x' PHYTOCHEMICAL INVESTIGATION OF

SURFACE EXUDATES OF
ANGUSTIFOLIA AND SENECIO ROSEfFLORUS
FOR BIOACTIVE PRINCIPLES

BY

LEON I DA 11 KERUBO OMOSA

A THESIS SUBMITTED IN FULFILMENT OF THE

DEGREE OF DOCTOR OF PHILOSOPHY OF THE

UNIVERSITY OF NAIROBI.

2009
University ot N AIRO B I Library

0378925 2
 I HEREBY DECLARE THAT THE MATERIAL CONTAINED IN THIS

THESIS IS MY ORIGINAL WORK AND HAS NEVER BEEN

PRESENTED FOR A DEGREE IN ANY UNIVERSITY.

LEONIDAH KERUBO OMOSA

We hereby certify that this thesis has been submitted for examination with

our approval as university supervisors

v ,

PROF. J. (). MIDIWO

DEPARTMENT OF CHEMISTRY DEPARTMENT OF CHEMISTRY

UNIVERSITY OF NAIROBI UNIVERSITY OF NAIROBI

n
 ACKNOWLEDGMENTS
It is with sincere gratitude that I would like to thank my supervisors Prof. ). (). Midiwo and

Dr. Solomon Derc.se for their guidance, dedication, support, inspiration throughout the course

ot this study.

I am indebted to Prof. Abi\ Yenesew tor his constant encouragement a d guidance and for

facilitating the analysis of my compounds, throughout my study. I am indebted to Dr.

Matthias Heydreich. Prof. Martin G. Peter. University of Potsdam. aru Dr. Langat, M. K.

University ol Surrey, for analyzing my samples on high resolution NMR and MS and

providing data for optical rotation of the new compounds. Dr. Matthias w s very instrumental

in providing the necessar\ literature for tnc compounds isolated. I acknowledge the support

of Deutsche I'orschungsgemeinschatt, Germany, grant no. Pc VV 14-5 and by the

Bundcsministerium feur Zusammenarbeit. Grant No. PI -254/i‘t 6.

Mr. Akala, I f and Dr.Waters, N. C., Liyala, P. of the United Stales Am. Medical Research

Unit-Keriya are acknowledged for assistance in testing for anti plasmodiai activity of the

crude extracts and the isolated compounds. I acknowledge Dr. Beatrice . ' mugune, School of

Pharmacy. University o f Nairobi, for testing of the anti-microbial activ ity of the extracts and

the isolated compounds. I acknowledge Dr. Jacques Kabaru. School of Biological Sciences

(SBS), University of Nairobi, for providing the facilities for testing of the mosquito larvicidal

activity of extracts and the isolated compounds in this study. My appr lation also goes to

Mr. Simon Maihenge o f the SBS, University of Nairobi, for the collect km and identification

of the plants investigated in this study.

i wish to li’.jr.k thi. German Academic Exchange Service (DAAD) f the award of the

scholarship without which I would not have been able to do my ,%tud\

iii
I would like to express my gratitude to the teaching and technical staff of the Department of

Chemistry, University of Nairobi, for creating an enabling working environment.

Many thanks go to the current members of the Natural Product Research group, University of

Nairobi, for their cooperation, encouragement and support during the course of this research

work.

Finally, 1 would like to express my heart felt thanks to my husband Bright Kadenge Mihiga,

my sons Ryan Kadenge, Emmanuel Kadenge and daughter Joy Keisha Kadenge who had to

endure to the loss of quality time in long working hours during the week and weekends and

even daily after working hours.

IV
THIS THESIS IS DEDICATED TO MY HUSBAND BRIGHT

KADENGE; SONS RYAN KADENGE AND EMMANUEL

KADENGE; DAUGHTER KEISHA KADENGE; AND MY

PARENTS
 TABLE OF CONTENTS
ACKNOWLEDGMENTS................................................................................................. iii
LIST OF FIGURES...........................................................................................................xii
LIST OF SCHEMES........................................................................................................ xiii
APPENDICES.................................................................................................................. xiv
LIST OF ABBREVIATIONS AND SYMBOLS........................................................... xv
LIST OF SUB-STRUCTURES.......................................................................................xvi
ABSTRACT.....................................................................................................................xviii

CHAPTER O N E .............................................................................................................................. 1
INTRODUCTION................................................................................................................ 1
1.0 GENERAL.......................................................................................................................I
1.1 Plant Resins..................................................................................................................... 4
1.1.1 Terpenoid R esins........................................................................................................ 5
1.1.2 Phenolic Resins............................................................................................................8
1.2 The Malaria burden..................................................................................................... 12
1.3. Botanical larvicides......................................................................................................14
1.4. Microbial Drug Resistance......................................................................................... 16
1.5 Oxidative Stress and Anti-oxidants.......................................................................... 18

CHAPTER T W O ............................................................................................................................ 21
LITERATURE REVIEW .................................................................................................. 21
2.1 BOTANICAL INFORMATION................................................................................ 21
2 .LI THE FAMILY SAPINDACEAE.............................................................................21
2 .1.1.1 THE GENUS DODONAEA.................................................................................. 22
2.1.1.1.1 D. VISCOSA.........................................................................................................23
2 .1.1.1.2 DODONAEA ANGUSTIFOLIA.........................................................................24
2.1.1.2 MORPHOLOGICAL DIFFERENCES BETWEEN D. VISC OSA AND D.
ANG USTIFOLIA....................................................................................................26
2.1.2 FAMILY ASTERACEAE......................................................................................27
2.1.2.1 THE GENUS SENECIO....................................................................................... 29
2.1.2.1.1 SENECIO R OSEIFL OR U S................................................................................29
2.2 ETHNO-MEDICAL APPLICATION AND PHARMCOLOGICAL
INFORMATION ON THE GENUS DODONAEA AND SENECIO............... 29
2.2.1.1 ETHNO-MEDICAL APPLICATION OF DODONAEA SPECIES............... 29
2.3 BIOLOGICAL ACTIVITIES OF COMPOUNDS AND EXTRACTS FROM
DODONAEA SPECIES........................................................................................ 31
2.3.1 CORRELATION OF PHARMACOLOGICAL ACTION AND BIOACTIVITY
OF METABOLITES............................................................................................. 35
2.2.1.2 ETHNO-MEDICAL APPLICATION OF T1 IE GENUS SENECIO............. 36
2.4 PHYTOCHEMISTRY OF THE GENUS DODONAEA......................................... 44
2.4.1 COMPOUNDS FROM DODONAEA SPECIES...................................................44
2.4.1.1 DITERPENES FROM DODONAEA SPECIES.................................................44
2.4.1.2 TRITERPENES FROM DODONAEA SPECIES...............................................47
2.4.1.3 SHIKIMATE DERIVED METABOLITES FROM DODONAEA SPECIES.48
2.4.1.4. FLAVONOIDS FROM DODONAEA SPECIES........................................... 49
2.4.1.4.1 FLAVANONES FROM THE GENUS DODONAEA.................................... 49
2.4.1.4.2 FLAVONES FROM THE GENUS DODONAEA.......................................... 50
2.4.2 GENERAL PHYTOCHEMICAL INFORMATION ON SENECIO SPECIES.51
2.4.2.1. PYRROLIZIDINE ALKALOIDS FROM SENECIO SPECIES...................52
2.4.2.2 SESQUITERPENOIDS FROM SEN EC NO SPECIES.......................................64
2.4.2.2.1 EREMOPHILANES SESQUITERPENOIDS............................................... 65
VI
 2A.2.2.2 FURANOEREMOPI1ILANES FROM SENECIO SPECIES......................73
2.4.2.2.3 CACALOL SESQUITERPENES FROM SENEC1 0 SPECIES...................79
2.4.2.2.4 BISABOLOL SESQUITERPENES FROM SENEC7 0 SPECIES...............82
.42.2.5 SECO AND ABEOEREMOPHILANES.......................................................86
u> to
.4.2.2.6 SIMPLE GERMACRANE SESQUITERPENOIDS.....................................89
2.4.2.2.7 OPLOPANE SESQUITERPENOIDS FROM SENEC IO SPECIES............93
2.4.2.2.8 EUDESMANE SESQULI ERPENOID...........................................................95
2.4.2.2.9 BENZOFURANOIDS FROM SENECIO SPECIES.....................................97

CHAPTER THREE.................................................................................................................... 100

RESUITS AND DISCUSSION..................................................................................... 100
3.0 PRELIMINARY BIOASSAY TEST RESULTS................................................... 100
3.1 COMPOUNDS FROM DODONAEA ANGUSTIFOLIA- NGONG FOREST.... 103
3.1.1. 5-HYDROXY-3,7,4'-TRIMETHOXYFLAVONE (1 )...................................... 103
3.1.2. 3.5-DIHYDROXY-7,4',-DIMETHOXYFLAVONE(2)................................... 106
3.1.3. KUMATAKENIN (3)............................................................................................108
3.1.4. SANTIN (4 )............................................................................................................ I l l
3.1.5. RHAMNOCITRIN (5)...........................................................................................113
3.1.6. ISOKAEMPFERIDE (6)....................................................................................... 115
3 .1.7. 6-METHOXYKAEMPFEROL (7)....................................................................... 118
3.1.8. PINOCEMBRIN (8)...............................................................................................120
3.1.9. 2P- HYDROXYHARDWICKIIC ACID (9)....................................................... 121
3.1.10. DODONIC ACID (10).........................................................................................125
3.1. 11. ENT-3 ft, 8a; 15,16-EPOXY-13(16), 14-LABDA DIENE 3, 8-DIOL (11).126
3.2 COMPOUNDS FROM DODONAEA ANG USTIFOUA-NO\.............................127
3.2.1. PENDULETIN (12)................................................................................................128
3.2.2. AYANIN (13)..........................................................................................................130
3.2.3. 5-HYDROXY-3,6,7,4'-TETRAMETHOXYFLAVONE (1 4 )..........................132
3.2.4. KAEMPFEROL (15)..............................................................................................134
3.2.5. SCOPOLETIN( 16)................................................................................................ 136
3.2.6. HAUTRIWAIC ACID (17).................................................................................. 137
3.2.7. VTOCLERODAN-3,13-D1EN-16.15: 18.19-DIOI.IDE (18)............................140
3.2.8. 15P-N£OCLERODAN-3J3-DIEN-16.l5: 18,19-DIOLIDI (19) AND 15a-
V£OCLERODAN-3,13-DIEN-16,15: 18,19-DIOLIDE (2 0 )......................... 144
3.2.9. PROPOSED BIOGENESIS OF COMPOUNDS 18. 19 AND 2 0 .................... 147
3.3 COMPOUNDS FROM SENECIO ROSEIFLORUS..............................................150
3.3.1. 5, 7-DIHYDROXY-3,4 '- DIMETHOXYFLAVONE (21)................................ 150
3.3.2. QUERCETIN-3, 4 ’-DIMETHYL ETHER (2 2 )................................................. 152
3.3.3. RHAMNAZIN (2 3 )...............................................................................................154
3.3.4. RETUSIN (24)........................................................................................................156
3.3.5. 5. 4 -DIHYDROXY-7-METHOXYFLAVANONE (25)................................... 158
3.3.6. METHYLPARABEN (26).................................................................................... 161
3.3.7. 5.4'-DIHYDROXY-3,7,3'-TRIMETHOXYFLAVONE ( 2 7 ) ...........................162
3.4 CIIEMOTAXONOMIC SIGNIFICANCE OF THE, ISO! A I I D COMPOUNDS
................................................................................................................................165
3.4.1 DODONAEA ANGUSTIFOLIA (FROM NGONG FORES I AND VOI)....... 165
3.4.2 SENECIO ROSEIFLORUS.....................................................................................169

3.5 BIOLOGICAL ACTIVITIES............................................................................ 170

3.5.1 ANTIPLASMOD1AL TESTS...............................................................................170
3.5.1.1 ANTI-PLASMODIAL ACTIVITY OF D. ANGUSTIFOU. -NGONG
FOREST............................................................................................................... 171
3.5.1.2 ANTI-PLASMODIAL ACTIVITIES OF D. ANGUSTIFOl IA-\IO I.............171
VII
 3.5.1.3 ANTI-PLASMODIAL ACTIVITIES OF SENECIO ROSE1FLORUS......172
3.5.4 ANTI-MICROBIAL ACTIVITY......................................................................... 172
3.5.2 LARVICIDAL ACTIVITY.................................................................................177
3.5.2.1 LARVICIDAL ACTIVITY OF D. ANGUSTIFOLIA-NGONG FOREST AND
ITS COMPOUNDS............................................................................................ 177
3.5.2.2 LARVICIDAL ACTIVITIES OF D. ANGUSTlFOLIA-NO\ AND ITS
COMPOUNDS.....................................................................................................180
3.5.2.3 LARVICIDAL ACTIVITIES OF SENECIO ROSEIFLORUS AND ITS
COMPOUNDS.....................................................................................................182
3.5.3 ANTI-OXIDANT ACTIVITIES.........................................................................182
3.5.3.1 ANTI-OXIDANT ACTIVITIES OF DODONAEA A NG ( IS/ IFOLIA -N GON G
FOREST................................................................................................................ 182
3.5.3.2 ANTI-OXIDANT ACTIVITIES OF DODONAEA ANGUSTIFOLIA-NO\.. 184
3.5.3.3 ANTI-OXIDANT ACTIVITIES OF SENECIO ROSEIFLORUS................... 184

CHAPTER FOUR......................................................................................................................186
CONCLUSIONS AND RECOMMENDATIONS........................................................186
4.1 CONCLUSIONS......................................................................................................... 186
4.2 RECOMMENDATIONS........................................................................................... 188

CHAPTER F IV E ......................................................................................................................... 190

EXPERIMENTAL............................................................................................................ 190
5.0 GENERAL...................................................................................................................190
5.1 PLANT MATERIALS............................................................................................... 190
5.1.1 DODONAEA ANG USTIFOLIA (NGONG FOREST)..........................................190
5.1.2 DODONAEA A NG USTIFOLIA (VOI).................................................................. 190
5.1.3 SENECIO ROSEIFLORUS.....................................................................................190
5.2 EXTRACTION AND ISOLATION OF COMPOUNDS.................................... 191

5.2.1 DODONAE AN G U STIEO U A (NGONG F O R E ST )........................................191

5.2.1.1 EXTRACTION AND ISOLATION OF C OMPOUNDS FROM FRESH
LEAVES (AERIAL PARTS)............................................................................. 191
5.2.1.2 PHYSICAL AND SPECTROSCOPIC PROPERTIESOF COMPOUNDS OF
D. A NG USTIFOLIA FROM NGONG FOREST............................................ 192
5-Hydroxy-3,7,4'-trimethoxyJlavone ( I ) ............................................................ 192
3,5-Dihydroxy- 7 , 4 -dimethoxyflavone (2).........................................................192
5,4'-Dihydroxy-3,7-dimethoxyflavone (kumatakenin) (3)................................ 192
5.7- Dihydroxy-3,6,4'-trimethoxyflavone (santin) (4).................................... 192
3,5,4'-Trihydroxy-7-methoxyflavone (rhamnocitrin) (5 )................................. 193
5,7,4\-Trihydroxy-3-methoxyflavone (isokaempferide) (6)............................ 193
3,5,7,4'-tetrahydroxy-6-methoxyflavorie (6-methoxykaempJerol) (7)..............193
5.7- Dihydroxyflavanone (pinocembrin) (8)................................................... 193
2(3-Hydroxy-15,16-epoxy-5 (3,8/3,9/3,1Oa-cleroda-3,13(16), 1 Ftrien-18-oic
acid. 2(3- hydroxyhardwickiic acid (9)................................................................193
15,16-epoxy-6-hydroxy-3,13(16), 14-clerodatrien-18-oic acid. (Dodonic acid)
(10).........................................................................................................................193
ent-3/3, 8 a -15,16-Epoxy-13(16). 14-labdadienc-3, 8-diol ( 11) ..................... 193

