Wanyama - Phytochemical Investigation of Tephrosia Purpurea and Eriosema Psoraleoides For Pharmacological and Antiplasmodial Activities PDF

U N I V E R S I T Y OF N A I R O B I
D E P A R T M E N T OF C H E M I S T R Y
PHYTOCHEMICAL INVESTIGATION OF TEPHROSIA

PURPUREA AND ERIOSEMA PSORALEOIDES FOR
PHARMACOLOGICAL AND ANTIPLASMODIAL
ACTIVITIES"
*>y
WANYAMA PETER JUMA
A THESIS SUBMITED IN PARTIAL FULFILMENT OF THE DEGREE OF MASTER OF SCIENCE OF

THE UNIVERSITY OF NAIROBI
DECLARATION
This thesis is my original work and has never been presented for a degree in any
university.
Date.....
WANYAMA PETER JUMA
This thesis has been submitted for examination with our approval as University
supervisors
...........Date....... 2 jJ lh « ± .....
PROFESSOR ABIY YENESEW
Department o f Chemistry,
University of Nairobi
Sign.... ..................................................... Dat ..................

DR. FAITH OKALEBO
Department of Pharmacology and Pharmacognosy,
Sign.,... tf&kar. .. -W l £\. .......... Date. l ft:9.LP..........................

PROFESSOR PETER M. GITU
Department o f Chemistry,
11
DEDICATION
This thesis is dedicated to my beloved mother, Florah and my

brothers: Tisa, Shivachi, James and my sisters: Irene and Robai.
Their support and encouragement enabled me to undertake this
piece o f work.
in
ACKNOWLEDGEMENT
I would like to express my sincere gratitude to my supervisors Prof. Abiy Yenesew, Dr.
Faith Okalebo and Prof. P. M. Gitu for their prompt guidance, support and inspiration
throughout my MSc work.
I am greatly indebted to the University o f Nairobi for giving me a scholarship which
enabled me to complete this work on time. My sincere appreciation also goes to Prof.
Martin G. Peter, University o f Potsdam, the Deutche Forschungemeinschaft (DFG),
Germany and the Germany Federal Ministry for Economic Cooperation and Development
(BMZ) within the DFG/BMZ Programme “Research Cooperation with Developing
Countries”, for sponsoring part of the study. I thank Dr. Matthias Heydenreich, University
o f Potsdam, for carrying out Nuclear Magnetic Resonance and Mass Spectrometric
analyses on the samples.
I would like to sincerely thank the academic and technical staff of the Department of
Chemistry, Mrs. R. Munenge, Mr. K. Maloba and Mr. Njihia of the Department of
Pharmacology and Pharmacognosy, Dr. Amugune and Mr. Mugo of the Deparment of
Pharmaceutical Chemistry, University of Nairobi for the assistance accorded to me
whenever I needed it. Many thanks to the former and current members of the Natural
Product Research group, University of Nairobi, for their cooperation and encouragement
during the course of this research.
My parents, brothers, sisters, friends and colleagues are sincerely thanked for their endless
support and encouragement. Finally, I wish to thank the Almighty God for giving me life,
strength and protection during the entire study period. To Him, I give all the Honour and
Glory, Amen.
IV
TABLE OF CONTENTS
DECLARATION........................................................................................ __ii
DEDICATION............................................................................................ ...iii
ACKNOWLEDGEMENT.......................................................................... ...iv
TABLE OF CONTENTS............................................................................ ,...v
LIST OF TABLES...................................................................................... .v iii
LIST OF FIGURES..................................................................................... ... ix
LIST OF SCHEMES................................................................................... ... xi
APPENDICES............................................................................................. .x ii
LIST OF ABBREVIATIONS..................................................................... x iii
ABSTRACT................................................................................................ xiv
CHAPTER ONE.........................................................................................
INTRODUCTION....................................................................................... ..1
GENERAL INTRODUCTION............................................................... ..1

1.2 PROBLEM STATEMENT............................................................... ..3
1.3 STUDY JUSTIFICATION............................................................... ..3
1.4 OBJECTIVES................................................................................... ..4
CHAPTER TW O........................................................................................ ..5
LITERATURE REVIEW............................................................................. ..5
2.1 ERECTILE DYSFUNCTION............................................................ ..5

2.1.1 Drugs used to treat erectile dysfunction....................................... ..6
2.1.2 Traditional plants used to treat erectile dysfunction.................... ..7
2.2 BOTANICAL INFORMATION ON ERIOSEMA AND TEPHROSIA 10
2.2.1 The genera of Eriosema and Tephrosia....................................... 10
2.2.2. Botanical description of Eriosema psoraleoides......................... 10
2.2.3 Botanical information of Tephrosia purpurea............................. II
2.3. ETHNOMEDICAL INFORMATION............................................... 13
2.3.1 Ethnomedical information of the genus Eriosema....................... 13
2.3.2 Ethnomedical information on the genus Tephrosia..................... 14
2.4 BIOSYNTHESIS OF FLAVONOIDS AND THEIR DERIVATIVES 15
2.4.1 Biosynthesis of flavonoids and their derivatives........................ 17
v
2.4.2 Biogenesis of tetrahydrofuran ring in flavonoids........................................................ 22
2.4.3 Compounds reported from Eriosema.......................................................................... 24
2.4.4 Compounds reported from Tephrosia......................................................................... 29
2.5 BIOLOGICAL ACTIVITIES.........................................................................................43
2.5.1 Biological activities of the genus Eriosema................................................................ 43
2.5.2 Biological activities of the genus Tephrosia............................................................... 44
CHAPTER THREE.....................................................................................................................45
MATERIALS AND METHODS................................................................................................ 45
3.1 GENERAL....................................................................................................................... 45
3.1.1 Reagents..................................................................................................................... 45
3.1.2 Instrumentation.......................................................................................................... 45
3.1.3 Collection of plant materials...................................................................................... 46
3.1.4 Chromatographic conditions...................................................................................... 46
3.1.5 Animals...................................................................................................................... 47
3.1.6 Sources of microorganisms for antiplasmodial test.................................................... 47
3.1.7 Sources of bacteria and fungi test strains.................................................................... 47
3.2 EXTRACTION AND ISOLATION OF COMPOUNDS................................................. 48
3.2.1 Extraction and isolation of compounds from Eriosema psoraleoides (roots).............. 48
3.2.2 Extraction and isolation of compounds from Tephrosia purpurea (stem)................... 48
3.3 PHYSICAL AND SPECTROSCOPIC DATA OF ISOLATED COMPOUNDS.............. 49
3.4 BIOLOGICAL ASSAY.................................................................................................... 53
3.4.1 Effects of plant extracts and isolated compounds on isolated Guinea pig Trachea.....53
3.4.2 Effects of plant extracts and isolated compounds on isolated aorta of a Sheep............54
3.4.3 Effects of plant extracts and isolated compounds on vas deferens of a rat and sheep .. 55
3.4.4 Effect of E. psoraleoides root extract on mating behavior..........................................55
3.5 IN-VITRO ANTIPLASMODIAL ACTIVITY ASSAY..................................................... 56
3.6 ANTIMICROBIAL TEST USING DISC DIFFUSION TECHNIQUE.............................57
CHAPTER FOUR.......................................................................................................................58
RESULTS AND DISCUSSION................................................................................................. 58

4.1 PHYTOCHEMICAL INVESTIGATION ON ERIOSEMA PSORALEOIDES...................58
4.1.1 Preliminary test results................................................................................................58
4.1.2 Characterization of compounds from Eriosema psoraleoides (roots).......................... 58
4.2 PHYTOCHEMICAL INVESTIGATION ON TEPHROSIA PURI'UREA.........................66
4.2.1 Preliminary test results................................................................................................66
4.2.2 Characterization of compounds from Tephrosia purpurea (stem)............................... 67
4.3 RESULTS OF THE BIOLOGICAL ASSAYS.................................................................. 79
4.3.1 Effects of plant extracts and isolated compounds on the Guinea pig trachea............79
vi
4.3.2 The effect of plant extracts and isolated compounds on isolated aorta....................... 82
4.3.3 Effects of plant extracts and pure compounds on the isolated vas deferens of sheep and
rat......................................................................................................................................83
4.3.4 Effect of Eriosemapsoraleoides root extract on the mating behavior....................... 84
4.4.1 Antiplasmodial activities of the plant extracts and pure compounds......................... 86
4.4.2 Antimicrobial activities of plant extracts and pure compounds.................................88
CHAPTER FIVE....................................................................................................................... 89
CONCLUSIONS AND RECOMMENDATIONS..................................................................... 89
5.1 CONCLUSIONS............................................................................................................. 89
5.2 RECOMMENDATIONS.................................................................................................90
REFERENCES.......................................................................................................................... 91
vn
LIST OF TABLES
Table 2.1: Cultivated Medicinal Plants used for treatment of Sexual Impotence
in Western Uganda...................................................................................................9
Table 2.2: Traditional uses of Eriosema species in Africa.................................. 13
Table 2.3: Ethnomedical use of some Tephrosia species of Kenya................... 14
Table 2.4: Occurrence of C-8 prenylated flavones in Tephrosia species.......... 30
Table 4.1: 'H and 1 C NMR data of compound 1 ............................................... 60
Table 4.2: *H and 1 C NMR data of compound 4 ............................................... 62
Table 4.3: ‘H and l3C NMR data of compound 3 ............................................... 64
Table 4.4: 'H and l3C NMR data of compound 2 ............................................... 66
fable 4.5: 'H and ljC NMR data of compound 6 ............................................... 68
Table 4.6: 'H and 13C NMR data of compound 9 ............................................... 70
Table 4.7: 1 H and 13 C NMR data of compound 7 ............................................. 72
Table 4.8: 'H and l3C NMR data and HMBC correlation for compound 8 ...... 75
Table 4.9: 'H and 13C NMR data of compound 5 and stigmasterol (5 )............ 78
T able 4.10: Effects o f plant extracts and pure compounds on isolated trachea of a
Guinea p ig .............................................................................................................. 79
Table 4.11: Effect o f plant extracts and isolated compounds on isolated aorta of
sheep.......................................................................................... ............................ 82
Table 4.12: In vitro IC50 values of the crude extracts o f E. psoraleoides, T.

purpurea and pure compounds against D6 and W2 strains of P. falciparum. .. 87
LIST OF FIGURES
Figure 1.1: Drugs used to manage erectile dysfunction................................................... 2
Figure 2.1: Second generation of PDE5 inhibitors........................................................... 6
Figure 2.2: Drugs that act by interacting with dopamine receptors................................ 7
Figure 2.3: Flowering E. psoraleoides plant..................................................................... 11
Figure 2.4: Tephrosia purpurea found in Kenya.............................................................. 12
Figure 2.5: Skeleta o f flavonoids based on the position of benzopyran ring................16
Figure 2.6: Examples of flavonoid (sensu stricto) subclasses.......................................16
Figure 2.7: Examples of isoflavonoid subclasses........................................................... 17
Figure 2.8: Basic skeleton of an isoflavone.....................................................................24
Figure 2.9: Isoflavones isolated from the roots of E. kraussianum.............................. 25
Figure 2.10: Isoflavone glycosides from E. tuberosum................................................. 26
Figure 2.11: Isoflavones from E. tuberosum....................................................................27
Figure 2.12: Chromones isolated from the roots of E. tuberosum..................................27
Figure 2.13a: Phenolic compounds from the roots o f E. tuberosum............................ 28
Figure 2.13b: Phenolic glycosides from the roots o f£ . tuberosum.............................. 29
Figure 2.14a: C-8 prenylated flavones isolated from Tephrosia species........................30
Figure 2.14b: C-8 prenylated flavones isolated from Tephrosia species........................31
Figure 2.14c: C-8 prenylated flavones isolated from Tephrosia species........................32
Figure 2.15: Isoflavones isolated from T. purpurea and T. maxima...............................33
Figure 2.16: Flavanone skeleton........................................................................................33
Figure 2.17: Prenylated flavanones isolated from Tephrosia species.............................34
Figure 2.18: Prenylated flavanones from Tephrosia species.......................................... 35
Figure 2.19: Furanoflavanones from the genus Tephrosia............................................. 36
Figure 2.20: Basic skeleton o f chalconoids...................................................................... 37
Figure 2.21: Chalcones isolated from T. purpurea and T. hamiltonii.............................38

ix
Figure 2.22: Chalcones isolated from T. toxicaria and T. crassifolia........................... 39
Figure 2.23: Rotenoids of the genus Tephrosia.............................................................. 40
Figure 2.24: Basic skeleton of most commonly occurring pterocarpan....................... 41
Figure 2.25: Pterocarpans from Tephrosia species......................................................... 42
Figure 4.1: Structure o f compound 1 ................................................................................59
Figure 4.3: Structure o f compound 4 ............................................................................... 62
Figure 4.5: Retro-Diels Alder fragmentationof compound 3a....................................... 64
Figure 4.9: Retro-Diels Alder fragmentationof compound 9......................................... 69
Figure 4.10: Structure o f compound 7 ...............................................................................71
Figure 4.11: Structure of compound 8 and tephrorin B ...................................................74
Figure 4.12: Structure of compound 5 ...............................................................................77
Figure 4.13: The effect of the pure compounds and plant extracts on isolated trachea-
volume o f Krebs’ solution displaced............................................................................... 80
Figure 4.14: A graph showing cumulative frequency ot male mice smelling the vagina84
Figure 4.15: The cumulative frequency of male mice licking thepenis........................ 85
Figure 4.16: A graph showing cumulative frequency o f erectionin male m ice............. 86
x
LIST OF SCHEMES
Scheme 2.1: Biosynthesis of flavonoids and isoflavonoids............................................... 19
Scheme 2.2: Biosynthetic interrelationships among different flavonoids..........................21
Scheme 2.3: Biosynthesis of furan ring in flavanones and flavones.................................. 23
Scheme 4.1: Proposed biogenesis of terpurinflavone 8 .......................................................76
xi
APPENDICES
Appendix A: Spectra for compound 1 .............................................................................. 104
Appendix B: spectra for compound 2 ............................................................................... 119
Appendix C: Spectra for compound 3............................................................................... 126
Appendix D: Spectra for compound 4 .............................................................................. 138
Appendix E: Spectra for compound 5 ............................................................................... 151
Appendix F: Spectra for compound 6 ............................................................................... 154
Appendix G: Spectra for compound 7 .............................................................................. 159
Appendix H: Spectra for compound 8 .............................................................................. 163
Appendix I: Spectra for compound 9 ................................................................................ 180
Appendix J: Physiograms of the effect of isolated compounds and extracts on isolated
aorta....................................................................................................................................... 187
Appendix K: Physiograms of the effect of isolated compounds and extracts on
vasdeferens........................................................................................................................... 191
xn
LIST OF ABBREVIATIONS
6 Chemical shift in delta values CoA Coenzyme A

COSY Correlated Spectroscopy t Triplet
J Coupling constant SD Seed
d Doublet brd Broad doublet
ddd Doublet o f a double doublet ,3C Carbon-13 isotope
dd Doublet of a doublet 'H Proton
EIMS Electron ionization mass CHI Chalcone isomerase
spectroscopy
Hz Hertz RT Root
HMBC Heteronuclear multiple bond CHS Chalcone synthase
correlation
HMQC Heteronuclear multiple quantum NADPH Nicotinamide adenine

coherence dinucleotide
HR-MS High Resolution Mass IFS Isoflavone sythase

spectroscopy
IFS Isoflavonoid synthase Ap Aerial parts
MS Mass spectroscopy PTLC Preparative thin layer
chromatography
m/z Mass to charge ration ax axial
^-max Maximum wavelength [O] Oxidation in schemes
of absorption
MHz Mega Hertz NOESY Nuclear overhauser

