiments. The COSY
correlations for P7 are summarised in fig. 7.
85
Fig.7 Correlations signals observed in IH_IH COSY spectrum OfP7 (79)
79
TheHMBC correlations for P7 are summarised in fig. 8.
Fig. 8 Correlations observed in HMBC spectrum OfP7 (79)
79
86
In the HMBC spectrum, the ester carbonyl at () 173.8 showed correlations to the
characteristic AB proton pattern ofH-20 (0 3.83, d, J = 11.9 Hz, H-20a; 4.77, d, J = 11.9
Hz, H-20b) as reported in literature (Tchinda et a/., 2003). DQF-COSY, HMQC and
HMBC spectra of P7 confirmed the presence of two ester moieties, dodecanoyl at C-13
and hexadecanoyl at C-20. Montanin-20-palmitate (79) has been isolated before from
Neoboutonia glabrescens (Tchinda et al., 2003) but it is being reported for the first time
inNeoboutonia macrocalyx.
5.1.9 12-Deoxyphorbol-13-pentadecanoate (Ps) (145)
The compound was isolated as a light yellow gum (9.3 mg, R, 0.59, Si02, 1:1 CHCh:
EtOAc). The 1R spectrum revealed bands at Umax 3675 (-OH) , 1711 (-C=C-C=O) and
1643(-C=C-).
IHNMR revealed the following signals (Table 20).
The IH NMR spectrum of P, revealed 5 methyl groups at () 0.89 (t, J = 5.6 Hz, 3H, H-
IS'); 0.93 (d, J = 6.5 Hz, 3H, H-~8); 1.78 (s, 3H, H-19); 1.19 (s, 3H, H-16); 1.06 (s, 3H,
H-17) and 2 olefinic signals at 0 7.59 (s, IH, H-l); 5.68 (d, J = 5.0 Hz, IH, H - 7). Two
allylic proton signals at 0 4.39 (d, J = 13.8, IH, H-20a) and 4.27 (d, J = l3.8, IH, H-20b)
indicated that the C-20 hydroxyl was free. Therefore the ester moiety was placed at C-13
of the 12-deoxyphorbol (Ma et al., 1997). It has been reported that in all tigliane
diterpenoids isolated from nature, H-8 is ~, 9-0H and H-I0 are a (Ma et a/., 1997).
87
Table20: IH NMR (600 MHz) and COSY data ofPs (145) in CDCh
Proton Chemical Multiplicity J (Hz) Integral COSY
shift(o)
1 7.59 s IH
5a 2.49 d 12.3 IH H-5b
5b 2.29 d 12.3 IH H-5a
7 5.68 d 4.0 IH H-8
8 3.00 dd 4.0,6.6 IH H-7, H-14
10 3.28 s IH
11 1.97 m IH H-12a, H-12b, H-18
12a 2.06 dd 7.1, 14.7 IH H-ll, H-12b
12b 1.55 dd 11.4,14.7 IH H-ll, H-12a
14 0.83 d 6.6 IH H-8
16 1.19 s 3H
17 1.06 s 3H
18 0.93 d 6.5 3H H-l1
19 1.78 s 3H
20a 4.39 d 13.8 IH H-20b
20b 4.27 d 13.8 IH H-20a
2' 2.30 t 7.5 IH H-3"
3' 1.59 m 2H H-2', H-4'
4'-14' 1.26 br,m 22H *
IS' 0.89 t 5.6 3H H-14'
OR 2.24 br, s IH
OR 2.91 s IH
OR 5.58 br, s IH
* Completeconnectivitiesnotdeterminedduetooverlapof IH NMR signalsin thisrange
On comparing the IH NMR spectral data of Pg with the literature values, close similarity
was observed with 12-deoxyphorbol-13-hexadecanoate (86) (Ma et aI., 1997; Tchinda et
al., 2003) except for additional signals at 0 0.89 (t, J = 5.6, 3H, H-151), 1.26 (br, s, 22H,
H- 4'-H-14'), 1.59 (m, 2H, H-3') and 2.30 (dd, J = 7.3, 7.5 Hz, 2H, 2') indicating that Pg is
an ester derivative of 12-deoxyphorbol-13-pentadecanoate (86) and the orientation ofH-
8, 9-0H and H-IO are f3,a and a, respectively (Ma et aI., 1997). Previous IH NMR work
showed that~o of the two protons (H-5a and H-5b) in the presence of 4a-OH is larger
than those with 4f3-0H (Gschwendt and Hecker, 1969). In IH NMR spectrum of'Ps, two
non-equivalent methylene protons appeared as two doublets at 0 2.49 (J = 12.3 Hz, IH,
R-5a) and 2.29 (J = 12.3 Hz, IH, H-5b) with ~o = 0.20, which confirmed presence of 4f3-
OH. Therefore, Pg was deduced to have a 4f3-0H, 8f3-H, 9a-OH, lOa-H, lla-Me, 13a-
OR and 14a-H. The signals due to the ester group were observed at 02.30 (t, J = 7.5 Hz,
88
2H, H-2'), 1.59 (m, 2H, H-3') and the broad multiplet at 0 1.26 (24H, H-4'-H-15'). The
datais in agreement with the literature values (Tchinda et aI., 2003; Zhao et aI., 1998; Ma
et aI., 1997).
The BC NMR spectra of Ps revealed 35 carbon signals consisting of 5 methyls, 16
methylenes, 6 methines and 8 quartenary carbons (Table 21).
Several characteristic carbon signals were determined including two carbonyls at 0 209.3
and 176.0, 2 trisubstituted double bonds at 0 161.2 (d, C-1); 139.8 (s, C-6); 132.8 (d, C-
2); 130.3 (s, C-7), 3 oxygenated quartenary carbons at 0 76.0 (s, C-9); 73.8 (s, C-4); 63.4
(s, C-13) and 1 oxygenated methylene at 0 68.3 (1, C-20). Comparison of the spectral
data of Ps with those of tigliane diterpenoids 75, 84, 86, 87 and 142, the cyclopropane
ringcharacteristic of these diterpenoids was present at C-13 and C-14.
89
Table 21: BC NMR (150 MHz), HMBC and HMQC data for Ps (145) in CDCh
Carbon Chemical shift DEPT HETCOR HMBC (HtoC)
(8)
1 161.2 CH 7.59 (s, H-l) 3,4,19
2 132.8 C
3 209.3 C
4 73.8 C
5 38.7 CH2 2.49 (d, H-5a) 3,7,10,20
2.29 (d, H-5b) 3,7,10,20
6 139.8 C
7 130.3 CH 5.68 (d,H-7) 5,9,14,20
8 39.2 CH 3.00 (dd, H-8) 6, 10,11, 13, 15
9 76.0 C
10 55.8 CH 3.28 (s, H-lO) 2,3,5,8,11
11 36.3 CH 1.97 (m, H-ll) 8, 10,13
12 31.8 CH2 2.06 (dd, H-12a) 9,14,15,18
1.55 (dd, H-12b) 9,14,15,18
13 63.4 C
14 32.6 CH 0.83 (d, H-14) 7,9,12,16,17
15 22.7 C
16 23.2 CH3 1.19 (s, H-16) 13,14,17
17 15.3 CH3 1.06 (s, H-17) 13,14,16
18 18.5 CH3 0.93 (d, H-18) 9, 12
19 10.1 CH3 1.78 (s, H-19) 1,3
20 68.3 CH2 4.39 (d, H-20a) 5,7
4.27 (d, H-20b) 5, 7
1' 176.0 C
2' 34.6 CH2 2.30 (t, H-2') 4'
3' 24.8 CH2 1.59 (m, H-3') l' -,, )
4' 29.1 CH2 1.26 (m, H-4') 2',6'
5' 29.2 CH2 1.26 (m, H-5') 3',7'
6' 29.3 CH2 1.26 (m, H-6') 4',8'
7' 29.3 CH2 1.26 (m, H-7') 5',9'
8' 29.4 CH2 1.26 (m, H-8') 6',10'
9' 29.6 CH2 1.26 (m, H-9') 7', 11'
10' 29.6 CH2 1.26 (m, H-I0') 8',12'
11' 29.6 CH2 1.26 (m, H-ll ') 9',13'
12' 29.6 CH2 l.26 (m, H-12') 10',14'
13' 29.7 CH2 l.26 (m, H-13') 11',15'
14' 22.7 CH2 l.26 (m, H-14') 12'
15' 14.1 CH3 0.89 (t, H-15') 13'
The positive ion mode CI mass spectrum gave quasimolecular ion adduct peak at m/z 591
[M+CHst and a quasimolecular ion peak at m/z 573 [M+Ht which indicated its
molecular mass to be 572. It was characterised by a unique signal appearing at m/z 295
amu (100010) which was 36 amu lower than the skeletal mass. This implied that there was
90
inductivecleavage of the hydroxyl groups leading to the loss of two molecules of water
andfurther ionisation leading to loss of the pentadecanoyl ion fragment. The presence of
anestergroup was confirmed by the CI molecular ion peak appearing at m/z 573 and the
pentadecanoylion fragment appearing at m/z 225 in the EI mass spectrum (Scheme 6).
Theother part of the molecule contained 20 carbons. The molecular formula of P, was
deducedas C35H5606.