5 .2.2 DODONAE ANGUSTIEOUA (V O \)................................................................. 194

5.2.2.1 EXTRACTION AND ISOLATION OF COMPOUNDS FROM FRESH
LEAVES (AERIAL PARTS)............................................................................. 194
5.2.2.2 ISOLATION AND PURIFICATION OF SURFACE COMPOUNDS FROM
DODONAEA ANGUSTIEOUA (V O I).............................................................. 194
viii
 5.2.2.3 PHYSICAL AND SPECTROSCOPIC PROPERTIES OF C OMPOUNDS
FROM D. ANGUSTIFOLIA FROM VOI.......................................................... 195
5,4'-Dihydroxy-3,6,7-trimehtoxyflavone (penduletin)(12)............................... 195
5,3'-Dihydroxy-3, 4', 7-trimethoxyflaxone (ayanin) (13)..................................195
5-Hydroxy-3,6,7,4'-tetramethoxyflavone (14)................................................... 195
3,5,7,4'-Tetrahydroxy-flavone (kaempferol) (15).............................................196
7-Hydroxy-6-methoxycoumarin (1 6 )................................................................. 196
ent-15,16-Epoxy-19-hydroxy-3,13(16), 14-clerodalrien-18~oic acid (hautriwaic
acid) (17)...............................................................................................................196
Neoclerodan-3,13-dien-16,15: 18,19-diolide (18)............................................196
/ 5(3-Neoclerodan-3,l 3-dien-16,l 5: 18,19-diolide (19) and I 5 a-neoclerodan-
3,13-dien-16,l 5: 18,19-diolide (20)....................................................................196

2.3 SENECIO R O SE IF LO R U S............................................................................... 196

5.2.3.1 EXTRACTION AND ISOLATION FROM FRESH LEAVES (AERIAL
PARTS)................................................................................................................. 196
5.2.3.2 ISOLATION AND PURIFICATION OF SURFACE COMPOUNDS FROM
SENECIO ROSEIFLORUS..................................................................................197
5.2.3.3 PHYSICAL AND SPECTROSCOPIC PROPERTIES 01 COMPOUNDS
FROM SENECIO ROSEIFLORUS.....................................................................198
5, 7-Dihydroxy-3, 4'-dimethoxyflavone (21)..................................................... 198
3',5,7-Trihydroxy-3, 4'-dimethoxyflavone (quercetin-3, 4'-dimethyl ether) (22)
................................................................................................................................ 198
3, 4', 5-Trihydroxy-3', 7-dimethoxyflavone (rhamnazin) (2 3 )...................... 198
5-Hydroxy-3,7,3',4'-tetramethoxyflavone (retusin) (24)..................................198
5,4'-Dihydroxy-7-methoxyflavanone (25)..........................................................198
4- Hydroxy benzoic acd methyl ester (2 6 )...........................................................198

5.4 BIOLOGICAL ACTIVITY STU D IES................................................................ 199

5.4.1 IN VITRO ANTI-PLASMODIAL TEST.............................................................. 199
5.4.2 LARVICIDAL ACTIVITY ASSAY.....................................................................199
5.4.3 RADICAL SCAVENGING TEST USING DPPH............................................. 200
5.4.4 ANTI-MICROBIAL TEST.................................................................................... 200

CHAPTER SIX ............................................................................................................................202

6.1 REFERENCES.........................................................................................................202

IX
 LIST OF TABLES

Table 2.1: Morphological differences of D.viscosa and I). angustifolia..................................27

Table 2.2: Ethno-botanical uses of Dodonaea species.............................................................. 30
Table 2.3: Ethno-medical application o fSenecio species..........................................................37
Table 2.4: Labdane and clerodane type diterpenoids from Dodonaea species.........................45
Table 2.6: Pyrrolizidine alkaloids from Senecio species............................................................52
Table 2.7: Eremophilanoides o f Senecio species........................................................................66
Table 2.8: Furanoeremophilanoides from Senecio species......................................................... 73
Table 2.9: Cacalol sesquiterpenes from Senecio species............................................................ 79
Table 2.10: Bisabolol sesquiterpenes from Senecio species.......................................................83
Table 2.11: Seco and Abeoeremophilanes sesquiterpenes from Senecio species.....................87
Table 2.12: Simple Germacrane sesquiterpenoids from Senecio species................................. 90
Table 2.13: Oplopane sesquiterpenoids from Senecio species...................................................94
Table 2.14: Simple Eudesmane from Senecio species.................................................................96
Table 2.15: Benzofuranoids from Senecio species.......................................................................98
Table 3.1: Anti-plasmodial activity of plant extracts................................................................. 100
Table 3.2: ID (CDC13: 300. 75.5 MHz) and 2D NMR data for 5-1 Iydroxy 3,7,4'-
trimethoxyflavone ( 1 ) ................................................................................................ 106
Table 3.3: ID (CDCI3: 300, 75.5 MHz) and 2D NMR data for 3,5-dihydroxy-7,4'-
dimethoxyflavone (2 )................................................................................................. 107
Table 3.4: ID (CI)CI3: 300. 75.5 MHz) and 2D NMR data for 5,4'-di hydroxy-3,7-
dimethoxyflavone (3 )................................................................................................. 110
Table 3.5: ID (CDC13: 300, 75.5 MHz) and 2D NMR data for 5,7-dihydroxy-3, 6, 4'-
trimethoxyflavone ( 4 ) ................................................................................................ 112
Table 3.6: ID (CD3OD: 300, 75.5 MHz) and 2D NMR data for 5,4'-dihydroxy-7-
dimethoxyflavonol ( 5 ) ............................................................................................... 1 15
Table 3.7: ID (CDC13: 300, 75.5 MHz)and 2D NMR data for isokaempferide (6)................117
Table 3.8: ID (CD3OD: 300, 75.5 MHz) and 2D NMRdata for 6-methoxykaempferol (7). 120
Table 3.9: 'll ((CD3)2CO, 200 MHz) and l3C(200 MHz) NMR for pinocembrin (8).............121
2(3-Hydroxyhardwickiic acid (9)............................................................................... 124
Table 3.10: ID (CDC13: 300, 75.5 MHz) and 2D NMR data for 2(3-hydro\yhardwickiic acid
(9).................................................................................................................................124
Table 3.11: ID(CDCI3: 300, 75.5 MHz) and 2D NMR data for penduletin (12)................... 130
Table 3.12: ID (CDCI3: 300, 75.5 MHz) and 2D NMR data for ayanin (13)......................... 132
Table 3 .13: ID (CDC13: 300, 75.5 MHz) and 2D NMR data for 5-hydroxy 3,6,7,4'-
tetramethoxyflavone (14)...........................................................................................134
Table 3.14: ID (CDCI3: 300. 75.5 MHz) and2D NMR data for kaempferol (1 5 ).................. 136
Table 3.15: ’ll (Acetone-deat 300 MHz) and l3C (75 MHz) NMR chemical shift data for 7-
hydroxy-6-methoxycournarin(16)............................................................................. 137
Table 3.16: 'l l (MeODat 300 MHz) and 13C (75 MHz) NMR chemical shift data for
hautriwaic acid (17)................................................................................................... 140
Table 3.17: ID (CDC13: 300. 75.5 MHz) and 2D NMR data for «eoclerodan-3,13-dien-16,15:
18.19-diolide (18)....................................................................................................... 143
Table 3.18: ID (CDC13: 300. 75.5 MHz) and 2D NMR data for the cpimcric mixture of 15P-
ra?oclerodan-3,13-dien-16,15: 18.19-diolidc (19) and 15a-«eoclerodan-3,13-d ien-
16,15: 18,19-diolide (2 0 ).......................................................................................... 147
Table 3.19: ID (CD3OD: 300, 75.5 MHz) and 2D NMR data for 5,7-dihydroxy-3, 4'-
dimethoxyflavone (21).............................................................................................. 151
fable 3.20: ID (CDCI3: 300, 75.5 MHz) and 2D NMR data for quercetin 3. 4'-dimethyl ether
(22)................................................................................................................................................. 153
x
 LIST OF FIGURES
Figure 2 .1: Distribution of D. angustifolia in Kenya.................................................................. 23
Figure 2.2: Distribution of D. viscosa in Kenya.......................................................................... 23
Figure 2.3: Dodonaea viscosa female flowers............................................................................. 24
Figure 2.4: Dodonaea viscosa male flowers................................................................................ 24
Figure 2.5: Dodonaea angustifolia shrub.....................................................................................25
Figure 2.6: Dodonaea angustifolia tree........................................................................................25
Figure 2.7: Fruits of D. angustifolia............................................................................................. 26
Figure 2.8: Representative necine bases........................................................................................52
Figure 3.1: TLC profiles of extracts of D. angustifolia from different populations...............102
Figure 3.2: NOESY interactions in 5-hydroxy-3,7,4'-trimethoxyflavone ( 1 ) ........................ 105
Figure 3.3: HMBC and HMQC o f 3,5-dihydroxy-7,4'-dimethoxyflavonc (2)........................108
Figure 3.4: HMBC and HMQC correlations for kumatakenin (3)............................................11 I
Figure 3.5: NOE correlation o f santin (4)....................................................................................113
Figure 3.6: HMBC correlations o f 5,4'-dihydroxy-7-dimethoxyflavonol (5)......................... 114
Figure 3.7: NOE correlations of the methoxy group o f 5.4'-dihydroxy-7-dimethoxyflavonol
(5)................................................................................................................................. 115
Figure 3.8: HMBC correlation of H-6 and H-8 of isokaempferide (6).....................................117
Figure 3.9: HMBC correlations in 3,5,7,4'-tetrahydroxy-6-methoxyflavonol (12)................ 129
Figure 3.10: HMBC correlations of ayanin (13).........................................................................131
Figure 3.11: HMBC of 5-hydroxy-3,6.7.4'-tetramethoxyflavone (14).................................... 133
Figure 3.12: HMBC correlations of kaempferol (15)................................................................ 135
Figure 3.13: HMBC of compound «eoclerodan-3,13-dien-16,l5: 18,19-diolide (18)............143
Figure 3.14: HMBC correlations of 15P-/7eoclerodan-3,13-dien-16,15: 18.19-diolide (19). 146
Figure 3.15: HMBC correlations of 15a-«eoclerodan-3,13-dien-16,15: 18,19-diolide (20) 146
Figure 3.16: 5.7-dihydroxy-3,4'-dimethoxyflavone (21)...........................................................152
Figure 3.17: NOE interactions of compound quercetin-3, 4'-dimethyl ether (22).................. 153
Figure 3.18: HMBC correlations of rhamnazin (2 3 )..................................................................155
Figure 3.19: NOE correlations of rhamnazin (23)......................................................................155
Figure 3.20: HMBC correlations of 5,4'-dihydroxy-3,7,3'-trimethoxyflavonc (27)...............165
Figure 3.21: Larvicidal activity of the extracts of Dodonaea angustifolia-Ngong Forest on the
second instar Aedes aegypti larvae........................................................................178
Figure 3.22: I.arvicidal activity of 3,5.4'-trihydroxy-7-methoxyflavone (5) on the second
instar Aedes aegypti larvae.......................................................................................179
Figure 3.23: I.arvicidal activity of the extracts of D. angustifolia (Ngong forest) and some of
its compounds after 24 hours.................................................................................. 179
Figure3.24: Larvicidal activity of hautriwaic acid on the second in star Aedes aegypti larvae
........................................................................................................................................................ 180
Figure 3.25: Larvicidal activity of hautriwaic acid lactone on the second instar larvae of
Aedes aegypti.......................................................................................................... 181
Figure 3.26: Larvicidal activity of extracts of Senecio roseiflorus on the second Aedes aegypti
instar larvae............................................................................................................. 182
Figure 3.27: Radical scavenging activity of the standard (quercetin), rhamnocitrin (5),
kaempferol (15) and quercetin-3, 4 ’-dimethyl ether (22)....................................185

xii
 LIST OF SCHEMES
Scheme 3.1: MS fragmentation pattern of 15-hydroxy-3,7,4'-trimethoxyflavone (1)...........104
Scheme 3.2: Fragmentation pattern of kumatakenin (3)...........................................................109
Scheme 3.3: Fragmentation pattern of isokaempferide (6)...................................................... 116
Scheme 3.4: Fragmentation pattern of 6-methoxykaempferol (7)...........................................118
Scheme 3.5: Fragmentation pattern of 5,4'-dihydroxy-7-methoxyflavanonc (25).................160
Scheme 3.6: Fragmentation pattern of 5,4'-dihydroxy-3,7,3'-trimethoxyflavone (27)..........163

xiii
 APPENDICES
APPENDIX I: SPECTRA FOR COMPOUND 1 ......................................................................225
APPENDIX II: SPECTRA FOR COMPOUND 2 .....................................................................231
APPENDIX III: SPECTRA FOR COMPOUND 3 ...................................................................236
APPENDIX IV: SPECTRA FOR COMPOUND 4 ...................................................................241
APPENDIX V: SPECTRA FOR COMPOUND 5.....................................................................245
APPENDIX VI: SPECTRA FOR COMPOUND 6 ...................................................................251
APPENDIX VII: SPECTRA FOR COMPOUND 7 ..................................................................257
APPENDIX VIII: SPECTRA FOR COMPOUND 8.................................................................263
APPENDIX IX: SPECTRA FOR COMPOUND 9 ...................................................................266
APPENDIX X: SPECTRA FOR COMPOUND 10...................................................................272
APPENDIX XI: SPECTRA FOR COMPOUND 1 1 .................................................................277
APPENDIX XII: SPECTRA FOR COMPOUND 12............................................................... 281
APPENDIX XIII: SPECTRA FOR COMPOUND 13.............................................................. 287
APPENDIX XIV: SPECTRA FOR COMPOUND 1 4 ............................................................. 293
APPENDIX XV: SPECTRA FOR COMPOUND 15................................................................299
APPENDIX XVI: SPECTRA FOR COMPOUND 1 6 ............................................................. 305
APPENDIX XVII: SPECTRA FOR COMPOUND 1 7 ............................................................ 309
APPENDIX XVIII: SPECTRA FOR COMPOUND 18........................................................... 315
APPENDIX XIX: SPECTRA FOR COMPOUND 19 AND 20...............................................322
APPENDIX XX: SPECTRA FOR COMPOUND 21................................................................329
APPENDIX XXI: SPECTRA FOR COMPOUND 22 ..............................................................335
APPENDIX XXII: SPECTRA FOR COMPOUND 2 3 ............................................................ 341
APPENDIX XXIII: SPECTRA FOR COMPOUND 2 4........................................................... 347
APPENDIX XXIV: SPECTRA FOR COMPOUND 25 .......................................................... 352
APPENDIX XXV: SPECTRA FOR COMPOUND 2 6 .............................................................357
APPENDIX XXVI: SPECTRA FOR COMPOUND 27 .......................................................... 362