Enhancement
spectroscopy
[M f Molecular ion Ph phenyl
m Multiplet PDE-5 Phosphodiesterase type-5
NMR Nuclear magnetic resonance TLC Thin layer
chromatography
NOE Nuclear Overhauser UV Ultra violet
Enhancement
s Singlet ED Erectile Dysfunction
25.3 Specific rotation measured with IC50 Concentration causing
[a ] sodium D-line liuht 50% inhibition
589 (589 nm) at 28.8* C
Xlll
ABSTRACT
The air dried and ground roots o f Eriosema psoraleoides were exhaustively extracted with
dichloromethane/methanol (1:1) by cold percolation. The extract was partitioned between
water and ethyl acetate. Chromatographic separation of the ethyl acetate layer led to the
isolation of four compounds. These were identified as 4',5-dihydroxy-2',7-
dimethoxyisoflavone (1), 4',5,7-trihydroxycoumaronochromone (2), 4’,7"-bisgenistein (3)
and 4',5,7-trihdroxy-2'-methoxyisoflavone (4). Similar treatment of the stem of Tephrosia
purpurea yielded five compounds: stigmasterol (5); lanceolatin B (6); semiglabrin (7);
terpurinflavone (8); lanceolatin A (9). Of these, compound 8 is a novel compound. The
characterization of these compounds was based on spectroscopic techniques ('H NMR, 13C
NMR, 2D NMR, UV and MS).
The crude extacts of E. psoraleoides and T. purpurea as well as some of the pure
compounds isolated from these extracts were tested for smooth muscle and blood vessel
relaxant activities. The methanol extract of the roots o f E. psoraleoides (at 88 jig/mL) had
the highest relaxant effect (13 mm) on isolated aorta. The ethyl acetate fraction of the
dichloromethane/methanol (1:1) extract of T. purpurea stem at 74 pg/mL, stigmasterol (5)
at 12 pg/mL, lanceolatin B (6) at 19 pg/mL relaxed the aorta by 9 mm, 6 mm and 10 mm,
respectively. This indicates that E. psoraleoides and T. purpurea may promote penile
erection.
The crude extract of E. psoraleoides (roots) did not show a significant effect on bronchial
muscle where as the ethyl acetate fraction of the dichloromethane/methanol (1:1) extract of
T. purpurea at 22.5 pg/mL and its pure compound semiglabrin (7) at 7.5 pg/mL relaxed
the bronchial smooth muscle by 1.5 pL and 15 pL respectively. This probably explains the
traditional use of T. purpurea for the management o f chest tightness.
The methanol extract of the roots of E. psoraleoides (2.5 g/Kg) was further tested for
mating behavior on mice using yohimbine (360 mg/Kg) and sildenafil (600 mg/Kg) as a
xiv
reference drugs. The results indicated that E. psoraleoides (roots) extract may promote
erection.
The crude extracts of E. psoraleoides, T. purpurea and some o f the compounds isolated
from T. purpurea were also tested for antiplasmodial activities. The crude extracts showed
antiplasmodial activities with IC 50 values o f 9.33 + 0.38 and 11.43 + 0.47 pg/mL for E.
psoraleoides, and 10.47 + 2.22 pg/mL and 12.06 + 5.53 pg/mL for T. purpurea, against
chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium
falciparum, respectively. The novel compound, terpurinflavone (8) showed the highest
antiplasmodial activity with an IC 50 value of 2.73 + 1 .1 6 pg/mL and 1.36 + 0.12 pg/mL
against W2 and D6 strains respectively. Laceolatin A (9) also showed significant activity
against the D6 and W2 strains of P. falciparum with IC 50 value of 3.82 + 1.00 and 3.82 +
1.04pg/mL, respectively.
The crude extracts of E. psoraleoides, T. purpurea and some of the pure compounds
isolated from T. purpurea were further tested for antimicrobial activity. The crude extract
of E. psoraleoides showed an inhibition zone of 12 mm against Candida albicans at a
concentration of 1.8 mg/disc while the novel compound terpurinflavone (8) showed an
inhibition zone of 16 mm against Microsporum gypsum at 50 pg/disc. The crude extract of
T. purpurea and the five pure compounds isolated from this extract were inactive against
Staphylococcus aureus, Pseudomonas eruginosa, Candida albicans, Cryptococcus
neoformans, Trichphyton mentagrophytes and Microsporum gypsum.
xv
CHAPTER ONE
INTRODUCTION
ENERALINTRODUCTION
on-infectious or intrinsic diseases are those that are not caused by a pathogen and are neither
>ntagious nor communicable. These diseases may arise as a result of environmental conditions.
Dr example skin cancer can be caused by radiation of the sun or dietary deficiencies. Other non-
)mmunicable diseases include impotency and cardiovascular diseases. One of the non-
fectious disease that has received little attention is the men’s reproductive health problem,
ectile dysfunction.
rectile dysfunction (ED) is the repeated inability to get or keep an erection firm enough for
:xual intercourse. The word “impotence” has also been used to describe other problems that
terfere with sexual intercourse and reproduction, such as lack of sexual desire and problems
ith ejaculation or orgasm (Pamplona-Roger, 2000; Roper, 2001). Since ethnobotanical
idigenous knowledge (IK) cannot clearly distinguish between these two terms, the terms
Erectile dysfunction” and “sexual impotence” have been used interchangeably. It is therefore
fficult to estimate the true prevelence of ED. The world prevalence of ED is estimated at 150
iillion and is expected to increase to 322 million by the year 2025 (Feldman et ai, 1994; Ayta et
'., 1999; Khalaf and Levinson, 2003).
he causes o f ED vary from one individual to another. These causes include: damage to nerves
id tissues. Seventy percent of ED cases are as a result o f other diseases (Derry et al., 1998).
ational Institute of Health (NIH, 2004) of the U.S.A. reported that between 35 and 50 percent
f men with diabetes experience ED (Melman and Christ, 2002). Many common medicines
Lis e ED as a side effect (Melman and Christ, 2002). Psychological factors cause 10 to 20
.r cent of ED cases (Melman and Christ, 2002). Smoking causes ED by varying hormonal
ilan ce and reducing blood flow in veins and arteries (Pak and Broderick, 2006).
r ectile dysfunction is managed using PDE-5 inhibitors such as sildenafil (10), PGEi analogs
lê alprostadil (11), ci2 antagonists like yohimbine (12) (Figure 1.1). An example of d| and ct2
titagonist drug is phentolamine (13). However sildenafil (10) is effective in less than 70% ol
atien ts and has side effects (Melman and Christ, 2002). Alprostadil (11) (Caverject 1M),
ohim bine (12), phentolamine (13) act by dilating blood vessels (Kametenesi-Mugisha and
)ryem -Origa, 2005). Unfortunately these medications for ED are very expensive for most people
n Kenya and other developing countries.
Alprostadil (11)
Sildenafil (10)
Phentolamine (13)
'igure 1.1: Drugs used to manage erectile dysfunction
2
-> PROBLEM STATEMENT
-ectile dysfunction is a major contributor to the global burden of disease and a significant
-i pediment to socio-economic development in poor countries. Modem medication to combat ED
very expensive for most people in Kenya and other developing countries. Yet, in traditional
LCtiicinal practice there are several plants that are used to treat ED (Kokwaro, 1993). There is
lerefore a need for continued efforts to study such plants in the search o f compounds that may
c useful for the management of erectile dysfunction so that these compounds can be developed
i to new, cheaper and more effective drugs.
1.3 STUDY JUSTIFICATION
Research on medicinal plants represents a major strategy for the discovery of compounds that
:a.n be developed into new drugs. Some compounds with penile muscle relaxant activity have
>ven isolated from the root stock of Eriosema krciussianum (Drcwes et al., 2002, 2003). There
ire several plants used traditionally to treat impotence in Kenya including Eriosema
?.\oraleoides. No phytochemical and biological work has been reported on this plant.
arious parts of Tephrosia purpurea are used to treat impotence, asthma, diarrhoea, gonorrhoea,
heumatism, ulcer and urinary disorders (Lodhi et al., 2006). F’revious phytochemical
■investigations on roots and aerial parts of Tephrosia purpurea have revealed the presence of
■otenoids, isoflavones, flavanones, chalcones, flavanols and sterols (Pelter et al., 1981).
Blowever, phytochemicals from the stem of T. purpurea with fertility promoting properties have
H o t been identified. It is therefore important that Eriosema psoraleoides and Tephrosia purpurea
L>ul<i be investigated for penile muscle relaxant activity. Previous studies have shown that
lV onoids and isoflavonoids have antiplasmodial activity (Yenesew et al., 2003). It is worth also
te s t the constituents of these plants for antiplasmodial activities.
. 4 OBJECTIVES
.4.1 General Objective
he general objective was to isolate and characterize phytochemicals from Eriosema
soraleoides and Tephrosiapurpurea with pharmacological and antiplasmodial activities.
.4.2: Specific Objectives
h e specific objectives were:
1. To isolate compounds from Eriosema psoraleoides and Tephrosia purpurea.
2. To determine the structures of the isolated compounds using spectroscopic techniques.
3. To establish the effects of the crude extracts and some of the isolated compounds on
isolated blood vessels and trachea.
4. To establish the effect of the plant extracts on mating behavior.
5. To establish the antiplasmodial activity of the crude extracts and the isolated compounds.
4
CHAPTER TWO
LITERATURE REVIEW
1 ERECTILE DYSFUNCTION
-tactile dysfunction (ED), which is also referred to as impotence, is the repeated inability to get
keep an erection firm enough for sexual intercourse (Moreland et al.; 2001, Klein and
hom pson, 2004). Impotence includes other problems that interfere with sexual intercourse and
production, such as lack o f sexual desire, problems with ejaculation or orgasm and a
/sfunctional organ system (Williams, 2007).
i n e e an erection requires a precise sequence of events, ED can occur when any o f the events is
isrupted. This sequence includes interruption of nerve impulses in the brain, spinal column, area
ro u n d the penis, and response in muscles, fibrous tissues, veins, and arteries in and near the
o rp o ra cavernosa (Melman and Christ, 2002). Causes of ED include, damage to nerves, arteries,
iT io o th muscles, and fibrous tissues (Williams, 2007). These are often as a result of diseases,
u c h as diabetes, kidney disease, chronic alcoholism, multiple sclerosis, atherosclerosis, vascular
lisease, and neurologic diseases that account for about 70 percent of ED cases (Rendell et al.,
9 9 9 ; Conti et al., 1999; Rosen et al., 2005).
Vntihypertensives, antihistamines, antidepressants, tranquilizers, appetite suppressants, and
ntiulcer drugs produce ED as a side effect. Psychological factors such as stress, anxiety, guilt,
Impression, low self-esteem, and fear of sexual failure cause 10 to 20 percent of El) cases
■Melman and Christ, 2002). Other possible causes are smoking, which affects blood flow in
Imins and arteries, and hormonal abnormalities, such as insufficient amounts of testosterone
■<osen et al, 2005; Pak and Broderick, 2006).

5
2.1.1 Drugs used to treat erectile dysfunction
Erectile dysfunction is managed by drugs that target the peripheral organs. The drugs act by
enhancing smooth muscle vasodilation or block the adrenergic or endothelin (ET)-mediated
vasoconstriction associated with penile flaccidity. Peripherally acting drugs are classified as
phosphodiesterase type 5 (PDE5) inhibitors. PDE5 inhibitors block the hydrolysis of cyclic
guanosine monophosphate (cGMP) produced by soluble guanylate cyclase (sGC) in response to
nitric oxide (NO), prolonging cGMP levels in corpus cavemosum smooth muscle causing
relaxation. First generation PDE5 inhibitors include sildenafil (10) while tadalafil (14) and
vardenafil (15) are second generation of PDE5 inhibitors (Figure 2.1). Sildenafil (10) is of little
value in men with cord injury and lack libido. Furthermore, it potentiates cardiovascular
diseases. Sildenafil (10) also has a negative effect on colour vision, causing difficulty in blue-
green discrimination.
Vardenafil (15)
Tadalafil (14)
Figure 2.1: Second generation of PDE5 inhibitors
Other drugs used to manage ED are prostaglandin Ej (PGE|) analogs. 1hese drugs act via PGE
receptors EP2 and EP4 on smooth muscle to increase intracellular cyclic adenosine
6
nionophosphate (cAMP) synthesis and potentiate smooth muscle relaxation. Alprostadil (11) that
causes priapism and phentolamine (13), nonselective a-adrenoceptor antagonist is an example of
prostaglandin E| (PGEi) analogs and a-antagonist respectively. The last group of ED drugs acts
on the central nervous system. The drugs include yohimbine (12) and apomorphine (16), ABT-
724 (17) that act by interacting with dopamine receptors (DA and D4) (Figure 2.2).
2-[(4-Pyridin-2-y lpiperazin-1-y l)methy 1]

Apomorphine (16)
-lH-benzimidazole (ABT-724) (17)
Figure 2.2: Drugs that act by interacting with dopamine receptors
2.1.2 Traditional plants used to treat erectile dysfunction
In traditional medicine, there are several medicinal plants used to treat ED. 1 his ethnobotanical
indigenous knowledge has not been documented. Plants used for management ol ED have not
been scientifically validated for efficacy and safety. World Health Organization (WHO, 2005)
estimates that 80% of the world population, primarily those in rural areas of developing
countries, depends on traditional medicines for their primary health care needs (Cunningham,
1993). In some cases such uses have been validated by isolation ol compounds that are active
against the causative agents of diseases.
Eriosema kraussianum (Leguminosae) is used for treatment of erectile dysfunction (Bryant,
1983; Hutchings, 1996a, Hutchings el al, 1996b). Phytochemicals isolated from Eriosema
7
.pecies include isoflavones with ability to promote penile erection (Drewes et al., 2003). The
»enus Eriosemci is represented in Kenya by E. bogdanii, E. glomeratum, E. psoraleoides, E.
^cinderystii, E. nutans, E. buchananii, E. montanum and E. robustum (Agnew and Agnew, 1994).
R.oots o f Eriosema psoraleoides are used for the treatment o f diarrhoea, dysentery and impotence
(Bryant, 1983).
One other plant which is also used widely for the treatment o f ED and other diseases is
Tephrosia purpurea (Papilionaceae). In Ayurvedic system of medicine various parts of T.
purpurea are used to treat impotence, asthma, diarrhoea, gonorrhoea, rheumatism, ulcer and
urinary disorders (Lodhi et al., 2006). The plant has been claimed to cure diseases of kidney,
liver spleen, heart and blood (Kirtikar and Basu, 1956; Despande et al, 2003). Previous
phytochemical investigations of Tephrosia purpurea have revealed the presence of rotenoids,
isoflavones, flavanones, chalcones, flavanols, and sterols (Pelter et al., 1981).
Modern medication for ED is very expensive for most of the people in developing countries. Yet,
in traditional medicine, there are several medicinal plants that have been relied on for the
treatment of ED. Erectile dysfunction is an old problem and traditionally the indigenous
knowledge had ways of treating or managing these conditions in many parts ol the world, for
example 33 medicinal plants both cultivated and wild-harvested are documented to be used
traditionally by men to manage ED in western Uganda. Some ol these plants are listed in I able
2.1. The herbal medicines used in the management of erectile dysfunction are mainly prepared
by pounding, chewing and boiling and are usually orally administered. I he traditional healers
treat sexual impotence by prescribing some o f these herbs lor use in tea, local beers, fermented
milk and porridge. Some herbs are roasted or smoked such as coffee before administration.
8
able 2.1: Cultivated Medicinal Plants used for treatment of Sexual Impotence in Western
Jganda (Kametenesi-Mugisha and Oryem-Origa, 2005)
Scientific Local Name Parts Preparation Administration

Name Used
Allium Katunguru (NY, KI, ST- Chewing, Oral in water and
cepa L. RU) Onion (Engl.) BU, L, cooking in food
RT
Allium Tungurusumu (KO) ST, Chewing, Oral in water and
sativum L. Garlic (ENG) BU, L, cooking in food
RT
Cannabis Njayi (GA), Njaga (NY) L Chewing, Oral, inhaling
sativa L. Marijuana (ENG), smoking fumes
Bangi (SW)
Cleome Esobyo/Amarera (KO) L, R, Chewing, Oral or as food
gynandra L. Eshogi (NY) FL cooking
Sida Keyoyo (RU) L Pounding, Oral

tenuicarpa boiling
Vollesen
Arachis Binyebwa (NY, RU) SE Roasting Oral as food
hypogaea L. Ground nuts (ENG)
Coffee Mwani (NY) Arabica SE Roasting, Oral as a

arabica L. Coffee (ENG) chewing beverage
Capsicum Kamurari (GA) FR Pounding, Oral in food

frutescens L. Eshenda (NY) Red boiling,
pepper (ENG) chewing
Key: SW (Swahili); ENG (English); KO (Rukonjo); RU (Runyaruguru); NY

(Runyankore); KI (Rukiga); GA (Luganda);L (leaves); R (roots); RT (root tuber); RH (rhizome);
FR (fruits); SE (seeds); ST-BU (stem-bulb).
Tephrosia purpurea has been documented to treat impotence (Lodhi et al., 2006) while the use
of E. psoraleoides to manage erectile dysfunction in Kenya has not been documented.
9
2 BOTANICAL INFORMATION ON ERIOSEMA AND TEPHROSIA
2.1 The genera of Eriosema and Tephrosia
he genera Tephrosia and Eriosema belong to the family Leguminosae, also known as Fabaceae.
his family comprises of 657 genera and 18,000 species of trees, shrubs and herbs, which are
ddely distributed in the temperate as well as tropical regions of the world. The family is the
econd largest of the dicotyledons after the Compositae, and plants in the family are known for
neir ability to support nitrogen fixation through symbiosis (Heywood, 1971). The family is
ubdivided into three sub-families: Mimosoideae, Papilionoideae and Caesalpinioideae. The
genera Tephrosia and Eriosema belongs to the Papilionoideae sub-family (Polhill, el al., 1981a).
2.2.2. Botanical description of Eriosema psoraleoides
The genus of Eriosema is distributed in tropical Africa, America, Asia and Australia. Thirty-
eight species are distributed in South America, primarily in Argentina, Brazil and Paraguay
(Polhill, et al., 1981b). The genus Eriosema is represented in Kenya by eight species, namely E.
bogdanii, E. glomeratum, E. psoraleoides, E. vanderystii, E. nutans, E. buchananii, E. monlanum
and E. robustum (Agnew and Agnew, 1994).
Eriosema psoraleoides is a herb or shrublet that is rarely erect with climbing characteristics. It
has branches that are strongly ribbed, brown, hairy and covered with small orange-red glands. It
has three leaflets that are pale beneath, venated with buff hairs and are narrow, elliptic and 2.3-
9.5 cm long and 0.8-3.5 cm wide. The apex of the leaf is round and micronulated. I he rest of the
leaflet surfaces are silvery. The plant has petiole of 1-5 mm long; rhachis of 1.8 mm length;
petiole of 1-3 mm length. The calyx is pubescented with triangular lobes. It has deep golden-
yellow corolla with a length of between 0.7-1.4 mm; and rarely with hairs outside. I he
10
suborbicular. oval, oblique pods with length between 1.1-1.8 mm and 0.9-1.1 mm width are
covered with long ferruginous hairs. The seeds are reddish-brown or pinkish with blue black
mottling that is shiny with length of 4.5-52 mm (Gillet el al., 1971a). Figure 2.3 shows a
flowering plant of E. psoraleoides.
Figure 2.3: Flowering E. psoraleoides plant (photograph taken by Peter Juma)
2.2.3 Botanical information of Tephrosia purpurea
Tephrosia is a large tropic and sub-tropic genus of perennial woody shrubs distributed in tropical
and sub-tropical regions. It is estimated to contain between 300 and 400 species, distributed all
over the world as follows; 35 species occur in India, 30 are native of South America, 70 are
found in South Africa, 50 in equatorial Africa of which 30 are found in Kenya (Tarus et al.,
2002; Beentje, 1994). Examples of some Kenyan Tephrosia species include; T. aequilala, T.
elata, T. hildebrandtii, T. holstii, T. inlerrupta, T. linearis, T. noctijlora, T. paucijuga, T.
pentaphylla, T. pumila, T. purpurea, T. villosa among others (Agnew and Agnew, 1994).
11
Tephrosia purpurea is a short lived perennial herb of up to 80-150 cm long with leaf rhachis of
approximate length of 8 cm. The petiole is about 1cm and it extends beyond the lateral leaflets. It
has approximately 9-17 leaflets that are elliptic with an estimated length of 2 cm and width of 6
mm. The leaflets apex is round, mucronated and pubescented at the base. The flowers are
reddish-purple or bright pink. It has brown calyx with appressed strigulose and spreading
pubescent. The upper filament is lightly attached, widened but not bent. The filament is 1.5 mm
above the base with filament sheath of approximate length of 0.3 mm and width of 6mm. The
style is glabrous, linear, gently curved with the length o f 2.5 mm. The pods are gently curved
towards the tip with a length of about 4-4.5 cm and width of 6.5 mm. It has 6-9 seeds that are
subcylindrical with a centrally placed hillum (Gillet et al., 1971b). The species is divided into the
following subspecies: purpurea, leptostachya, and dunensis. All the subspecies are found in
different parts of Kenya.
Figure 2.4: Tephrosia purpurea found in Kenya (photo taken by Loise Muiva)
12
.3 . ETHNOMEDICAL INFORMATION
.3.1 Ethnomedical information of the genus Eriosema
Hants belonging to the genus Eriosema have been used traditionally in various communities for
h e treatment of various ailments (Pretorius et al., 2002). The traditional uses of Eriosema
pecies in Africa is summarized in table 2.2.
Tsible 2.2: Traditional uses of Eriosema species in Africa
C ountry P arts used Medical use Reference

E. burkei Malawi Dried roots Water extract used Msonthi and
orally to cure pain in Magombo, 1983
adults and treat
lymphoid disorders
E . Cordatum South Africa Roots Hot milk infusion Bryant, 1966
taken as aphrodisiac
E . glomeratum Gabon Petiole Syncope, fish Akendengue and
poisoning Louis, 1994
E . krausianum South Africa Dried Root Used orally to Ojewolw and

alleviate impotence in Drawes, 2007
men
E . psoraleoides Central Africa Leeves Water extract used Sillans, 1953
orally as an oxytocic
during pregnant
Tanganyika Roots Water extract Haerdi, 1964
combined with
Piliostigma
trionningii threaten
miscarriage
Tanzania Dried Used as chewing stick Khan et al., 2000
twigs
E. salignum South Africa Roots Hot milk infussion Bryant, 1966
taken as aphrodisiac
E. tisseratii Central Africa Roots Aphrodisiac Sillans, 1953
E. benthamianum Brazil Roots Used as an anti Hirschhorn, 1982
inflammatory
E. diffusum Mexico Entire Unspecified female Hastings, 1990
Guatemala Plant diseases
13
1.3.2 Ethnomedical information on the genus Tephrosia
n Kenya, several plants o f the genus Tephrosia are used for the treatment of various ailments,
fa b le 2.3 below gives some representative Kenyan Tephrosia species and their respective
-jiedicinal use.
I'iib le 2.3: Ethnomedical use of some Tephrosia species o f Kenya
ipecies Plant p a rt and Ethno Locality/Commun References

medical use ity
r. aequilata Roots dug out, boiled and Makueni (Kamba), Kokwaro,
mixed with milk and drunk Kajiado (Maasai), 1993; Agnew
for pain in liver and spleen. Coast (Pare, Digo) and Agnew,
Roots chewed with salt as a 1994.
cure for venereal diseases.
r. elata Roots chewed as a cure for Machakos, Lwande,
stomach pains, fever and (Kamba), Kajiado 1985 ; Agnew
general weakness. (Maasai) and Agnew,
1994.
T. paucijuga Roots and leaves dried, Coastal region Kokwaro,