Fromthe above observations, together with consideration of its molecular formula, Pg
was proposed to have a tigliane-type diterpenoid skeleton. In addition to an a,~-
unsaturated cyclopentenone, Ps should have two hydroxyls at C-4 and C-9 in the
proposed skeleton (Ma et al., 1997). From IH and l3C information together with
literatureNMR data of mellerin A (75) (Zhao et al., 1998) and 12-deoxyphorbol-13-
hexadecanoate (86) (Ma et a/., 1997), a diterpenoid with a tigliane skeleton 12-
deoxyphorbol-13-pentadecanoate (145) was proposed.
15' l'
CH3(CHV13C«2 16
, I
r.:::--'...••' 1_3--.,,;..-',' 17
15
19
o
145
Themass fragmentation pattern (Scheme 6) supported the proposed structure 145.
91
+
+ O==C(CH2)13CH3
m/z 225
m/z 330
145 HO +
~+
~ o OH CH20H
m/z 239 m/z 109
m/z 189
The structure was confirmed by further 2D NMR experiments. The correlations observed
in the COSY spectrum of'P, are summarised in fig. 9.
Fig. 9: Correlations observed in IH_IH COSY spectrum ofPs (145)
92
HMBC(Fig. 10) also supported the proposed structure (145).
Fig10: Correlations observed in HMBC spectrum of Ps (145)
COSY, HMQC and HMBC spectra of Ps confirmed the presence of an ester group at C-
13 thus confirming the structure. To our best knowledge, this diterpenoid has been
isolatedfor the first time though it only differs slightly from 12-deoxyphorbol-13-
hexaethanoate(67) (Zhao et al., 1998). Other similar diterpenoids have previously been
isolatedfrom Neoboutonia glabrescens (Euphorbiaceae) (Tchinda et al., 2003) and
Euphorbiafischeriana (Euphorbiaceae) (Ma et aI., 1997). The compound is being
reportedfor the first time.
5.2Structural elucidation of compounds isolated from Fagaropsis angolensis
5.2.1Kl
This compound was isolated as light green oil (209.1 mg, Rf 0.56, Si02, 19:2
C6H14:EtOAc). Spectral analysis is still going on.
93
5.2.2FASD 2
Thiscompound was isolated as a yellow solid (18.0 mg, mp 253-254 "c, R, 0.63, Si02,
3% MeOH:EtOAc). Attempts to obtain spectroscopic data on FASD 2 were
unsuccessfu1.
5.2.3FASD 3
This compound was isolated as white crystalline needles (8.0 mg, mp 246-248 °c, Rr
0.28, Si02, 80:1 CHCh:C6HI4). Attempts to obtain spectroscopic data on FASD 3 were
unsuccessful.
5.2.4 FASD 4
Thiscompound was isolated as an orange solid (18.0 mg, mp 172-174 "c, RfO.70, Si02,
3% MeOH:EtOAc). Attempts to obtain spectroscopic data on FASD 4 were
unsuccessful.
5.2.5FASM 1
This compound was isolated as a white crystalline solid (25.6 mg, mp 290-291 °c, Rr
0.64, Si02,2% MeOH:CH2Cb). Attempts to obtain spectroscopic data on FASM 1 were
unsuccessful.
5.2.6FASM2
This compound was isolated as a white crystalline solid (39.7 mg, mp 283-285 °c, R,
0.59, Si02,2% MeOH:CH2Ch). Attempts to obtain spectroscopic data on FASM 2 were
unsuccessful.
5.2.7FASM3
This compound was isolated as a white crystalline solid (16.9 mg, mp 276-278 "c, Rr
0.58, SiOl, 1:20 MeOH:CHCb). Attempts to obtain spectroscopic data on FASM 3 were
unsuccessfu1.
94
5.2.8 FASM 4
Thiscompound was isolated as white crystalline solid (13.6 rng, mp 286-287 °c, Rf 0.47,
Si02, 4:1 CHCh:n-C6H14). Attempts to obtain spectroscopic data on FASM 4 were
unsuccessful.
5.2.9 FASC 1
Thiscompound was isolated as white crystalline solid (10.5 mg, mp 148-150 °c, Rr 0.36,
Si02, 15:1 CH2Ch:EtOAc). Attempts to obtain spectroscopic data on FASC 1 were
unsuccessful.
95
CHAPTER 6
CONCLUSION AND RECOMMENDATIONS
6.1 Conclusion
It was found that two plants Fagaropsis angolensis and Zanthoxylum usambarense used
bytraditional healers in Meru and Kilifi districts to treat malaria exhibited good in vitro
anti-plasmodial activity. The methanol extracts are the most active. Cytotoxicity studies
showedthat only N macrocalyx and F angolensis had toxic effects.
Thelow in vitro anti-plasmodial activity in some plants despite the fact that they have
been used as traditional anti-malarials for centuries could partly be explained by the
circumstances under which many plants are used in the treatment of malaria. They may
be useful in managing other manifestations associated with malaria but may have low
therapeutic (anti-parasitic) activity. These would include reducing fever, comforting
convulsions and headache, and possibly even immunostimulatory effects (Rasoanaivo et
al., 1992). Another possible explanation is that traditional healers give a concotion of
plants for the treatment and therefore they may only be active in combination, due to
synergistic effects of several compounds that are inactive singly (Gessler et al., 1994). It
is also possible that some of the compounds that are in active ill vitro could exhibit
activity in vivo due to enzyme catalysed transformation into potent derivatives and
therefore are playing the IDle of prodrugs. This phenomenon has been demonstrated for
A. indica extracts (Parida et al., 2002).
The fact that Z. usambarense exhibited good in vitro anti-plasmodial activity (IC5(l < 6
ug/ml) but was classified to have low activity (LDso > 90 flglml) in brine shrimp test
shows that the latter should not be used alone to determine biological activity. This
It was also observed that the compounds isolated from N. macrocalyx had lower in vitro
anti-plasmodial potency than the crude extracts which showed that activity was lost on
96
purification. This suggests that activity in the crude extracts may be due to synergistic
effectsof the different compounds therein.
Eight compounds were isolated from N. macrocalyx extracts of which 5 are tigliane and
daphnane diterpenoids, (79, 140-142, 145). Four of these diterpenoids are being reported
for the first time. A new diester of oleanolic acid (143) was also isolated in addition to
stigmasterol (U6). One more compound (P3) remains unidentified Coincidentally, the
samecompound exhibits toxicity to brine shrimp nauplii. This compound (P:.;) should be
identified to understand the basis of toxicity of this plant. Although the isolated
compounds did not show any activity, their combinations still need to be investigated to
confirm their synergism or othef\~lis.e~
Interestingly, ethyl acetate and dichloromethane extracts of N. macrocalyx showed mild
in vitro anti-plasmodial activity while methanol extract was inactive. This puts ta test the
useof N. macrocalyx for malaria therapy in traditional systems since invariably water IS
used in such decoctions. However, it \vould be interesting to investigate the process of
traditional drug preparation and its effects on efficacy. IIis KIIO\\'D that the herbal drug
preparation process 1S crucial for efficacy. If not prepared well actrvrty may be lost or
toxicity enhanced and even artifacts produced (Kim et al., 2004).
During powdering of materials of N macrocalyx and isolation of compounds, a pungent
and irritating feeling in the nose and painful rashes on the face were noticed. It is well
documented that tigliane and daphnane diterpenoids from plants 111 ..rue family
10()f.;.\• ~ ~~l·1998; Oksuz et al.,
F'TTt r' 'h.o rr dii "1 -n 4A1fi ""'4"' "'4-' .•......- . ., . ., r- .1·",rnererore, L.N nve nerpenoros /7, .I."Ju,.1. .I., 1 ~ ana .1"1;:'isosarec rrom nus pram may
be responsible for these effects.
Nine compounds were isolated from F. angolensis; Kl, FASD2, FASD3, FASD4,
FASMl, FASM2, FASM3, FASM4 and FASCI. It \WS not possible to determine their
structures since they were isolated in small amounts and could not dissolve entirely in
97
this study and it could have been important to determine the compounds present as well
as their in vitro anti-plasmodial activity. However, some spectral data of'K, was obtained
but the information was not sufficient to elucidate the structure. In vitro anti-plasmodial
as well as toxicological studies on K1 showed that it was not the active principle in this
plant. Therefore, biological studies of the oilier compounds are important to ascertain me
most active compound. It is important to note that nitidine (66) a compound reported as a
constituent of this plant was not isolated but has been recorded to exhibit anti-plasmodial
activity (Gakunju et al., 1995). This suggests that oilier constituents may also be
responsible for its activity in addition to nitidine (06).
The hypothesis set out at the onset of this study which stated that there are Kenyan plants
used in traditional malaria therapy in Kilifi and Meru districts that may provide stable,
isolable and identifiable compounds retaining their anti-plasmodial activity singly or
J\Zmacrocalyx resulted in loss of in vitro anti-plasmodial activity.
6.2 Reeommendatiens
It is recommended that further in vivo anti-plasmodial efficacy tests with Plasmodium
berghe! in mice art! important in order to validate the l~VJ vitro results. LFJ vitro assays
Similarly, drug interaction studies should be carried out on the crude as \~le!las the
isolated compounds using standard dose-response assays over a range of individual
drug concentrations to establish their potentiating effects. Since no smgle compound
necessary to confirm synergism.
and identifying the active principlets]. It may be necessary to analyse the crude
98
extracts for the presence of nitidine and structurally related compounds using standards
or LC-MS.