X IV
LIST OF ABBREVIATIONS AND SYMBOLS

APT Attached proton test IZ Inhibition /.one

brs Broad singlet ./ Coupling constant

CC Column chromatography LC50 Concentration of 50% lethality

CDC13 Deuterated chloroform Lit. Literature

CH2CI2 Dichloromethane MeOH Methanol

,3c -n m r Carbon NMR MHz Mega Hert/

d Doublet MS Mass Spectroscopy

dd Doublet o f doublet m Multiplet (multiplicity)

DMSO Dimethyl sulphoxide [M f Molecular ion

COSY Correlation Spectroscopy m/z Mass to charge ratio

DEPT Distortionless Enhancement by NOE Nuclear Ovcrhauser Effect

Polarization Transfer

DPPH Diphenyl picryl hydrazine NOESY Nuclear Ovcrhauser and

Exchange Spectroscopy

EIMS Electron Ionization Mass NMR Nuclear Magnetic Resonance

Spectroscopy

6 Chemical shift PTLC Preparative Thin Layer

Chromatography
HPLC High Perfomance Liquid ^max Maximum wavelength of
Chromatography absorption

HPTLC High Perfomance Thin Layer s Singlet

Chromatography

HMBC Heteronuclear Multiple Bond t Triplet

Correlation (2Jch, 3Jch)

HMQC 1leteronuclear Multiple TLC Thin Layer Chromatography

Quantum Coherence ('.leu)

'H N M R Proton NMR UV Ultra Violent

Hz Hertz mp** The melting point could not be
determined due to the low
yields of the compound
IC50 Concentration of 50%
inhibition

XV
LIST OF SUB STRUCTURES

Sent* =

() o

3-M e-2-lniten =

2-Hyd roxyme-2-buten

2,3-Dihydroxy-2-methylbu

3-Acetoxy-2-hvdroxy-2-mebu OH
3-Acetoxy-2,2-dihydroxy bu =
O
 o
5-Tigloyloxy-2Z-hexenoyl =

()

4-Hex-2Z-enoyloxy-2Z-hexenoyl

4-Angeloyloxy-2Z-hexenoyl =

5-Angeloyloxy-2Z-hexenoyl

0-2-(Acetoxymethyl)-2-buten) (Z) =

O O

XVI I
 ABSTRACT
The phytochemistry o f the surface exudates of Dodonaea angustifolia (from two locations
Ngong forest and Voi) and Senecio roseiflorus, as well as the antiplasmodial activity against
chloroquine-sensitive (D6) and chloroquine-resistant (W2) strain of Plasmodium falciparum,
for some of the isolated compounds, larvicidal activity against Aedes aegypti larvae, anti­
microbial activity against three strains of bacteria: Staphyloccocus aureus (ATCC29737),
Escherichia coli (ATCC25922) and Bacillus pumilus (local strain) and a local strain of
fungus, Saccharamyces cerevisiae and radical scavenging activity towards l,l-diphenyl-2-
picrylhydrazyl (DPPH) were carried out. The structures of the isolated compounds were
determined using a combination o f spectroscopic techniques |NMR, MS and UV].

The surface exudates o f the fresh leaves of D. angustifolia growing in Ngong were extracted
by successive dipping into fresh portions o f acetone for short periods (15 seconds). The
surface exudates showed anti-plasmodial activity with IC50 values of 4 1.5 1 3.9 pg/ml against
chloroquine-sensitive (D6) strain of the P. falciparum. The larvicidal activity of this extract
was not good, as its LC50 value against the larvae o ['Aedes aegypti was > 60 gg/ml after 24
hours. The radical scavenging activity (RSA) of the extract towards DPPI1 was 54.6% at 11.4
pg/ml. The surface exudates showed activity against Staphyloccocus aureus (ATCC29737),
Escherichia coli (ATCC25922) bacteria and Saccharamyces cerevisiae fungus.

The extract was subjected to a combination of chromatographic techniques leading to

isolation of the flavonoids, 5-hydroxy-3,7,4'-trimethoxyflavonc (1). 3,5-dihydroxy-7,4',-
dimethoxyflavone (2). kumatakenin (3), santin (4), rhamnocilrin (5). isokaempferide (6), 6-
methoxykaempferol (7), and pinocembrin (8); and the diterpenoids, 2\\ hydroxyhardwickic
acid (9), dodonic acid (10) and £«/-3p,8a; 15,16-epoxy-13(16), 14-labdadiene-3,8-diol (11).
All the compounds except santin (4) are reported from D. angustifolia for the first time from
this plant. The flavonoids. 5-hydroxy-3,7,4'-trimethoxyflavone ( 1) . 3,5-dihydroxy-7,4',-
dimethoxyflavone (2), kumatakenin (3). rhamnocitrin (5) were also isolated from the two
collections o f D. angustifolia (Taita hill near Voi town). However, the rest of the compounds
w'ere present only from the D. angustifolia (Ngong forest) reflecting geographical variability
in this species.

Most of the isolated compounds showed moderate anti-plasmodial activity against the D6
strain of Plasmodium falciparum with IC50 values between 7.6 ) 2.3 for kumatakenin and
18.4 ± 4.8 for 6-methoxykaempferol. Among the compounds tested for larvicidal activity
against Aedes aegypti; rhamnocitrin (5) and santin (4) showed good dose dependent activity
with an LC50 value of 1.75 and 5.1 pg/ml, respectively, after 24 hours. Some of the isolated
flavonoids showed radical scavenging activity at 50 pM with rhamnocitrin (5) showing an
activity o f 96.2% followed by 3,5-dihydroxy-7,4'-dimethoxyf1avonc (2) with an activity of
25.5%.

Compounds 1, 3, 4, 5, 8, 9 and 10 were tested for anti-microbial activity. Compounds 5, 8, 9

and 10, were active against Staphylococcus aureus. A number of compounds 4, 5, 8, 9 and 10
were identified as the active principles of the extracts against Bacillus pumilus. Compounds
4, 8, 9, 10 were active against the local strain of fungus, Saccharomyces cerevisiae with
santin (4) being the most active having an inhibition zone of 11.15 mm at 31.25 pg/ml.

The fresh leaves of D. angustifolia growing in Voi were washed off the exudate in a similar
way and tested for anti-plasmodial activity. The surface exudates showed moderate anti-
plasmodial activity with IC50 values of 56.3 ± 4.2 pg/ml against chloroquine-sensitive (D6)
strain of the P. falciparum. This is comparable with the activity o f the extract of the variety
growing in Ngong forest. The radical scavenging activity (RSA) of the crude extract at I 1.4
pg/ml was found to be 34.7% lower than the extract from D. angustifolia growing in Ngong
forest. The surface exudates showed activity against all the three strains of bacteria and one
strain of fungus.

Chromatographic separation o f this extract gave the flavonoids 1, 2, 3 rhamnocitrin (5),

penduletin (12). ayanin (13), 5-hydroxy-3,6,7,4'-tetramethoxyflavone (14), and kaempferol
(15) ; the shikimate acid derivative 7-hydroxy-6-methoxycoumarin (16); and the diterpenoids
hautriwaic acid (17). 15a-methoxy-/7co-clerodan-3,13-diene-18.19 :16.15 diolide (18), 15(3-
methoxy-/7co-clerodan-3,13-diene-18 .19 :16 ,15-diolide (19) and w'o-clerodane-3,13-dien-
18.19:16,15-diolide (20).

The flavonoids. 12, 13, 14, 15; the shikimate acid derivative 7-hydrox) 6 methoxycoumarin
(16) ; and the diterpenoids 17, 15a-methoxy-«eo-clerodan-3,13-diene 18.19:16,15-diolide
(18), 15(3-methoxy-«eo-clerodan-3,l3-diene-18,19:16,15-diolide (19) and weo-clerodane-
3,13-dien-l 8 .19:16.15-diolide (20) were only isolated from D. angustifolia collected from
Taita hills near Voi town.

xix
Among the compounds isolated the flavonoids, 12, 13, 14, 15: the coumarin 16 and the
diterpenoid 17 have not been previously described from this plant. However, most of these
compounds except 13 and 16 have been reported from other Dodonaea species. This is,
however, the first report of 18, 19 and 20 in nature.

Hautriwaic acid (17) showed antiplasmodial activities against chloroquine-sensitive (D6)

strains o f Plasmodium falciparum with an IC50 value of 10.2 pg/ml. Its activity was
compared with that of its lactone, which had IC50 values o f 23.6 » 2.6 and 23.0 ± 2.3 fig/ml
against chloroquine-sensitive (D6) and resistant (W2) strains of Plasmodium falciparum
respectively. ). Hautriwaic acid (17) showed good larvicidal activity ( L ( \0 10.2 j_ig/inl, after
24 hours) against Aedes aegy’pti larvae, while its lactone had an LC50 value > 100 pg/ml after
24 hours and therefore inactive. The radical scavenging activity of kaempferol (15) was
found to be the highest w ith % RSA of 96.8 at 50 pM comparable to that o f quercetin at this
concentration.

Only two compounds 12 and 17 were tested for activity anti-microbial activity. Compound 12
showed activity against Bacillus pumilus (local strain) and Saccharomyces cerevisiae (local
strain) Hautriwaic acid (17) showed activity against two strains o f bacteria, Staphylococcus
aureus (ATCC 29737), Bacillus pumilus (local strain) and one strain of Saccharomyces
cerevisiae (local strain) no activity against E. coli was observed for these compounds.

The surface exudates of the leaves of Senecio roseijlorus was extracted similarly. The extract
showed an antiplasmodial activity with IC50 values o f 90.0 \ 9.8 pg/ml against chloroquine-
sensitive (D6) strain o f Plasmodium falciparum. Anti-microbial activity against the three
strains of bacteria and one strain of fungus was observed with the surface exudates.

The extract was then subjected to a combination of chromatographic techniques leading to

the isolation of the flavonoids 1, 2, 3, 5. 6, 14, 5,7-dihydroxy-3,4'-dimethoxyflavone (21),
quercetin-3,4'-dimethyl ether (22), rhamnazin (23). retusin (24). 5,4'-dihydroxy-7-
dimethoxyflavanone (25). 5,4'-dihydroxy-3,7,3'-trimethoxyflavone (27), 3,5-Dihydoxy-
3',4',7-trimethoxyflavone (28) and the phenol, 4-hydroxy-methylben/oate (26). All these
compounds had not been isolated previously from this plant.

The flavanone, 5,4'-dihydroxy-7-methoxyflavanone(25) is the most potent against

chloroquine-sensitive (D6) and resistant (W2) strains of Plasmodium falciparum, with IC50

xx
values o f 3.2 ± 0.8 and 4.4 ± 0.01 pg/ml respectively. The other compounds showed
moderate activities. The extract did not show good larvicidal activity against the larvae of
Aedes aegypli, as its LC50 values was >100 pg/ml after 24 hours. The llavonoids, 25 and 21
showed moderate and dose dependent activity with an LC50 value o f 14.3 and 15.5pg/ml
respectively. The highest RSA activity was observed in quercetin-3,4'-dimethyl ether with %
RSA of 77.1 at 50 pM. however, the activity of quercetin was higher.

Compound 22 showed activity against the three strains of bacteria; Escherichia coli,
Staphylococcus aureus and Bacillus pumilus and no activity against the fungus. The
flavanone, 5. showed activity against Staphylococcus aureus (ATCC 29737), Bacillus
pumilus (local strain) and Saccharomyces cerevisiae (local strain).

xxi
 CHAPTER ONE

INTRODUCTION

1.0 GENERAL

A substantial fraction o f the world's population continues to use natural products, especially

medical plant extracts to control infectious diseases and combat pests. Today, the

pharmaceutical, food, beverage, flavour and fragrance companies have invested a lot of

resources in the development o f products that incorporate ingredients from plant sources, and

have acquired considerable wealth from them.

The use of plants for the treatment of disease goes back to early man. Before development of

synthetic chemistry in the nineteenth century, nearly 80% of all medicines were obtained

from plant materials [Farnsworth, I985J. Most developing countries are endowed with vast

resources o f medicinal and aromatic plants. These plants have been used over the millennia

for human welfare in the promotion of health and as drugs and fragrance materials. This close

relationship between man and the environment continues even today since a large proportion

of people in the developing countries still live in rural areas. Furthermore, these people are

excluded from the luxury of access to modern therapy, mainly for economic and cultural

reasons. In some countries where modern drugs are available, people still use the long

traditional socio-cultural practices. Presently many people in developing countries, especially

those in the sub-Saharan Africa, still depend on plant sources for their primary health care

needs.

In a number of countries such as India, China, Sri Lanka and Australia, pharmaceutical

companies are already marketing preparations of tablets, suspensions and capsules made

directly from plant extracts for the treatment of specific diseases c.g. hepatitis, malaria,

cancer, allergy and AIDS [Abrams, 1990; Decosterd et al., 1991 ].

Plants and other organisms have been recognised as being the most efllcient synthetic tools,

capable o f making a diversity of organic molecules (natural products that have complex

structures and a variety of physical, chemical and biological properties). Most of these

complex structures serve as templates (models) for the development of more effective

synthetic agents. Some of these natural products may have complex chemical structures, and

therefore may not be easily synthesised in the laboratory. For example, the two anti-cancer

agents vinblastine (27) and vincristine (28) isolated from the Madagascan periwinkle,

Catharantus roseus (Farnsworth, 1990; Cragg & Newman, 1997) are among the most widely

used plant-derived natural products in the pharmaceutical industry and have not been

substituted by new synthetic compounds. In fact, approximately 60 % o f the available anti­

cancer chemotherapeutic drugs are of plant origin [Kinghorn el a/., 19991. Similarly 25% of

the available modern anti-malarial drugs are of plant origin. Quinine (29) [Warhurst el al.,

2003J, which was initially obtained from the species o f Cinchona originating from South

America, remains a vital drug in the treatment of malaria. Except for anti folate anti-malarial

drugs, all the other commonly used anti-malarial compounds are based plant derived

compounds [Geoffrey, 1996]. Recent examples of development of drugs of plant origin

include the new anti-malarial agent artemisin (30) |Mueller el al., 2000] from Artemisia

annua and the anti-cancer drug taxol (31) [Strobel el a/., 1992: Sticrle el al., 1993; Kim el al.,

1999; Kumaran el al., 2008a; Kumaran el al., 2008b] from some Yew species. Many of the

structures discovered for the first time serve as models for the synthesis of biologically active

compounds and have promoted research into the activity of analogous structures.

2
 29

()

H =

The development of scientific knowledge with the present day environmental awareness,

selective and biodegradable compounds must replace the highly toxic and persistent

chemicals in the environment. For this reason interest on insecticides and pesticides of plant

origin is being revived. The most important pesticides of plant origin are pyrethrin (32),

rotenone (66) and nicotine (33) [Curtis el al., 19901.