pounded or ground into (Nyika) 1993.
power form and applied on
wounds.
T. pumila The roots are chewed as a Coastal region Kokwaro,
remedy for cold in the chest. (Digo),Kibwezi 1993; Agnew
Roots are also boiled and the Kamba) Kajiado and Agnew,
infusion taken as a broth as a (Maasai) 1994.
cure for venereal diseases.
T. purpurea Roots used as a medicine for Kilifi Kokwaro,
stomach pains. Leaves used (Giriama), 1993;
for snake bite and for Machakos, Agnew and
headache. Aerial parts are (Kamba) Agnew, 1994;
used as laxative, deobstruent Ahmad et al.,
and diuretic, treatment of 1999.
cough, biliary febrile attacks,
obstructions of the liver,
spleen and kidneys.
Anthelmintic for children and
chronic diarrhoea.
14
-4 BIOSYNTHESIS OF FLAVONOIDS AND THEIR DERIVATIVES
^vonoids sensu lato are natural products that have a C6-C3-C 6 carbon frame work or more
>ecifically a phenyl benzopyran functionality. They constitute one o f the largest groups of
iturally occurring phenols (Markham, 1982). All species of terrestrial plants and the relatively
Ivanced Algae families contain tlavonoids (Induli, 2006). It is estimated that about 2% of all
irbon photosynthesized by plants is converted into flavonoids or closely related compounds
s/larkham, 1982). The growing interest in plant flavonoids is due to their use as human dietary
im pounds and as pharmacological agents (Harbome et al., 1986; Harbone, 1998).
n plants, flavonoids occur in different structural forms. All flavonoid aglycones contain fifteen
cirbon atoms in their basic nucleus and these are arranged in a C 6 -C 3-C 6 configuration. Each C6
epresents an aromatic ring. These aromatic rings are linked by a three carbon unit which form a
hurd heterocyclic ring via cyclization with one of the aromatic ring via an oxygen atom. I he
Lromatic rings are labelled as ring A and B and heterocyclic ring as ring C (figure 2.5).
depending on the position of the linkage o f the aromatic ring to the benzopyrano moiety, the
lavonoids sensu lato are divided into flavonoids sensu stricto (2-phenylbenzopyrans) (18),
soflavonoids (3-benzopyrans) (19) and the neoflavonoids (4-benzopyrans) (20) (figure 2.5).
Fhese groups usually share a common chalcone precursor, and thus are biogenetically and
structurally related (Agrawal, 1989).
15
Flavonoids {Sensu stricto) (18)
B
S /
Neoflavonoids (20)
Figure 2.5: Skeleta of flavonoids based on the position of benzopyran ring
Oxidation and degree o f saturation in the heterocyclic ring-C ot the flavonoid (18) and
isoflavonoid (19) results into different subclasses of flavonoids sensu stricto (Markham, 1982).
Examples of these flavonoids subclasses are flavanones (21) and flavones (22) (Figure 2.6).
Subclasses of isoflavonoids include isoflavanone (23) and isoflavone (24) (Figure 2.7) (Agrawal,
1989).
Figure 2.6: Examples o f flavonoid (sensu stricto) subclasses
16
Isoflavanone (23)
f ig u r e 2.7: Examples o f isoflavonoid subclasses
Natural flavonoids and isoflavonoids are usually oxygenated and bear hydroxyl or methoxyl
substituents. A large number of flavonoids occur as O-glycosides in which one or more of the
hydroxyl groups of the flavonoid are bound to a sugar or sugars via an acid labile hemiacetal
bond (Agrawal, 1989). In some cases, isoflavonoids skeleton may get further modified by
cyclization of C-2 and C-4 of ring C with aryl ring B through an oxygen atom to generate a
tetracyclic ring system called a coumaronochromone (Agrawal, 1989).
2.4.1 Biosynthesis of flavonoids and their derivatives
All flavonoids sensu lato are biosynthesized from common precursors which incorporate both
shikimate and acetate malonate pathways (Scheme 2.1). The flavonoids initially formed in the
biosynthetic pathway are chalcones and all other forms are derived from these by a variety ol
routes.
Flavonoids are biosynthesized by extension of /7-hydroxycoumaroyl CoA with three molecules
of malonyl CoA in a head-to-tail manner in order to form a tetraketide intermediate. The process
is catalyzed by the enzyme chalcone synthase (CHS). The intermediate then folds and condenses
further to give the chalcones naringeninchalcone (25) and isoliquiritigenin (26). I he biosynthesis
17
, f isoliquiritigenin (26) is catalysed by CHS with NADPH as a co-factor and these reactions are
f-je first committed steps in flavonoid biosynthesis.
fine first compounds derived from chalcones are the flavanones, (2S)-naringenin (5,7,4'-
rihydroxyflavanone, 27) and (2S)-liquiritigenin (7,4'-dihydroxyflavanone, 28), from
âringeninchalcone (25) and isoliquiritigenin (26) respectively. The biosynthesis of the two
flavanones is catalysed by chalcone isomerase (CHI) (Dewick, 2002)
T h e enzyme isoflavone synthase (IFS) converts the flavanone substrates naringenin (27) and
1 iquiritigenin (28) to the isoflavones genistein (29) and daidzein (30), respectively. This reaction
i s proposed to involve two steps: 2 -hydroxylation and aryl migration from flavanone substrates
t o yield 2-hydroxyisoflavanone. Dehydration of the corresponding isoflavanone derivative is the
la s t step. Genistein (29) and daidzein (30) are then further metabolised to give the various classes
o f isoflavonoids (Dewick, 2002).

OH
Tetraketide intermediate
(2S)-Narigenin (27) (2S) Liquiritigenin (28)

(flavanone) (flavanone)
J IFS IFS
Genistein (29)
(isoflavone)
I 1
Legume and non-legume Legume isoflavonoids
isoflavonoids
Scheme 2.1: Biosynthesis of flavonoids and isoflavonoids (Dewick, 2002)

19
fie biosynthetic interrelationships among different flavonoid types are summarized in Scheme
2 (Markham, 1982; Induli, 2006). The variation in structure among the various flavonoid and
oflavonoid classes is achieved by the loss and addition of hydroxyl groups. The flavonoid and
.oflavonoid can further be methylated or prenylated. 1 he methyl and prenyl group can be
lodified to give dimethylpyrano and furano rings (Dewick, 2002; Induli, 2006).
20
Anthocyanidin
Catechin
Scheme 2.2: Biosynthetic interrelationships among different flavonoids (Dewick, 2002; Indnh
2006)
21
> ,4.2 Biogenesis of tetrahydrofuran ring in flavonoids
> renylation at C - 8 of flavanones, flavones and chalcones through a series of steps forms unique
com pounds with tetrahydrofuran moiety. The existence o f the furan ring attached on ring A of a
11 avanone and a flavone has been reported in some closely related taxa of the Leguminosae. The
^ e n u s Tephrosia is of interest because it is able to sythesise furan rings with varying degrees of
saturation. The biogenesis of the furan ring is presented in Scheme 2.3 (Pelter et al., 1981). The
co m p lex substituents at C - 8 arise from the ability of some Tephrosia species to oxidize the 7-
rnethoxy group.
-OH
-0
} .
/'ll
Cyclisation
6 \ ^
Oxidation V l >
----------------- * l ^ > /
j
water
Addition of
acetate grpup O
Enzymes
and
water
HO
•Scheme 2.3: Biosynthesis of furan ring in flavanones and tlavones (I’elter al„ 1981)
UJ
2
-4.3 Compounds reported from Eriosenui
.-4.3.1 Isoflavones reported from Eriosenui
,<j>flavones possess 3-phenylchromone skeleton. The heterocyclic ring-C in isoflavones is made
p of an oxyolefinic methine (C-2), olefmic quaternary carbon (C-3) and a carbonyl carbon (C-4)
- igure 2 .8 ).
Figure 2.8: Basic skeleton of an isoflavone
soflavones from E. kraussianum contain prenyl groups and its cyclized derivatives involving
id ja c e n t hydroxyl group to give pyranoisoflavones. All pyranoisoflavones from the roots of E.
cr-aussianum have the pyrano ring substituted at either C-6/7 of ring A or at C-374' of ring B or
:>oth (Drewes et al., 2002). Kraussianone 2 (31), kraussianone 3 (32) and kraussianone 5 (34)
l a v e a prenyl group substituted at C - 6 position. Kraussianone 4 (33) and Kraussianone 5 (34) are
Tiodified isoflavones, dihydrochromeno-chromones, isolated from E. kraussianum. I he
isoflavone elongatin (35) isolated from T. elongata is identical to kraussianone 1 (30) in terms of
r i ng A, B and E. The dimethylpyran ring D is however, absent.
24
HiC
HO
Kraussianone 1 (30) kraussianone 2 (31)
Kraussianone 3 (32) Kraussianone 4 (33)
F ig u r e 2.9: Isoflavones isolated from the roots of E. kraussiamim
S i x isoflavones isolated from the roots of E. tuberosum contain sugar moieties linked to oxygen
C isoflavone-O-glycoside) through acetal linkage (Figure 2.10, Ma et al., 1998). Genistein (33)
zin d 5-0-methylgenistein (35) from the same plant lack a sugar moiety (Figure 2.11).
25
5-0-M ethylgenistein-7-0-P -D -
5-O-M ethylgenistein 7-O-P-D apiofuranosyl-
glucopyranoside (36)
(1—* 6)-0-P-D -glucopyranoside (37)
G enistein-7-O -P -D -A piofuranosyl- Genistin (39)

(l-^6)-0-P ~ D -gIucopyranoside (38)
5-O-M ethylgenistein 7-O -P-D -A piofuranosyl-
(1 — 2)-0-P*D -G lucopyranoside (40)
igure 2.10: Isoflavone glycosides from E. tu b e r o s u m
26
Genistein (42) 5-0-Methylgenistein (43)
i g u r e 2.11: Isoflavones from E. tuberosum
.4 .3 .2 Chromones from Eriosema
w chromone (1, 4-benzopyrone) is a benzopyran derivative with a substituted keto group on the
^zran ring. All the chromones isolated from the roots of E. tuberosum have a pyrano ring
i_ibstituted at C-6/7 or C-7/8 (Ma et a l, 1996). These chromones include lupinifolin (44),
riosem atin E (45) and eriosematin D (46) (Figure 2.12).
OH
Eriosematin D (46)
f i g u r e 2.12: Chromones isolated from the roots of E. tuberosum
27
-4.3.3 Phenols reported from Eriosema
^ 'v en phenolic derivatives have been isolated from the roots of E. tuberosum (Ma et al., 1999).
j-i.e simple phenols include 4-hydroxybenzoic acid (46), hydroquinone (47) vanillic acid (48),
- id eriosematin F (49) (Figure 2.13a). The other three phenols are glycosides and include arbutin
>C)), eriosemaside A (51) and eriosemaside B (52) (Figure 2.13b).
OH
^4-Hydroxybenzoic acid (46) Hydroquinone (47)
E riosem atin F (49) Vanillic acid (48)
R" igure 2.13a: Phenolic compounds from the roots of E. tuberosum
28
OH
OH
Eriosemaside A (51)
Arbutin (50)
t
OH
IO
Eriosemaside B (52)
igure 2.13b: Phenolic glycosides from the roots of E. tuberosum
2 .4.4 Compounds reported from Tephrosia
2 .4.4.1 Flavones reported from Tephrosia
t lavones are 2,3-dehydro derivatives of the flavanones (Agrawal, 1989). C-8 prenylated flavones
c v ith extensive modifications have been reported from T. purpurea, *71 apollinea and T.
p>olystachyoides (Table 2.4). The structures of the flavones are shown in Figure 2.14a-214c. Such
modification has not been observed in other Tephrosia species showing that the three taxa are
~ losely related.
29
F>le 2.4: Occurrence of C - 8 prenylated flavones in Tephrosia species
vones Source Reference

illinine (53) T. apollinea (SD) Waterman and Khalid, 1980
T. purpurea (RT) Pelter etal., 1981
Semiglabrin (54) T. apollinea (SD) Waterman and Khalid, 1980
T. purpurea (RT) Pelter e ta l, 1981
T. apollinea (RT) Abou-Douh el al., 2005
-niglabrinol (55) T. purpurea (RT) Pelter el al., 1981
riceolatin A (56) T. apolline (SD) Waterman and Khalid, 1980
T. purpurea (RT) Pelter et al., 1981
T. apolline (RT) Abou-Douh et al., 2005
nceolatin B (57) T.purpurea (RT) Pelter et al., 1981
phroglabrin (58) T.purpurea (RT) Pelter et al., 1981
rpurindiol (59) T.purpurea (RT) Pelter etal., 1981
.ngaglabol (60) T. purpurea (AP) Ahmad et al., 1999
rphropurpulin (61) T. purpurea (AP) Hegazy et al., 2009
^glabratephrin (62) T. purpurea (AP) Hegazy et al., 2009
ichrosin (63) T. polystachyoides (SD) Waterman and Khalid, 1980
)- Pseudosemiglabrin T. apolline (SD) Waterman and Khalid, 1980
4) T. apolline (RT) Abou-Douh et al., 2005
-)-Glabratephrin (65) T. apolline (SD) Waterman and Khalid, 1980
T. pupurea (AP) Hegazy et al., 2009
ookerianin ( 6 6 ) T. hookeriana (RT) Prabhakar et al., 1996
Cey: SD-seeds; RT-Root s; AP-Aerial parts
o^ \ £ - ok
w
O
Apollinine, R=H (53) Semiglabrin, R=COCH 3 (54)
Hookerianin, R=OCH 3 (6 6 ) Semiglabrinol, R=H (55)
Figure 2.14a: C-8 prenylated flavones isolated from T e p h ro sia species

OH
Lanceolatin B, R=H (57)
Pongaglabol, R=OH (60)
Tephroglabrin, R=H (58)
Tachrosin, R=OCH 3 (63)
Tephropurpulin (61)
Figure 2.14b: C-8 prenylated flavones isolated from T e p h ro sia species
31
ig u re 2.14c: C - 8 prenylated flavones isolated from Tephrosia species
.4.4.2 Isoflavones isolated from Tephrosia species
>oflavones constitute the largest group of natural isoflavonoids. Several isoflavones have been
sported in the genus Tephrosia. At least two isoflavones have been reported from T. purpurea.
'hese isoflavones are 4 ',7 -dihydroxy-3 ',5 '-dimethoxyisoflavone (6 6 ) and purpuranin A (67) from
h e aerial parts and pods respectively (Figure 2.13) (Rao and Raju, 1984, Chang et al., 2000).
The isoflavone purpuranin A (67) is similar to maximaisoflavone C (6 8 ), maximaisoflavone E
69) and maximaisoflavone G (69) which have been isolated from the roots and pods ol T.
maxima (Rao et al., 1985).
32
Purpuranin A, R|=OCH3, R2=CH3, R3=H (67)
Maximmaisoflavone C, Rj=H, R2=Prenyl
R3=OCH3 (6 8 )
Maximaisoflavone E, R Ô C fy, R2=H, R3=H (69)
Maximaisoflavone G, R|=H, R2=H, R3=OCH3 (70)
igure 2.15: Isoflavones isolated from T. purpurea and T. maxima
; .4.4.3 Flavanones of Tephrosia
7lavanones (dihydroflavones) possess 2 -phenylchromanone as the parent skeleton. Since carbon-
. of the flavanones molecule is a centre of asymmetry, two isomeric forms ol each structure are
possible but most of the naturally occurring flavanones have a phenyl substituent at C-2 position
n pseudoequatorial orientation (Agrawal, 1989). Fig. 2.16 shows the basic skeleton of
flavanones and the numbering system.
Figure 2.16: Flavanone skeleton
33
j-iple prenylated flavanones have been isolated from Tephrosia species. The pyranoflavanones
c>vatin (71) and obovatin methyl ether (72) were isolated from the stem of T. obovata and the
yts of T. elata (Chen, 1978; Lwande, 1985; Gomez-Garibay et al, 1986). The pyranoflavanone
, lonchocarpin (76) isolated from the roots of T. purpurea differs from obovatin (71) and its
-thyl ether (72) in substitution at C-5 carbon (Pelter et al., 1981). The prenylated flavanones
^.branin (73), methylglabranin (74) were isolated from the whole plant of T. abbottiae (Gomez-
stribay et a l, 1986). Methylglabranin (74) has also been isolated from the whole plant of T.
illophylla, the roots of T. nitena and T. polyphylla (Gomez-Garibay et a l, 2002; Dagne et al,
>92). The flavanone candidone (75) isolated from the roots of T. elata is a methyl ether of
abranin (73) (Lwande, 1985).
Obovatin, R=H (71) Glabranin, R=R|=H (73)

Obovatin Me ether, R=CH3 (72) Methylglabranin, R= CH3, R|=H (74)
Candidone, R=R|=CH3 (75)
Isolonchocarpin (76)
fig u re 2.17: Prenylated flavanones isolated from Tephrosia species
34
T.er prenylated flavanones include tephroleocarpin A (77) and tephroleocarpin B (79) isolated
rn the aerial parts of T. leiocarpa (Gomez-Garibay et al., 1991); quercetol (78) isolated from
? roots of T. quercetorum (Gomez-Garibay et al., 1988); epoxycandidone (71) isolated from
- whole plant of T. hamiltonii (Hussaini and Shoeb, 1987) and falciformin (81) isolated from
5 pods of T. Falciformis (Figure 2.18) (Khan et al., 1986).
Tephroleocarpin A, R=H (77)
Quercetol C, R=CH 3 (78)
Falciformin (81)
Epoxycandidone (80)
F ig u re 2.18: Prenylated flavanones from Tephrosia species
Complex 7 / 8 -furanoflavanones have been isolated trom the seeds ol T. purpurea. Ihesc aie
purpurin (82), tephrorin A (83) and tephrorin B (84) (Gupta et al., 1980; Chang et al., 2000).
f-ne flavanones like emoroidenone from the roots of T. emoroide have a single furan unlike
f-purin which has two furan rings (Machocho et al., 1995).
Tephrorin A (83)
Purpurin (82)
Emoroidenone (85)
Tephrorin B (84)
fig u r e 2.19: Furanoflavanones from the genus Tephrosia
36
_4.4.4 Chalconoids of Tephrosia
T.alconoids are preflavonoids that exhibit the basic C6-C3-C 6 skeleton of flavonoids but the C 3
>rtion of the molecule is acyclic. Other ‘Acyclic’ flavonoids besides include, dihydrochalcones
i d re/ro-chalcones (Figure 2.20); all of which may or may not contain oxygenated substituents at
th e r the a - or j3 -positions (Agrawal, 1989; Gomez-Garibay et al., 2002; Dewick, 2002). The
rm re/ro-chalcone is used to indicate that the typical substitution pattern of the A- and B-rings of
i e chalcone has been inverted. Some chalcones with oxygen substituition at /^-position occur in the
t^nus Tephrosia.
Chalcone Dihydrochalcones
Retro-chalcone
F ig u re 2.20: Basic skeleton of chalconoids
IMore than twenty chalcones have been reported from the genus tephrosia (Al-Hazimi et al.,
2005). At least seven chalcones, purpuritenin (86), tephrosone (87), O-methylpongamol (88),
X>ongamol (89), purpurenone (90), purpuritenin B (91) and (+)-tephropurpurin (92), have been
isolated from T. purpurea roots, seeds and the whole plant (Pelter et al., 1981; Sinha et al., 1982,
t o and Raju 1984; Saxena and Choubey 1997; Chang el al., 2000). O-Methylpongamol (8 8 ),
,rigamol (89), purpurenone (90) are chalcones with oxygen substituition at P-position. The
^leones O-methylpongamol ( 8 8 ) and pongamol (89) have also been reported in the roots of T.
i/niltonii (Rao and Prasad, 1992).
CHi
Purpuritenin (8 6 )
Pongamol, R=H (89) H

o \£i\\O A c
H""V— v'"<H
0
och3 0
Purpuritenin B (91) (+)-Tephropurpurin (92)
Figure 2.21: Chalcones isolated from T. purpurea and T. hamiltonii
38
simplest chalcones, isoliquiritigenin (93) was reported from the stem of T. toxicaria while
jsichalcone (94) occurs in the roots and aerial parts of T. crassifolia (Gomez-Garibay el al.t
><?).
Isoliquiritigenin (93) Crassichalcone (9 4 )
«»ure 2.22: Chalcones isolated from T. toxicaria and T. crassifolia
4.4.5 Rotenoids of Tephrosia
otenoids are subclass of isoflavonoids containing an extra carbon in an additional heterocyclic
n g . The C-2 of the isoflavanone skeleton bears an extra methylene carbon (C-11), which get
^clised with C-2' o f ring B to form a tetracyclic ring system (Agrawal, 1989).
least twenty nine rotenoids have been reported by 2005 from twenty Tephrosia species (Al-
l azimi el al, 2005). Rotenone (95) has been repeatedly isolated from the rotenoid containing
pecies (Figure 2.23, Al-Hazimi el al., 2005). The rotenoid a-toxicarol (96) has been reported
ro m the aerial parts of T. purpurea (Rao and Raju 1984) and the roots of T. Candida (Andrei el
*1, 1997). The dimethylchromene rotenoids tephrosin (97), deguelin (98) and 1 2a-hydroxy-a-
oxicarol (99) have been reported in the roots of T. Candida (Andrei el al., 1997). Deguelin (98)
'las also been isolated from the whole plant of T. abhottiae, roots of T. falciformis and T.
^>entaphylla (Al-Hazimi el al., 2005)