Further toxicity studies (acute, chronic and cytotoxicity on cells) should be carried out
on the crude active fractions to establish their safe levels for use by humans. This
should help in calculation of the safety dosage required for parasite clearance. The
dosage if calculated would help in improving the safety of F. angolensis-based herbal
drugs for malaria treatment.
Since N macrocalyx is used in traditional therapy as anti-septic, it would be interesting
to test the isolated compounds for anti-bacterial and anti-fungal activity. This is
particularly important for the management of resistant isolates of pathogenic micro-
organisms associated with the opportunistic infections in immuno-compromised
persons.
Plants which exhibited moderate [Z usambarense) and mild (M salicifolia, A. nilotica,
S. heningsii and H. abyssinica) in vitro anti-plasmodial activity in this study need
further biochemical investigations.
99
CHAPTER 7
EXPERIMENTAL
7.1Materials, reagents and equipment
7.1.1 Reagents
Aceticacid, citric acid, dextrose, Giemsa stain, glycerol and N-2-hydroxyethylpiperizine
N-2-ethanesulfonic acid (HEPES) were purchased from Sigma Chemical Company, St.
Louis, US.A. eH]-Hypoxanthine, methanol, sodium hydrogen carbonate, sodium
chloride, Rosewell Park Memorial Institute 1640 (RPMI 1640) powdered medium were
purchased from Gibco Laboratories, California, US.A. Culture gas mixture (92% N, 5%
C02,3% O2) was purchased from British Oxygen Company (BOC), Nairobi, Kenya. The
organic solvents like acetone, n-hexane, ethyl acetate, dichloromethane, chloroform,
ethanol, butanol and methanol were purchased from, Kobian, Nairobi, Kenya. All
solvents used for the extraction, fractionation and crystallization were double distilled to
remove impurities, stabilizers, or other organic components present in small amounts and
were stored in amber 2.5 Ibottles. Ethanol was also used in sterilization while methanol
wasalso used in fixing of thin dry blood smears.
7.1.2 Equipments (01' the in vitro culture
Laminar flow hood, liquid scintillation counter, microscopes, refrigerators (-4 and -80
"C), incubator, gas-tight box, cell harvester, analytical balance with sensitivity of 0.1 mg,
vacuum pump, centrifugal machine, adjustable volume Eppendorf micro-pipette,
automatic pipet pump, vibro mixer and electrically heated water bath were used.
7.1.3 Disposable plastics and glassware
Anti-coagulant-free blood collecting bags and sterile gloves (Triflex®), 15 and 50 ml
centrifuge tubes, (Brinkmann Instruments Company, Westbury, US.A), 50 and 150 ml
culture flasks, (Corning® US.A), microscope slides and cover slips (Sigma Chemicals
Company, US.A), 0.45 and 0.22 11mfilter units (Naglene®) Naglene Company, US.A),
serological Pasteur pipettes (Fischer Scientific, Pittsburgh, US.A) were acquired through
Baxter Diagnostics, US.A.
100
7.1.4Recycled glassware
Re-usable glassware was soaked in hot water with liquid detergent. They were then
washedand rinsed thoroughly with tap water, several times with distilled water, ethanol
andacetone. They were then dried at 110°C in an electric oven for at least 1 hour and
allowedto cool slowly to room temperature before use.
7.1.5Sterilizing materials
Greatcare was taken to keep all the materials for culture sterile. All culture experiments
werecarried out in a laminar flow hood. Sterilised Pasteur pipettes, lids and disposable
pipettes were passed over a Bunsen burner flame several times before use. Ethanol
(70%) was used to sterilize the hood and other equipment. The used Pasteur pipettes
wereput in 20% sodium hypochlorite to disinfect before washing. Disposable apparatus
weresimilarly disinfected before being discarded.
7.2Plant Material
7.2.1Sampling
Plant selection was based on ethno-botanical information combined with information in
the literature Kokwaro (1993). They included Strychnos heningsii, Zanthoxylum
usambarensis, Carissa edulis, Withania somnifora, Fagaropsis angolensis, Neoboutonia
macrocalyx, Harrissonia abyssinica, Azadirachta indica, Myrica salicifolia and Acacia
nilotica. They were identified, authenticated by Mr. G.M. Mungai of East African
Herbarium based at National Museum of Kenya (NMK). The voucher specimens were
deposited in the East African Herbarium.
7.3 General procedures
7.3.1 Extraction
Each plant material was air dried at room temperature, pulverised and weighed. A known
amount of sample was extracted by maceration in organic solvents in increasing polarity.
The extracts were decanted and filtered through Whatman filter paper and the marcerate
and steeped again for 24 hours. The extraction process was repeated until a clear extract
101
was obtained. The filtered extracts were combined and solvent removed under reduced
pressure. The dried samples were weighed, mass noted down and stored in a freezer.
7.3.2Brine shrimp assay
7.3.2.1Hatching shrimp
Brine shrimp eggs, Artemia salina Leach, were hatched in artificial sea water prepared by
dissolving 38 g of sea salt (Sigma Chemicals Co., UK.) in 1 litre of distilled water. After
48 hours incubation at room temperature (22-29 °C), the larvae (nauplii) were attracted to
one side of the vessel using a light source and collected with pipette. Nauplii were
separated from eggs by aliquoting them three times in small beakers containing sea water.
7.3.2.2 Test protocol
Samples were dissolved in 50 J.lI dimethylsulphoxide (DMSO) and diluted with artificial
sea salt water. Sea water 50 ul was placed in all the wells of the 96-well micro-titre plate
and 50 J.lI of 4000 ppm of the plant extract placed in row 1 and two-fold dilution carried
out down the column. The last row was left with sea water only and served as drug-free
control. Subsequently, 50 J.lI suspension of nauplii containing about 10 larvae was added
into each well and incubated for 24 hours. The micro-titre plates were then examined
under a microscope (x12.5) and the number of dead nauplii in each well counted.
Lethality concentration fifty (LCso) values were then calculated by probit computer
program method developed by Finney (Finney, 1964).
7.3.3 In vitro anti-plasmodial assay
7.3.3.1 Parasites
In this study, two strains of P. falciparum parasites (ENT30 and NF54) were used.
ENT30 is chloroquine-resistant while NF54 is a chloroquine-sensitive strain. All these
parasites were obtained from the Centre for Biotechnology Research and Development
(CBRD) malaria parasite bank at KEMRI, Kenya. ENT30 (field isolate) was originally
collected from Entosopukia, Kenya (Sabah, Personnal communication) whereas NF54
was collected from Amsterdam Airport (ponnudurai et al., 1981; Delemarre and Van der
Kaay, 1979).
102
7.3.3.2Parasite Cultivation
Parasite cultivation was based on the in vitro technique described by Trager and Jensen
(1976). Cultivation was carried out aseptically in a laminar flow hood.
7.3.4Culture preparations
7.3.4.1RPMI 1640lHEPES medium
This was prepared according to Trager and Jensen (1976). Briefly, it contained 25 nM
Hepes (5.94 g/l) and 10.5 g RPMI 1640-powdered medium (withoutp-aminobenzoic acid
(PABA) and lactic acid (LA) dissolved in 960 ml of distilled and autoclaved water. It
was filtered and sterilized using a vacuum pump and 0.22 urn filter, and stored at 4 °C
beforeuse within 4 weeks.
7.3.4.2Wash medium (WM)
It was prepared according to Rowe et al (1968) by mixing 95.8%v/v of the RPMI 1640
andHEPES medium and 4.2% v/v of 5% w/v sodium carbonate.
7.3.4.3Uninfected erythrocyte
The methods used were adopted from Trager and Jensen (1976). Briefly, uninfected
blood group 0 Rhesus positive from Kenya Medical Research Institute (KEMRI)
recruited volunteers was drawn into 15% (v/v) acid-citrate-dextrose (ACD). Enzyme-
linked immunosorbent assay (ELISA) method was used to screen blood for human
immune virus (HIV) and hepatitis B infections at Centre for Virology Research (CVR),
KEMRI. Prior to blood donation, it was ascertained that the individuals had not
contracted malaria or visited a malaria endemic area in the past two months. It was also
ascertained that the donor had not taken any anti-malarial or anti-biotic drugs.
The blood was washed free of plasma and white blood cells before use in the culture by
centrifugation at 3600 rpm for 10 min at 4°C. The plasma and buffy coat at the top of
the cell pellet was aspirated and discarded. The red cell pellet was washed twice with 2
volumes of wash medium (WM) and the resulting suspension centrifuged at 3600 rpm for
103
IOminat 4 "C. After the last wash, the packed cells were re-suspended in an equal
volumeofWM to obtain a haematocrit of 50%. The cells were exposed to a gas mixture
(92% N2, 5% C02 and 3% 02) and stored at 4°C and used within two weeks.
7.3.4.4Preparation of human serum
Themethods used were adopted from Trager and Jensen (1976). Briefly, blood from
donorswas collected aseptically into blood bags without anti-coagulants and allowed to
clotby leaving it at room temperature for 90 minutes followed by an overnight storage at
4°C. The following day, the serum was carefully dispensed into sterile 50 ml centrifuge
tubesand centrifuged at 3000 rpm for 10 minutes at 4 "C. The serum was aseptically
aliquoted into sterile 10 ml snap-top tubes and heat inactivated in a water bath at 56°C
for50 minutes. The tubes were placed in upright position at -20°C overnight and then
keptat -70 "C until they were used.