3
 ()
if
32
()

33
o
There is a trend in the World today to turn to natural substances due to various side effects by

some synthetic drugs. Currently, about 200 plant-derived chemical compounds of known

structures are being used as drugs or as agents that lead to improvement of human health

[Farnsworth & Saejarto, 1991]. In Europe, about 50% and in the US more than 25% of all the

prescriptions dispensed from pharmacies have their origin in plants. It is estimated that the

trade in herbal medicines reached approximately US$500 billion by the year 2000 [Craig,

1999].

During the past decade, the World Health Organisation (WHO) and the governments in

developing countries have been campaigning for the promotion and integration of herbal

remedies in health care, as supplementary contribution to modern medical facilities especially

in rural areas where modern health facilities and resources are inadequate or completely

unavailable and if they are. they often prove to be too expensive.

1.1 Plant Resins

Plant resins, are lipid-soluble mixture of volatile and non-volatile terpenoid and/or phenolic

secondary compounds that are usually secreted in specialized structures located either

internally or on the surface of the plant and are of potential significance in ecological

interactions.

4
Terpenoid resin occurs in most conifer families but is widely scattered among the major

evolutionary lineages of Angiosperms. Specific terpenoid skeletal types, however, often

characterize taxa such as particular families and genera |Gershenzon & Mabry, 1983].

Conifers only produce internally secreted terpenoid resins whereas Angiosperms produce

both terpenoid and phenolic resins, which may be secreted internally or on the surface of the

plant. The complexity of the mixture of compounds constituting a resin is important for

ecological interactions. In general, among the 20-50 or more compounds that constitute a

resin, only a few occur in high concentration. Surface resins are usually eliminated from the

cellular sites of synthesis into different kinds of structures, and thence, exudation to the

outside of the plant with or without injury. There are two types of extracellular resinous

secretions, endogenous and exogenous secretions [Fahn, 1988]. Endogenous secretions

accumulates in various internal structures (canals, pockets or cysts, cavities), which

essentially are intercellular spaces surrounded by secretory cells. Normally, such material

only exudes from the plant when it is injured. Exogenous secretion, on the other hand, occurs

in various types of epidermal secretory cells (glandular hairs, bud trichomes) that may

discharge the material to the outside of the organ either directly or llrst into a sub-cuticular

space before further secretion. Resins that are secreted externally from these specialized

structures usually coat the surfaces of stipules (which sheathe leaf buds), young leaves stems

and /or the floral calyx.

1.1.1 Terpenoid Resins

The volatile fraction of some angiosperm resins, usually consists of mono- and/or

sesquiterpene hydrocarbons with some oxygenated forms and, occasionally, diterpene

hydrocarbons. Structures of some of the most common volatile monoterpenes in various

resins include campene (34), limonene (35), (3-myrcene (36), p-phdlandrene (37) and

5
sesquiterpenes common in various resins include a-bisabolene (38). b-cadinene (39), P-

caryophyllene (40), y-humulene (41), y-muurolene (42) 5-selinene (43).

Diterpenes are the dominant components in the nonvolatile fraction o f most angiosperm

resins. They form the very hard copals used for varnishes because of the presence of

labdadienc-type acids such as ozoic acid (47) (or alcohols). Angiosperm resins also contain

numerous other diterpenoids such as the clerodane-type hardvvickiic acid (48), labdane-type:

communic acid (46) and ozoic acid (47), abietane-type: abietic acid (44), pimarane-type:

6
pimaric acid (45) and trachylobane-type: trachylobanic acid (49) [Richmond & Ghisalberti,

1994].

44 45

COOH

In some angiosperm families, triterpenes rather than diterpenes dominate the non-volatile

composition of the resin. For example, triterpenes primarily with tctra- or pentacyclic

skeletons characterize resins from the tropical families Burseraceae, Dipterocarpaceae, and

Anacardiaceae. Resins from Burseraceae typically have pentacyclic lupanc (50), ursane (51).

and oleanane (52) triterpene skeletal types [Khalid, 1985], Triterpenes with dammarane type

skeleton such as dammaradienol (53) also characterize plant resin. The non-volatile fraction

increases the viscosity of the resin, w hich can enhance the possibility of engulfing herbivores

7
and other organisms visiting the tree. I'he relative proportion of volatile to nonvolatile

compounds, which can vary even between species o f the same genus, determines a resin's

fluidity, viscosity, and polymerization rate. These in turn influence its ecological properties

[Langenheim. 1994; Cates, 1996].

1.1.2 Phenolic Resins

Phenolic resins consist mostly o f flavonoid aglycones with reduced number of hydroxyl

substituents and a variable number of phenolic groups that are a methyl substituted

(methoxylated) or with methylenedioxy substituents. Penduletin (12) from Dodonaea species,

5-hydroxy-6.7-dimethoxyflavanone (54) found in farina o{'Primula speciec and myrigalon B

8
(55) found in the leaf exudates of Comptonia peregrina are such compounds (Harborne,

1980).

Such lipophilic flavonoids commonly co-occur with terpenoids, particularly sesquiterpenes

and triterpenes [Wollenweber & Dietz, 1981; Wollenweber & Jay. 1988]. Lipophilic

flavonoids are best characterized in bud exudates from northern temperate zone Angiosperm

trees such as birches (Betula) and poplars (Populus) and in leaf resins of arid-zone shrubs like

monkey flower (Mimulus) and yerba santa (Erioclictyon). Diplacone (56) and diplacol (57)

are some o f the lipophilic flavonoids from Mimulus (I lare, 2002). Some phenolic resins have

hydrophobic side chains which increase their solubility in the terpenoid milieu. The

allergenic phenolics pentadecylcatechol (58) and heptadecylcatcchol (59) found in resins of

Anacardiaceae are of this type (Paseshnichenko, 1995). Alternatively, one or more terpene

(prenyl) residues may be attached to phenolic compounds to form prenylated phenolics like

tetrahydrocannabinol (60) (Paseshnichenko. 1995).

9
The surface resins consisting mostly of a mixture of terpenoid and phenolic compounds are a

prominent feature o f genera in numerous plant families, including Boraginaceae,

Euphorbiaceae, Goodeniaceae, Lamiaceae, Myoporaceae. Sapindaceac. Scrophulariaceae,

and Solanaceae in Australia, and the Asteraceae. Boraginaceae, Scrophulariaceae, and

Zygophyllaceae (Wollenweber, 1981). Arid-zone shrubs of these plant families produce

copious leaf surface resins that may constitute 17-30% of the dry weight of the leaf in

western Australian shrubs [Dell & McComb, 1978].

Some of the genera of Asteraceae that produce terpenoid and phenolic surface resins, include,

Baccharis, Chrysolhamnus, Grindelia, Gutierrezia, Balsamorhiza, Flourensia, Heliunthus,

Madia, Tagetes Haplopappus, and Xanthocephalum [McLaughlin & Hoffmann, 1982]. Some

genera such as Grindelia, have been studied extensively for economic use of their resins.

Surface resins may be produced on plants growing in diverse habitats, but resin coatings are

particularly prominent in plants of semi-arid regions. The greatest amount of resin produced

by organ weight may be that coating leaves and stems o f shrubs and some herbs in semi-arid

to arid regions |l)ell & McComb, 19781. Surface resins occur commonly >n shrubs and some

herbs o f diverse shrub taxa in Western Australia [Dell and McComb. 1978], in the deserts of

the American Southwest [McLaughlin & Hoffmann, 1982], and in the chaparral of

California. In European Mediterranean vegetation, the shrubs primarily produce essential oils

rather than resins. In fact 49% o f essential-oil producing genera (frequently in the families

10
Lamiaceae and Asteraceae) occur in regions with a Mediterranean climate [Ross &

Sombrero, 1991].

The localization of flavonoids and terpenoids on leaf surface appear especially suited for

protective mechanisms. The most evident role for this lipophilic material is an important

reduction o f water loss by cuticular transpiration [Rhoades, 1977b |. Another function for leaf

resins, especially for desert plants, is partial reflection o f light at the surface of these coatings,

thus reducing excessive heating o f the leaves. They also serve as screen against excess UV-

radiation, due to the UV absorption by the dissolved flavonoids | Rhoades, 1977a].

Furthermore, the resin components, terpenoids as well as flavonoids possess anti-fungal, anti­

bacterial, acti-viral activity [Chinou et al., 1994] and also serve as insect deterrants when

eaten or after penetration of the insect cuticle [Lincoln et al., 1982]. For plants that fight for

survival under extreme conditions it appears advantageous that micro-organisms are repelled

at the leaf surface and that phytophagous insects that want to chew, suck or mine, will only

eat a very small amount of leaf material before they realize a deterrent reaction [Finch, 1977].

Field observations at Aura Valley indicated that Semiothisa colorata (Lepidoptera:

Geometridae), a moth larva and the grasshopper (Ibolacris parviceps Bruner (Orthoptera.

Acrididae), a more generalized feeder, preferably consumed mature leaves and avoided

young leaves of Larrea tridentata cav of the high resin content of the young leaves. The high

resinous phenolic constituents o f creosatebush, Larrea tridentata cav. and L. cuneifolia cav

(Zygophyllaceae) also deters herbivore grazing | Meyer & Karazov, 1989]. Psiadia

punctulata is also avoided by browsing herbivores like giraffe and goats, even during severe

drought [Midiwo et al, 1997]. This is due to the fact that the leaves, especially when young,

are covered by gummy exudates which may be responsible for the deterrent effects. All the

functions discussed would be of special importance for plants that light for survival in

extreme climatic conditions or xeric habitats.

The leaf surfaces o f Dodonaea angustifolia and Senecio roseiflorus are covered with sticky

resinous exudate, which makes about 4-13% of the dry weight of the leaves [Ghisalberti.

1998]. In this study the crude extracts and some isolated pure compounds were tested for

their activity against Plasmodium falciparum, Aedes aegypti larvae, Escherichia coli

(ATCC25922), Staphyloccocus aureus (ATCC29737), Bacillus pumilus (local strain) and a

local strain of fungus, Saccharamyces cerevisiae. and fungi. The extracts and pure

compounds were also tested for anti-oxidant activity to determine their potential use as

medicines. In this section the literature on the malaria burden, botanical larvicides, microbial

drug resistance, oxidative stress and anti-oxidants will be reviewed.

1.2 The M alaria burden

Malaria is one of the most important infectious diseases, which affects more than a third of

the world's population (about 2 billion people) who live in endemic areas. In Africa alone,

there are an estimated 200-^150 million cases infected with malaria parasites each year

[Breman, 2001]. Estimates for annual malaria mortality range from 0-5 to 3-0 million people

(Marsh, 19981. The disease has probably accounted for more deaths and influenced the

course of history more than any other disease [Roberts et ai, 2004]. It has had a disastrous

effect on economic development throughout the world and continues to do so in some of the

world's poorest developing countries. Most of the drugs used for treatment of malaria are of

plant origin.

The first drug used for treatment of malaria, quinine (29), was obtained from the bark of a

Cinchona tree. Quinine (29) still plays an important part in the treatment of malaria and in

many countries is the drug of choice for complicated or severe malaria. Quinine (29) has

strong unpleasant side effects and it is therefore often administered intravenously to

hospitalised patients |Roberts et al., 2004]. Up to 70% o f patients who take quinine, for

example, can experience tinnitus, vertigo and nausea that lasts throughout the dosage period

12
[Roberts et a/., 20041. As a result of this chloroquine (61), which is structurally related

quinine, was developed for a preferred prophylaxis and treatment of malaria.

61

Chloroquine (61) was initially preferred ow ing to the fact it was well tolerated and cheap to

manufacture. However, the parasite developed resistance against chloroquine in the early

1960s in South East Asia and South America and has subsequently spread to most other

malarial countries, which made the drug ineffective. Consequently, the malaria problem is

more complicated now than before. The parasite has continued to develop resistance to

current first-line anti-malarial drugs leading to escalating mortality rates | I rape et al., I998J

imposing considerable pressures on health care systems.

As has been the case in the development of quinine and its derivatives, traditional medicinal

plants continue to be the source o f new anti-malarial drugs. In this connection artemisinin

(30) was isolated from the Chinese traditional anti-malarial herb Artemisia annua. Currently,

artemisinin (30) derivatives are used for the treatment o f uncomplicated and severe forms of

malaria. They reduce parasitaemia more rapidly than any other known anti-malarial

compound, and are effective against multidrug-resistant parasites [Olliaro et a l., 2001].

It is therefore necessary to continue to the search for new anti-malarial drugs to manage

parasite resistance. The search for anti-malarial drugs from plants can be expedited by

focusing on plants that have been used traditionally in the management oi' the disease or those

13
that produce compounds that are active against the malaria parasite or the symptoms caused

by the parasite. In this vein this study looks for anti-plasmodial activity o f the leaf surface

exudates compounds of D. angustiJolia-'Ngong forest, D. augustifolia -Voi and S.

roseiflorous.

1.3. Botanical larvicides

Larval control involves the control of mosquito populations they develop into adults. For

most malaria vectors, reducing mosquito population densities by means of larvae eradiction is

generally an efficient way of controlling malaria transmissions. Organophosphates, larvicidal

oils, arsenical compounds, and larval development inhibitors have all been used with varying

degrees of success [Gratz & Pal, 1988].

Constant applications o f organophosphates such as methoprene (62) and temephos (63) and

insect growth regulators such as diflubenzuron (64) and fenthion (65) arc generally practised

for the control of mosquito larvae [Yang et al., 2002]. Although these insecticides are

effective, their repeated use can disrupt natural biological control systems and result in the

widespread development of resistance leading to large population o f mosquitoes. The practice

can also have undesirable effects on non-target organisms, creating environmental and human

health concerns [Yang et al., 2002]. These problems have highlighted the need for the

development of new strategies for selective mosquito larval control. In the search for cost

effective, environmental friendly alternatives for the control of disease s ector insects, plant

extracts and pure compounds have been screened for larvicidal activities [Mwangi &

Mukiama. 1988: Mwangi & Rembold, 1988: Gikonyo et a!., 1998).

14
Rotenone (66), one o f the most extensively used natural insecticide, active against fourth-

instar larvae o f Aedes aegypti L [Abe et al., 1985], The insecticidal activity of rotenone (66)

and some other rotenoids including deguelin (67) and tephrosin (68) against a variety of

insect species is well known (Dewick, 1994). Commercially, rotenone (66) is mainly

extracted from roots o f Derris species in Asia and Lonchocarpus species in South America.

These and other rotenoids are also known to occur in the related genera including Milletia

and Tephrosia [Dewick, 1994].

15
 ()

Identification of cheap, readily available, biodegradable and environmentally friendly

botanical larvicides as an alternative to synthetic larvicides is very important. Locally grown

botanical larvicides could, not only be used, by local communities, for immediate control of

mosquitoes but can also save precious foreign currency used on importation of synthetic

insecticides. T he identification o f such botanical insecticides among the local flora may

eventually lead to the cultivation and commercialization of these plants by the local farmers

as an extra source of income.