rotenoids 6 a , 1 2 a-dehydrodeguelin (100) and 6 a , 12 a-dehydro-a-toxicarol (101) have been
p o r t e d from T. Candida (Andrei et al., 1997). These two rotenoids are unsaturated at C-6 a and
l F
X 2 a(F igure2.23)
a-Toxicarol, R|=H, R2=OH (96)
Rotenone (95) Tephrosin, Rj=OH, R2=H (97)
Deguelin, R |=R 2=H (98)
12a-Hydroxy-a-toxocarol
R,=R2=OH (99)
6 a , 1 2 a-dehydrodeguelin, R-H (100)
6 a , 1 2 a-dehydro-a-toxicarol, R=OH (101)
'igure 2.23: Rotenoids o f the genus Tephrosia
40
V.4.6 Pterocarpanoids of the genus Tephrosia
rrocarpanoids are a group of naturally occurring heterocycles containing a 6 a ,lla -
i^drofurobenzopyran nucleus which has two asymmetric centers (Figure 2.24). Although
: rocarpans contain two chiral centers, only the configurations, 6 aR ,llaR and 6 aS,llS , are
•rically possible. The absolute configuration of the majority of the naturally occurring
-rocarpans is 6 aR,l laR. This designation is constistent with those pterocarpans which have no
fc>stituents at carbons 6 a and 11 a.
Figure 2.24: Basic skeleton of most commonly occurring pterocarpan
Îany of the pterocarpans are phytoalexins that are produced in plants during infection by lungi,
•acteria or viruses. A total of forty-four nonprenylated pterocarpans have been obtained from
different leguminous plants (Al-Hazimi et al., 2005). Among these, the compounds maackiain
102), pterocarpin (103) and medicarpin (107) (Figure 2.25) widely occur in plants belonging to
various genera (Al-Hazimi and Alkhathlan 2000). Both prenylated and non-prenylated
Pterocarpans have been reported in the genus Tephrosia. Maackiain (102) has been isolated lrom
Fie roots of T. purpurea, T. maxima T. elata, T. fulvinervis, T. hamiltoni and T. bidwilli (Al-
I
I
dazimi et al., 2005). A derivative of pterocarpin, 2-hydroxypterocarpin (104) has been reported
41
I
the pods of T. pentaphylla (Al-Hazimi et al., 2005) while 2-methoxymaackiain (105), a
p-r'ivative o f maackiain has bee reported in aerial parts o f T. bidwilli (Maximo and Lourenco
, <^<98). Pterocarpans reported in the roots of T. hilderbrandtii and pods of T. pentaphylla are
^ j 1 dercarpin and 2-hydroxypterocarpin respectively (Lwande et al., 1987). The pterocarpin
3 *4 -:8 ,9 -dimethylenedioxypterocarpan (108) isolated from the roots o f T. aequilata is the only
p t^ ro c a rp a n with 6 aR and 1 laS configuration (Tarns et al., 2002).
Ro
OR,
Maackiain, Ri=H, R2=H (102)
Pterocarpin, R i=CH3, R 2=H (103)
2-hydroxypterocarpin, R i=CH3, R2=OH (104)
2- Methoxymaackiain, Ri=H, R2=OCH3 (105)
HO
3 ,4 :8 ,9 -D im e th y le n e d io x y p te ro ca rp a n (108)
igure 2.25: Pterocarpans from Tephrosia species
42
S BIOLOGICAL ACTIVITIES
3.1 Biological activities of the genus Eriosema
's/estigations of Eriosema species has resulted to the isolation of bioactive compounds,
cam ples o f such bioactive compounds are five pyrano - isoflavones isolated from the roots of
r-iosema kraussianum with penile relaxant activity (Drewes et al., 2002). The compounds were
-ven trivial names kraussianone 1 (30), kraussianone 2 (31), kraussianone 3 (32) and
-aussianone 5 (34) (Figure 2.9). The major components o f the extract of E. kraussianum that
i_used relaxation of penile smooth muscle were kraussianone 1 and kraussianone 2.
raussianone 3 and kraussianone 5 caused contraction of penile smooth muscle. The biological
^tivity of the roots of this plant was mainly due to kraussianone 1 and kraussianone 2 .
! hromones and phenolic compounds with antifungal activity have been isolated from the roots
E E. tuberosum (Ma et a l, 1996). The chromones, eriosematin D (44) and eriosematin E (45)
✓ 'ere active against Candida cucumerinum and C. albicans (Figure 2.12). I he phenolic
om pounds, 4-hydroxybenzoic acid (46), hydroquinone (47) and vanillic acid (48), were active
gainst Cladosporium herbarum (Figure 2.13a). An isoflavonoid glycoside 5 -(9 -methylgenistein
r -O-p-D-glucopyranoside (37) isolated from the roots ot E. tuberosum has antiviral activity in
”itro assay (Ma et al., 1998).
43
5.2 Biological activities of the genus Tephrosia
jnemical investigations o f plants of the genus Tephrosia has resulted to the isolation of active
im pounds with anticancer, insecticidal and anitumour activities. In the case of T. purpurea the
-tivities are attributed to lipophilic flavonoid aglycones such as flavanones, flavonols, flavones
rtd chalcones present in the extracts (Santram et al., 2006). Rotenoids which have insecticidal
ctivity also exhibit strong ictiotoxic activity (Andrei et al., 1997). Rotenone (95, Figure 2.23)
a s insecticidal properties (Ramen et al., 1992). Tephrosin (97) is active against tumours
deluding skin cancer (Andrei et al., 1997). The roots of T. emoroides yielded emoroidenone (85,
'igure 2.19) which has insect anti-feedant activity against the larvae of stalk borer, Chillo
uartellus (Machocho et al., 1995). The roots o f T. hildebrandtii yielded hildecarpin (106, Figure
l .25) which has insect anti-feedant activity against the legume pod-borer, Maruca testulalis as
v'ell as anti-fungal properties (Tarus et al., 2002; Lwande et al., 1986).
> ongamol (89, Figure 2.21), isolated from T. lanceolata and T. purpurea, is used in insecticide
rnd pesticide manufacture (Parmar et al., 1989). Pseudosemiglabrin (64, Figure 2.14), isolated
From T. apollinea is a platelet aggregation inhibitor (Waterman and Khalid, 1980). I he
pterocarpan 2-methoxymaackiain (105, Figure 2.25), which is a constituent of T. bidwilli, has
antifungal activity (Tarus et al., 2002). a-toxicarol (96, Figure 2.23) obtained from the stem ol T.
adorata, T. toxicaria and aerial parts of T. purpurea is used as a fish poison and its biological
activities are comparable to rotenone (95) (Jang et al., 2003).
44
CHAPTER THREE
MATERIALS AND METHODS

. 1 GENERAL
. 1.1 Reagents
echnical grade organic solvents which included A7-hexane, dichroromethane, ethyl acetate,
iethanol and acetone were used.
alts used to prepare physiological solutions were of analytic grade. Sodium hydrogen carbonate,
alcium chloride and sodium chloride were sourced from Loba Chemie, Mumbai, India,
’otasium chloride was obtained from S. D. Fine-Chem, Mumbai India; glucose was obtained
rom Fisher Scientific UK limited; sodium dihydrogen orthophosphate was sourced from BDH
Chemicals Ltd. Poole England and magnesium sulphate was sourced from Howse & McGeorge
imited, Nairobi, Kenya. Oxygen and carbon dioxide was obtained from BOC, Kenya.
5.1.2 Instrumentation
The 'H NMR (600, 500 or 200 MHz) and 13C NMR (150, 125 or 50 MHz) spectra were recorded
z>n Bruker or Varian-Mercury spectrometers using tetramethylsilane ( I MS) as the internal
standard. Homonuclear Correlation spectroscopy (COSY), Nuclear Overhauser Enhancement
spectroscopy (NOESY), Heteronuclear Multiple Quantum Coherence (HMQC) and
Lleteronuclear Multiple bond Correlation (HMBC) spectra were acquired using the standard
Bruker software. EI-MS spectra were recorded on a direct inlet, 70 eV, on SSq 710, Einnigan
N A T mass spectrometer. Melting points were determined using a Gallenkamp melting point
45
r
apparatus w ith capillary tubes. UV/VIS spectra were recorded using a Pye-Unican SPS-150
spectrophotometer. The plant materials were ground using a Willymill.
1 1.3 Collection o f plant materials
Eriosema psoraleoides and Tephrosia purpurea were collected from the wild. E psoraleoides
from Kilungu hills in Makueni district in January, 2009; while T. purpurea was collected from
Kilifi district in August, 2007. The plant species were identified by Mr. Patrick C. Mutiso of the
University Herbarium, School of Biological Sciences, University of Nairobi. Ihe voucher
specimen (Mutiso- 015 / January 2009) for E. psoraleoides and (Mutiso - 520/ August 2007) for
T. purpurea were deposited in the University Herbarium. The plant parts were dried under a
shade before being milled.
3.1.4 Chromatographic conditions
Column chromatography was carried out using silica gel 60 (70-230 mesh) and Sephadex Ml 20.
The fractions were monitored by TLC using Merck pre-coated silica gel 60 1254 plates, with UY
11(254, 366 nm) and iodine vapour used for detection. Preparative thin layer chromatogruf h>
ll(PTLC) was done on silica gel (Merck). PTLC plates were prepared by adding 200 ml ol water
j j t o 80 g of silica gel. Slurry that formed was allowed to stand for 43 mm. Ihe slurry was then
poured and spread evenly on clean 20 cm2 glass plates and was left to dr\ at mum tem|
Activation of the silica gel was done in an oven for 30 min at 383 K, removed and allowed to
cool to room temperature before use.
46
_1.5 Animals
lice weighing about 25 g and a Guinea pig weighing 300-400 g were obtained from the Animal
louse o f the Department o f Pharmacology and Pharmacognosy, School of Pharmacy and the
department o f Public Health Pharmacology and Toxicology, Kabete Campus. The animals were
loused in plastic cages and maintained at standard conditions (12 hour light/dark cycle, 25 ± 5
C ; 35 - 60% relative humidity). The mice and guinea pig were fed on commercial pelleted feed
Lnd tap water ad libitum. The feeds were obtained from Unga Feeds, Limited Nairobi, Kenya.
The aorta and vas deferens of the sheep was obtained from slaughter house in Dagoretti,
Ciambu, Nairobi.
3.1.6 Sources of microorganisms for antiplasmodial test
The D6 and W2 strains of P. falciparum were obtained from the United States Army Medical
Research Unit-Kenya, Walter Reed Project, Kisumu (courtesy of Mr Hoseah M. Akala), all
preserved at -20°C.
3.1.7 Sources of bacteria and fungi test strains
Antibacteria activity was tested against standard strains of bacteria: Staphylococcus aureus
ATCC 25922 and Pseudomonas aeruginosa ATCC 25923. The antifungal activity was tested
against standard strains, Candida albicans ATCC 90028, Cryptococcus neoformans, Trichphyton
mentagrophytes and Microsporum gypsum fungi. The standard drugs used were chloramphenicol
and fluconazole for antibacterial and antifungal activities, respectively.
47
2 EXTRACTION AND ISOLATION OF COMPOUNDS
2.1 Extraction and isolation of compounds from Eriosemapsoraleoides
oots)
ne air dried and ground roots (164 g) of E. psoraleodes were extracted with
chloromethane/methanol (1:1) by cold percolation at room temperature (4 x 800 ml). The
>lvent was removed under vacuum by use of a rotary evaporator at 35°C. This afforded a dark
ily extract that was partitioned between water and ethyl acetate. The organic layer (4 g) was
objected to CC on silica gel (61 g) eluting with hexane containing increasing amounts (1%, 2%,
5%, 7%, 10%, 14%, 16%, 20%, 30%, 40% and 50%) of EtOAc.
Tie fraction eluted with 10% EtOAc in n-hexane was separated on Sephadex LH-20 column
sin g CH 2Cl2/MeOH (1:1) to yield yellow solid o f 1 (4.0 mg). The fractions eluted with 14% and
6 % EtOAc in hexane were combined and separated on Sephadex LH-20 column using
- H 2Cl2/MeOH (1:1) and gave an orange solid of 2 (2.6 mg). The fraction eluted with 20%
ItO A c in hexane gave seven fractions after CC on Sephadex. Further separation ol fraction one
,ave amorphous brown powder of 3 (5.6 mg). The remaining six fractions were combined and
njrified by PTLC (dichloromethane/ethyl acetate/hexane (30:50:20) to yield compound 4 (1.4
^g)-
V2.2 Extraction and isolation of compounds from Tephrosiapurpurea (stem)
^\ir dried ground stems of Tephrosia. purpurea (2 Kg) were extracted with
i ichloromethane/methanol (1:1) by cold percolation at room temperature (3 x 1.5 L). 1he extract
^vas filtered and the solvent removed under vacuum using a rotary evaporator at 35 C. I his gave
48
trk oily extract that was partitioned between water and ethyl acetate. The organic layer (36 g)
3 js subjected to CC on silica gel (400 g) eluting with hexane containing increasing amounts of
fryl acetate (2%, 4%, 6%, 8%, 10%, 12.5%, 15%, 20%, 25%, 30%, 40%, and 50% ethyl acetate
hexane).
om pound 5 (108.6 mg) crystallized from the fraction eluted with 4% ethyl acetate in hexane,
hie fraction eluted with 6% Ethyl acetate in hexane was crystallized from
jxane/dichloromethane to yield 6 (317.1 mg).
fie fractions eluted with 10% and 12.5 % ethyl acetate in hexane were mixed, and subjected to
:>lumn chromatography on Sephadex LH-20 from which 7 (46.3 mg) crystallized from one of
i e fraction. The second fraction (1.12 g) was further purified by CC [on silica gel (92 g) eluting
rith hexane/dichloromethane (40:60, 30:70, 10:90) followed by dichloromethane containing
icreasing amounts of ethyl acetate (2% and 10%)] to give Solid 7 (41.2 mg),
ractions eluted using 15% ethyl acetate was crystallized in hexane/dichloromethane to give a
ro w n stick solid and colourless filtrate. The filtrate was separated on Sephadex to give a white
>owder o f 8 (33.5 mg). The fraction that was eluted with 25% ethyl acetate was crystallized trom
lexane/dichloromethane to yield a pure amorphous powder ol 9 (106.1 mg).
*.3 PHYSICAL AND SPECTROSCOPIC DATA OF ISOLATED
COMPOUNDS
£ ompound 1
Y ellow solid. Rf = 0.64 (CH2Cl2/(CH3) 2CO, 9:1). UV ^ ax (MeOH) nm: 208, 286 nm. ‘H NMR
C200 MHz, acetone-d6): 8H 12.34 (.s, 5-OH), 6.96 (d, .7=8.4 Hz, H-6'), 6.60 {d, 2=2.4 Hz, H-3'),
49
2 (77, .7=8.2,2.2 Hz, H-5’), 6.05 (d, 7=2.4 Hz, H-6), 6.02 (7, 7=1.6 Hz, H-8), 4.55 (77, 7=10.6,
0 Hz, H -2« ), 4.41 (dd, 7=5.2, 10.6 Hz, H-2*,), 4.26 (77, 7=5.4, 11.0 Hz, H-3 ), 3.86 (s,
:Hj-7), 3.76 (^, OCHj-2"). 13C NMR (150 MHz, acetone-d*): 8c 199.3 (C-4), 165.2 (C-5),
5.0 (C-8a), 162.4 (C-7), 160.1, (C-4’), 160.0 (C-2'), 132.5 (C-6'), 115.7 (C-T), 108.7 (C-5'),
4.3 (C-4a), 101.1 (C-3'), 96.1 (C-6), 95.0 (C-8), 71.9 (C-2), 56.9 (OCHj-7), 56.6 (OCHj-2’),
1 (C-3). EI-MS m/z(rel. int.): 316 (37, [M] *), 167 (100), 149 (26).
om pound 2
rown-yellow powder. Rf = 0.4 (n-CeHiVC^Ch/EtoAc, 2:4:4). UV Xmax (MeOH) nm: 208, 257
m . 'H NM R (200 MHz, acetone-d6): 5H 12.98 (s, 5-OH), 7.82 (7, 7=8.6 Hz, H-6'), 7.14 (7,
=1.8 Hz, H-3'), 7.02 (77,7=2.2, 8.6 Hz, H-5'), 6.60 (7,7=2.2 Hz, H-6), 6.37 (7,7=2.0 Hz, H-8).
3CNMR (50 MHz, acetone d6): SH 183.2 (C-4), 168.4 (C-2), 163.6 (C-5), 163.0 (C-7), 158.7 (C-
!'), 156.2 (C-8a), 152.9 (C-4'), 121.8 (C-6'), 115.5 (C-1% 113.8 (C-5’), 110.5 (C-30, 105.0 (C-
4a), 100.2 (C-3'), 98.9 (C-6), 95.0 (C-8). EI-MS m/z (rel. int.): 285 (38, [M+H] *), 153 (22), 125
: 14).
Compound 3
Amorphous light brown powder. Rf = 0.6 (n-C6 Hi 4/CH 2Cl2/EtOAc, 8:7:5). UV X.max (MeOH)
nm: 207, 262 nm. 'H NMR (600 MHz, acetone-d6): 8H 13.04 (s, 5-OH), 8.16 H-2), 7.46 (7,
> 8 .4 Hz, H-6'), 7.46 (7,7=8.4 Hz, H-2'), 6.91 (7,7=8.4 Hz, H-5'), 6.91 (7,7=8.4 Hz, H-3'), 6.42
(7,7=1.8 Hz, H-8), 6.29 (7, 7=1.8 Hz, H-6). I3C NMR (150 MHz, acetone-d6): 6C 182.22/182.30
(C-4, 4"), 165.58/165.63 (C-5, 5"), 164.27/164.57 (C-7, 7"), 159.32/159.70 (C-8a, 8a"),
159.04/159.70 (C-4', 4"’), 154.91/154.95 (C-2, 2"), 131.83/131.83 (C-2', 2'"),124.68/124.73 (C-3,
50
123.70/123.73 (C -l', 1 "'), 116.58 (C-3', 3"'), 106.78/106.81 (C-4a, 4a"), 100.41/100.46 (C-6 ,
, 95.10/95.17 (C-8 , 8 "). EI-MS m/z (rel. int.): 270 (100), 153 (75), 118 (22).
rnpound 4
.lourless oily substance. Rf = 0.6 (n-C 6 Hi4/CH 2Cl2/EtOAc, 1:4:5). 'H NMR (600 MHz,
stone-d6): 6H 12.38 (s,5-OH), 6.97 (7, 7=8.4 Hz, H-6 '), 6.52 (d, 7=2.4 Hz, H
2.4, 8.4 Hz, H-5’), 5.97 ( d,7=2.4 Hz, H-6 ), 5.95 (7, 7=1.8 Hz, H-8 ), 4.52 (dd
-2ax), 4.42 (dd, 7=5.4, 11.4 Hz, H-2e,), 4.27 (dd, 7=5.4, 10.8 Hz, H-3), 3.77 (s, OCH3-2'). I3C
M R (150 MHz, acetone-d6): 8 C 198.0 (C-4), 167.9 (C-5), 166.3 (C-7), 165.3 (C-8 a), 160.2 (C-
) , 159.9 (C-2')> 132.4 (C-6 '), 115.9 (C -l'), 108.7 (C-5'), 104.0 (C-4), 101.0 (C-3 '), 97.6 (C-6 ),
6.3 (C-8 ), 71.9 (C-2 ), 56.5 (OCHj-2’), 48.0 (C-3).
Compound 5
Colourless amorphous powder. Rf = 0.69 (CHiC’E/EtOAc, 9:1). 1H NMR (200 MHz, CDCI3): 8 h
>.34 (d, 5.2 Hz, H-6 ), 5.17 (77,7=8.2, 15.2 Hz, H-22), 4.98 (77,7=7.8, 15.4 Hz, H-23), 3.49 (m,
1-3), 1.02 (7,7=5.6 Hz, H-21), 1.00 (s, H-19), 0.91 (7,7=6.2, H-27), 0.81 (t, 7=5.6,11-29), 0.69
> , H-18). I3C NMR (50 MHz, CDCI3): 5C 141.0 (C-5), 138.6 (C-22), 129.5 (C-23), 121.9 (C-6 ),
72.0 (C-3), 57.1 (C-14), 56.2 (C-17), 51.5 (C-24), 50.4 (C-9), 42.5 (C-l 30), 42.4 (C-4), 39.9 (C-
12), 37.5 (C -l), 36.7 (C-10), 32.1 (C-7, 8 , 24), 31.9 (C-2), 29.2 (C-15), 25.6 (C-28), 24.5 (C-15),
21.3 (C -l 1, 21), 19.6 (C-27), 19.2 (C-26), 12.5 (C-29).
51
im pound 6
am orphous powder. Rf = 0.42 (n-C 6 H | 4/EtOAc, 9:1). UV Xmax (MeOH) nm: 280, 310 nm. 'H
\ rM R (500 MHz, CDC13): 5H 8.18 (7, 7=9.0 Hz, H-5), 7.97 (m, H-2', 6'), 7.78 (7, 7=2.5 Hz, H-
7.56 (m, H-3', 4', 5’, 6), 7.22 (rf, rf=2.5 Hz, H-3"), 6.94 (j, H-3). I3C NMR (50 MHz, CDCIj):
178.8 (C-4), 163.3 (C-2), 158.6 (C-7), 151.1 (C-8a), 146.1 (C-2"), 131.9(0-1*, 4*), 129.4 (C-3',
• > , 126.5 (C-2', 6'), 122.0 (C-5), 119.3 (C-4a), 117.7 (C-8), 110.6 (C-6), 108.0 (C-3), 104.4 (C-
• * > EI-MS m/z (rel. int.): 263 (15, [M+H] *), 161 (10), 132 (20).
37 o m p o u n d 7
X^morphous white powder. R f= 0.47 (CH2Cl2/EtOAc, 9:1). UV Xâx (MeOH) nm: 295, 310 nm.
I H NMR (200 MHz, CDC13): 5H 8.16 (7,7=8.4 Hz, H-5), 7.91 (m, H-2’, 6’), 7.54 (m, H-3', 5’, 4’),
> . 94 (rf, rf=8.4 Hz, H-6), 6.83 (s, H-3), 6.64 (rf, rf=6.4 Hz, H-2”), 5.64 (s, H-4"), 4.30 (rf, rf=6.4 Hz,
r ^ - 3 " ) , 2.22 (s, COCHj-4"), 1.32 (5 , CH3-5"), 1.09 (i, CH3-5). I3C NMR (50 MHz, CDC13): 6C
1 "78.3 (C-4), 170.4 (COCH3-4”), 164.7 (C-7), 164.0 (C-2), 154.1 (C-8a), 132.6 (C-5), 132.2 (C-
1 *), 129.9 (C-4'), 129.7 (C-3', 5'), 127.3 (C-2', 6'), 119.9 (C-4a), 113.4 (C-8), 113.2 (C-2"), 110.0
CC - 6 ) , 108.3 (C-3), 88.7 (C-5"), 80.9 (C-4"), 53.6 (C-3"), 28.3 (CH3-5"), 23.9 (CH3-5").
CUTompound 8
. [«]25'3
N ^/hite amorphous powder. Rf = 0.45 (n-C6 Hi 4/EtOAc, 3:2), melting point 144-145°c.
^-58.2399. UV X.max (MeOH) nm: 295, 325 nm. ‘H NMR (500 MHz, acetone-d6): 5H 8.18 (m, H-
p , 6'), 8.00 (7, J=8.5 Hz, H-5), 7.63 (m, H-3', 4', 5'), 6.94 (7, 7=8.5 Hz, H-6), 6.79 (s, H-3), 5.35
: «/, 7=8.5, H-4”), 5.02 (77, 7=2.0, 9.5 Hz, H-2”), 4.84 (77,7=8.0, 9.5 Hz, H-2"), 4.44 (777, 7=2.0,
52
8.0,8.5 Hz, H-3"), 2.00 (s,COCH3-7), 1.76 (s, CH3-5"), 1.61 (i, CHj-5''), 1.
NMR (75 MHz, acetone-d6): 8C 177.6 (C-4), 170.8 (COCH3-7), 170.3 (COCHj-4''), 167.9 (C-7),
163.9 (C-2), 155.9 (C-8a), 133.6 (C -l'), 133.1 (C-4'), 130.7 (C-3', 5'), 129.3 (C-5), 127.9 (C-2\
6'), 119.9 (C-4a), 116.0 (C-8), 110.1 (C-6), 108.5 (C-3), 84.0 (C-5"), 79.1 (C-2"), 78.7 (C-4").
42.3 (C-3"), 24.4 (CH3-5"), 23.0 (COCH3-7), 22.4 (CH3-5"), 20.9 (COCH3-4"). EI-MS (rel.
int ): 437 (7, [M+H] *), 319 (6), 317 (60), 316 (100), 263 (76), 161 (14), 102 (5).
Compound 9
Amorphous powder. Rf = 0.54 (CH2C12/(CH3)2C0, 8:2). UV Xmax (MeOH) nm: 295 nm. ‘H NMR
(200 MHz, acetone-d6): 5H 8.12 (m , H-2', 6'), 8.02 (d, J= 9.0 Hz, H-5), 7.61 (m, H-3', 4', 5'), 7.25
(d, J=9.0 Hz, H-6), 7.09 (d, J=\6.6 Hz, H-l"), 6.93 (<d, 7=16.2 Hz, H-2"), 6.81 (.s, H-3), 4.05 (s,
OCH3-7), 1.46 (s, 2CH3-3"). I3C NMR (50 MHz, acetone-d6): 5C 177.0 (C-4), 163.1 (C-2), 161.9
(C-7), 161.5 (C-8a), 145.6 (C-l"), 132.4 (C -l’), 131.7 (C-4'), 129.3 (C-3’, 5’), 126.7 (C-2', 6'),
124.9 (C-5), 115.0 (C-4a, 8), 114.6 (C-2"), 110.0 (C-6), 106.8 (C-3), 70.5 (C-3"), 56.1 (OCH3-7),
29.5 (CH3-3"), 29.2 (CH3-3"). EI-MS m/z (rel. int.): 336 (14, [M] +), 318 (51 ([M-H20), 102
( 100).
3.4 BIOLOGICAL ASSAY
3.4.1 Effects of plant extracts and isolated compounds on isolated Guinea pig
Trachea
fThe method used is described in literature (Leal et al., 2006). A Guinea pig was killed by
cervical dislocation. The whole isolated trachea of Guinea pig with the nerve supply was
removed and attached to capillary tube in an organ bath containing Kreb’s solution of the
following composition (mM): NaCl 118.0; KC1 4.4; CaCl2 2.5; MgS04 1.1; KH POj 12*
NaHC03 25.0 and glucose 11.1. The organ bath was maintained at a temperature of 37°C and
Kreb’s solution was continuously aerated with oxygen - carbon dioxide mixture (95:5).
Each of the plant extract and pure compound was dissolved in water and introduced into the
organ bath. The displacement of Krebs solution in the capillary tube was recorded after adding
each of the extract and pure compound. The organ bath was drained and the tissue rinsed twice.
It was let to rest for two to three minutes before adding another drug.
3.4.2 Effects of plant extracts and isolated compounds on isolated aorta of a
Sheep
A sheep was killed by cervical dislocation. The whole isolated aorta of a sheep with the nerve
supply was removed. The aorta was cut transversely between the segments so as to give rings
which were tied together with cotton to form chains. The chains were mounted in an organ bath
containing Krebs solution at a tension of 1 g maintained at 37°C.
organ bath. The isomeric muscle twitch-tension after adding each o f the extract and pure
compound was recorded using a force displacement transducer coupled to a physiograph. The
organ bath was drained and the tissue rinsed twice. It was let to rest for two to three minutes
before adding another drug.
54
3.4.3 Effects of plant extracts and isolated compounds on vas deferens of a rat
and sheep
A rat was killed by cervical dislocation. The whole isolated vas deferens with the nerve supply
was removed. Each of the vas deferens was cut transversely between the segments so as to give
rings which were tied together with cotton to form chains. Each of the chains was set up in an
organ bath containing Krebs solution.
organ bath. The isomeric muscle twitch-tension after adding each of the extract and pure
compound was recorded using a force displacement transducer coupled to a physiograph. The
organ bath was drained and the tissue rinsed twice. It was let to rest for two to three minutes
before adding another drug.
3.4.4 Effect of E. psoraleoides root extract on mating behavior
I he method used is described in literature (Carro-Juarez et al., 2004). The male mice were
devided into four groups o f five each. Group one was treated with the roots o f E. psoraleoides
(2.5 g/kg of body weight) extract orally, group two was treated with sildenafil (600 mg/kg of
body weight) orally, group three was treated with yohimbine (360 mg/kg of body weight) orally
and group five acted as control. Twelve females were treated with stilboestrol (40 mg/ kg of
body weight) intraperitoneally 24 hours before experiment.
About 30 minutes later three females were introduced into each of the male group. The time that
elapsed from the introduction of the female into a cage until the first mount, the frequency of
mounts in a period of 3 hours, the intromission frequency and latency, the number of mounts and
55
intromission preceding ejaculation, frequency of penile erection (PE) and the number of times
the mouse bends down to lick the penis were observed and recorded.
3.5 IN-VITRO ANTIPLASMODIAL ACTIVITY ASSAY
Antiplasmodial activity was tested against chloroquine-sensitive Sierra Leone I (D6) and
chloroquine-resistant Indochina I (W2) strains of Plasmodium falciparum. They were cultured as
described in literature (Johnson et al., 2007). The crude extract and pure compounds were
assayed using a non-radioactive assay technique with modifications to determine 50% growth
inhibition of cultured parasites (Smilkstein et al, 2004). The in vitro drug susceptibility method
that uses the fluorochrome called “SYBR Green I”, a non-radioactive intercalating DNA marker
that accurately depicts in vitro parasite replication was applied. Concurrently, twofold serial
dilutions of the drugs chloroquine (1.953 to 1,000 ng/ml), mefloquine (0.488 to 250 ng/ml) and
test sample (97.7 - 50,000 ngm f1) were prepared on a 96 well plate. The culture-adapted P.
falciparum were added on to the plate containing dose range of drugs and incubated in gas
mixture (5% CO 2 , 5% O2 , and 90% N 2) at 37°C. The assay was terminated 72 hrs later by
freezing at -80°C. After thawing, lysis buffer containing SYBR Green I (lx final concentration)
were added directly to the plates and gently mixed by using the Beckman Coulter Biomek 2000
automated laboratory workstation (Beckman Coulter, Inc., Fullerton, CA). The plates were
incubated for 5 - 15 minutes at room temperature in the dark. Parasite growth inhibition was
quantified by measuring the per-well relative fluorescence units (RFU) of SYBR green 1 dye
using the Tecan Genios Plus (Tecan US, Inc., Durham, NC) with excitation and emission
wavelengths of 485 nm and 535 nm, respectively, and with the gain set at 60. Differential counts
ol relative fluorescence units (RFUs) were used in calculating ICso’s for each drug using Prism
56
4.0 software for Windows (Graphpad Software, San Diego, CA). A minimum of three separate
determinations was carried out for each sample. Replicates had narrow data ranges hence
presented as mean + SD. The antiplasmodial tests were done in collaboration with Mr. Hosea M.
Akala of Kenya Medical Research Institute and United States Medical Research Unit, Kenya.
3.6 ANTIMICROBIAL TEST USING DISC DIFFUSION TECHNIQUE
All glassware was sterilized by dry heat at 200 °C for one hour. Ten milliliters of water was
sterilized autoclaving at 121 °C, 2 bar pressure for 15 minutes. All work was carried out using
aseptic techniques. All bacteria were grown on Mueller Hinton medium while. All fungi were
grown on Sabouraud’s Dextrose Agar medium.
The crude extracts of E. psoraleoides, T. purpurea and pure compounds from T. purpurea were
tested for activity against Candida albicans, Microsporum gypsum, Staphylococcus aureus,
Pseudomonas aeruginosa, Cryptococcus neoformans, Trichophyton mentagrophytes and
Microsporum gypsum.
Working cultures were grown using the slope technique. A microbial suspension of each
organism was prepared by gently suspending the organism in the slope culture in about 10 ml of
sterile water. Three milliliters of the microbial suspension was added to one liter of sterile molten
medium at a temperature of about 45 °C. Twenty milliliters o f seeded molten medium was added
to petri dishes and medium and was left to set for about one hour. Whatman filter paper no. 1
disks o f 6-mm diameter were used to screen the antimicrobial activity. Each sterile disc was
impregnated with 20 pL o f the extract and pure compound. Sterile water was used as the
negative control. Chloramphenicol and fluconazole were used as reference drugs for antibacterial
activity and antifungal activity respectively. All bacteria were incubated at 37.4 °C for 24 hours
and the fungi were cultured at 24 °C for three days. After incubation, the diameter of zones of
inhibition of the control and test was measured (mm) using an automated zone reader.
57
CHAPTER FOUR
RESULTS AND DISCUSSION