7.3.4.5Complete culture medium with serum (CMS)
Themethods used were adopted from Trager and Jensen (1976). Briefly, the eMS was
prepared by mixing 86.22% (v/v) of RPMI 1640/HEPES, 3.78% (v/v) of 5% NaHC03
and 10% (v/v) human serum. The eMS was stored at 4°C and used within one week of
preparation.
7.3.4.6Thawing of the malaria parasites
The methods used were adopted from Rowe et al (1968). Briefly, laboratory strains of
malaria parasites preserved in liquid nitrogen were removed quickly thawed in a water
bathmaintained at 37°C. The ampoules were surface sterilized with 70% ethanol. The
stabilites were then gently agitated and transferred to a sterile 15 ml centrifuge plastic
tubewhile still cold. The cells were centrifuged in a thermostated centrifugal machine at
1500 rpm for 5 minutes at 20°C. The supernatant (SN) was aspirated and discarded.
Thepacked cells were immediately re-suspended in 0.3 ml of filter sterilized 3.5 (w/v)
sodiumchloride in distilled autoclaved water (DAW) and immediately re-centrifuged and
the supernatant aspirated and discarded again. This was to prevent osmotic lysis during
104
theremoval of the glycerol from cells. The parasites were finally re-suspended in 1 ml of
CMS,centrifuged and supernatant aspirated and discarded.
7.3.4.7Setting of the culture
Themethod described by Trager and Jensen (1976) was used. Briefly, after washing the
parasites,the packed cell volume (pcv) of the parasitized erythrocytes was estimated and
thevolume of the RBC adjusted to 6% (v/v) (6% haematocrit (hct) by the addition of the
CMS. The culture flasks were exposed to a gas mixture (92% N2, 5% CO2 and 3% O2)
andincubated at 37°C for 24 hours. The medium was changed daily to remove the toxic
compounds and smears prepared after every 48 hours to determine the percentage
parasitaemia(% P), the growth rate and to monitor contamination.
7.3.4.8Determination of parasitaemia and parasite growth rates
Thiswas done according to Trager and Jensen (1976). Briefly, thin blood smears were
prepared using sterile plugged Pasteur pipettes after carefully aspirating and discarding
thespent medium. A small drop of cell suspension was placed on a clean frosted glass
microscopeslide and a thin film made by touching the drop with the edge of another slide
heldat 45°C to the first. This spread the cells across the width of the slide and a smear
wasmade along the length of the slide with quick smooth movement. The blood films
wereair-dried, fixed with absolute methanol and stained with 10% Giemsa stain for 10
minutes. The slides were rinsed gently under flowing tap water, air-dried and observed in
oil immersion under microscope (xl 00). Dilution or sub-culturing was usually done
when the percentage parasitaemia was high, and no contamination found on examining
theslide under the microscope. The necessary volumes of culture 50010 fresh erythrocytes
andmedium needed for 5 ml, 6% hematocrit culture were calculated from the formulae:
CultureVolume (CV) = 51D
50%Erythrocyte Volume (EV) = 6/(50-CV)
MediumVolume = (CV+EV)
Where D is the reciprocal factor of the desired dilution factor (e.g. D=10 for 1:10
dilution). The appropriate volume of 500/0RBC and medium were mixed together in new
25 cm3 culture flasks using sterile technique, gassed (92% N2, 5% C02 and 3% 02) and
105
incubatedfor 20 minutes at 37°C. The desired volume of old culture was then added,
gassedand incubated.
7.3.4.9Freezmg of parasites (cryopreservation)
Themethod of Rowe et at (1968) was adapted for cryopreservation of parasites to ensure
enoughsupply of laboratory-adapted isolates as well as having manageable culture flasks.
Thicksmear was usually made to ascertain the cultures to be frozen are not contaminated.
Briefly,the culture to be cryopreserved was transferred into 15 ml centrifuge tube and
centrifuged at 1500 r.p.m (400 g) for 5 minutes at 20°C. After aspirating the
supernatant, packed cell volume (pCY) was estimated and one PCV of Rowe's
cryosolution added. Aliquotes of 0.25 ml were then put into 2 ml cryovials (Nunc®,
U.S.A)placed in aluminium canes, which were placed into liquid nitrogen freezer.
7.3.4.10In vitro drug sensitivity test
Thesemi-automated micro-dilution technique of Desjardins et al (1979) for assessing in
vitro anti-malarial activity as modified by Le Bras and Deloron (1983) was adapted in the
drugsensitivity studies for chloroquine and plant extracts against P. falciparum isolates.
Briefly,the 96 flat-bottom well micro-titre plates (8 rows x 12 columns) were set such
thatall wells except control contain 25 J.lI of doubling concentrations of drug solutions.
Parasitisized red blood cells (200 J.lI) were added so that the total volume per well is 225
~1.
7.3.4.10.1Preparation of plant extracts and dlIoroquioe for in vitro bioassays
Thedry plant extract samples were retrieved from 4 °C and dissolved in distilled water so
that the final highest concentration in the micro-titre plates was 250 ug/ml. For these
experiments, 0.045 g of the plant extract was dissolved to a final volume of20 ml (stock
solutionof2,250 ug/rnl).
Since the final volume in each well was 225 J.lL this stock solution was meant to give the
firstrow concentration of250 ug/ml using the formula:
CN1=C2V2
106
WhereC1 = initial concentration, VI = initial volume, C2 = final concentration, V 2 = final
volume
Takinginto account that the volume of each drug in each well was 25 ul, the highest
concentration(250 ug/ml) was calculated so that:
2,250ug/ml XVI = 250 ug/ml x 225 JlI
VI = 250 x 225/2250 = 25 ul
Thismeant that 25 JlI of stock solution (2250 ug/ml) was used in the first row (Elueze et
al., 1996). Each drug was filter sterilized with syringe adaptable 0.22 urn filters into
sterileBijoux bottles and stored at -2°C.
9 ug/ml x VI = 1 ug/ml x 225 fll
VI = 1 ug/ml x 225 Jlll9 ug/ml
= 25 ul
C1 = 25 JlI in the first row of the micro-titre plate
Fordrugs that were not readily soluble in water, especially non-polar extracts of hexane
and chloroform, they were dissolved in 50 !ll of dimethyl sulfoxide (DMSO) (solvent
concentration in tests did not exceed 0.02%) and the volume adjusted to 20 ml with
distilledwater (Elueze et aI., 19%).
73.4.10.2 Preparation of micro-titre plates
Thiswas done according to Desjardins et al (1979). Briefly, the 96 well flat-bottomed
micro-titre plates (Nunc®, U.S.A) with covers were used for drug sensitivity tests.
Under sterile conditions in the laminar flow hood (Bellco Glass Inc., U.S.A), the plates
were laid along the columns (l-12). Sterile deionised water, (25 ~l) was added with a
multi-channel pipetter from row B to H, exempting row A. The drugs (50 ul) were added
111 duphcate mto wells of row A (each drug held two columns and one plate therefore
fll from G wells discarded. Row H wells were exempted since they served 3S controls.
row A wells had a concentration of 250 ug/m], B wells 125 ug/rnl as
107
concentrationshalved down to G, which had the lowest concentration of 3.90625 ug/ml.
Thefinal volume per well was 25 ul. The plates were covered and kept at 4 "C.
7.3.4.1-0.3 Addition of parasites to the pre-dosed plates
Thiswas done according to Desjardins et al (1979). Briefly, the test culture at ring stage,
havinga percentage parasitaemia (% P) 2: 4% and growth rate (GR) 2: 3 % was used for
sensitivitytests. After examining the parasites under a microscope, the % P of the test
cultureto be added to the wells of predosed plates was adjusted to 0.4 % and haematocrit
(het)adjusted to 1.5 % with 50 % RBC. The mixture (200 ul) was then added into each
well except tor H7 to Hn If tor instance the % P of the test culture (Vi) was 4 % and the
numberof elates to be set was 1 (n =1), the following calculations were done to the. \ ~
ralmres maintained at 5 ml and 6 % haematocrit.
WhereC &. Cf= initial and final concentrations respectively.
Viand Vr = initial and final volumes respectively
C=4%
Cf= 0.4 %
Thevolume of the plate (Vr) was calculated as follows approximating 96 wells to 100
wells.
Vf= 1 plate x 100 wells x 200 J..ll (volume of culture per well) = 20000 ul
=20ml
The volume of the test culture (5 ml, 6 % hct) which was used (Vi) was calculated as
follows:
cs;= CrVr
4%xVj =4%x20 ml
Vi = 0.4 % x 20 ml/4 %
=2ml
Since 5 ml has 6 % hct, or 6/100 x 5 ml = 0.3 ml (100 % MC)
2ml culture has 0.12 ml (100 % RBC)
To adjust haematocrit to 1.5 % erv,
1.5/100 x 20mI = 0.3 ml (100 % RBC)
108
Butthe Vi (2 ml) has 0.12 ml (100 % RBC) and (0.3-0.12) ml = 0.18 ml (100 % RBC)
arerequired. This requires the addition of 50 % RBC. Since the remaining 0.18 ml
haenatocritis 100 % RBC, 0.18 ml x 2 = 0.36 ml of 50 % RBC is needed.