1.4. Microbial Drug Resistance

Since their discovery during the 20lh century, anti-microbial agents (antibiotics and related

medicinal drugs) have substantially reduced the threat posed by infectious diseases [WHO,

2002]. There is a growing concern about the emergence and spread o f microbes that are

resistant to cheap and effective first-line drugs [Baron, 1982],

Resistance to anti-microbial agents has become an increasingly important and pressing global

problem. Seventy percent (70%) of the people who acquire bacterial infections each year

involve strains that are resistant to at least one drug [Cushnie & l amb. 2005]. When

infections become resistant to first-line anti-microbials, treatment has to be switched to

second- or third-line drugs which are much more expensive and sometimes more toxic, as

well. An example can be found in the drugs used to treat multidrug.-resistant forms of

tuberculosis which are over 100 times more expensive than the first-line drugs used to treat
16
non-resistant forms [WHO, 2002]. In many countries, the high cost o f such replacement

drugs is prohibitive, with the result that some diseases can no longer be treated in areas where

resistance to first-line drugs is widespread. Most alarming of all are diseases where resistance

is developing for virtually all currently available drugs, thus raising the spectre of a post­

antibiotic era [WHO, 2002]. Substantial investment and research in the Held of anti-infectives

are now desperately needed if a public health crisis is to be averted. Structural modification

of anti-microbial drugs to which resistance has developed has proven to be an effective

means of extending the lifespan of anti-fungal agents such as the azoles |Jeu et al., 20031,

anti-viral agents such as the non-nucleoside reverse transcriptase inhibitors |De Clerq, 2001],

and various anti-bacterial agents including lactams and quinolones [Poole, 2001]. It is not

surprising then, that in response to anti-microbial resistance, major pharmaceutical companies

have concentrated their efforts on improving anti-microbial agents in established classes

[Taylor et al., 2002].

However, with the portfolio of chemotherapeutics currently available, it has been

acknowledged that researchers are getting close to the end game in terms of parent structure

alterations. A call has therefore been made for the development o f new classes of drug that

work on different target sites to those in current use |Kimberlin & Whitley, 1996]. Rational

drug design does not always yield effective anti-microbials. Broad empirical screening of

chemical entities for anti-microbial activity represents an alternative strategy for the

development of novel drugs. Natural products have been a particularly rich source of anti-

infective agents, yielding, for example, the penicillins in 1940. the tetracyclines in 1948 and

the glycopeptides in 1955 [Silver & Bostian, 1990]. Dodonaea species arc traditionally used

for their anti-microbial properties in different parts o f the world and have revealed several

anti-microbial compounds, mostly flavonoids [Ahmed et al., 1994: Rojas et a\., 1992;

Sukkawala & Desai, 1962], Increasingly, flavonoids are becoming the subject of medical

research. They have been reported to possess many useful properties, including anti-

17
inflammatory, oestrogenic, enzyme inhibition, anti-microbial [Havstecn, 1983; Cushnie &

Lamb, 2005]. anti-allergic, anti-oxidant (Middleton & K andaswam i. 1993], vascular and

cytotoxic anti-tumour activity [Harborne & Williams. 20001. Owing to the widespread ability

of flavonoids to inhibit spore germination of plant pathogens, they have been proposed for

use against fungal pathogens o f man |Harborne & Williams, 2000]. In this project the

biological activity of the crude extracts and pure compounds of D. cmgnstifol/#-Ngong Forest,

D. angustifo/ia-Voi and Senecio roseiflorus against bacteria and fungi were studied.

1.5 Oxidative Stress and Anti-oxidants

Under normal conditions, a dynamic equilibrium exists between the production of reactive

oxygen species (ROS) and the anti-oxidant capacity of the cell [Granot & Kohen, 2004;

Stohs, 1995]. Oxidative stress is caused by an imbalance between the generation of ROS by

endogenous/exogenous pro-oxidants and the defence mechanism against ROS by anti­

oxidants. ROS are highly reactive molecules which arc naturally occurring by-products of

normal biological processes within the body or from exogenous factors. When anti-oxidants

are not enough or during diseases, the amount of ROS increase and react with DNA, lipids

and proteins causing cell death due to necrosis [Kannan & Jain, 2000],

Excess levels of ROS and the resulting oxidative stress have been implicated in a number of

human diseases including diseases, cancer, atherosclerosis, asthma, arthritis, autism,

cardiovascular and neurodegenerative diseases such as Parkinson's disease and Alzheimer's

disease (Knight, 1998: Kehrer, 1993]. Increasing evidence suggests that aging may be a

consequence of the normal, long-term exposure to ROS and the accumulation of oxidized,

damaged molecules within the cell. A potent scavenger of free radicals (anti-oxidants) may

serve as a possible preventative intervention for the diseases [Gyamfi el a/., 1999].

Anti-oxidants act as radical scavengers through the donation of one or more electrons thereby

neutralizing the free radical and thus terminating the chain reactions, which could other wise
18
damage cells and tissues leading to diseases. Anti-oxidants can be classified into natural and

synthetic. Natural anti-oxidants can be classified further into en/ymatic (superoxide

dismutase, ascorbate peroxidase, glutathione reductase, glutathione peroxidase, etc) and non-

enzymatic anti-oxidants (glutathione, ascorbic acid, vitamin E, vitamin A) [Luis & Nombela,

1999]

The most commonly used synthetic anti-oxidants are butylated hydroxyl anisole (BHA),

butylated hydroxyl toluene (BUT), tertiary butylated hydroquinone and gallic acid esters,

which are incorporated in food products to stop the oxidation of polysaturated fatty acids that

leads to the degradation of the food. However, these synthetic anti oxidants have been

implicated in liver damage and carcinogenesis in laboratory animals |Wang et al., 2000].

Strong restrictions have been placed on their application and there is a trend to substitute

them with naturally occurring anti-oxidants. Moreover, the synthetic anti oxidants show low

solubility and moderate anti-oxidant activity [Barlow. 1990; Branen, 19751

Plant species with high amount of flavonoid and phenolic compounds such Mellilotus

officinalis extract have shown higher potency than B iff in scavenging free radicals

[Pourmorad et al, 2006].

Recently there has been an upsurge of interest in the therapeutic potentials of medicinal

plants as anti-oxidants, in reducing free radical induced tissue injury. Besides well known and

traditionally used natural anti-oxidants from tea, wine, fruits, vegetables and spices, some

natural anti-oxidant (rosemary and sage) are already exploited commercially either as anti­

oxidant additives or a nutritional supplements [Schuler, 1990]. Also mans other plant species

have been investigated in the search for novel anti-oxidants |Chu. 2000: Koleva et al., 2002;

19
Mantle et al., 2000; Oke & Hamburger, 2002] but generally there is still a demand to find

more information concerning the anti-oxidant potential o f plant species.

I he anti-oxidant activity of plants might be due to their phenolic compounds [Cook and

Samman, 1996] which act as free radical terminators [Shahidi & Wanasundara, 1992].

Mavonoids are a group ot polyphenolic compounds with known properties which include free

radical scavenging, inhibition o f hydrolytic and oxidative enzymes and anti-inflammatory

action [Frankel, 1995]. Some evidence suggests that the biological actions of these

compounds are related to their anti-oxidant activity (Gryglcwski ct al., 1987]. The

mechanisms of action o f flavonoids are through scavenging or chelating process [Kessler et

a l 2003, Cook & Samman, 1996],

Dodonuea and Senecio plants have been known to elaborate flavonoids and phenolics some

of which could have anti-oxidant activities. The crude extracts and some flavonoids from

these plants were screened for their potential as anti-oxidants in the present work. The crude

(or their active constituents) identified as having high levels of anti-oxidant activity in vitro

may be o f value in the design o f further studies to unravel novel treatment strategies for

disorders associated with free radicals induced tissue damage.

20
 CHAPTER TWO

LITERATURE REVIEW

2.1 BOTANICAL INFORMATION

2.1.1 THE FAMILY SAPINDACEAE
The genus D odonaea belongs to the family Sapindaceae. Among flowering plants, the family

Sapindaceae is one of the most widely distributed ones. It comprises between 140-150 genera

and between 1,500-2,230 species, typically tropical and strongly represented in the American,

African and Asian tropical zones, also in Japan and widespread in Australia. Members of the

family are mostly trees and shrubs, or sometimes vines (cardiosperm) climbing with the help

of simple or branched tendril Is, which are modified in fluorescence axes, or lianas.

The East African genera of Sapindaceae belong to the following sub families and tribes:

Subfamily Dodonaeoideae with three tribes D odonaeeae {D odonaea), Doraloxyleae

(F ilicium ) and H arpullieae {M ajidea)\ and the subfamily Sapindoideae with eight tribes

Cupanieae (Aporriza), N ephelieae ( Stadm ania ), Schleichereae {M acphersonia), M elicocceae

(Tris d ro p s is), Lepisantheae (L episanlhes ), Sapindeae (D einbollia ). Thouinieae (.Allophylus )

and P aullinieae (C ardiosperm um ) [Davis & Verdcourt., 1998].

In East Africa the family is represented by 61 species in 25 genera, f ourteen (14) of these

species are endemic to the region. The 25 genera o f East Africa arc: Dodonaea (2 sp),

F ilicium ( 1 sp), Zanha (2 spp), Mcijidea (2 spp) A porrhiza (1 sp), Blighia (2 spp), Eriocoelum

(1 sp), H aplocoelopsis (1 sp), Stadmania (1 sp), Pappea (I sp) M acphersonia (I sp),

Lecaniodiscus (2 spp), H aplocoelum (2 spp), C am ptolepis (I sp), Lepisanthes (1 sp),

Glermiea ( I sp), Placodiscus (2 spp), Pancovia (6 ,v/?p), Chytranthus (4 spp), Deinbollia (3

spp). Sapindus (2 spp), Allophylus (18 spp) Cardiosperm um (3 spp) Paullinia (1 sp). [Davis

& Verdcourt. 1998]

21
Several speeies from this family have been cultivated in E. Africa for their edible fleshy fruits

including several well known fruits such as Litchi chinensis (Lychec), which has been

cultivated in Kenya (Kiambu & Kilifi district). Tanzania (Lushoto & Morogoro District) and

Uganda (Mengo District). N epheliium lappaceum (rambutan) is grown for edible fruit in

Tanzania (Lushoto District) and Uganda (Mengo District) while M elicoccus bijugatus also

known as honey berry or Spanish lime, a native of S. America has been grown in Tanzania

(Lushoto District) and Uganda (Entebbe). Tristinopsis acuteangula, a native of Malaysia to

Queensland. Solomon Island, Guam and Palau Island is grown in Tanzania (Zanzibar) for

timber [West. 1984].

2.1.1.1 THE GENUS D O D O N A E A

The genus D odonaea is predominantly Australian, comprising of 68 species of which 61

species are found in that sub-continent. Of the 61 species found in Australia, 59 are endemic.

Dodonaea p a lyn d ra extends to New Guinea and D odonaea viscosa is pantropical extending

to Southern Africa and the Pacific. D. viscosa is a polymorphic species containing at least 7

sub-species [West, 1985]. All D odonaea species are evergreen woody, perennials, most are

erect, multi-stemmed shrubs 1-2 m in height but there is considerable variation in size.

Dodonaea hum ifosa and D odonaea procum bens are prostrate shrubs (10 cm high), whereas

Dodonaea viscosa and D odonaeaplatyptera can reach a height of 8m. In Australia, the genus

is widespread especially in the inland regions [West, 1985]. Many of the western Australian

species possess viscid resinous exudates on the leaves as is common in many other xerophitic

species found in the region and elsewhere. It is widely grown for horticultural purposes in

Australia, and in other countries it is used as a hedge plant, sand binder and for marshland

reclamation [West, 1985]. In East Africa the genus D odonaea is represented by two species

D. angustifolia (Fig. 2.1) and D. viscosa (Fig. 2.2) [Beentje, 19941.

22
Figure 2.1: Distribution ofZ). angustifolia in Figure 2.2: Distribution o f D. viscosa in
Kenya [Beentje, 1994] Kenya [Beentje, 1994]

2.1. 1.1.1 D. VISCOSA

Dodonaea viscosa is an extremely variable species throughout its natural range. There are

many distinctive populations that have been described as separate species from D.

angustifolia by some authorities. Wagner el al 1990 considers the Hawaiian D. viscosa

populations as one species.

Dodonaea viscosa can be a small tree or shrub 1.5 to 4 in tall. Leaves are lance-shaped to

elliptic (6 - 12.5 cm) long (1.8 - 4.2 cm) wide (mostly widest above the middle) and relatively

broader. Flowers are whitish, mostly bisexual; scar of fallen sepals beneath fruit strongly

bilobed. Fruits are white or brown to straw-coloured or greenish white, sometimes yellow or

reddish, mostly two winged. (1.5 - 2.3 cm. long and 1.8 - 2.5 cm. wide. Seeds are sub-

globose. 3 mm diameter, 2 - 3 mm thick and mostly rolling easily when dropped on a flat

surface [Davies & Verdcourt, 1998].

23
Figure 2.3: D odonaea viscosa female flowers Figure 2.4: D odonaea viscosa male
[Carr, 1998] flowers | Carr, 1998]

2.1.1.1.2 D O D O N A E A A N G U S T IF O L IA
Dodonaea angustifolia belonging to the Sapindaceae family is variously considered

synonymous with, sub-species of, variety of or distinct species from D. viscosa [West, 1984];

[Vedcourt, 1994] and [Leenhouts, 1983] respectively depending on the particular authority. It

is widely distributed in four continents - Australia, Africa, Asia and South America. In Africa

it exists in Eastern, Southern and Western Africa. In Kenya, it exists along with D. viscosa

which otherwise has exogenous origin in India [Beentje, 19941. West (1984) revised the

taxonomy o f D odonaea of Australia [ICRAF, 1991] and morphologically determined that D.

viscosa had up to four sub-species existing in that country: D. viscosa ssp. viscosa , ssp.

burmanniana, ssp. angustifolia and ssp. spatulata. No such work has been done for the

African D. angustifolia other than the declaration that the coastal population is synonymous

with D. viscosa from Australasia (Figure 2.2) [Leenhouts, 1983]. The main phytochemical

feature in D. angustifolia is the leaf surface exudate (up to 13% dry leaf weight) composed of

methylated ilavones and flavonols in a diterpenoid (clerodane) milieu. In this work the

phytochemical investigation of the upland (Ngong forest) and coastal (Voi) populations of D.

24
angustifolia was done. Dodonaea angustifolia can be a medium-sized shrub (Fig. 2.5) or

small tree up to 9 m tall (Fig. 2.6), but most often 0.5-7.5 m in height. I he plant may have

one or several main trunks, which have reddish-brown to blackish - grey bark. There is a lot

of variation in leaf size and shape, but the leaves are generally longer than they are wide and

most often pointed (Lance-shaped to elliptic). Most often, the leaves are 1.5-13 cm. long, 0.6-

3.5 cm. wide, narrowed gradually and rounded to an acute and weakly apiculate tip. They are

usually glossy green and often have reddish midribs or stems. All the leaves are covered by a

sticky substances but new leaves are stickier than the old ones. Generally, older leaves have a

rough, sandpapery texture.

Oodcm wA Jinpv*Utotin

Figure 2.5: Dodonaea angustifolia shrub (Carr, Figure 2.6: Dodonaea angustifolia tree
1998] [Carr, 1998]

25
 The indi\idual flowers are

small, greenish yellow, 0.6

cm in diameter, and occur in

branched clusters. They are

mostly unisexual and only

the female flowers develop

into the decorative capsules.