4.1 PHYTOCHEMICAL INVESTIGATION ON ERIOSEMA
PSORALEOIDES
4.1.1 Preliminary test results
The root extract of E. psoraleoides was tested for anti-microbial, anti-plasmodia!. vasorelaxant,
anti-asthmatic and penile relaxant activities and showed significant activities. TLC analysis of
the crude extract showed the presence of several UV (254, 366 nm) sensitive components. From
biogenetic point o f view most o f these compounds were assumed to be flavonoids and
isoflavanoids. The extract was subjected to a combination of chromatographic separations that
led to the isolation of four compounds. The identity of the isolated compounds was established
by the use of a combination of spectroscopic methods. The characterization of the compounds is
discussed below.
4.1.2 Characterization of compounds from Eriosemapsoraleoides (roots)
4.1.2.1 4', 5-Dihydroxy-2\ 7-dimethoxyisoflavanone (1)
EI-MS of compound 1 showed [M]+ at m/z 316, corresponding to the molecular formula
C 17H 16O 6 . The presence of an isoflavanone skeleton was deduced from UV (Xmax 286 nm ), 'H (5
4.55, dd, 7=10.6, 11.0 Hz, for H-2ax; 6 4.41, dd, 7=5.2, 10.6 Hz for H-2cq; 8 4.26, dd, 7=5.4, 11.0
Hz for H-3) and 1 C (8 71.9 for C-2; 48.1 for C-3 and 199.3 for C-4) NMR spectroscopic data
(Yenesew et al., 2000; Tanaka et al., 2003; Zhao et al., 2007). The 'll NMR (Table 4.1) further
58
revealed the presence of a chelated hydroxyl (8H 12.34) and two methoxyl (8H3.86 and 6 3.76)
substituents.
In the 'H NMR spectrum, the me/a-coupled protons at 6 6.02 and 6.05 were assigned to H-8 and
H-6 with C-5 and C-7 of ring-A being oxygenated, as expected from biogenetic considerations.
In the EI-MS, the fragment at m/z 167 (la, Figure 4.2) allowed the placement of one of the two
methoxyl groups in ring-A (at C-7). In agreement with this, the HMBC spectrum showed
correlation of the methoxyl protons (5 3.86) with C-7 and the hydroxy proton (6 12.34) with C-5.
An ABX spin system (8 6.42 dd, J= 8.2, 2.2 Hz for H-5'; 8 6.60 d, 7=2.4 Hz for H-3'; 6 6.96 7,
J=8.4 Hz for H-6') was attributed to 2', 4-dioxygenation ring B. The placement of methoxyl
group at C-2' rather than C-4' was established from the HMBC spectrum which showed
correlation of methoxyl protons (8 3.76) with C-2'. The (rel) R- configuration at C-3 was
deduced from the presence of a trans-diaxial relationship between H-2ax and H-3 (7=11.0 Hz) in
the ’H NMR spectrum (Yenesew et al., 2000). This compound was therefore identified as 4',5-
dihydroxy-2',7-dimethoxyisoflavanone (1), whose trivial name is cajanol.
This compound has earlier been reported from the roots and leaves of Cajanus cajan (Duker-
Eshun et al, 2004). However this is the first report of its occurrence in the genus Eriosema.
Figure 4.1: Structure of compound 1
59
+
1a
Figure 4.2: Retro-Diels Alder fragmentation of compound 1
Table 4.1: ‘H and 13C NMR data o f compound 1 in acetone-d6.
Position 'H (200 MHz) IJ C (150 MHz) HMBC (l 7,> J)

5 h (J in Hz) 8c
2 ax 4.55 dd (10.6,11.0) 71.9 C -r ,3 ,4 ,8 a
2 eq 4.41 d(5.2, 10.6)
3 4.26 dd (5.4, 11.0) 48.1 C -r, 2', 4, 6'
4 199.3
4a 104.3
5 165.2
6 6.05 d (2.4) 96.1 C-4a, 5, 8
7 162.4
8 6.02 d (1.6) 95.0 C-6, 8a
8a 165.0
r 115.7
2’ 160.0
3' 6.60(7(2.4) 101.1 c - r , 5'
4' 160.1
5' 6.42 (7(7 (8.2, 2.2) 108.7 C-1', 3', 4'
6' 6.96 d (8.4) 132.5 C-2',3 '
2'-OCH3 3.7 6 s 56.6 C-2'
7-OCH3 3.865 56.9 C-7
1 5- OH 12.34 5 C -4a, 5, 6
4.1.2.2 4', 5, T-Trihydroxy-I'-methoxyisoflavanone (4)
Compund 4 was isolated as a colourless oily substance with an Rf value of 0.6 in
hexane/dichloromethane/ethyi acetate (1:4:5). The presence of an isoflavanone skeleton was
deduced from the 'H (8 4.52, dd, 7=10.8 and 11.4 Hz, for H ^ ; 6 4.42, 77,7=5.4, 11.4 Hz for H-
2eq; 8 4.27, dd, 7=5.4, 10.8 Hz for H-3) and 13C (8 71.9 for C-2; 48.0 for C-3 and 198.0 for C-4)
NMR spectroscopic data (Yenesew et al., 2000; Tanaka et al., 2003; Zhao et al, 2007). The
NMR spectrum (Table 4.2) further revealed the presence of a chelated hydroxy (8H 12.38) and
one methoxy (8H 3.77) substituents. In the 'H NMR spectrum, the presence of meta-coupled
protons at 8 5.95 and 5.97 was consistent with oxygenation at C-5 and C-7 of ring-A, which is
expected from biogenetic considerations.
The HMBC spectrum showed correlation of methoxy (8 3.77) with the carbon at 8 159.9,
suggesting that the methoxy is attached on either C-2' or C-4'. This was resolved from NOESY
spectrum which showed spatial interaction of methoxy protons at 8h 3.77 with the proton at 8h
6.52 but not proton at 8h 6.42. This allowed the placement of the methoxy at C-2' leaving the
hydroxyl to be attached on C-4' because of the existence of an ABX system (8 6.97 d, 7=8.4 Hz;
5 6.52 d, 7=2.4 Hz; 8 6.42 dd, 7=2.4 and 8.4 Hz) on ring-B. The presence o f //ww-diaxial
relationship between H-2ax and H-3 (7=10.8 Hz) was consistent with (rel) R- configuration at C-3
(Yenesew et al., 2000). The compound was identified as 4',5,7-trihydroxy-2'-methoxy
isoflavanone. This is the first report o f the occurrence of this compound in the genus Eriosema.
61
Table 4.2: 'H and l3C NMR data o f compound 4 in a c e to n e ^
Position 'H (600 MHz) 13 C (150 MHz) HMBC ( l J, 3J