Thefinal volume of 20 ml, needed is achieved by addition of CMS to 2 ml test culture
and0.36 ml of (50 % RBC).
eMS needed = 20 ml- (2 +0.36) ml
= (20 - 2.36) ml = 17.64 ml
Thismeans that to set 1 plate using a culture whose % P = 4, you require 17.64 ml CMS,
0.36 ml (50 % RBC) and 2 ml test culture, to achieve 0.4 % P and 1.5 % haematocrit.
Thepre-warmed CMS (37 DC)was put into 25 cm2 flask, the appropriate volume of 50
% RBC added, flushed with 92 % N2, 5 % O2 and 3 % CO2 gas mixture (BOC, Kenya)
andkept at 37 DCincubator for 5 minutes.
Usingsterile technique in a laminar flow hood, the appropriate volume of test culture was
added into the flask containing CMS and 50 % RBC, and gently swirled in a circular
motion to mix. The pre-dosed plates were warmed at 37 DC for about 20 minutes,
retrieved, placed in the laminar flow hood and the test culture put into sterile tissue
culturedishes (Lux®, U.S.A). Using 1-200 III pipette tips (Fisherbrand®, U.S.A) and a
multi-channel pipette, aliquots of 200 III were dispensed into the wells except for H7 to
H12 (6 wells). To these, unparasitized red blood cells (URBC) were added (negative
control) so that HI-H6 served as parasitized red blood cells (PRBC) control (positive
control) since they had no drug and the former served as UPRBC control. For 1 plate (6
wells):
Volume= 6 wells x 200 III = 1200 III
= 1.2 ml
1.5% hct = 1.51100 x 1.2 = 0.018 ml (100 % RBC)
= 0.036 (50 % RBC)
CMS = (1.2 - 0.036) ml
= 1.164 ml
109
0.36 ml of 50 % RBC was mixed with 1.164 ml CMS and 200 /-llwere aliquoted into
wellsH7 to H12using a multichannel pipette. The same procedure and calculations were
donefor n number of plates (n = 2,3 .... etc).
7.3.4.10.4 Incubation of the plates
This was done according to Desjardins et al (1979). Briefly, after replacing the lids of
micro-titre plates and agitating the plates gently, they were placed into gas-tight box,
which had a damp tissue to maintain a humid atmosphere in the chamber. The gas box
lid was replaced and the air-tight box flushed with 92 % N2, 5 % C02 and 3 % O2 and
incubated at 37°C. After 48 hours, [G-3H] hypoxanthine (1 /-lCi/well) was pulsed in
aliquots of25 /-llinto each well and the plates incubated for a further 18 hours.
7.3.4.10.5 Harvesting of cells and scintillation counting
This was done according to Desjardins et al (1979). Briefly, the cells were harvested
using a multiple semi-automatic cell harvester (Skatron@, Norway) onto glass fibre
filters (Skatron@, Norway) for each row, from A to H. The filters were then dried at 37
°C overnight, introduced into scintillation vials, and 1 ml of scintillation fluid (ecolume)
added and the vials loaded into a liquid scintillation f3-counter (1211 Minibeta, England).
Disintegrations per minute were calculated for each sample. The count per minute
(CPM) for each sample represented the incorporation of [G-3H] hypoxanthine into the
parasite nucleic acids.
73.4.10.6 Inhibitory concentration 50 (ICso)
This was done according to Sixsmith et al., (1984). Briefly, the 50% inhibitory
concentration (IC50) refers to the drug concentration inhibiting 50% of the parasite
incorporation of [G_3H] hypoxanthine found in the drug-free PRBC wells. The UPRBC
CPM values were taken as the background count and corrected CPM values of each well
by substracting UPRBC CPM values from each wells CPM values. To calculate IC50,the
mid-point (Y50)was calculated by the formula:
Y50= (pRBC CPM values - UPRBC CPM value)/2
IC50was calculated from the formula:
110
IC50 = Antilog (LOgXI + [(Log Yso - Log Yl)(LOgX2 - LOgXl)/ (LOgY2 - LogY!)],
Where: IC50 = inhibitory concentration 50, Xj and X2 = lower and higher concentrations
respectively, YI = CPM values which correspond with Xi, Y2 = CPM values which
correspond with X2.
7.4 Isolation of compounds from the stem bark of Neoboutonia macrocalyx
The petroleum ether extract (3.0 g) was subjected to fractionation by column
chromatography on silica gel with a petroleum ether:ethylacetate gradient (100:0-0: 100)
giving 142 x 10 ml fractions. These were pooled on the basis of their Rf values and
concentrated in vacuo to give 8 fractions (F13, F23, F70, F89, F93,F102,Fus and FI42). Only
fractions F13, F89 and F93 were available in reasonable amounts and were therefore
subjected to further purification. Five compounds: PI, P2, Ps, P6 and P7 were obtained
through preparative thin layer and column chromatography coupled with re-
crystallization (Fig. 11). Through NMR analysis, P2 was found to contain some
impurities and was re-purified to yield P2a and P2b.
Fig 11: Isolation of compounds from the petroleum ether extract of N macrocalyx.
Petroleum ether extract (3.0 g)
l fC ~
F13(179.1 mg)
+ Ptlc
F2 (P6) (27mg)
F89 (1.5593 g)
_____ Icc
+ -+
F96(565.1 mg) F17(P2) (42.5mg) +Ptlc
I cc.-- + ~ F,(P,)(60mg)
F20 (106 mg) F36 (PI) (10 mg) FI (P2J(6 mg) F2 (P2~ (24 mg)I Ptlc
F93 (264.6 mg)
+ cc
F35 (85.4 mg)
Fl (Ps) (34.7 mg)
III
Stigmasterol (126) (PI)
KBr 1White needles. Found: IR Vmax 3424 (-OH), 1688 (C=C) ern" ; mp 166-168 °C (lit. 170
DC,Budavari, 1996); IH NMR (200 MHz, CDCh) (Table 8); l3C NMR (50 MHz, CDCh)
(Table 9).
6,7-Epoxy-4,5,9-b'ihydroxy-13-pentadecanoate-20-dodecanoate-l-tiglien-3-one (140)
(P2b)
KBrLight orange oil (Rr0.45, 3:1 n-C6H14:EtOAe). Found: IR Vmax 3389 (-OH), 1689 (C=O),
CH CI
1624 (C=C) em"; uv ).mix2 347 (sh), 250 nm; CI-MS m/z 801.2 (100) [M+Ht C48H8109
requires 801.2, 857 (12), 830 (35), 829 (80),802 (45), 783 (10), 774 (24), 773 (56),537
(6), 327 (21), 309 (22), 281 (15); EI-MS m/z 293 (5) [M-C28Hss07]', 256 (2), 223 (2),
213 (3), 191 (4),185 (6),167 (19),149 (82),129 (11),111 (19),97 (25), 85 (31), 83 (39),
71 (78),55 (100),43 (85).; IH NMR (600 MHz, CDCh) (Table 10); l3C NMR (150 MHz,
CDCh) (Table 11).
4,9-Dihydroxy-13-hexadecanoa te-20-dodecanoate-l-tigliadien-3-one (142) (P5)
Light orange oil (Rf 0.70,3:1 n-C6HI4:EtOAe). Found: IR v~~~3379 (-OH), 1707 (C=O),
CH Cl
( ) -1 ). 2 2 ! +1612 C=C em ; UV max 325 nm; CI-MS m/z 495.0 (30) [M-C12H2402] C32~704
requires 495.0, 481 (25), 477 (10), 465 (20), 327 (6), 313 (18), 311 (11), 299 (25), 295
(100), 281 (20), 277 (19), 265 (14), 253 (12), 239 (6), 294 (2); EI-MS m/z 526 (1) [M-
C1Ji3102t, 294 (2),256 (3), 239 (1), 228 (4),200 (11),185 (20),171 (18),157 (30),
149 (12), 143 (19), 129 (71), 115 (25),97 (24), 85 (50), 73 (94),60 (100), 55 (100),43
(87), 41 (91); IH NMR (600 MHz, CDCh) (Table 14); l3C NMR (150 MHz, CDCb)
(Table 15).
Methyl3-heptaeicosanoyloxyoleanoate (P6) (143)
Light orange oil (27 mg, Rf 0.40, 20:1 n-CJIwEtOAe). Found: IR v~~ 3054 (-OH) ,
CH Cl
1684 (C=C), 1264 (C-O) em"; uv A",ix2 380 (sh), 325 nm; CI-MS m/z: 858.6 (100) [M-
112
Hzt CS8H9804requires 858.6, 859 (13), 881 (4),872 (56), 855 (56), 831 (17),747 (12),
692 (13), 663 (24), 607 (13), 563 (12),453 (80),429 (17), 393 (24),203 (28), 189 (30),
165 (35); EI-MS m/z 453 (1) [M-C27HS302]', 452 (3),393 (1),316 (2), 293 (2),262 (4),
203(7), 191 (3), 167 (2), 149 (30), 111 (8),97 (20), 85 (22), 83 (27), 71 (55),69 (61),57
(100),43 (82); IH NMR (600 MHz, CDCh) (Table 16); BC NMR (150 MHz, CDCh)
(Table17).