Some plants have both male

and female flowers in which

Figure 2.7: Fruits of D. angustifolia [Carr. 1998J

case only a single plant is required to produce capsules |Bornhorst 1996; Koob, 2001; Obata,

1997; Rauch, 1997; Wagner, 1990]. Scar of fallen sepals beneath fruit usually annualar or

slightly lobed. The fruit is red-tinged or mottled or crimson to purplish but can be white,

brown or reddish green, predominantly two winged, but often with 3 or even 4 wings. Seeds

compressed globose, ± 3 mm in diameter, 1.5 to 1.8 mm thick, with obtuse dorsal keel, not

easily rolling when dropped on flat surface [Davies & Verdcourt, 1998]

2.1.1.2 MORPHOLOGICAL DIFFERENCES BETWEEN IX V ISC O SA AND D.

A N G U S T IF O L IA
From the morphological point o f view it is not easy to distinguish the two species from each

other because most features overlap (Table 2.1). In this study the phytochemistry of the

Dodonaea populations from Ngong Forest representing D. angustifolia and from Taita hills

near Voi town representing D. viscosa [Leenhouts, 1983; Davies & Verdcourt, 1998 ] was

done to compare the compounds from the two populations and see if differences could be

discerned.

26
Table 2.1: Morphological differences of D.viscosa and D. angustifolia
D. VISCOSA D. ANGUSTIFOLIA
Small tree or shrub 1 .5 -4 metres tall.Medium-sized shrub or small tree up to 9 metres
tall.
Leaves 6 - 12.5 cm. long, 1.8 - 4.2 cm Leaves are 1 .5 -1 3 cm. long and 0.6 to 3.5 cm.
wide. wide.
Flowers are whitish, mostly bisexual. Flowers are greenish yellow mostly unisexual.
Fruit mostly two winged 1.5 - 2.3 cm. The fruit predominantly two winged, but often
long and 1.8 - 2.5 cm. wide. with 3 or even 4 wings
Seeds sub-globose, 3 mm diameter, 2 - 3 Seeds compressed globose, i 3 mm diameter,
mm. thick, mostly rolling easily when 1.5 - 1.8 mm thick, with obtuse dorsal keel, not
dropped on a flat surface. easily rolling when dropped on flat surface

2.1.2 FAMILY ASTERACEAE

The genus Senecio belongs to the family Asteraceae or Compositae. known as the aster, daisy

or sunflower family, the largest family of flowering plants in terms o f number of species

[Cronquist, 1981]. Plants in the Asteraceae family are mostly herbs, but some shrubs, trees

and climbers do exist. According to the information from Royal Botanical Gardens of Kew,

the family comprises more than 1,600 genera and 23.000 species 11leywood et al., 1977]. The

largest genera are Senecio (1,500 species), Vernonia (1,000 species), C ousinia (600 species),

Centaurea (600 species). This family is characterized by having the (lowers reduced and

organized into an involucrate pseudanthium in the form of a head or capitulum [Stevens,

2007]. Asteraceae are cosmopolitan, but most common in the temperate regions and tropical

mountains. The fruit o f the Asteraceae has one seed per fruit, it may sometimes be flat,

winged or spiny. The fruit morphology is often used to help determine plant relationships at

the genus and species level [Heywood et a l ., 1977]. The seeds usually have little or lack

endosperm (Stevens, 2007].

The Asteraceae family is exceptionally rich in a range of secondary metabolites and also in

the number of complex structures known one class [Heywood et a /., 19771. These secondary

metabolites include iso/chlorogenic acid, different classes of sesquiterpenoids lactones,

pentacyclic triterpene alcohols, various alkaloids, acetylenes (cyclic, aromatic, with vinyl end
27
groups), flavonoids and tannins. They have terpenoid essential oils which never contain

iridoids [Usher. I966J. It is one of the most economically important families, for it is the

source o f food crops such as L a ctu ca saliva (lettuce), C ichorium (chicory), Cynara scolymus

(globe artichoke), H elianlhus annuus (sunflower). Sm allanthus sonchifolius (yacon),

C artham us tinctorius (safflower) and Helianthus tuberosus (Jerusalem artichoke). A study of

early herbals reveals that a surprisingly large number of Asteraceae were used for their

curative properties [Hind el a l ., 1995J. It is the source of medicinally important herbal teas

such as chamomile tea from M atricaria recutita or G erm an cham om ile and the perennial

Chamaemelum nobile , also called Roman chamomile. Other herbal teas include C alendula ,

(pot marigold), Fchinacea (Echinacea purpurea), Tagetes lucida which is commonly grown

and used as a tarragon substitute in climates where tarragon cannot survive. Finally, the

wormwood genus A rtem isia includes absinthe (A. absinthium ) and tarragon (A. dracunculus).

This family is an important source of medicine especially in areas where there is no access to

Western medicine. Many members of the family are grown as ornamental plants for their

flowers and some are important ornamental crops for the cut (lower industry. Some examples

are C hrysanthem um , G erbera , C alendula , D endranthem a , A rgyranthem um , Dahlia , Tagetes ,

Zinnia among many others. H elianthus annuus (domestic sunflower), and some species of

Solidago (golden rod) are major "honey plants" for beekeepers. Solidago produces relatively

high protein pollen, which helps honey bees over winter [Croat. 1972].

Some members of the Asteraceae which are economically important as weeds include the

ragwort. Senecio ja co b a ea , groundsel Senecio vulgaris and Taraxacum (dandelion). The

genera Tanacetum , C hrysanthem um and P ulicaria contain species with insecticidal

properties. Parthenium argentatum (Guayule) is a source of hypoallergenic latex [Cornish &.

Brichta, 2002].

28
2.1.2.1 THE GENUS S E N E C IO
Senecio is the largest genus o f the Asteraceae family with over 1500 species distributed all

over the World [Hind el a l ., 1995]. A large number o f these species are common perennial or

annual weeds, but some are succulent and caudiciforms from tropical and sub-tropical areas.

A number o f succulent relatives have now been moved to the genus K leinia. The flowers of

Senecio are arranged in clusters at the top of the plants, they vary in color from white and

yellow, to red and purple. Most succulent species tolerate no frost. Some species produce

natural pesticides (especially alkaloids) to deter or even kill animals that would eat them.

Senecio species are used as food plants by the larvae of some Lepidoptera species and as

medicine by many communities.

2.1.2.1.1 S E N E C IO R O S E IF L O R U S
Senecio roseiflorus is an erect herb or weak shrub, densely glandular on ail parts; leaves are

oblong-lanceolate, lobed, stalkless, to 8 cm long: heads in a terminal corymb, with about 15

purple rays; phyllaries 8-10 mm long; achenes hairy. In Kenya Senecio roseiflorus is

common in the drier alpine zone, 3100-4200 m [Agnew. 19941.

2.2 ETHNO-MEDICAL APPLICATION AND PHARMCOLOGICAL

INFORMATION ON THE GENUS D O D O N A E A AND S E N E C IO .

2.2.1.1 ETHNO-MEDICAL APPLICATION OF D O D O N A E A SPECIES

Members of D odonaea have been used for medicinal purposes by indigenous people in

several continents and to a remarkable extent, for similar complaints, fable 2.2 gives a

summary of different D odonaea species, their origin and uses by different communities. The

information given in the table is adapted from NAPRALERT, 2008 data base.

29
Table 2.2: Ethno-botanical uses of Dodonaea species
Species (Origin) Plant part Uses Reference
D odonaea viscosa
East Africa Entire plant Fish poison Hcdberg et al., 1983
Stomach pain
Leaves Hemorrhoids Chhabra et al., 1991a.
Antipruritic in skin rashes 1ledberg e ta l., 1983
Febrifuge
Sore throat.
Antirheumatic
Hemorrhoids Stimulant
Anaesthetic Vermifuge
Dermatitis
Root Increase lactation Kokwaro, 1976
Irregular menstruation Hcdberg e ta l., 1983
Tanzania Leaf Antipruritic in skin rashes Chhabra et al., 1991a

Root Indigestion Vasileva, 1969

Irregular menstruation
Peptic ulcer
Galactogogue in animals Chhabra et a l., 1991a
and humans
Indigestion I ledberg et al., 1983
Peptic ulcers
Ethiopia Leaves Skin lesions Dcsta, 1993
Fever Asres et al., 2001
Malaria
Papua-New Guinea Leaf To make woman sterile. Holdsworth, 1989
For poultice
Guinea Root Augment lactation goats Vasileva, 1969

India Fresh leaf Wounds in cattle Davyt et al., 1991

Fractures in cattle Reddy e ta l., 1988
Swellings in cattle
Febrifuge 1lope et al., 1993
Rheumatism Sukkawala & Desai,
1962
Laxative
Menstruation
Mexico Entire plant Stomach pain Ro jas et al., 1995
Hepatic or splenic pain
Uterine colic
Leaves Rheumatism Rojas et al., 1996
Wounds
Diarrhea
Skin infections
Fractures.
Used in a poultice
Postpartum recovery

30
Table 2.2: Ethno-botanical uses o f Dodonaea species
Species (Origin) Plant part Uses Reference
Leaves Menorrhagia Browner, 1985
Hemorrhage
Infertility
Prevent miscarriage
Fever
Uruguay Fruit Rheumatism Gonzalez et al., 1993
Dried leaf Laxative Davyt et al., 1991
Dried leaf+ Astringent Gonzalez et al., 1993
root Rheumatism
Used as a febrifuge Ramirez et al., 1988
Hawaii Leaf Asthma Gonzalez et al., 1993
Peru Dried leaf + Increase lactation in cows Ramirez et al., 1988
stem
Dodonaea viscosa var. angustifolia
Mexico Dried aerial Fevers Dominguez et al.,
parts 1980
South Africa Pneumonia and Watt & Breyer-
tuberculosis Brandwijik, 1981
Malaysia Wood Flatulence and cholic
Dodonaea viscosa var. angustissim a
Australia Leaves Used to relieve fevers l.at/, 1995
Dodonaea lanceolata
Australia Leaves Pain and snake bite 1.assak et al., 1990
Dodonaea m adagascariensis
Madagascar Leaves Hypotensive and Trot in et al., 1972
antispasmodic properties
Dodonaea ph ysocarpa
Australia Leaves+twigs Alleviate symptoms of Barr, 1993
colds and flu
Dodonaea po lyzyga
Australia Leaves+twigs Alleviate symptoms of Barr, 1993
colds and flu
Dodonaea triquetra
Australia Root Wounds and toothache Dominguez et al.,
1980

2.3 BIOLOGICAL ACTIVITIES OF COMPOUNDS AND EXTRACTS FROM

D O D O N A E A SPECIES
Docionaea species extracts have been reported to have some bioactivities. An ethanolic

extract of the leaves of D. viscosa showed anti-bacterial activity against various Micrococcus,

Bacillus, Salm onella species and C orynebacterium diphtheriae, Sarcina /idea and

Escherichia coli [Sukkawala and Desai. 1962]. In another study, a methanol extract of the

aerial parts of D. viscosa collected in Mexico inhibited the growth of Siaphylococcus aureus,

Bacillus subtilis, Escherichia co li, Pseudomonas aeruginosa and ( andida albicans at

31
concentrations of 20 fig ml"1 [Rojas et a! ., 1992). Extracts of the leaves of D.

m adagascariensis showed minimal anti-microbial activity [Trotin et al. , 1972]. The aqueous

and ethanolic extracts obtained from the leaves of I), viscosa showed hypotensive properties

[Sukkavvala & Desai, 1962].

Many o f the triterpenes present in D odonaea have been shown to have bioactivity in separate

studies. Lupeol (69) (25-250 mg Kg’1) isolated from D. attenuatta var. linearis exerted anti-

inflammatroy activity in a range of acute and chronic test models in rats. Oleonolic acid (70)

obtained from the leaves of D. m adagascariensis [Trotin, 19721 has been shown to have

hepatoprotective, anti-hyperlipidemic and anti-inflammatory properties in laboratory animals

[Lui, 1995].

Triterpene saponins have long been known as fish poisons and the monodcsmosidic saponins,

with a free carboxylic acid at C-28, exhibit important molluscidal activity [Marston et a !.,

1985]. Some saponins been tested for the control of schistosomiasis, a disease which affects

millions o f people living in Africa, Asia and South American countries. The saponins 71 and

72 isolated from D. viscosa showed 100% lethality at 25 ppm in the molluscicidal test using

the bilharzia a vector snail Biom phalaria glabrata [Wagner et a/., 1987]. Many saponin

preparations also have antitussive and expectorant properties as well as analgesic properties

[Lacailledubois et a l ., 1996].
32
 30 29

Barringtogenol C-21-angelate (73) from the seeds, flowers and stems of I). viscosa was found

to be cytotoxic in the in vitro to human nasopharygeal carcinoma (9-KB) cultured cell system

(ED50 3.6 jag m l1), while the parent, triterpene was inactive. However, the 15, 16-21, 22-

diacetonidc from different Dodonaea species had equivalent activity [Konoshima, 1986].

Hydroxycinnamic acids like caffeic acid (74), and chlorogenic acid (75) isolated from

different Docionaea viscosa, have anti-oxidant and tumour inhibiting activity [Johns et a/.,

33
1995]. The coumarin. fraxetin (16) from Dodonaea viscosa has attracted some attention as an

anti-oxidant [Yanishlieva et al., 1996; Hoult and Paya 1996|.

Fraxetin (16) also displayed analgesic properties in the acetic acid-induced writhing test on

mice [Okuyama et a l ., 1996]. The coumarin cleomiscosin A (76) from D. viscosa was

recognized early as an anti-leukemic compound [Handa et al., 1983]. However, more recent

studies have shown that it is ineffective in induction of cell differentiation with human

promyeolocytic leukemia (HL-60) cells [Luyeng/ et al., 1996].

Kaempferol (15) isolated from D. viscosa has been reported to have anti-ulcer and anti­

inflammatory activity [Goel et al., 1988; Izzo et al., 1994]. The isorhamnetin 3-glycoside

(77) from D. viscosa has been shown to have blood sugar lowering effects [Zabroham et al.,

1986]. Quercetin (78) from D. viscosa and its glycosides have also been shown to exhibit

anti-inflammatory and anti-spasmodic activity in a number of tests.

A number of 3-methoxyflavones, quercetin (78) and kaempferol (15) derivatives from D.

viscosa have been shown to exhibit pronounced anti-viral activity and to be active against

[Vlietinak et al., 1995] picornavirus in tissue cultures. From a large screening programme,
34
some 3-niethoxyflavones, including penduletin (21), from D. viscosa cxhibitecd in vitro

activity against polio and rhinovirus.

R, r2 R3 r.

77 ORut OH OH OH

78 Oil OH OH OH

Rut = rutinosidc

2.3.1 CORRELATION OF PHARMACOLOGICAL ACTION AND BIOACTIVITY

OF METABOLITES
The reported medicinal uses o f Dodonaea species by indigenous people in different parts of

the world show considerable similarities. In a broad sense, preparations arc employed largely

as analgesic, anti-inflammatory, spasmolytic, anti-viral and hypotensive agents. Various

gastro-intestinal disorders, skin conditions and healing of wounds are also managed by these

preparations. Considering the spectrum of activities, it is possible to find a correlation with

constituents or groups of constituents. Consequenty, the triterpenc saponins and the

coumarin, fraxetin (16) have analgesic properties. Anti-inflammatory activity could be

associated with some o f the diterpenes, lupeol (69), oleonolic acid (70), the saponins and the

flavanoids. Spasmolytic activity could arise from the presence o f some diterpenes,

sakuranetin (79). quercetin (78) and rutin (80).