5 h (*/ in Hz) 5c
2 ax 4.52 dd (10.8, 11.4) 71.9 C-r, 4 ,8 a ,
2 eq 4.42 dd (5.4, 11.4)
3 4.27 d5.4,
( 10.8) 48.0
4 198.0
4a 104.0
5 167.9
6 5.97 d (2.4) 97.6 C-4a
7 166.3
8 5.95 <7(1.8) 96.3 C-6, 4a
8a 165.3
r 115.9
2' 159.9
3' 6.52 d (2.4) 101.0 C -r, 5', 4
4’ 160.2
5' 6.42 dd (8.4, 2.4) 108.7 C - r , 3 ’, 4 '
6’ 6.97 d (8.4) 132.4 C-2', 4
2'-OCH3 3.77 5 56.5 C-2'
5-OH 12.38 s
4.1.2.3 4’,7"- Bisgenistein (3)
Compound 3 was isolated as an amorphous light brown powder with an Rf value of 0.6 in
hexane/dichloromethane/ethyl acetate (8:7:5). The presence of an isoflavone skeleton was
deduced from UV (A.max 207. and 262 nm), ‘H (5 8.16 for H-2) and l3C (6 154.91/154.95 for C-
2/2", 124.68/124.73 for C-3/3” and 182.22/182.30 for C-4/4"). The 'H NMR spectrum (Table
62
4.3) showed meta-coupled protons at 8 6.29 and 5 6.42 which were assigned to H-6 and H-8
respectively on ring-A. The 4'-oxygenated ring-B was readily deduced from the 'H NMR
spectrum forming an AA'XX' spin system (5 6.91 d, .7=8.4 Hz for H-37H-5'; 5 7.46 d, 7=8.4 Hz
for H-2'/H-6') (Table 4.3).
In agreement with this, the NOESY spectrum showed the protons at 5 6.91 (H-37H-5') correlated
with protons at 8 7.46 (H-27H-6') that also correlated with proton at 5 8.16 (H-2'). These data are
in agreement with 4',5,7-trihydroxyisoflavone with trivial name genistein. However the doubling
of l3C NMR signals were observed which suggested that this compound could be a non-
symmetrical dimer such as 3. The EI-MS (70 eV) did not show the molecular ion peak, but rather
showed a peak at m/z 270 (Figure 4.5), which is due to the monomeric genistein (3a). Compound
3 was provisionally identified as 4',7"-bisgenistein. The identity of this compound needs to be
confirmed through further experiments involving determination of the molecular mass through
chemical ionization (Cl) method.
63
+
Figure 4.5: Retro-Diels Alder fragmentation of compound 3a
Table 4.3: ’H and 1 'C NMR data o f compound 3 in a c e to n e ^
Position ‘H (600 MHz) UC (150 MHz) H M B C ( V /J )

5 h {J in Hz) 5c
2 /2 " 8.16 ^ 154.91/154.95 C-3, 4, 8 a
3/3" 124.68/124.73
4/4" 182.22/182.30
4a/4"a 106.78/106.81
5/5" 165.58/165.63
6 /6 " 6.29 £? (1.8) 100.41/100.46 C-4a, 5, 7, 8
7/7" 164.27/164.57
8 /8 " 6.42 d (1.8) 95.10/95.17 C-4a, 6 , 8 a
8 a/8 "a 159.32/159.70
r/r 123.70/123.73
272’" 7.46 d (8.4) 131.83/131.83 C-3, 3’, 4’
373’" 6.91 d (8.4) 116.58/116.58 C-T, 4’
474"’ 159.04/159.70
575’" 6.91 </(8.4) C-T, 4'
676’" 7.46 d (8.4) C-3, 3', 4’
5-OH 13.04 s C-4a, 5, 6
4.1.2.4 4’, 5, 7-Trihydroxycoumaronochromone (2)
Compound 2 was isolated as a brown-yellow powder with an Rf value of 0.6 in
hexane/dichloromethane/ethyl acetate (2:4:4). The positive ESI-MS of compound 2 showed
fM+H]f at m/z 285, corresponding to the molecular formular C 15H8O6 . A coumaronochromone
system was inferred from UV ( J w 257 nm), l3C (5 183.2 for C-4, 164.0 for C-2 and 110.5 for
C-3) NMR spectrum (Franco and Irene, 1991). Lack of a characteristic isoflavone H-2 singlet in
the !H NMR spectrum further proved the existence of a coumaronochromone ring system
64
(Tahara et a l, 1985). Aromatic signals in the 'H NMR spectrum of compound 2 (Table 4.4) were
evident as two meta-coupled doublets (8 6.37, 7= 2.0 Hz and 8 6.60, .7=2.2 Hz) which were
assigned to H-8 and H-6, respecively on ring-A (Franco and Irene, 1991). This was confirmed
from HMBC spectrum which showed correlation of the proton at 8 6.37 (H-8) with C-6 and
proton at 8 6.60 (H-6) with C-4a, 5 and C-8.
An ABX spin system (8 7.02 dd, J= 8.6 Hz and 7=2.2 Hz for H-5'; 8 7.14 d, 7=1.8 Hz for H-3';
57.82 d, 7=8.6 Hz for H-6') were asigned to the ring-B protons of a coumaronochromone system
with oxygenation at C-2' (8c 158.7) and C-4' (8c 152.9). Compound 2 was therefore identified as
4',5,7-trihydroxycoumaronochromone. This compound has earlier been reported from the roots
of lupinus albus (Tahara et al, 1985). However this is the first report of its occurrence in the
genus Eriosema.
OH 0
2
65
Table 4.4: ‘H and l3C NMR data o f compound 2 in acetone-d6
Position ‘H (200 MHz) u C (50 MHz) HMBC

5 H («/ in Hz) 5c
164.0
3 110.5
183.2
4
4a 105.0
5 163.6
6.60 d {2.2) 98.9 C-4a, 5, 8
6
7
163.0
8 6.37 d (2.0) 95.0 C-6
8a 156.2
r 115.5
2’ 158.7
3' 7.14 d (1.8) 100.2 C-4'
4' 152.9
5’ 7.02 dd (2.2, 8.6) 113.8 C -l’, 3 ’
6' 7.82 d (8.6) 121.8 C-2', 4'
5-OH 12.98 5 C- 4a, 5, 6
4.2 PHYTOCHEMICAL INVESTIGATION ON TEPHROSIA PURPUREA
4.2.1 Preliminary test results
Anti-microbial, anti-plasmodial, anti-hypertensive, anti-asthmatic and penile relaxant tests were
done on the stem extract of T. purpurea and showed appreciable activities. The presence of
several UV (254, 366 nm) sensitive components of the crude extract was detected on TLC. From
biogenesis, these compounds were assumed to be flavonoids and isoflavanoids. The extract was
subjected to a combination of chromatographic separation that led to the isolation of five
compounds. Spectroscopic methods were used to identify the isolated compounds. The
characterization o f the compounds is presented below.
66
4.2.2 Characterization of compounds from Tephrosia purpurea (stem)
4.2.2.1 Lanceolatin B (6)
Compound 6 was isolated as an amorphous powder with an Rf value of 0.42 in hexane/ethyl
acetate (9:1). The positive ESI-MS of compound 6 showed [M+H]+ at m/z 263, corresponding to
the molecular formular C 17H 10O 3 . The UV (Xmax 310 and 280 nm), 'H (5 6.94 s for H-3) and ,3C
(163.3 for C-2, 108.0 for C-3 and 178.8 for C-4) NMR provided evidence of a flavone skeleton
(Aneja et al., 1963; Talapatra et al., 1980; Huang and Chen, 2003). Unsubstituted aromatic ring-B
was deduced from ‘H (5 7.97 m for H-276'; 7.56 m for H-37475' and l3C (5 131.9 for C-174’;
129.4 for C-375'; 126.5 for C-276') NMR spectrum (Table 4.5). The presence of ortho-coupled
protons at 5 8.18 and 8 7.56 (7=9.0 Hz) in ring-A suggested that C-7 and C- 8 are substituted. In
the 'H NMR spectrum, an AX doublets at 8 7.22 and 8 7.78 (J= 3.0 Hz) revealed that the substituent
at C-7/8 is a furano ring.
The UV, 'H, l3C NMR and MS data for compound 6 was identical with those previously
published (Table 4.5) for lanceolatin B (Tanaka et al., 1992; Lee et al., 1995; Huang and Chen,
2003). Therefore compound 6 was identified as lanceolatin B.
6
67
Table 4.5: 'H and l3C NMR data o f compound 6 and Ianceolatin B (6)
Compound 6 (CDCI3) Lanceolatin B (6 , CDCIj)(Huang and Chen

2003)
Position 1 H (500 MHz) C (50 MHz) H (300 MHz) (75 MHz)
8 HC/in Hz) SH(J in Hz)
\2 ~ 163.3 161.7
3 6.94 s 108.0 6.89 5 107.6
178.8 178.3
4
4a 119.3 118.9
5 8.18 (9.0) 1 2 2 .0 8.17 £/ (8 .6 ) 121.7
6 1.56 m 1 1 0 .6 1.52 m 110.5
7 158.6 158.4
8 117.7 117.2
8a 151.1 151.0
r 131.9 131.8
2’ 1.91m 126.5 1.91 m 126.3
3' 1.56 m 129.4 1.52 m 129.2
4’ 1.56 m 131.9 1.52 m 131.8
5' 1.56 m 129.4 1.52 m 129.2
6' 7.97 m 126.5 1.91 m 126.3
2" 7.78 d (3.0) 146.1 7.78 d (1.8) 145.9
3" 7.22 d (3.0) 104.4 7.21 d ( 1.8) 104.2
42.2.2 Lanceolatin A (9)
Compound 9 was isolated as an amorphous powder with an Rt value of 0.54 in 20% acetone in
dichloromethane. The positive-ion EI-MS of compound 9 showed a molecular ion peak at m/z
336 indicating a molecular formula o f C 21H20O4 . A flavone skeleton was deduced from UV (Aâ*
295 nm), 'H (5 6.81 s for H-3) and l3C (163.1 for C-2, 106.8 for C-3 and 177.0 for C-4) NMR
spectra. Unsubstituted ring-B was inferred from 1H (5 8.12 m for H-2V6'; 7.61 m for H-3V475')
an d l3C (5 132.4 for C-T, 131.7 for C-4', 129.3 for C-375', 126.7 for C-276') NMR spectra (Table
4.6). The presence of a methoxy and a 3"-hydroxy-3"-methylbut-l"-enyl substituent was evident
from NMR (Table 4.6). The appearance of a pair of ortho-coupled protons (8 7.25 and 8 8.02
J-9.0 Hz) requires the placement o f the methoxy (8 4.05 s) at C-7 and the 3"-hydroxy-3"-
methylbut-l"-enyl substituent at C-8 .

68
In the EI-MS, the appearance o f a fragment ion at m/z 102 (9a) resulting from Wro-Diels-Alder
(RDA) cleavage o f the ring-C (Figure 4.9) was in agreement with the placement of these groups
in ring-A. This compound was therefore identified as 7-methoxy-8-(3"-hydroxy-3"-methylbut-1
enyl)-flavone, trivial name lanceolatin A (9) (Pelter ai, 1981, Abou-Douh el al 2005)
m/z 336
Figure 4.9: Retro-Diels Alder fragmentation of compound 9
69
Table 4.6: ‘H and l3C NMR data o f compound 9 and lanceolatin A (9)
9 (acetone-d 6) Lanceolatin A (9, CDCI3)(Abou-Douh el

al.. 2005)
Position 1 H (600 MHz) UC (50 MHz) H (500 MHz) ,J C (125 MHz)
8 h ( J in Hz) 5 h ( J in Hz)
163.1 163.3
P
3 6.81 5 106.8 6.7 6 s 107.1
177.0 178.3
4
14a 115.0 118.1
5 8.02 7 (9.0) 124.9 8.12 7 ( 8 .8 ) 125.5
7.25 7 (9.0) 1 1 0 .0 7.02 7(8.8) 109.0
6
7 161.9 162.7
8 115.0 114.3
8a 161.5 154.6
IT 132.4 132.1
2' 8 .1 2 m 126.7 7.90-7.92 m 126.4
3' 7.61 m 129.3 7.50-7.51 m 129.0
4' 7.61 m 131.7 7.50-7.51 m 131.5
5' 7.61 m 129.3 7.50-7.51 m 129.0
6' 8 .1 2 m 126.7 7.90-7.92 m 126.4
r 7.09 7 (16.6) 145.6 6.97 7(16.5) 144.1
2" 6.93 d (16.2) 114.6 6.82 7(16.5) 115.2
3" 70.5 71.6
3"-2CH3 1.46 s 29.2 1.55 s 30.0
1.46 s 29.5 1.50 s 30.0
7-OCH3 4.05 s 56.1 3.98 5 56.2
4.2.2.3 Semiglabrin (7)
Compound 7 was obtained as a white amorphous powder. The UV (>»max 310, 295 nm), *H ( 8
6.83 s for H-3), l3C (8 164.0 for C-2, 108.3 for C-3 and 178.3 for C-4) NMR indicated a flavone
skeleton (Waterman and Khalid, 1980; Huang and Chen, 2003; Abou-Douh et al., 2005). The
presence of an unsubstituted ring-B was again deduced from 'H (8 7.91 m for H-276’; 7.54 m for
H-37 47 5') and l3C (8 132.2 for C-T, 127.3 for C-27 6 ', 129.7 for C-37 5' and 129.9 for C-4’)
NMR spectra.
The *H NMR spectrum further revealed the presence of a pair of or/Zw-coupled protons (8 6.94,
and 8 8.16, 7=8.4 Hz) on ring-A, indicating the presence o f substituents at C-7 and C-8 . The
70
substituent at these positions was found to be two fused furan rings (Table 4 .7 ) derived from a
prenyl group at C - 8 and a methoxy at C-7 as in lanceolatin A. Thus the presence of a gem-
dimethyl group at C-5" was evident from two singlets (5 1.09 and 8 1.32) attached to oxygenated
sp' carbon atom (5C88.7). The presence of an acetoxy group at C-4" was also evident from NMR
(Table 4.7). The 'H NMR spectrum further showed a pair of mutually coupled doublets at 8 4.30
and 6.64 (7=6.4 Hz) were assigned to H-3" and H-2" respectively. The coupling constant (7=6.4
Hz) was constistent with cis orientation between these hydrogen atoms (Abou-Douh et al..
2005). Furthermore a singlet at 8 5.64 was assigned to the acetoxymethine proton at C-4 ". This
proton appeared as a singlet despite the presence of a vicinal proton (H-3M

). This indicated that
the relative stereochemical orientation of the former to the latter was trans as in semiglabrin
(Abou-Douh et al., 2005).
Comparison of the NMR data of this compound with literature report indeed confirmed that 7
was 2,",2"'-dimethyl-3"'-acetoxy-tetrahydrofurano-[3",2"-b]-dihydrofurano-[5",4”-h]-flavone,
trivial name semiglabrin (Table 4.7) (Smalberger et al, 1973; Waterman and Khalid, 1980;
Pelter et al., 1981; Ahmad et al., 1999; Abou-Douh et al., 2005).

71
Table 4.7: 1 H and lj C NMR data o f compound 7 and semiglabrin (7)
7 (CDC13) Semiglabrin (7, <-UCIj)(Abou-Douh el al

2005)
Position 'H (200 MHz) ,JC (50 MHz) H (500 MHz) ^ 0 ( 1 2 5 MHz)
5h(*/ in Hz) 5h(7 in Hz)
P 164.0 162.9
3 6.83 s 108.3 6.76 s 108.8
178.3 177.4
4
j 4a 119.9 118.7
5 8.16 £/(8.4) 132.6 8.15 ef (8.5) 128.9
6 6.94 d (8.4) 1 1 0 .0 6.93 rf (8.5) 109.0
7 164.7 163.7
8 113.4 112.4
8a 154.1 153.1
r 132.2 131.6
276' 7.91 m 127.3 7.90 m 126.4
375' 7.54 m 129.7 1.52 m 129.0
4' 1.54 m 129.9 7.51 m 131.6
2" 6.64 d (6.4) 113.2 6.62d(6.5) 112.4
3” 4.30 d (6.4) 53.6 4.30 d (6.5) 52.8
4" 5.64 s 80.9 5.63 s 80.2
4"-COCH3 170.4 169.6
4''-COCH3 2.22 s 2 1 .6 2.22 s 2 0 .8
5" 88.7 87.8
5"-CH3 1.09 s 28.3 1.08 s 27.5
5"-CH3 1.32 s 23.9 1.30 s 23.1
4.2.2.4 Terpurinflavone (8)
Compound 8 was obtained as a white amorphous powder with an Rf value of 0 .4 5 in hexane/
ethyl acetate (3:2). Compound 8 showed [M+H]f peak at mJz 437.1593 in its positive
electrospray ionization time o f flight mass spectrum (ESI-TOF-MS) constituting the molecular
formula C25H24O 7 . The presence of a flavone skeleton was deduced from the UV (A.max 295 nm),
'H (5 6.79 s for H-3) and l3C (163.9 for C-2, 108.5 for C-3 and 177.6 for C-4) NMR
spectroscopic data (Table 4.8). Unsubstituted ring-B was clearly shown in 'H (5 7.63 m for H-
7475', 8 8.18 /w for H-2V6') and 13 C (5 127.9 for C-276', 5 130.7 for C-375', 5 133.1 for C-4'and
72
133.6 for C -l') NMR spectra. In ring-A, an AX protons which are ortho-coupled at 6 6.94 and
8.00 (J=S.5 Hz) were assigned to H-6 and H-5 respectively, with C-7 and C-8 being substituted
with an acetoxy group (at C-7) and a furan ring (at C-8) derived from modified prenyl group as
in tephrorin B (84, Chang et al., 2000) (Table 4.8). The presence of a second acetate group was
also evident from the NMR spectrum (Table 4.8) and placed at C-4" of the furan group.
The 'H and 13C NMR chemical shift values of the furan ring of 8 were quite similar to those of
tephrorin B (84, Table 4.9). The coupling constant (J= 8.5 Hz) between H-3" and H-4" indicated
that the relative orientation of H-3" and H-4" is cis as in tephrorin B (84). NOE interaction of H-
3" with H-4” from NOESY spectrum supported the cis orientation.
The HMBC spectrum showed correlation of H-3" (6 4.44) with C-2" (79.1), C-4" (78.7), C-5"
(84.0), C-7 (167.9), C-8 (116.0) and C-8a (155.9) confirming that the furan ring is attached at C-
8. The HMBC spectrum further showed that H-4" (5 5.35) correlated with acetoxy carbon
(170.3) and the two methyl carbon (22.4 and 24.4) which confirmed the placement of one of the
acetoxy groups at C-4" and the two methyl groups at C-5".
Ihe new compound was therefore characterized as 7-acetoxy-8-[3-acetoxy-2,2-dimethyl
tetrahydro-4-furanyl] flavone (8) for which the trivial name terpurinflavone was assigned.
73
0
Tephrorin B (84)
Figure 4.11: Structure of compound 8 and tephrorin B
74
Table 4.8: 'l l and n C N M R data and H M B C correlatio n for com pound 8 and tephrorin B (84).
8 (acetone-d6) Tephrorin B (84 CDClj) (Chang et al.. 2000)