Montanin-20-palmitate (P7) (79)
KBrColourless oil (Rf 0.59, 3:1 n-CJII4:EtOAe). Found: IR Vmax 3522 (-OH), 1735 (C=O),
CH C\
1693 (C=C) em"; uv A~x 2 230, 245, 325 (sh) nm; CI-MS m/z 799.2 (100) [M+Ht
C4sH7909requires 799.2, 827 (36), 781.2 (16), 743 (23), 581 (8), 553 (6), 543 (3), 525
(7), 507 (3), 342 (8), 325 (20), 307 (14), 279 (15), 253 (14); EI-MS m/z 543 (4) [M-
ClJi3102t, 527 (11), 499 (20), 495 (10), 414 (3), 324 (3), 295 (3),283 (3), 269 (3), 256
(3),239 (2), 213 (2),185 (10),157 (12),149 (13),129 (28),111 (15),97 (27),.83 (35),73
(75),60 (86), 55 (100),43 (96); IH NMR (600 MHz, CDCh) (Table 18); 13CNMR (150
MHz, CDCh) (Table 19). The NMR data was identical to that ofmontanin-20-palmitate
previously isolated from N glabrescens (Tchinda et a!., 2003).
The CH2Clz extract of N. macrocalyx (6.0 g) was subjected to vacuum liquid
chromatography to yield 12 fractions. These were pooled on the basis of their Rt values
and concentrated in vacuo to give 5 fractions (FI, Fs, F6, F9 and F12). FI and Fs were
found to contain Pi and P2 which had already been isolated while F6 and F12were in
small amounts. Therefore, fraction F9 was subjected to further column chromatography
and preparative thin layer chromatography to yield two compounds, P4 and Ps (Fig.12).
113
Fig12: Isolation of compounds from the dichloromethane extract of N macrocalyx
CH2Cl2 extract (6.0 g)
+ Vle
F9 (90S.S mg)
~cc
F46 (128.2 mg)
~ CC
F13 (36.2 mg)
IPtlc
~
F2 (Ps) (9.3 mg)
6-7-Epoxy-4,5,9,20-tetrahydroxy-13-tetradecanoate-1-tiglien-3-one (141) (P4)
KBrOrangeoil (Rf 0.24,2:1 CHCh:EtOAc). Found: IR Vmax 3397 (-OH), 1684 (C=O), 1623
CH Ci
(C=C)ern"; uv A.mix2 325 nm; CI-MS m/z 590.9 (100) [M+Ht C3JI550g,requires 590,9,
618(13), 572 (13), 562 (28), 555 (28), 527 (9), 362 (16), 344 (61), 327 (50), 309 (35),
297(18), 281 (21),269 (20), 255 (15), 253 (7); EI-MS m/z 590.9 (4) ~], 362 (2), 344
(2),255 (3),246 (4), 229 (3), 211 (10),189 (7),183 (7), 163 (7), 151 (7),149 (17),123
(6), 111 (7), 109 (8), 95 (16), 83 (66), 69 (69), 57 (95), 43 (100); IH NMR (600 MHz,
CDCb) (Table 12); l3C NMR(150 MHz, CDCh) (Table 13).
12-Deoxyphorbol-13-pentadecanoate (145) (Ps)
Light yellow oil (Rf 0.59, 1:1 CHCh:EtOAc). Found: IR v~~~3675 (-OH), 1711 (C=O),
CH Cl
I A 2 2 +1543(C=C) em' ; UV max 325 nm; CI-MS m/z 573.0 (10) [M+H] C35Hs706requires
573.0,591 (15),561 (11),555 (16), 548 (11), 527 (20),513 (18), 509 (10),495 (5),453
(6),395 (5), 345 (11), 329 (11), 327 (29), 313 (57),309 (30), 295 (100), 293 (21),281
(19), 277 (17), 253 (15), 239 (8), 225 (9), 209 (7), 197 (7); EI-MS m/z 330 (2) [M-
C1sH2702t, 328 (2), 312 (4), 294 (4),293 (3),225 (3), 211 (10), 197 (6), 183 (8), 179 (7),
114
149(9),129 (10),109 (11), 95 (11), 85 (16), 83 (50),69 (47),57 (98), 43 (100); IHNMR
(600 MHz, CDCI3) (Table 20); 13C Nl\1R (150 MHz, CDCI3) (Table 21).
The ethyl acetate extract (2.50 g) was subjected to column chromatography on silica gel
with petroleum ether: ethylacetate: methanol gradient (100:0:0-0:100:0-0:0:100) giving
87 x 10 ml fractions. These were pooled on the basis of Rr values and concentrated in
vacuo to give 6 fractions (F6,F27,F59,F77,Fso). Only fractions F59and F77were available
in reasonable amounts and were subjected to further purification. Purification of F77to
obtain a single compound was unsuccessful due to further decrease in amount. F59
yielded a pure compound, P3 (Fig. 13).
Fig 13: Isolation of compounds from the ethyl acetate extract of N macrocalyx
EtOAc extract (2.50 g)r+ ~ ~ +F6 F27 FS9 (223.9 mg) F77 Fso+ Recrystallization
P3 (61.7mg)
o KBrOrange crystals (RfO.4, 2:1 CHCh:MeOH). Found: mp 289-290 C; IR Vmax 3153 (-OH),
MeOH
1713 (C=O), 1639 (C=C) ern"; UV Amax (log E) 248, 334 nm; EI-MS m/z 254 (93), 226
(68),211 (l00), 198 (31), 183 (40), 165 (20), 153 (21), 152 (19),89 (12),82 (20),77
(28),76 (30), 63 (25),51 (31),39 (32); IH NMR (200 MHz, MeOD) 1.26 (s, 6H); 2.55 (s,
3H); 6.35 (d, lA, IH); 6.90 (d, lA, III); 7.29 (s, IH); 7.87 (s, IH); l3C NMR (50 MHz,
MeOD) 16.7 (CH3), 23.7 (CH3), 23.7 (CH3), 46.9 (C), 112.2 (CH), 121.8 (CH), 125.0
(C), 128.7 (CH), 130.9 (CH), 131.8 (C), 156.8 (C), 163A (C).
7.5 Isolation of compounds from the stem bark of Fagaropsis angolensis
Hexane extract (2.0 g) was subjected to fractionation by column chromatography on
silica gel with a petroleum ether: ethyl acetate gradient (100:0-0:100) to yield 150 x 10
ml fractions. On the basis of thin layer chromatography R.- values, 3 fractions (F19, F91
115
and F107) were obtained. F19 was available in small amounts while F91 gave a single spot
and was therefore subjected to spectroscopic analysis (Fig. 14). F107 and the other
fractions 108-150 were not pursued further since they contained decomposition products.
Fig. 14: Isolation of compounds from the n-hexane extract of F. angolensis.
____ n_-_c_1~~extract (2.0 g)t i L
F91 (Kt) (209.1 mg)
Kl
Lightgreen oil (Rf 0.56, 19:2 n-CJIt4:EtOAc). Found: IR v~:3690 (-OH), 1736 (C=O),
CH ClA 2 21543(C=C), 1264 (C-O) em"; uv max 325 nm; EI-MS m/z 344 (3), 255 (3), 246 (4),
229 (3), 211 (10),189 (5),183 (5),163 (5), 151 (6),149 (17),109 (9), 95 (17), 83 (65),
71 (67),69 (69), 57 (55), 43 (l00); IH NMR (200 MHz, CDCh) 5.32 (dd, J = 5,7 Hz,
5H), 4.30 (dd, J = 4,6 Hz, 1H), 4.40 (dd, J = 4,6 Hz, 1H), 2.81 (q, J = 5.2 Hz, 2H), 2.31
(t,J = 7.5 Hz, 3R); 2.03 (m, 5R), 1.65 (br s, 4R), 1.27 (d, J = 9.6 Hz, 24R), 0.94 (m, 5H);
J3C NMR (50 MHz, CDCh) 14.3 (CH3), 20.5 (CH2), 22.6 (CH2), 22.7 (CH2), 24.9 (CH2),
25.5 (CH2), 25.6 (CH2), 27.2 (CH2), 29.1 (CH2), 29.2 (CH2), 29.3 (CH2), 29.5 (CH2), 29.6
(CH2), 29.7 (CH2), 31.5 (CH2), 31.9 (CH2), 34.0 (CH2), 34.2 (CH2), 62.1 (CH2), 68.8
(CH), 127.1 (CR), 127 .7 (CR), 127.9 (CH), 128.0 (CR), 128.2 (CH), 128.3 (CR), 129.7
(CH), 130.0 (CH), 130.2 (CH), 131.9 (CR), 172.9 (C), 173.3 (C).
Dichloromethane extract (7.60 g) was subjected to fractionation by vacuum liquid
chromatography on silica gel with petroleum ether.dichloromethane.ethyl acetate
gradient (100:0:0-0:100:0-0:0:100) giving 30 x 200 ml fractions. These were pooled on
the basis of their Rr values and concentrated in vacuo to give 5 fractions (F7, F13, F19, F22
and F29). Only fractions F22and F29 were selected for further purification since the rest
were obtained in small amounts. Three compounds, F ASD 2, FASD 3 and FASD 4, were
obtained through preparative thin layer and column chromatography coupled with re-
116
crystallization (Fig. 15). The remaining fractions were pooled together and subjected to
columnchromatography to avail compound FASCI (Fig. 16).