35
This activity could also explain the use of Dodonaea preparations to alleviate gastro­

intestinal disorders. The 3-methoxyflavones could contribute to the anti viral activity. The

tlavone hyperoside (77), quercetin (78) and rutin (80) have been shown to exert a

hypotensive effect [Schlusser, 1995; Pathak, 1991]. The use of Dodonaea preparations in the

treatment o f a variety of skin disorders, ulcers and for wound healing is interesting. The

presence o f a variety of flavones including quercetin (78) from the leaves and oleanene

glycosides (34) and (35) from the seeds of D. viscosa may contribute to the wound healing

activity ascribed to preparations from these plants. In conclusion, there is significant

circumstantial evidence for the pharmacological basis of the traditional medicinal uses of

Dodonaea species. It seems likely that a number o f compounds from these species may

provide interesting leads for pharmacological evaluation.

2.2.1.2 ETHNO-MEDICAL APPLICATION OF THE GENUS SENECIO.

Senecio is known to elaborate pyrrolizidine alkaloids. Pyrrolizidine alkaloid-containing plants

are widely distributed in many geographical regions in the world [Mattocks, 1986]. They

exhibit hepatotoxic, mutagenic, carcinogenic and antitumor activities | Mattocks 1986; Rizk

1991; Wink, 1998].

Livestock are poisoned by grazing on plants containing pyrrolizidine alkaloids (PAs), causing

livestock loss due to liver and pulmonary lesions |Roeder 1995; 2000; Smith & Culvenor

1981]. Pyrrolizidine alkaloids have also been found to contaminate human food sources, such

as wheat, milk, honey, herbal medicines, and herbal teas, and may potentially cause

worldwide human health problems [Betz et al., 1994; lluxtable 1980; lluxtable et al., 1983

Winship 1991; Byron 1998; Prakash et al., 1999]. Hydrolysis products of a pyrrolizidine

alkaloid are a necine base and a necic acid. The necic acids are four to six carbon-containing

mono- or di-carboxylic acids, and are mostly branched-chained. unsaturated, hydroxylated. or

epoxidized. Most of the pyrrolizidine alkaloids derived from esters of basic alcohols, the

36
necine bases, have been found to exhibit toxic effects. In most plants, they occur in their N-

oxide form

PAs are a typical group of plant secondary compounds which arc constitutively produced by

plants as a defense against herbivores and insects [Hartmann & Ober, 2000]. They are part of

a complex system o f chemical ecological interactions between the plant and insect

herbivores. Some adapted herbivores have even developed specific mechanisms to use these

plant derived compounds for their own defense against predators [Ober, 2003; Hartmann,

2004].

Members of Senecio species has been used by indigenous people in several continents, to

treat a number of ailments. Table 2.3 below summarizes the different Senecio species, their

origin, plant parts and uses by different communities. The information given in Table 2.3 is

adapted from NAPRALERT, 2006 data base.

Table 2.3: Ethno-medical application of Senecio species

Species (Origin) Plant part Use Reference
S. abyssinicus
Nigeria Roots Treat rheumatism Akah et al., 1995
Treat syphilis
Treat bruises
Used as a stomachic
Used as a blood purifier
S. acanthifolius
Egyp' . Flower + leaf Treat amenorrhea | Dragendorff, 1898
S. albican!is
Argentina Aerial parts Used to promote Manfred, 1947
menstruation
S. appendical at us
Canary islands Dried aerial Used for dysmenorrhea Darias et al., 1989
parts
S. aureus
USA Aerial parts Treat amenorrhea Schmid, 1976

Used as a diuretic Christopher,

Used as a diaphoretic 1976

Used as a tonic
Used as an emmenagogue Krochmal and
krochmal., 1973

37
Table 2.3: Ethno-medical appIication o f Senecio species
Species (Origin) Plant part Use Reference
Entire plant Used to hasten delayed Krochmal and
childbirth krochmal, 1973

Used as female regulator

Used for painful and Fielder, 1975

spasmodic menstruation

Used to hasten childbirth Lewis el al., 1977

India Dried aerial Used for nervous disorders Krochmal &
part krochmal, 1973

England Entire plant Used for abortion Speck, 1944

Used ergot during parturiton Shcmluck, 1982
Used as emmenagogue Krag, 1976
55 Used as sedative

Rhizome Used as diuretic Mausert, 1932

Used to promote menstrual
discharge
Root Used for dysmenorrhea Novitch &
Schweiker, 1982
S. biafrae
Nigeria Fresh leaf Used for wound dressing Akah, 1995
Used as an antiseptic
Used for indigestion
S. brasiliensis
Brazil Dried entire Treat fevers Brandao et a l.,
plant 1985
Dried leaf Treat malaria Giberti, 1983
Argentina Used as a febrifuge
S. caniiicans
Chile Dried aerial Said to be toxic Ur/ua et al., 1989
parts
S. canicida
Mexico Aerial parts Used to kill dogs and Mendez, 1937
coyotes
55 Has been used in
homeopathy to treat epilepsy
S. cannabifolius
Japan Dried aerial Eaten as a food Hirono et al.,
parts 1983
S. chenopodioides
Honduras Dried entire Used for aches and pains Lentz et al., 1998
plant
S. chionogeton
Peru Dried flowers Used as a diaphoretic Ramirez et al.,
1988
Used as an expectorant
Used for bronchitis

38
Table 2.3: Ethno-medical application o f Senecio species
Species (Origin) Plant part Use Reference
Dried leaf Used as an expectorant
Used for bronchitis
Used as a diaphoretic
S. chrysanthem oides
India Dried entire Used by physicians's as a Koelz, 1979
plant medicine for debility
Nepal Dried root Used for indigestion Manandhar. 1986

S. cineraria
South America Entire plant Used as an emmenagogue Dragendorff, 1898
Ben/.anger-
France Dried entire Used as an emmenagogue Beauquesne et al,
plant 1980
S. cydoniifolius
Rwanda Dried leaf Used for diarrhea Maikere-Faniyo et
al, 1989
S. disc ifol ius
East Africa Entire plant Used to stimulate milk (low Kokwaro, 1976
Leaf Used as an anthelmintic
S. discolor
Jamaica Entire plant Used to make a tea for fever Asprey &
Thornton, 1955
S. divers ifol ins
Nepal Leaf juice Used as a hemostatic. Bhattarai, 1997
99 Used as an anti-bacterial
Used for bleeding wounds
and cuts as a hemostatic
agent
Used for bleeding wounds
and cuts as an antiseptic
S. douglasii
USA Aerial parts Used as a cough medicine Stillman el al.,
1977
Used for pulmonary diseases 1lux table, 1989;
1990
Used for infected sores, or Bocek, 1984
for cuts
Dried entire Used for a "cold in the
plant idneys
Used for puerperal tetanus.

S. eriophyton
Bolivia Dried leaf Used as an emmenagogue. Gonzalez & Silva,
1987
Chile 99 99 99

S. erosus
Peru Leaf Used for pains in the kidney. Yclasco-
Ncgueruela et al.,
L . 1995

39
Table 2.3: Ethno-medical application o f Senecio species
Species (Origin) Plant part Use j Reference
S .g ig a s
Ethiopia Dried leaf Treat typhus. Desta, 1993
Dried root Treat typhoid fever Asrcs el a l ., 2001
Treat rheumatism
S. glaucus
Kuwait Entire plant Used as an emmenagogue in Alami el al., 1975
case of amenorrhea
S. g ra veo lem
Argentina Dried aerial Used against diarrhea Perez & Anesini,
1994
Treat respiratory tract
infections
Treat urinary tract infections

Treat stomach pains Giberti, 1983

Used as an antitussive
Used for altitude sickness
Chile Used for altitude sickness Loyola el al.,
1985
Bolivia Used as a tranquilizer for Bastien, 1983
gastritis
Used as an expectorant for
chronic cough
S. grisebachii
Paraguay Fresh Used to counteract Schmeda &
inflorescence misfortune, thought to be Cespedes, 1986
prevalent in August
S. hartw egii
Mexico Fresh root Rubbed on affected areas to Ishikura, 1982
kill lice and/or ticks
S. inornatus
Mexico Dried aerial Used for cardiac ailments Wicdenfeld el al.,
parts 1996
Used for respiratory
ailments
S. integerrim us
USA Entire plant Used as a female regulator Fielder, 1975
Used as an emmenagogue Krag, 1976
S. integrifolius
China Entire plant Used as an anticancer agent Duke and Ayensu,
1985
S. ja co b a e a
England Aerial plant If eaten by sheep their wool Culpeper, 1650
parts grows loose
Used as an emmenagogue
Used against cancerous
lesions
Dried entire Used as an emmenagogue Dragendorff, 1898
plant

40
Table 2.3: Ethno-medical application ot'Senecio species
Species (Origin) Plant part Use Reference
France Dried entire Used as an emmenagogue Culpeper, 1650
plant
S. kaem pferi
Canary islands Dried entire Used for dysmenorrhea Bcn/anger-
plant Beauquesne et al.,
1980
S. latifolius
South Africa Dried entire Used for wounds Simon & Lamia,
plant 1991
Used for sores
S. linifolius
Spain Dried aerial Used in folks medicine. Torres et al., 1988
parts
S. mannii
Rwanda Dried leaf Used for malaria 1laki/.amungu,
1992
S. m aranguensis
Tanzania Dried entire A Haya remedy for yaws Watt & Breyer-
plant and syphilis Brandwijk, 1962
S. rnoorei
Kenya Fresh aerial Suspected of being toxic to Mugera, 1970
parts cows when eaten
S. nem oralis ssp.fuchsii
Germany Aerial parts Used as a diabetic tea Habs et al., 1982a
Dried entire Used for diabetes I labs. 1982b
plant
S. nitidus
Colombia Entire plant Used to treat menstrual Garcia-Barriga,
perturbations 1975
Used as an emmenagogue Gon/alez & Silva,
1987
S. nudicaulis
India Dried entire Used to treat itching Jain & Puri, 1984
plant
Fresh leaf Used for gonorrhea
juice Jain e t al., 1994
Root Used as an anti­
in tlammatorv agent
S. obovatus
USA Aerial parts Used as an emmenagogue Burlage, 1968
(hepatotoxic and/or
carcinogenic)
S. oreophyton
Chile Aerial parts Used as an emmenagogue Gon/alez & Silva,
1987
S. oryzetorum
China Entire plant Used as an anticancer agent Duke, 1985
S. oxyriaefolius
South Africa Dried root Used by a Mpondo female Watt & Breyer-
herbalist as a remedy for Brandwijk, 1962
41
Table 2.3: Ethno-medical application o fS en e c io species
Species (Origin) Plant part Use Reference
sterility in a married woman
S. p a l m a t us
China Dried entire Used for amenorrhea Manual, 1974
plant Used for abdominal
distention and cramps.
S. p a uperculus
USA-TX Aerial parts Used as an emmenagogue Burlage, 1968
(hepatotoxic and/or
carcinogenic)
S. pseud-otites
Bolivia Dried leaf Used as an emmenagogue Gon/alez & Silva,
1987
Peru Known as an abortive Debelmas, 1975
properties
S. quinquelobus
India Dried seed Used for colic Joshi e t a l ., 1982
S. rhizom atus
Peru Dried leaf Used as an astringent Ramirez et a l.,
1988
Used as a diuretic
Used as a eupeptic
Used for pneumonia
Used in cases o f sterility
Used for acne
S. rudbeckiaefolius
Peru Leaf Used as an antitussive Yelasco-
Negueruela et a l.,
1995
Used to cure dislocations
S. salignus
Mexico Leaf Used as a tonic Zamora-Martinez
& Pola, 1992
Quatemala Dried leaf Used for ringworm Caceres et a l.,
1987
Used for pimples and
pustules
Used for conjunctivitis
S. sc an dens
India Aerial part Used for jaundice Srivastava, 1993
95 Used for malaria
China Entire plant Used as an anti-cancer agent Duke & Ayensu.,
1985
Used for fever
China Dried entire Used for ophthalmic lam e t al., 2000 &
plant disorders Malsuda et al.,
1995

India Dried leaf Used for malaria Manual, 1977

Used for eye troubles Srivastava, 1993
S. schim peri

42
Table 2.3: E thno-medical application of Senecio species
Species (Origin) Plant part Use Reference
Yemen Dried leaf Used medicinally Fleu rent in et a l.,
1983
S. serratuloides var. serrutuloides
South Africa Dried aerial Used for tuberculosis Fall & Meyer,
parts 1999
S. sonchifolius
Indonesia Fresh root Used for mild diarrhea. Hirschhorn, 1983
S. species
Tanzania Dried aerial Used to treat fever Chhabra & Uiso,
parts 1991b
Used to treat sores.
Used to treat colic
Used to treat skin rashes
Yemen Used as bush tea Scott et a l ., 1962
Used medicinally Fleurentin et al.,
1983
S. spegazzinii
Argentina Dried aerial Used to treat earache Giberti, 1983
parts
S. tenuifolius
India Entire plant Used for amenorrhea Nagaraju & Rao,
1990
Used for dysmenorrhea
S. triangularis
India Dried entire Used as a sedative. Rucgcr & Benn,
plant 1983
Used to treat chest pain
USA Dried leaf + Used as a sedative Hart, 1981
root Shcmluck, 1982
S. tussilaginis
Canary islands Dried aerial Used as an anti-tussive Darias et al., 1986
parts
Used for catarrh in children
S. uspallatensis
Argentina Dried root Used as a substitute for mate Pestchanker el al .,
infusion (ilex 1985b
paraguariensis)
S. vaccinioides
Ecuador Dried entire Used as an emmenagogue. Gon/alez & Silva,
plant 1987
S. viridis var. viridis
Argentina Dried leaf Chewed to calm tooth pains | Giberti, 1983
S. volckm annii
Argentina Dried aerial Used to treat shock Giberti, 1983
parts
S. vulgaris
England Aerial parts Used for internal ulcer Culpeper, 1650
healing
Used for sciatica
Used as an antiepileptic Dragendorff, 1898
43
Table 2.3: Ethno-medical application o f Senecio species
Species (Origin) Plant part Use Reference
Used for menstrual 55

troubles/complaints 55

Used as an anthelmintic 55

Used as an Chopra et al.,

em menagogue( hepatotoxic 1949
India Sap and/or carcinogenic
Used as an emmenagogue Al-Rawi &
Iraq )) Chakravarty, 1964
Italy 55 De l eo el al.,
Used as a washing to treat 1992.
varicose veins
55 Entire plant Used to treat internal
varices.
Ecuador 55 Used as an emmenagogue. Gonzalez & Silva,
1987
Europe 55 Used for amenorrhea Watt & Breyer-
Brandwijk, 1962
France Dried entire Used as an emmenagogue Ben/.anger-
plant (hepatotoxic and/or Beauquesne el al.,
carcinogenic) 1980
Tunisia Dried entire Used as an emmenagogue Lcmordant et al.,
plant 1978
USA Root Used as a uterine sedative
Used as an emmenagogue ”

Dried entire Used as an emmenagogue Watt and Breyer-

plant Brandwijk, 1962

2.4 PHYTOCHEMISTRY OF THE GENUS D O D O N A E A

Review on the chemical information of the Dodonaea species reveals that the most

commonly reported secondary metabolites are diterpenes, flavonoids, triterpenes, and

shikimate-derived metabolites. Others compounds include essential oils, carbohydrates and

sterols. These classes of compounds are the main constituents of plant resins [Jefferies &

Payne, 19731.