Position 'H (500 MHz) ,JC (75 MHz) HMBC 'H (300 MHz) 13C (75 MHz) HMBC ( 2
8 h (•/ in Hz) 5c 5 „ (J in Hz) 5c JM
2 163.9 5.42 dd (3.2, 12.6) 80.8 c -r, 4,
276'
3 6.79 s 108.5 C - r , 2, 4, 4a 2.83 m 44.7 C-2, 1 \ 4
4 177.6 190.7
4a 119.9 115.9
5 8.00 d (8.5) 129.3 C-4, 7, 8 a 7.83 d ( 8 .6 ) 131.1 C-4 a, 4,
8 a, 7
6 6.94 d (8.5) 110.1 C-4a, 8 6.57 d ( 8 .6 ) 105.9 C- 8 , 4a, 7
7 167.9 168.9
8 116.0 114.0
8a 155.9 159.5
r 133.6 138.9
276* 8.18 m 127.9 C -l', 2 7.46 m 126.0 C-2, 3', 4*
375' 7.63 m 130.7 C-276', 4* 7.36 m 128.6 C -l', 2 ',
4’ 7.63 m 133.1 7.36 m 130.7 C-3', 5',
276'
2 " 4.84 dd (8.0, 9.5) 79.1 C-4" 4.66 dd (9.3, 8.5) 78.5 C-4", 3", 8
5.02 dd (2.0, 9.5) C-3", 8
3" 4.44 ddd (2.0, 8.0, 8.5) 42.3 C-2", 4", 5",7, 8 , 4.07 m 40.6 C-2", 4",
8a 5",7, 8
4" 5.35 d (8.5) 78.7 COCH 3-4 ", 2", 5.10 d (6.3) 80.1 2CHj-5",
5" 3". 5", 8 ,
1
5" 84.0 72.9
4"-COCH 3 170.3
4"-C0CH j 1.61 5 20.9 COCHj-4"
5”-CHj 1.76 s 22.4 CH 3-5", 4”, 5" 1.24 s 26.6
5"-CHj 1.61 s 24.4 Me-5", 4”, 5” 1.24 s 27.1 C-5", 6 ",
7-COCHj * 170.8
7-COCHj 2 .0 0 s 23.0 COCHj-7
r 166.3
2"' 6 .2 4 ^ (1 6 .0 ) 117.4 c - r , 3*"
3"' 7 .4 5 ^ (1 6 .0 ) 146.0 c - r . 2-
4“ 134.2
5 - / 9 ... 7.36 m 129.1
6“78,M 7.46 m 129.3
7~ 7.36 m 131.1
The biogenesis of terpurinflavone (8) appears to be derived from lanceolatin A as shown
Scheme 4.1
Terpurinflavone (8)
Scheme 4.1: Proposed biogenesis of terpurinflavone 8
76
•J.2.2.5 Stigmasterol (5)
Compound 5 was isolated as a colourless amorphous powder with an Rf value of 0 69 in
CHjCh/EtOAc (9:1). The compound on TLC plate was not sensitive to UV light (254 and 366
nm) and was detected by exposure to iodine vapour. Steroidal skeleton was deduced from 'H (8
0.69 s; 0.81 t, > 5.6 Hz; 0.91 d, 7=6.2 Hz; 1.00 r; 1.02 d; 7=5.6, 3.49 m; 5.34 brd, > 5 .2 Hz) and
11 (8 72.0 for C-3; 121.9 lor C-6; 141.0 for C-5). The presence of two double bond were
inferred from 'H (8 4.98 dd,7=7.8, 15.4 Hz; 5.17 dd, > 8 .2 , 15.2 Hz) and
121.1 for C-6, 129.5 for C-23 and 138.6 forC-22).
Comparison of the NMR data of this compound with literature (Gomez-Garibay et al. 2002)
confirmed that 5 is stigmasterol. TLC comparison with authentic sample further confirmed that 5
was stigmasterol. Stigmasterol has been reported from the aerial parts of T. pumila
(Pethakamsetty et al., 2009).
27
Figure 4.12: Structure o f compound 5
77
Iable 4.9: ‘H and ,3C NMR data o f compound 5 and stigmasterol (5)
5 (CDCh) Stigmasterol (5, CDCh) (Patch

etal., 2009)
Position 'H (200 MHz) IJC (50 MHz) H (400 MHz) UC(100 MHz)
8 h U in Hz) 5c 5h (J in Hz) Sc
1 37.5 37.3
2 31.9 31.6
3 3.49 m 72.0 3.52 m 71.8
4 42.4 42.3
5 141.0 140.8
6 5.34 d (5.2) 121.9 5.36 br s 121.7
7 32.1 31.9
8 32.1 31.9
9 50.4 51.2
10 36.7 36.5
11 21.3 2 1.1
12 39.9 39.7
13 42.5 42.3
14 57.1 56.9
15 24.5 24.4
16 29.2 28.4
17 56.2 56.1
18 0.69 s 1 2 .2 0.69 5 1 1 .0
19 1 .0 0 5 21.4 1 .0 1 5 2 1.2
20 40.7 40.5
21 1.02 d (5.6) 21.3 1.02 d (7.5) 21.2
22 5.17 d d (8.2, 15.2) 138.6 138.3

23 4.98 del (7.8, 15.4) 129.5 129.3
24 51.5 51.2
25 32.1 31.9
26 19.2 0.80 cl (6.5) 2 1.2
27 0.91 d(6.2) 19.6 0.85 d (6.5) 19

28 25.6 25.4
29 0.8\ t (5.6) 12.5 0.80 t (7.5) 12.1
78
4.3 RESULTS OF THE BIOLOGICAL ASSAYS
4.3.1 Effects of plant extracts and isolated compounds on the Guinea pi»
trachea
The effects of Eriosema psoraleoides (roots), Tephrosia purpurea (stem) extracts and pure
compounds from Tephrosia purpurea (stem) on isolated trachea of a Guinea pig were tested and
the results are presented in table 4.10 and figure 4.13.
Table 4.10: Effects of plant extracts and pure compounds on isolated trachea of a Guinea pig
Test sample Organ bath Volume of Krebs Physiological

concentration solution effect
(pg/mL) displaced(pL)
Eriosema MeOH extract 31.5 0 No effect

psoraleoides
(Root) EtOAc fraction of 20.3 -1 Relaxation
CH 2Cl2/MeOH
extract
Tephrosia EtOAc fraction from 22.5 -1.5 Relaxation
purpurea CH 2Cl2/MeOH
(stem) extract
Stigmasterol (5) 12 ho- No effect
Lanceolatin B (6 ) 9 0 No effect
Semiglabrin (7) 7.5 -15 Relaxation
Lanceolatin A (9) 2 1 .8 0 No effect
Reference Acetylcholine 0.5 +6 Contraction

contractile drug
Reference Adrenaline 0.25 -7.5 Relaxation

1 relaxant drug
79
Key
MR-Methanol extracts o f E.psoraleoides roots. SEMI-Semiglabrin (7)
OGl-EtOAc layer o f E.psoraleoides roots LA-Lanceolatin A (9)
OG2-EtOAc layer o f T.purpurea stems Ach-Acetylcholine
STER-Stigmasterol (5) ADR-Adrenaline
LB-Lanceolatin B (6)
Figure 4.13: The effect of the pure compounds and plant extracts on isolated trachea-volume of
Krebs’ solution displaced
The trachea has P2 adrenoreceptors as well as M3 muscarinic receptors (Katzung, 2007).
Stimulation of the P2 adrenoreceptors by adrenaline resulted in relaxation of the bronchial
smooth muscle (Katzung, 2007). This is observed experimentally as a decrease in the volume of
Krebs solution in the capillary tube attached to the trachea (Table 4.10). On the other hand
stimulation of M 3 receptors resulted in contraction of the bronchial smooth muscle which is
observed as an increase in the volume of Krebs solution in the capillary tube attached to the
trachea (Katzung, 2007).

80
rhe root powder of Eriosemci psoraleoides was extracted with CH2Cl2/MeOH (1:1) and
methanol separately. The methanol extract showed no activity on the isolated trachea. The
dichloromethane/methanol (1:1) extract could not be subjected to test for biological activity
because of its poor water solubility. It was therefore partitioned between water and EtOAc. The
EtOAc fraction caused relaxation o f the trachea (Table 4.10). This finding highlights the
importance of screening fractions o f plant extract that seem to be inactive. The inactivity of the
methanol extract may have been due to the presence o f the components that have both a
contractive and relaxant effects. Thus the net effect was lack of activity. The EtOAc fraction
could have acted by either stimulating the p2 receptor or antagonizing the muscarinic receptors or
possibly by other novel mechanism.
The EtOAc fraction of the CH2Cl2/MeOH extract of Tephrosia purpurea caused relaxation of the
Guinea pig trachea. O f the five compounds isolated from T. purpurea, only four were tested.
Semiglabrin (7) showed a potent relaxant effect. This compound could therefore be responsible
for the relaxant effect of the EtOAc fraction of the extract o f T. purpurea. This compound has
two structural features required for p2 receptor stimulation. These features are a bulk aromatic
group and a free OH group that is likely to arise from the hydrolysis of the ester group. The free
OH group is required for interaction with p2 receptors. However receptor binding assays are
required to confirm whether semiglabrin (7) interact with the p-receptor or not. The relaxant
effect of the extract and semiglabrin (7) provides scientific justification for the traditional use of
T. purpurea for the management of tightness of the chest (Ahmad et al., 1999). Thymoquinone
from Nigella saliva and isokaempferide from Amhurana cearensis have been reported to have
relaxant effect on the trachea (Boskabady and Aslani, 2005; Leal et al., 2006).
81
4.3.2 The effect of plant extracts and isolated compounds on isolated aorta
The effects of Eriosema psoraleoides (roots), Tephrosia purpurea (stem) extracts and isolated
compounds on isolated aorta of sheep were tested and the results are presented in table 4.11
Table 4.11: Effect of plant extracts and isolated compounds on isolated aorta of sheep
Sample Organ bath Displacement Physiological

concentration (mm) from effect
(pg/mL) baseline
Eriosema MeOH extract 88 -13 Relaxation

psoraleoides
'•(Root) EtOAc fraction of 60 +7.5 Contraction
CH2Cl2/MeOH
extract
Tephrosia EtOAc fraction 74 -9 Relaxation
purpurea from CH2Cl2/MeOH
(stem) extract
Stigmasterol (5) 12 -6 Relaxation
Lanceolatin B (6) 19 -10 Relaxation
Lanceolatin A (9) 21 +2 Contraction
Referrcnce Adrenaline 0.5 +54 Contraction

Contractile
drug
Reference
Relaxant Acetylcholine 0.25 -5 Relaxation
drug
Compounds that cause vasorelaxation initiates penile erection by dilating the penile carvenosa.
The methanol extract o f E. psoraleoides (roots) caused relaxation (Table 4.11). This may
indicate that it can cause relaxation of the penile carvenosa vessels. The EtOAc fraction of the
82
CH:Cl2/MeOH extract of E. psoraleoides caused vasoconstriction. This may indicate that
compounds in E. psoraleoides (roots) responsible for penile erection may reside in the methanol
fraction. Relaxation may have been due to stimulation o f muscarinic receptors found in the
endothelium of vascular smooth muscle of the aorta.
The EtOAc fraction of CH2Cl2/MeOH of Teprosia purpurea caused relaxation which indicates
that it may contain vasoactive component that may be useful for the management of ED.
Stigmasterol (5) and lanceolatin B (6) isolated from this fraction caused vasodilation (Table
4.11). The leaves extracts of Brillantaisia nitens and Epimedium brevicornum Maxima have been
reported to have relaxant effect on isolated aorta o f sheep, rat and a rabbit (Chien et al., 2006;
Dimo et al., 2007).
4.3.3 Effects of plant extracts and pure compounds on the isolated vas
deferens of sheep and rat
Ihe crude extracts o f E. psoraleoides, T. purpurea and isolated compounds did not have an
effect on isolated vas deferens of a rat and sheep. This shows that neither the crude extract nor
the isolated compounds can promote ejaculation. This indicates lack of a-adrenoceptor stimulant
activity.
83
4.3.4 Effect of E rio sem a p so ra le o id e s root extract on the mating behavior
The male mice were divided into four groups of six each. The groups were treated with methanol
extract of Eriosema psoraleoides (roots) (2.5 g/kg), sildenafil (600 mg/kg), yohimbine (360
mg/kg) orally and the remaining group acted as a control. Three female injected with stilboestrol
(40 mg/kg) were introduced into each group. Cumulative frequencies of male mice smelling the
vagina, licking their penis and number of times erection present was represented on graphs.
Cumulative frequency of male mice smelling the vagina after being given the Eriosema
psoraleoides root extract (2.5 g/kg) and yohimbine (360 mg/kg) was represented in Figure 4.14.
Cumulative frequency of male mice smelling the

vagina against time(minutes)
figure 4.14: A graph showing cumulative frequency of male mice smelling the vagina.
Nohimbine showed the highest cumulative frequency of male mice smelling the vagina.
Eriosema psoraleoides (roots) extract showed a lower cumulative frequency of male mice
smelling the vagina as compared to yohimbine but slightly higher than control. This shows that
£ psoraleoides (roots) extract may promote sexual desire by probably acting on central nervous
system.
Cumulative frequency of male mice licking the penis after being given the Eriosema
psoraleoides extract and yohimbine is shown in Figure 4.15.
Cumulative frequency of male mice licking the penis

against time (minutes)
Figure 4.15: The cumulative frequency of male mice licking the penis
Yohimbine treated mice showed the highest cumulative frequency of male mice licking the
penis. Eriosema psoraleoides (roots) extract showed a lower cumulative frequency of male mice
licking the penis as compared to yohimbine but higher than the control. Licking the penis was
used as an indicator of erection.
t umulative frequency of erection in male mice after being given Eriosema psoraleoides roots
extract, yohimbine and sildenafil is presented in Figure 4.16.
85
Cumulative frequency of erection in male mice present
against time (minutes)
Figure 4.16: A graph showing cumulative frequency of erection in male mice
Sildenafil treated mice showed the highest number of erection in male mice. Eriosemu
psoraleoides (roots) extract showed a lower cumulative frequency of erection in male mice as
compared to sildenafil but higher than yohimbine. E. psoraleoides root extract may have penile
erectile activity. This supports its traditional use as an aphrodisiac.
4.4.1 Antiplasmodial activities of the plant extracts and pure compounds
I he F.tOAc fraction of the CFhCh/MeOH (1:1) extracts of E. psoraleoides, T. purpurea and the
pure compounds were screened for antiplasmodial activities chloroquine-sensitive (D6) and
chloroquine-resistant (W2) strains, of Plasmodium falciparum. The results obtained indicated
good antiplasmodial activities against both D6 and W2 strains of P. falciparum. Table 4.12
summarizes the in vitro antiplasmodial activity for the crude extracts and pure compounds.
I he new compound terpurinflavone (8) exhibited the highest antiplasmodial activity with IC50
values of 1.36 + 0.12 and 2.73 + 1.16 pg/ml against D6 and W2 strains of P. falciparum,
86
respectively. Lanceolatin A (9) also showed good activity with IC50 values of 3.82+1.00 and
6.03+1.04 against D6 and W2 strains o f P. falciparum, respectively. Terpurinflavone (8) and
lanceolatin A (9) appears to be responsible for the antiplasmodial activities observed in the crude
extract of T. purpurea. It is worth noting that all the compounds are more active against the
chloroquine-sensitive strain (D6) than the chloroquine-resistant strain (W2).
Table 4.12: In vitro IC50 values of the crude extracts of E. psoraleoides, T. purpurea and pure
compounds against D6 and W2 strains of P. falciparum.
Sample IC5o (|ig/ml+ SD) Resistance index (RI)

D6 W2
£ psoraleoides root 9.33 + 0.38 11.43 + 0.47 1.23
extract
T. purpurea stem 10.47 + 2.22 12.06 ±5.54 1.15
extract
Lanceolatin B (6) 7.08 + 0.59 9.43 ± 1.11 1.33
Semiglabrin (7) 9.33 + 2.20 12.88+ 1.96 1.38
Terpurinflavone (8) 1.36 + 0.12 2.73+1.16 2.00
Lanceolatin A (9) 3.82+ 1.00 6.03 ± 1.04 1.58
Mefloquine - 0.0036 + 0.0006 -
Chloroquine 0.011+0.001 - -
87
There was reduced activity against chloroquine-resistant (W2) strain with resistance indices of
1.10*2.00. Activities were inferior as compared to mefloquine and structure modification are
required to improve the acivity. Terpurinflavone (8) had the highest resistance value which may
indicate high risk of future cross resistance development.
4.4.2 Antimicrobial activities of plant extracts and pure compounds
The crude extracts o f E. psoraleoides, T. purpurea and pure compounds from T. purpurea were
tested for antimicrobial activity. The crude extract of E. psoraleoides showed an inhibition zone
of 12 mm against Candida albicans at a concentration of 1.8 mg/disc while the new compound
terpurinflavone (8) showed an inhibition zone of 16 mm against Microsporum gypsum at a
concentration of 50 pg/disc. The crude extract of T. purpurea, lanceolatin B (6), semiglabrin (7)
and lanceolatin A (9) were inactive against Staphylococcus aureus, Pseudomonas aeruginosa,
Candida albicans, Cryptococcus neo f or mans, Trichophyton mentagrophytes and Microsporum
gypsum.
88
X"
CHAPTER FIVE
CONCLUSIONS AND RECOMMENDATIONS

5.1 CONCLUSIONS
I. The phytochemical study on the E. psoraleoides (roots) led to the isolation of four
compounds and these were characterized as the isoflavanone derivatives, 4',5-dihydroxy-
2',7-dimethoxyisoflavanone ( 1), 4,,5,7-trihdroxy-2'-methoxyisoflavanone (4 ). an
isoflavone dimer, 4',7"-bisgenistein (3), and the coumaronochromone 4’,5,7-
trihydroxycoumaronochromone (2). 4',7"-Bisgenistein (3) appear to be new.
2. The study on T. purpurea (stem) led to the isolation o f five compounds which included a
steroid, stigmasterol (5), four prenylated flavone derivatives, lanceolatin B (6),
semiglabrin (7), terpurinflavone (8) and lanceolatin A (9). Of these, compound 8 is novel.
3. E. psoraleoides (roots) and T. purpurea (stem) showed significant relaxant effect on the
aorta and therefore they may be used for the management of erectile dysfunction (ED).
4. Semiglabrin (7) had the highest relaxant effect on the bronchial smooth muscle of an
isolated trachea. This provides scientific justification for the use of T. purpurea for the
management o f tightness of the chest.
5. The crude extracts and the prenylated flavones from T. purpurea (stem) showed
antiplasmodial activities against chloroquine-sensitive strain (D6) and the chloroquine-
resistant strain (W2) of P. falciparum parasite for malaria. Among the prenylated
flavones tested, the terpurinflavone (8) had the highest activity.
6. The crude extract of E. psoraleoides (roots) was active against Candida albicans while
terpurinflavone (8) was active against Microsporum gypsum.
89
RECOMMENDATIONS
1. Further phytochemical investigation on E. psoraleoides (roots) and T purpurea
(stem) should be carried out in order to establish the chemical profiles of these
medicinal plants.
2. In vivo penile relaxant activity of E. psoraleoides (roots) extract should be carried out
in order to establish its potency and efficacy.
3. In vivo anti-asthmatic activity should be carried out on T. purpurea (stem) extract and
semiglabrin (7) in order to establish the potency and efficacy.
4. In vivo antiplasmodial activity tests should be carried out on the extracts and isolated
compounds from these medicinal plants in order to establish their potency and
efficacy.
5. Toxicity study of the extracts and compounds from these medicinal plants should be
done in order to establish their safety in long term use, as might be required for long
term infection management.
6. The mechanism of action of the compounds needs to be fully examined so that
targeted proteins by these compounds can be clearly demonstrated for differentiation
with those of already established drugs. This information is important for developing
the compounds as therapies for management of infections arising from resistant
strains of P. falciparum malaria parasite.