Fig. 15: Isolation of compounds from the dichloromethane extract of F angolensis.
CH2C12 extract (7.60 g)+_I_·V1C_~
F22 (0.819 g) F29 (2.20 g)
Ice
+
F" (180mg)j_V_1_c _ +--+
F2 (216 mg) F3 (1.126 g)
+ cc
F36 (20 mg)
F15 (60 mg)
+Fl (120 mg)
+r----l-cc -+
FI1(FASD4)(l8mg) F17 (FASD3)(5mg) F30(FASD2)(l4mg)
Precipitate Filtrate (FASD2)(4 mg)
~ Sephadex LH 20
F 16 (FASD3) (3 mg)
FASD2
Yellowsolid (18.0 mg, RfO.63, 3% MeOH:EtOAc). Found: mp 253-254 DC;uv ~2 245
(sh),284, 325 (sh), 380 nm. Attempts to obtain spectroscopic data on FASD 2 were
unsuccessfuldue to its insolubility in the available NMR solvents.
FASD3
hi D KBrW rte needles (8.0 mg, Rf 0.28, 80:1 CHCh:n-C6HI4). Found: mp 246-248 C; IR Vmax
CH Cl
3392(-OH), 1740 (C=O), 1576 (C=C) em"; UV Amh'381,326 nm. Attempts to obtain
117
spectroscopic data on FASD 3 were unsuccessful due to its insolubility in the available
NMR solvents.
FASD4
CH C\o 1 2 2Orange solid (18.0 mg, Rr 0.70,3% MeOH:EtOAc). Found: mp 172-174 C; UV "wax
263,325 nm. Attempts to obtain spectroscopic data on FASD 4 were unsuccessful due to
itsinsolubility in the available NMR solvents.
Fig. 16: Isolation of compounds from the pooled dichloromethane extracts of F.
angolensis after isolation ofF ASD 2-4.
Combined CH2C12 fractions (2.690 g:
~ CC
F25 (55 mg)
11.Ptlc2. Recrystallization
FASC 1 (10.5 mg)
FASC 1
o MfOHWhite crystals (10.5 mg, Rf 0.36,30:2 CH2Ch:EtOAc). Found: mp 148-150 C; UV A.lffil(
325, 380 nm. Attempts to obtain spectroscopic data on FASC 1 were unsuccessful due to
its insolubility in the available NMR solvents.
Methanol extract (29.0 g) was dissolved in water (100%) and was subjected to multiple
gradient partitioning on a 500 ml separating funnel with chloroform, ethyl acetate and
butanol to give 3 x 300 ml fractions. The EtOAc and n-BuOH extracts were obtained in
small amounts and therefore not pursued further. The extracts were dried and
concentrated in vacuo. Four compounds (FASM 1, FASM 2, FASM 3 and FASM 4)
were obtained through preparative thin layer and column chromatography coupled with
re-crystallization of the chloroform extract (Fig. 17).
118
Fig.17: Isolation of compounds from the methanol extract of F angoiensis.
MeOH extract
29 gI partitioning
+n-BuOHCHCl3 (1.69 g) EtOAc
+_t_cc __ ~
F6 (150 mg) F9(1.97 g)
_____ 1 re-crystallization I vie
~ + ~ --+
Precipitate(FASM 1) (25.6 mg) Filtrate (FASM 2) (39.7 mg) F4 (20 mg) F (578 mg)+ re-crystallization 17CC
Precipitate (FASM 4)(13.6 mg) t
F9 (220 mg)
~ CC
FZ6 (FASM 3) (16.9 mg)
FASMl
White solid (25.6 mg, RfO.64, 2% MeOH:CH2Ch). Found: mp 290-291 DC;IR v~~ 3451
MeOH
(-OH), 1740 (C=O), 1505 (C=C) ern"; UV Amax 242, 311 nm. Attempts to obtain
spectroscopic data on FASM 1 were unsuccessful due to its insolubility in the available
NMR solvents.
FASM2
White solid (39.7 mg, Rr 0.59, 2% MeOH:CH2Ch). Found: mp 283-285 Dc. Attempts to
obtain spectroscopic data on FASM 2 were unsuccessful due to its insolubility in the
available NMR solvents.
FASM3
White solid (16.9 mg, Rf 0.58, 1:20 MeOH:CHCh). Found: mp 276-278 DC;IR v~~~3458
MeOH
(-OH), 1740 (C=O), 1506 (C=C) em"; UV Amax 325 nm. Attempts to obtain
119
spectroscopic data on FASM 3 were unsuccessful due to its insolubility in the available
NMRsolvents.
FASM4
White crystals (13.6 mg, Rf 0.47, 20:5 CHCh:n-C6H14). Found: mp 286-287 0c.
Attempts to obtain spectroscopic data on FASM 4 were unsuccessful due to its
insolubility in the available NMR solvents.
120
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ENYAT~l\ U~\HVERS1TYLIBRARY
140
APPENDICES
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ANMDALAI: p1 1/em
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Type: HYPER IR User: CHEMRE Detector: standard
Abscissa: 1/an Ordinate: %T Apodlzatlon: Happ
Min: 400.20 Max: 3999.12 Range: 1/cm
Nclp: 3733 Data Interval: 0.96434 Resolution: 2.0
Goln: auto Aperture: auto Mirror Speed: 2.8(1ow)
IH NMR spectrum of 6, 7-Epoxy-4,5, 9-trihydroxy-13-hexadecanoate-20-dodecanoate_l_tiglien_1-3-one (140)
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P28
UnlverSlty Of CctSwarla
CnemlSlry DePdrt~enl
NA8SA NMR Serv : c c
.. In cr <D '"C' tr. r-. ~ ,..,a~ l.O=: '" r-;'f.( :d ~r:
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Curren: Data Para'!1e~p.,,~
NAME Dell
EXPNO ~
PROeM) 1
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N\JCI IlC
PI ,01 00 usee
I'll ·2 00 OB
5F'01 150 9193478 MHZ
••• " •••••••• CI-IANNtL 12 •••••••••••••
CPQPAG2
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PCPOI
Pt.1
Pt."
PLlJ
51'02
••• It 116
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<J 00 08
600 1324005 MHt
F"2 • PI"OCus If'lQ oara •••eters
51 JU6B
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wow EN
ssa 0
L8 I 00 1-11
G8 0
PC 1 '-0
10,..:a plot plrallftel"S
ex 20 00 em
riP 215 000 DOfil
F"I )24 •• 10 Hl
F 2P -5 000 DOWI
F2 -75" !II HI
pC;>M(:N II 00000 oo./c_
MzeN 18~9 93030 Hl/c_
DEPT spectrum of 6,'7-Epoxy-4,5, 9-trihydraxy-13-hexadecanaate_ 20-dadecanaate-l-tiglien_I_3 -one (140)
P28
UnIverSIty of 80tswana
ChemIstry Deoartment
NABSA NMR ServIce ~~~ru~~W~~-W~~_~~_~~~om~~nMru~ru~~MroM_~ru~ruMwruw~~M~~~W~OOWM~~M __ W~_O~o~
m-o~~~ommoo~wwMru_Mru~_~M. . . . ..
m~~oo~~o~m~mmm~~Mmw~o\V~~v;7P; II
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r-~-~-'I-~-~'-- I I I
1dO 120 100 80 I i I I i I50 40 20
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rf'olSIRI.JM soeel
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rlar:t(S 0 ,.a811 HI
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j:lG 11~85 .?
011 Il.900 US!!C
DE 6 00 uSI!C
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01 2 00000000 ItC
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012 0.00002000 see
DEL'" 0 0000t183 see
•.••••••••••••• CHANNELfl·············
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P\.I ·Z.OO ee
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CPOPAUc .,ltll!
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PCP02 85.00 vsec
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~12 21 00 08
SfO~ 600. I )2'OO~ ""
f2 ~ Procn,.I'IO pollraflfte"t
51 32768
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F I 258004 62 HZ
Fcp -3.612 Utlfll
r2 ~~45 12 HI
PPHCN 8.1)010 OOfll/clfI
liltM 1317 48694 1i,/clII
HMBC spectrum of 6,7-Epoxy-4,5,9-trihydroxy-13-hexadecanoate-20-dodecanoate-I-tiglien-] -3-one (140)
" II
----1.-1 I " dol I 1~11
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ppm B 6 4 2
P28~'-
univerSIty of Botswana
Cnemlstry Oepartment
NABSA NM~ ServIce
c•••.~••.•t a.t ••••. _1•..'....• ...,.... ,......, ,
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fJMlt"" ,.. •• " ••• /0:_
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HMQC spectrum of 6,7-Epoxy..:4,5,9-trihydroxy-13-hexadecanoate-20-dodecanoate_l_tiglien_] -3-one (] 40)
t"-on...-<
tJ.379
l.d06~
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UniverSity of 80tswana
Cnemlstry Department
NA8SA NMR Service
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CI-MS spectrum of 6,7 -Epoxy-4, 5,9-trihydroxy-13-hexadecanoate- 20-dodecanoate-l-tiglien-l- 3-one (140)
O:'lNllblla04\Nabsa32
APel
i'iiibsa32 #1 RT: 0.01 AV:1 NL:f:04E9
T: + c: Full ms ( 200.00-860.00)
l()O'~
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2004/06/08 12:48:52 P2a
801.2
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745.2 765.3111 I 811.2 85 2
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El-MS spectrum of 6,7_Epoxy_4,S,9-trihydroxy-13-hexadecanoate-20-dodecanoate-l-tiglien-1-3-one (140)
_ .._----_. - ---
Hllh': 19- ..\utt-2'H •., Thm': JJ:.u):~H
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JR spectrum of 6,7 -Epoxy-4,5, 9-trihydro::y-13-hexadecanoate-20-dodecanoate-I_tiglien-1-3-one (140)
100.0 -..,-_.