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a p p e n d ic e s
Appendix A: Spectra for compound
E I -M S S P E C T R U M F O R C O M P O U N D 1
Heydtnr*ich_44 #34-255 R T: 0.31-1.10 A V: 222 NL: 1 0OE7
T : + c F u ll m s [ 35.00-500.00]
105
' H N M R S P E C T R U M F O R C O M P O U N D 1 (200 M H z , A C E T O N E - d 6)
12 . 3 4 0
- i— i— |— i— i— ■— i— [ - “ 1---------1 I " l'~ ■ !'
12 11 10 7 ppm
0. 6 1 1.00 2. 1 6 4.24 0.00

0. 56 1. 67 1 . 7’ 6. 1 6 0.00 106
C N M R S P E C T R U M F O R C O M P O U N D 1 (125 M H z , A C E T O N E -d,,)
180 160 140 120 100 80 60 40 pp m
7 2. 063
71. 9 26
7 1. 779
C NM R S P E C T R U M E X PA N SIO N F O R C O M P O U N D 1
-------61.735
__ _
-------61.203
57.498
57.442
56.980
56.940
56.874
56.696
56.628
56.557
56.316
------- 51.982
-------50.615
------- 50.195
------- 49.434
46.616
48.230
48.188
48.110
48.067
O
00
‘h ^ h cosy spectrum fo r COMPOUND 1
109
111-1H C O S Y S P E C T R U M E X P A N S I O N F O R C O M P O U N D 1
JUL J L _____ WL i i ___ AA_ ppm
110
I t ltvi U UI I JO N
HMQCSPFCTRt'M FOR COMPOI'NM I
112
H M Q C SPECTRUM EXPANSION FO R COM POUND 1
113
I IM B C S P E C T R U M F O R C O M P O U N D 1
jj__L Jll . ppm
114
H M B C S P C T R U M E X P A N S IO N F O R C O M P O U N D 1
11
H M B C S P E C T R U M E X P A N S IO N F O R C O M P O U N D 1
116
H M B C S P E C T R U M E X P A N S IO N F O R C O M P U N I ) 1
HM13C S P E C T R U M E X P A N S IO N F O R C O M P O U N D 1
118
Appendix B: spectra for compound 2
RelatKre A b u n d a n c e -t‘ ixt
+
3p-
0
to
Oi ^
%
§S
8
§:
E I-M S F O R C O M P O U N D 2
bJ
O
" N M R S P E C T R U M F O R C O M P O U N D 2 (200 M H z , a ceto n e -^)
1-- r—-r—t - «—r T—r t r”»— —r “ I—r T ' —r-T —1—i-- v- ' T '
13 12 11 10 7 6 4 3 2 1 ppm
— ------ - ». . ,-----
0.28 0.99 1 . 0S 12.90 1.30 0.36
1.00 1.01 0.91 1.S4 17. S7 • .S3
121
'H N M R S P E C T R U M E X P A N S IO N F O R C O M P O U N D 2
ro
cn
rs.
co
no
00
v
W—
A
1 >"t • n T-t . ;-p -i T 1—I 1—T r S I • -T-J- I I I —r i I I I I > l -r T-f1r 1 t 1 r T _ r r r | *

8.0 7.8 7.6 7.0 6.8 6.6 6.4 6.2 ppm
0.98 1.00 0.92 1 OS

0. 28 0. 99
187. 623
HM QC SPECTRUM FOR COM POUND 2
124
HM BC SPECTRUM FOR COM POUND 2
125
Appendix C: Spectra for compound 3
E I-M S S P E C T R U M F O R C O M P O U N D 3
Heydenreich_46 #120-270 RT: 0 60-1.11 AV: 151 NL: 1.22E7
T: + c Full m s [ 35.00-650.00]
127
' l l N M R S P E C T R U M F O R C O M P O U N D 3 (600 M H z , A C E T O N E -d * )
n <s >N( N- . . . ^ ^ , : , : " ' i ' 7; 0; 0: ,I; 0; ^ o ; “ ^ « ' r '," r , r m N i N N w a i w » o » > o ^ o , to<0 r - N ^ ” ^ “ S " o o ? ? “ f ! 3 S S S S S
7 n V n n n n n n n n n n n n n n n H H H H H H H H
H N M R S P E C T R U M E X P A N S IO N F O R C O M P O U N D 3
8.167
Ml/ 1/ ^ W // V N m W 'V I
182.298
182.216
CO
o
r 165.626
165.583
o £ 164.569
C N M R S P E C T R U M F O R C O M P O U N D 3 (150 M H z , A C E T O N E - d 6)
— 164.272
___ 159.704
"^ 1 5 9 .3 2 0
g ^^-159.037
V-1 54.953
V 154.908
132.425
131.827
131.432
129.691
124.727
124.675
w -3 J V r 123.727
o J /r 123.704
2 ^ - 122.986
r 116.792
WH / > 1 16.582
o — 115.221
o -3
o
(0
o
oo
o
>1J
o
01 J
o ■56.528
01 J
o
44.192
— 44.136
■u
o
g I
oMJ1
T3 :
11
U>
O
131
HMBC SPECTRUM FOR COMPOUND 3
132
\
HMBC SPECTRUM EXPANSION FOR COMPOUND 3
133
H M B C S P E C 1 R U M E X P A N S IO N F O R C O M P O U N D 3
T------------ 1-------------1-------------1-------------1------- I ' I ' I

7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 ppm
134
H M B C S P E C T R U M E X P A N S IO N F O R C O M P O U N D 3
135
ppm
136
N O E S Y S P E C T R U M E X P A N S IO N F O R C O M P O U N D 3
137
Appendix D: Spectra for compound 4
'll N M R S P E C T R U M F O R C O M P O U N D 4 (600 M H z , ACETONE-d6)
I *“ • •“ * H O O'* VO to r*- vO in in «>• <—■» r-t\ • <N 'T O O O*
*-«aoaovovOvOvOvOvOvOvOu"iir>iou">%r «r ^ r v v ^ r v n f O f O f n n n f O f o n
139
4.641
4.622
4.597
4.591
4.582
4. 579
4.564
4. 556
4.547
■t- 4.534
CT> 4.528
4. 516
4. 510
4.498
4.472
4.463
•fc»
4.459
4.449
in 4.441
4.431
4.425
4.415
II N M R S P E C T R U M E X P A N S I O N F O R C O M P O U N D 4
'
4.406
4.397
4.358
•C* 4.339
4.333
4.274
4.265
4.256
4.247
4.227
GO 4.217
4.213
4.203
4.192
4.183
4.173
4— 4.135
ro 4.126
4.117
4.109
4.101
4.051
4.040
4.029
3.987
3.964
3.939
3.933
3.926
3.915
3.892
3.886
3.876
3.871
3.867
3.859
3.851
3.848
oo 3.841
to 3.838
3.835
3.829
3.819
3.816
3.813
CO 3.810
ot> 3.805
3.801
3.794
3.787
3.784
3.765
co 3.754
-J 3.728
3.725
3.707
3.678
3.661
3.643
3.633
3.6
3.628
3.615
3.605
3.597
ppm
3.393
-U
o
II NMR SPECTRUM EXPANSION FOR COMPOUND 4
OOB»ffir* T Mn H M A «lA «V
O O»o>(T*tf'O'®ao®coiC<r>®®cor-r-r‘ r'r'r-'r->e'£>v*>s0N0'O*>'4>^v*>«n*n»n*n*n»n»nin'r«r'r<r»r«rxr*r«r«r'«rv*r<ncocNCwC'* M »-• *-• o o o o o o o
i w^mwwwwwi ^ m \ \ / / / \ \ w \// i w//1\i/1 \\i w
141
H - H COSY SPECTRUM FOR COMPOUND 4
,.J. . i i . . I »J s-Jk-JI-^ *JAU p p m
4. 0
-4 .5
-5 .0
-5 .5
- 6.0
6. 5
7. 0
-7 .5
8.0
ppm
h - ’h c o s y s p e c t r u m e x p a n s io n FOR COM POUND 4
ppm
522
y
167.892
166.339
165.207
160.200
< 159.953
C N M R S P E C T R U M F O R C O M P O U N D 4 (150 M H z , A C E T O N E - d 6)
-----132.418
-----123.415
-----115.859
114.351
----- 108.670
----- 101.010
----- 97.580
— 96.299
-----71.854
57.010
56.536
----- 48.004
33.301
32.118
31.051
30.882
30.755
30.626
30.499
30.369
30.242
30.113
23.990
15.018
■U
146
1IMQC SPECTRUM EXPANSION FOR COMPOUND 4
i—^
........... .
miMiW
147
IIlVfBC S P E C T R U M F O R C O M P O U N D 4
148
IIM B C S P E C T R U M E X P A N S I O N F O R C O M P O U N D 4
149
H M B C SPE C T R U M EX PA N SIO N FO R C O M PO U N D 4
150
Appendix E: Spectra for compound 5
II N M K s n a I KUIVI P U K i . u i v i m u i > w :> ^ u u iv m z , v i n i . jj
u>
Appendix F: Spectra for compound 6
154
E I-M S S P E C T R U M F O R C O M P O U N D 6
N M R S P E C T R U M F O R C O M P O U N D 6 (5 0 0 M IIz , C D C L ,)
11.110
---- 1.246
•
is*
o
----------8 . 1 8 1
----------8 . 1 6 9
7 .9 8 6
7 .9 8 2
7 .9 7 7
A 7 .9 7 3
7 .9 7 0
\ 7 .9 6 6
7 .9 6 4
7 .9 5 8
II N M R S P E C T R U M E X P A N S I O N F O R C O M P O U N D 6
7 .7 8 0
7 .7 7 5
7 .5 7 5
7 .5 7 4
7 .5 6 5
7 .5 6 1
7 .5 5 6
7 .5 5 5
7. 5 5 1
7 .5 4 3
--------- 7 . 2 6 2
y 7 .2 1 8
^>7.216
^ - 7.213
N . 7 .2 1 2
7.002
"^ -7 .0 0 1
--------- 6 . 9 3 6
LA
u
c N M R S P E C T R U M F O R <- oiVfV O U N U 6 (SO M H 1 , l u l l j)
\
178.762
29. 921
100 60 ppm
158
Appendix G: Spectra for compound 7
091
I S C SO 0 69'0
sc c 00*
• *1 - 1- *
- ,
uidd I
J. . _ X. J ____
O
96 0
00 0 SOI u t T 66'0
<
S 8
O
134
.138
929
.937
8.180
1 6 4 .0 2 9
2 8 .2 5 9
1
Appendix H: Spectra for compound
TOF-MASS SPECTRUM FOR COMPOUND H
H EYC B SFG C H JE9 _ 22_ E> Q \/I 5 (0.101) PM (Gan,5,80.00,14,50000,490.89,0.00); Q n (2 5 ) T C F IV B E & f

76 .3
100 4371503
%■
438.1620
4334900434 ^ 435.0110
\
439.15834404473 4422478 4427646 445.0746 446.8333
0 l_____ j l i, ,, l I I ______ J_____ oJ-----L— rrfz
432 433 434435436 437 438439440 441 442 443444445446 447 448
164
E I-M S S P E C T R U M F O R C O M P O U N D H
00
-J
cn
II N M H S P E C T R U M F O R C O M P O U N D 8 ( 5 0 0 M H z , A C E T O N E - d t )
In
>1 -i
b
*
\
O)
cn
cn
01
cn
Ul
o
u
In
u
o
w
cn
4.414
2.884
2.852
2.100
2.071
2.064
jo 2.060
cn 2.055
2.051
2.047
ro 1.998
o 1.992
1.859
1.760
1.750
1.680
cn 1.627
1.608
1.605
1.588
1.542
1.429
O
O
' l l N M R S P E C T R U M E X P A N S IO N F O R C O M P O U N D 8
167
'll NMR SPECTRUM EXPANSION FOR COMPOUND H
r» Ov i f l h <m «n o n o n Nm r-
5.344
m (n o o in (n(n ifi mrowo«»
o o o o* ® «d® r*
\/ \l \l
8. 196
8.194
8. 190
8.187
8.184
8. 183
8. 180
8. 178
8. 175
8.094
8. 090
----------8. 005
----------7.987
II N M R S P E C T R U M E X P A N S IO N F O R C O M P O U N D K
7. 639
i 7.638
7. 635
7.631
7.627
7.620
7.617
7.609
--------- 6.944
--------- 6.927
---------- 6.881
--------- 6.864
-------- 6.848
--------- 6.792
--------- 6.745
O
vO
180
-------- 177.649
170.768
*s~~— 170.297
— 167.857
C N M R S P E C T R U M F O R C O M P O U N D 8 (7 5 M llx , A C E T O N E -d j)
-------- 163.898
160
-------- 155.870
140
133.601
133.077
130.662
129.330
127.950
127.882
120
-------- 119.914
-------- 115.975
-------- 110.136
-------- 108.488
100
-------- 83.949
— 79.081
80
78.666
60
-------- 42.303
40
24.396
22.974
22.383
20
20.887
ppm
8. 0 7.5 7.0 6.5 6. 0 5. 5 5. 0 4.5 ppm
IIM Q C S P E C T R U M FO R C O M PO U N D N
ppm
20
30
40
50
60
■ 70
- 80
- 90
-100
-110
-120
-130
ppm
172
IIMBC SPECTRUM FOR COMPOUND S
JU L J___ c J l X PPm
-154
-156
-1 5 8
-160
-162
-164
-166
I -168
0 -178
' I ' 1 ' ' I ......I ........ I .......I ' "T"' ■ i .......v r ■1" —T *—
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 ppm
173
IIM H C S P E C T R U M E X P A N S IO N F O R C O M P O U N D 8
174
H M H C S P E C T R U M E X PA N SIO N FO R C O M P O U N D 8
175
1IM B C S P E C T R U M E X P A N S I O N F O R C O M P O U N D X
176
NOESY SPE CTR U M FOR CO M PO U N D 8
177
NOESY SP E C T R U M EXPANSION FOR C O M P O U N D H
178
NOESY SPE CTR U M EXPANSION FO R C O M PO U N D S
JU
o
a
<s>
" I ........." T ............. I ...............I ............... I ' .............i ...............r

7.8 7.7 7.6 7.5 7.4 7.3 7.2
179
Appendix I: Spectra for compound 9
E I -M S S P E C T R U M F O R C O M P O U N 13 9
Heyd«nr*ich_2S #105-134 R T: 0.55-0.64 A V. 30 NL: 8 00E5
T : + c Full mt [ 35.00-500.00]
181
8.008
7.622
1.962 7.621
CO 7.614
1.007 7.611
o
7.608
7.605
2.958 7.600
>l 7.598
b 7.596
7.585
1.000
H N M R S P E C T R U M F O R C O M P O U N D 9 (600 M H z, A C E T O N E -d 6)
7.581
vl 0.959 7.254
o 1.009 7.239
7.100
TT056 7.073
6.939
p> 6.912
b 6.812
01
o
w
b
oi
o
JSi
b
--------4.050
3.148
o
w
b
2.856
2.823
2.068
2.062
2.059
2.055
2.051
2.048
-------- 1.456
5.981
3 ]
x
NJ
8. 127
8 .1 25
£ 8 .1 2 1
8. 117
8. 114
8.1 1 1
---------- 8 .0 23
---------- 8.0 08
00
o
-vl
CO
H N M R S P E C T R U M EXPANSION FOR CO M PO U N D 9
7.622
7.621
7.614
7.611
7.608
7.605
7. 600
7.598
7. 596
7. 585
7.581
--------- 7.254
---------- 7.239
---------- 7.100
---------- 7.073
---------- 6.939
---------- 6.912
00
u>
1 6 3 . 11 «
161. 494
177.009
Appendix J: Physiograms of the effect of isolated compounds and extracts on
isolated aorta
187
E F F E C T O F ETH YL ACETATE F R A C T IO N O F E. PSORALEOIDES (R O O T ) A N D /.
1 T
r P U R P U R E A (STEM ) ON ISO LA TED AO RTA O F SH EEP
- ~
EFFEC T OF ETH Y L A C ETA TE FR A CTIO N O F E. P S O R A L E O I D E S (RO O T) AND T.
---------------------------------
A
Lanceol?,
U O O B A S IL E
E FF E C T O F LA N CEO LA TIN B (6), ST IG M A STER O L (5) AND LA N C E O LA TIN A (9)
ON ISO LA TED AORTA OF SH EEP
-f-T —
E FFEC T OF LA N CEO LA TIN B (6), STIG M A STER O L (5) AND L A N C E O LA TIN A (9)
ON ISOLA TED AO RTA O F SH EEP
$
L anccolatin A (9 )
w JskL
\
EFFECT OF METHAIVOLJC EXTRACT OF (ROOT) OJ\
ISOLATED AORTA OF SHEEP

I______L
EFFECT OF METHANOLIC EXTRACT OF PSORALEOIDES (ROOT)

ISOLATED AORTA OF SHEEP
\ \ T V
ZZT 7ZT 7 T~L
I I
awTiii
j T_
___ Methanol extract of
E. psoraleoides __[------------- o
Acetylcholine
(Root extract)
—1 190
V A f \T ~ Y
UGO BASILE I I
i i I I I I I I
the effect of isolated compounds and extracts on
Appendix K : P h y s io g r a m s
vasdeferens
191
< EFFECT OF ADRENALINE, A C E T YCUOLINK,
C H O rr^ TAND
^ ETHYL ACETATE FRACTION
° f /:' K o / 0 U £ o /d ^ ^ k m e x t r a c t on is o l a t e
lTED VAS DEFERENS OF A RAT
.. I_ ______ 1 _J I T I
EFFECT OF ADRENALINE, ACETYCHOLINE, AND ETHYL ACETATE FRACTION
----- 01' E- PSORALEOIDES STEM EXTRACT ON ISOLATED VASS DEFERENS

DEF OF A RAT
\ ' 1 1 in
A__ l_ V —V r l
\ \ I V
T
\____ \
EH i— /- UHL
1____ x
1__ L
i_
T
Adrenaline!- Acetylcholine
A d re n a lin e
— \— V
E. psoraleoides'
Acetylcholine r \ (Stem extract)
92
w m
L / \ v m \ \ \ UUO f >11
! » '•

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U N I V E R S I T Y OF N A I R O B I

PHYTOCHEMICAL INVESTIGATION OF TEPHROSIA

WANYAMA PETER JUMA

A THESIS SUBMITED IN PARTIAL FULFILMENT OF THE DEGREE OF MASTER OF SCIENCE OF

Sign.... ..................................................... Dat ..................

Sign.,... tf&kar. .. -W l £\. .......... Date. l ft:9.LP..........................

This thesis is dedicated to my beloved mother, Florah and my

TABLE OF CONTENTS............................................................................ ,...v

LIST OF TABLES...................................................................................... .v iii

LIST OF FIGURES..................................................................................... ... ix

LIST OF SCHEMES................................................................................... ... xi

LIST OF ABBREVIATIONS..................................................................... x iii

GENERAL INTRODUCTION............................................................... ..1

LITERATURE REVIEW............................................................................. ..5

2.1 ERECTILE DYSFUNCTION............................................................ ..5

MATERIALS AND METHODS................................................................................................ 45

RESULTS AND DISCUSSION................................................................................................. 58

CONCLUSIONS AND RECOMMENDATIONS..................................................................... 89

Table 2.2: Traditional uses of Eriosema species in Africa.................................. 13

Table 2.3: Ethnomedical use of some Tephrosia species of Kenya................... 14

Table 2.4: Occurrence of C-8 prenylated flavones in Tephrosia species.......... 30

Table 4.1: 'H and 1 C NMR data of compound 1 ............................................... 60

Table 4.2: *H and 1 C NMR data of compound 4 ............................................... 62

Table 4.3: ‘H and l3C NMR data of compound 3 ............................................... 64

Table 4.4: 'H and l3C NMR data of compound 2 ............................................... 66

fable 4.5: 'H and ljC NMR data of compound 6 ............................................... 68

Table 4.6: 'H and 13C NMR data of compound 9 ............................................... 70

Table 4.7: 1 H and 13 C NMR data of compound 7 ............................................. 72

Table 4.12: In vitro IC50 values of the crude extracts o f E. psoraleoides, T.

Figure 1.1: Drugs used to manage erectile dysfunction................................................... 2

Figure 2.1: Second generation of PDE5 inhibitors........................................................... 6

Figure 2.2: Drugs that act by interacting with dopamine receptors................................ 7

Figure 2.3: Flowering E. psoraleoides plant..................................................................... 11

Figure 2.4: Tephrosia purpurea found in Kenya.............................................................. 12

Figure 2.5: Skeleta o f flavonoids based on the position of benzopyran ring................16

Figure 2.6: Examples of flavonoid (sensu stricto) subclasses.......................................16

Figure 2.7: Examples of isoflavonoid subclasses........................................................... 17

Figure 2.8: Basic skeleton of an isoflavone.....................................................................24

Figure 2.9: Isoflavones isolated from the roots of E. kraussianum.............................. 25

Figure 2.10: Isoflavone glycosides from E. tuberosum................................................. 26

Figure 2.11: Isoflavones from E. tuberosum....................................................................27

Figure 2.12: Chromones isolated from the roots of E. tuberosum..................................27

Figure 2.13a: Phenolic compounds from the roots o f E. tuberosum............................ 28

Figure 2.13b: Phenolic glycosides from the roots o f£ . tuberosum.............................. 29

Figure 2.14a: C-8 prenylated flavones isolated from Tephrosia species........................30

Figure 2.14b: C-8 prenylated flavones isolated from Tephrosia species........................31

Figure 2.14c: C-8 prenylated flavones isolated from Tephrosia species........................32

Figure 2.15: Isoflavones isolated from T. purpurea and T. maxima...............................33

Figure 2.16: Flavanone skeleton........................................................................................33

Figure 2.17: Prenylated flavanones isolated from Tephrosia species.............................34

Figure 2.18: Prenylated flavanones from Tephrosia species.......................................... 35

Figure 2.19: Furanoflavanones from the genus Tephrosia............................................. 36

Figure 2.20: Basic skeleton o f chalconoids...................................................................... 37

Figure 2.21: Chalcones isolated from T. purpurea and T. hamiltonii.............................38

Figure 2.23: Rotenoids of the genus Tephrosia.............................................................. 40

Figure 2.24: Basic skeleton of most commonly occurring pterocarpan....................... 41

Figure 2.25: Pterocarpans from Tephrosia species......................................................... 42

Figure 4.1: Structure o f compound 1 ................................................................................59

Figure 4.3: Structure o f compound 4 ............................................................................... 62

Figure 4.4: Structure o f compound 3 ............................................................................... 63

Figure 4.5: Retro-Diels Alder fragmentationof compound 3a....................................... 64

Figure 4.6: Structure o f compound 2 ............................................................................... 65

Figure 4.7: Structure o f compound 6 ............................................................................... 67