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~fOOO.O 3500.0 3000.0
ANDALAIR: 2b
Date: 09/01/2004
Type: HYPER IR
Abscissa: 1/cm
Min: 400.~!O
Ndp: 3733
Gain: auto
go.o -
80.0 -
o
\0,......
2500.0 2000.0
Time:
User:
Ordinate:
Max:
Data Interval:
Aperture:
'11:47.:49
CHEMRE
%T
'3999.12
0.96434
auto
1750.0 1000.0 750.01500.0 1250.0
NScans:
Detector:
Apodlzation:
Range:
Resolution:
Mirror Speed:
20
standard
Happ
1/em
2.0
2.8(low)
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500.0
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161
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150 140 130 120 70 40 309. 80
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4000.0 3500.0 3000.0
ANDALAlR:p3
Date: ()811812004
Type: HYPER IR
Abscissa: 1/cm
Min: l100.20
Ndp: :1733
Gain: auto
2500.0 2000.0 1750.0
Time:
User:
Ordinate:
Max:
Data Interval:
Aperture:
13:24:53
CHEMRE
%T
3999.12
0.96434
auto
1500.0 1250.0 1000.0 750.0 500.0
1/cm
NScans: 20
Detector: standard
Apodization: liapp
Range: 1/an
Resolution: 2.0
Mirror Speed: 2.8(low)
168
BECK!'1AN DU-6 SF'ECTr-:ClPHOTOME'T ER
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IH NMR spectrum of 4,9-Dihycircxy-2 )-hc:cadc<:aIloate-B·,dod(:canoatl~-l ,6-tigli adien-3 -one (142)
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DC NMR spectrum of 4,9-Dihydroxy-20-hexadecanoate-13-dodecanoate-l ,6-tigliadien-3-one (142)
e
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DEPT spectrum of 4,9-Dihydroxy<W-hexadecanoate-13-dodecanoate-1 ,6-tigliadien-3-one (142)
P5
tJ~l\'er'sl~Y of Bot~d'iane
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HMBC spectrum of 4.9-Dihydroxy-20-hexadecanoate-13-dodecanoate-l.6-tigliadien-3-one (142)
r.-..
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29 ~9\'-- ~
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of 4,9-Dihydroxy-20-hexadecanoate-13-dodecanoate- i:,6-tigliadien-3-one
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UnIverSIty of Botswana
[hem I S try Deoartment
NAllSA NMR Serv Ice
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Ci-MS spectrum or 4,9-Dihydroxy-20-hexadecanoate-13-dodecanoate-l ,6-tigliadien-3-one (142)
[):~'~ab"~lNabaa30
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EI-MS spectrum of 4,9-Dihydroxy-20-hexadecanoate-13-dodecanoate-l ,6-tigliadien-3-one (142)
: Inll: '(; I'llttrunn II (;(.:tI.("·'IS
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IR spectrum of 4,9-Dihydroxy-20-hexadecanoate-13-dodecanoate-1 .o-tigl iadien-3-one (142)
100.0 -.-- I I
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20.0
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4000.0 3500.0 3000.0 2500.0 2000.0 1750.0 1500.0 1250.0 1000.0 750.0
ANDALAIR: p5
Date: 0~'/0112004
Type: HYPER IR
Abscissa: 1/cm
Min: 400.20
Ndp: 3733
G;ain: auto
80.0
70.0
60.0
50.0
r-oo,......
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90.0
Time:
U.er:
Ordinate:
Max:
Data Interval:
Aperture:
11:34:50
CHEMRE
%T
3999.12
0.96434
auto
500.0
1/cm
NScans: 20
Detector: standard
Apodlzatlon: Happ
Range: 1/cm
Resolution: 2.0
Mirror Speed: 2.8(1ow)
,)t>:3
.:!:. ('(II)
188
BECt<l"lf-lN l)U-h C;r::'EL-1F,ClF't'II.lTDI'1ETER
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SOURCES, UV/VIS ~
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DATE: __~J~:J.~._"_~A~--
OF'ERATOf\: _t<l~.:.X_~~..!~__
SAMPLE: f£ _
REFERENCE: ~l~~~
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11-1NMR spectrum of Methyl3-heptaeicosanoyloxyoleanoate (143)
p~
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0:CO-1Current Data ParametersNA~E 06EXPNO I
PROCNO I
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0.1868'6 t'z
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10 NMR plot ue- eeet er s
CX 20.00 c.
rtP 14.391 0011'I
FI 6636.50 HI
F?P 0 177 ace
f2 106 19 HI
PPHCH 0.71070 OO,"/(m
H/C" 426 51538 HI/cm
COSy spectrum of Methyl 3-heptaeicosanoyloxyoleanoate (143)
I· /( i IIIJ
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I:lC NMR spectrum of Methyl 3-heptaeicosanoyloxyoleanoate (143)
~
Un!~e~slly of 80~5wana
ChE~!stry Department
N~8SA NMR Seev, ce
C.U""ent o.t. P.re~e~I!"S
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DEPT spectrum of Methyl 3-heptaeicosanoyloxyoleanoate (143)
tIt:,
un i ver s i t v l'l eNswana
ChemIstry D~oa •..tment
NABSA ~'m Ser"ce
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HMBC spectrum of Methyl 3-heptaeicosanoyloxycleanoate (143)
~ll----------~,----~~~
I .
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100
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I-IMQC spectrum of Methyl 3-heptaeicosanoyloxyoleanoate (143)
~0\,.....,
r- ! ,---~ r----+ I I .1 I
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C1-MS spectrum of Methyl 3-heptaeicosanoyloxyoleanoate (143)
D:lNabll04lNlIbal27 2004106/08 11:52:29 P6
APel
Nabs82i #n~T:o.60AV: 1NL: 1.23E8---' .--------. -----.--- - -.----.----.-------------.- - .
T: + c Full ms [ 150.00-900.00)
100:; 858.6
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150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900
mlz
El-MS spectrum of Methyl 3-heptaeicosanoyloxyoleanoate (143)
.,(, tl,~4 ~·1\.:9~h
1'h':(,IlluJHflll
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IR spectrum of Methyl J-heptaeicosanoyloxyoleanoate (143)
100.0
5().O -
60.0 -1 , 3054.1 i 2360.7) ~ I: .~_v.....T 1 ; J~1:9 J I ;
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80.0
70.0
30.0
t"-o-..,....; 20.0
10.0
0.0 I I I I I i
4000.0 3500.0 3000.0
ANDALAIR: p6
Date: 09/01/2004
Type: HYPER IR
Abscissa: 1/cm
Min: 400.20
Ndp: 3733
Gain: auto
2500.0 2000.0 1750.0
Time:
User:
Ordinate:
Max:
Data Interval:
Ap",rtu~e:
11:51 :23
CHEMRE
%T
3999.12
0.96434
auto
NScans: 20
Detector: standard
Apodlzatlon: Happ
Range: 1/cm
Resolution: 2.0
Mirror Speed: 2.8(low)
1500.0 1250.0 1000.0 750.0 500.0
1/em
198
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IHNMR spectrum of Montanin-20-palmitate (79)
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HMBC spectrum of Montanin-20-palmitate (79)
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Type: HYPER IR
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Mln: 400.20
Ndl>: 3733
Gain: auto
IR spectrum of Montanin-20-palmitate (79)
2500.0 2000.0 1750.0 1500.0 1250.0
Time:
User:
Ordinate:
Max:
Data In&erval:
Aperture:
11 :55:15
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I 327.0 •309"\) I
I II I
I II '293.1
20 '-111 Ii 527.0
2711 281.0 I 'I 'I! ,-- 555.1 591 0
15 253.1 I I I III I 513.0 ' , .
" !I I 541.0 561.1 I
10 225.1 I!! I I, I n9-·0 14.4.9 509,9 iii' 573.0 !
209 0 ,239.1 , 1 I I ~ 453 1 ' 'I '1971 I I' 395.2' 4951,' i, 15750'
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T: + C Full m. ( 180,00·eoo.00]
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r-•.....•
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12-Deoxyphorbol-13-pentadecanoate (145)
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Time:
User:
Ordinate:
Max:
Data Interval:
Aperture:
.11:38:47
CHEMRE
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3999.12
0.96434
auto
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4000.0 3500.0 3000.0
ANDALAIR: p8
Date: 09/01/2004
Type: HYPER IR
Abscissa: 1/em
Min: 400.20
Ndp: 3733
Gain: auto
-_ ]" [ .
1750.0 1250.0 750.0 500.0
1/em
1500.0 1000.0
NScans: 20
Detector: standard
Apodlzatlon: Happ
Range: 1/em
Resolution: 2.0
Mirror Speed: 2.8(low)
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218
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SCAN SHED: 3')(' Nfol/MIN
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SOURCES: UV/VIS
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:A~IF'LE: £1. _
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