2.4.1 COMPOUNDS FROM D O D O N A E A SPECIES

2.4.1.1 DITERPENES FROM D O D O N A E A SPECIES
Many Dodonaea species possess viscid resinous exudate on the leaves surfaces. The resins

are composed mainly of bicyclic diterpenes, with the occasional inclusion of flavones

[Jefferies & Payne, I973|. Bicyclic diterpenes (69-87) (Table 2.4) based on the e7?/-labdane

44
and e/!/-clerodane skeleton have been isolated from various D odonaea species. From D.

viscosa, the most widespread member of the genus, the diterpenoids, huutriwaic acid (17)

[Mata et a l ., 1991]. methyl dodonate A (92) [Ortega el al., 20011, methyl dodonate B (93)

[Ortega e t a l ., 2001],, methyl dodonate C (94) [Ortega el al., 2001 ], the e«/-labdane furan

(76) [Mata el a l ., 1991] and the e/7/-clerodane furan (82) (Sadchev et at., 1984) have been

reported.

Table 2.4: Labdane and clerodane type diterpenoids from D odonaea species
Labdane diterpenoids
6,8-Dihydroxy-15-labdanoic acid; D. inaequifolia Dawson e t al., 1966
(e/?/-6a,8a,13£)-form, 6-Ac (81)
8-Hydroxy-15-labdanoic acid; (e n t - D. lobulata Dawson e t al., 1966
6a,8a,13£)-form (82) D. ptarm icaefolia
6,8-Dihydroxy-15-labdanoic acid; D. lobulata Dawson et al., 1966
(en t-6 a ,S a ,\ 3£)-form (83) D. ptarm icaefolia
7.8-Dihydroxy-15-labdanoic acid; D. lobulata Dawson et al., 1966
(e72/-6a,8a,13^)-form (84) D. ptarm icaefolia
15,16-Epoxy-3-hydroxy- D. petiolaris Jefferies et al., 1981
8( 17), 13( 16), 14-labdatrien-18-oic
acid; (ewf-3(3)-form, 3-Ac (85)
7.13-Labdadiene-2,15-diol; 15- D. m icrozyga Jefferies et al., 1974
Carboxylic acid (86)
2.17-Dihydroxy-7,13-labdadien-15- D. m icrozyga Jefferies et al., 1974
oic acid; (e/?/-2a, 13£)-form (87)
15.16-Epoxy-13( 16), 14-labdadiene- D. viscosa Dawson et al., 1966
3,8-diol.(e/?6-3(3,8a)-form (11) Mata et al., 1991
e/7/-Labdanolic acid (91) D. lobulata Dawson et al., 1966
Clerodane type diterpenoids from Dodonaea species
Hautriwaic acid (17) D. attenuata var linearis Jefferies & Payne, 1967
I), viscosa Jefferies & Payne, 1973
7a-Hydroxy- hautriwaic lactone D. attenuata Dawson el al., 1966
(88)
Hautriwaic lactone, 7-Ac (89) D. attenuata Dawson et al., 1966
Hautriwaic lactone (90) D. attenuata var. linearis Jefferies & Payne, 1967
Abdel-Mogib et al., 2001
2P-Hydroxy hardwickiic acid (9) D. boroniifolia Jefferies et al., 1973
Dodonic acid (10) D. viscosa Sachdcv e t al., 1984
Methyl dodonate A (92) D. viscosa Ortega et al., 2001
Methyl dodonate A (93) D. viscosa Ortega et al., 2001
Methyl dodonate C (94) D. viscosa Ortega et al., 2001
Dodonolide (95) D. viscosa Ortega et al., 2001

45
 12

81 OAc
83 OH

88 OH
89 OAc
90 H

46
2.4.1.2 TRITERPENES FROM DODONAEA SPECIES.
Lupeol (69) and the 11 (3-hydroxy derivative (96) were isolated from D odonaea attenuatta

var. linearis [Ghisalberti et al., 1973], Oleonic acid (70) and hederogcnol (97) have been

obtained from the leaves o f D. m adagascariensis [Trotin. 1972|. A number of

polyhydroxylate triterpenes containing the oleone skeleton have also been isolated. Among

these jegosapogenol (98) and Rrbarrigenol (99) were obtained from the stem bark of D.

viscosa [Dimbi et a l 1985], The seeds (Khan et a l ., 1992), flowers and stems [Dimbi et a l .,

1985] o f D. viscosa contain derivatives of Rrbarrigenol (99) esterified by angelic acid at C-

16, C-21 and C-22. From the seeds of D. attenuata , the two 21, 22-diesters (100) and (101)

and the monoester (102) have been obtained after mild acid hydrolysis of the sapogenins.

47
2.4.1.3 SHIKIMATE DERIVED METABOLITES FROM DO D O N A EA SPECIES

Shikimic acid (108) [Khan et a l 1992] and a group of shikimic acid derived aromatic

compounds. 74-76 and 109-112, have been isolated from Dodonaea species.

CHO

OMe
OH

108
110

48
2.4.1.4. FLAVONOIDS FROM D O D O N A E A SPECIES
Many flavones I, 4-6, 8. 12. 14-15, 21. 77-80 and 113-123 ( Table 2.5), have also been

isolated from the seeds, bark, flowers and leaves of Dodonaea species. A significant number

contain a methoxy group at C-3 and C-6. In D. madagascariensis, flavonoids account for up

to 3.3% o f the dry weight of dry leaves [Trotin et al., 1970| and in D. lobulata [Dawson et

al., 1966] and D. attenuatei var. linearis [Jefferies et ah 1973] up to 1%.

2.4.1.4.1 FLAVANONES FROM THE GENUS D O D O N A E A .

The flavanones 5,7-dihydroxyflavanone (8) [Sachdev & Kulshreshlha, 1983] and 5,4-

dihydroxy 7-methoxyflavanone (79) [Mata et al., 19911 have been isolated from D.viscosa.

K, k2

8 H H
79 Me OH

49
2 .4 .1 .4 .2 FLA V O N ES FR O M TH E GENUS DODONAEA
R|

Table 2.5: Flavones from D odonaea species

3-Methoxyflavones
3-methoxyflavone Plant source R. r2 Rj R4 Rs Reference
5.7-Dihydroxy-3,6,4'- D. attenuate! OMe OMe OH H OMe Jefferies &
trimethoxyflavone (4) var. linearis Payne, 1973
D. viscosa
D. viscosa
var.
angustifolia
5-Hydroxy-3,6.7,4’- D. lobidata OMe OMe OMe H OMe Sachdev &
tetramethoxyllavone D. viscosa Kulshreshtha,
(14) 1983
5-Hydroxy-3,7.4'- D. viscosa OMe H OMe H OMe Dreyer, 1978
trimethoxyflavone (1)
5,7,4 -Trihydroxy-3- OMe H OH H OH Wollenvveber el
methoxyflavone (6) a l ., 1986
5,4'-Dihydroxy-3,6,7- OMe OMe OMe "ll OH Khan el a l ., 1992
trimethoxvtlavone
(12)
5.7-Dihydroxy-3,4'- OMe H OH H OMe
dimethoxyflavone
(21).
5,7,4'-Trihydroxy- OMe OMe OH H OH Sachdev &
3.6-dimethoxyllavone Kulshreshtha,
(113) 1983
5,7-Dihydroxy-3.6,4'- OMe OMe OH Pre OMe
trimethoxy-2'-
prenylflavone (114)
5, 7-Dihydroxy-3,6- OMe OMe OH [A] on
dimethoxy-2'-(3-
hydroxymethyl butyl)
flavone (115)
5, 7-Dihydroxy- OMe OMe OH [A] OMe
3,6,4'-trimethoxy-2'-
(3-
hydroxy methy 1buty 1)
flavone (116).
5,4'-Dihydroxy-3.6,7- OMe OMe OMe [A] OH “
trimethoxy-2'-(3-
hydroxymethylbutyl)
50
Table 2.5: Plavones from D odonaea species
flavone (1 1 7 ) .
5-Hydroxy-3,6.7,4'- OMe OMe OMe [A] OMe
tetraniethoxy-2'-(3-
hydroxymethylbutyl)
flavone (1 1 8 ) .
5.6,4'-Trihydroxy-7- OMe OH OMe 11 Oil Khan et al., 1992
methoxyflavone
( L 1 ? ) , _______________________________

Flavonols
Flavonol Plant R. r 2 Rj R-i Rs Reference
source
5.4'-Dihydroxy-7- D. viscosa OH H OMe H OH Wollenweber et al.,
methoxyflavonol (5 ) 1986

5,7,4'- OH H OH H OH Paris & Nothis,

Trihydroxyflavonol 1970
(15)
5,7,2'.4'- OH H OH OH OH Khan et al., 1992
Tetrahydroxyflavonol.
(78) _
5. 7,4'-trihydroxy-2'- OH H OH OMe OH
methoxyflavonol.(l 20)
Glycosides
Glvcosides Plant source R. r 2 R3 R4 Rs Reference
[77] D. m adagascariensis ORut H OH OH OH Wollenweber et al.,
D. viscosa 1986
[80] Ogal H OH OH OH Ram ac hand ran &
Subramanian, 1978
[121] D. m adagascariensis OGal H OG1 OMe OH Trot in et al., 1972
u
[122] D. viscosa ORha H OH OMe OH Khan et al., 1992
-gal
[1 2 3 ] ORut H OH OMe OH Ramachandran &
Subramanian, 1978
[A] = 3-Hydroxymethylbutyl; Pre = prenyl; Ogal = galactoside; OGIu g ucoside; ORha =
rhamnoside; ORut = rutinoside.

2.4.2 G E N E R A L P H Y T O C H E M IC A L IN F O R M A T IO N O N SENECIO S P E C I E S
A large variety of sesquiterpenoids [Bohlmann et a l ., 1985; Dupre et al.. 1991], triterpenoids

[Torres et al., 1998], diterpenoids [Dong- Liang et al., 1992], pyrrolizidine alkaloids

| Bohlmann et al.. 1986b] and shikimic acid and its derivatives [Cardoso et al., 1987] have

been characterized from Senecio species [Ndom et al., 2006].

51
2 .4 .2 .1 . P Y R R O L IZ ID IN E A L K A L O ID S F R O M SENECIO S P E C I E S
Pyrrolizidine alkaloids (PAs) are a class of phytochemicals found in several genera of the

plant families, the occurrence o f PAs is restricted to certain unrelated families within the

angiosperm. particularly the three plant families, Boraginaceae, Compositae (Asteraceae),

and Legumionsae (Fabaceae) and in more than 350 plant species, mainly the Heliotroprium,

Senecio , C ra ta l aria, and Sym phytum species.

The structures and numbering system of the four types of representative necine bases,

platynecine (124), retronecine (125), heliotridine (126). and otonecinc (127) are shown in

Figure 2.9. The platynecine type pyrrolizidine alkaloids do not contain a double bond in the

base, and retronecine and heliotridine are enantiomers. Because of their abundance and

toxicities, including hepatotoxicity and carcinogenicity, the retronecine- and heliotridine-

derived pyrrolizidine alkaloids have received the most attention.

HO H C H 2OH CH ,O II 01,0 1 1 IIO „ CH ,O H

H° \ » H9 ij
\ 9
\ \
V -N ^ y \^ -N - < ^ H
v y
Platynecine Retronecine H eliotridine CH,
O to n e c in e

F i g u r e 2 .8 : R e p r e s e n t a t i v e n e c in e b a s e s .

Table 2.6: Pyrrolizidine alkaloids from Senecio species

A lk a lo id S o u rc e R e fe re n c e
Bulgarseninc (124) S. ahrotanifalius Boeder et al., 1984
S. doronicum
S. nem orensis
Platyphylline (125) S. adnatus Koekemoer et al., 1951
S. hygrophyllus
S. platyphyllus
Platyphyl 1ine A-oxide (126) S. adnatus Koekcmoer e ta l., 1951
S. hygrophyllus
S. platyphyllus
Adonifoline (127) S. adonidifolius Witte et al., 1992b
Erucifoline (128) S. aegypticus Witte et al., 1992a; Boeder
52
Table 2.6: Pyrrolizidine alkaloids from Senecio species
Alkaloid Source Reference
S. erucifolius et al.. 1993
S. erraticus
S. ja c o b a e a
S. perso o n ii
Jacozine (129) S. alpinus Klasek e t a l 1968
S. jacobaea
S. incanus
Angularine (130) S. angulatus Porter et al., 1962
Rosmarinine (131) S. angulatus Roitman, 1983
S. braychypodus
S. hygrophyllus
S. pauciligulatus
S. ta iw a n em is
S. triangularis
S. rosm arinifolius
9-Angeloylhastanecine (132) S. aquations ssp. Christov et al., 2002a
barbareifolius
S. chrysocom a
Eruciflorine (133) S. argunensis Liu et a/., 1991
S. erucifolius
S. ja c o b a e a
14J5-/ram-Senaetnine (134) S. aucheri Boh 1mann et al., 1977;
1978c; 1979a
Dehydrosenaetnine (135) S. barbertonicus Boh 1mann et al., 1977;
1978c; 1979a
Jacobine (136) S. brasi/iensis Bradbury et al., 1954
S. cineraria
S. ja c o b a e a
Sencalenine (137) S. cacaliaster Boeder et al., 1984
11-O-Acetylbulgarsenine (138) S. callosus Romo de Vivar et al., 2007
11-O-Acetylbulgarsenine A^-oxide (139) S. callosus Romo dc Vivar et al., 2007
Af-Chloromethylbulgarsenine (140) S. callosus Romo de Vivar et al., 2007
Callosine (141) S. callosus Perez-Castorena. et al.,
1998
0 J-Senecioylmacronecine (142) S. caudatus Bohlmann et al., 1986b
O ’-Senecioylmacronecine (143) S. caudatus Boh 1mann et al., 1986b

Retronecine 9-(2,3-dihydroxy-2- S. caudatus Bohlmann et al., 1986b

hydroxymethylbutanoate) 7-senecioate
(144) '
Retronecine 9-(2,3-dihydroxy-2- S. caudatus Bohlmann et al., 1986b
methylbutanoate) 7-senecioate N-oxide
(145)
Retronecine 9-(3-acetoxy-2-hydroxy-2- S. caudatus Bohlmann et al., 1986b
methylbutanoate) 7-senecioate (146)
Retronecine 9-(3-acetoxy-2-hydroxy-2- S. caudatus Bohlmann et al., 1986b
methylbutanoate) 7-senecioate (147)
Senecicaudatin O-isopentanoate (148) S. caudatus Bohlmann et al., 1986b
Senecicaudatin O-senecioate (149) S. caudatus Bohlmann et a l, 1986b
Senecicaudatinal hemiacetal (150) S. caudatus Bohlmann et a l, 1986b

53
Table 2.6: Pyrrolizidine alkaloids from Senecio species
Alkaloid Source Reference
Retronecine 9-sarracinate 7- senecioate A- S. caudal us Bohlmann et al., 1986b
oxide (151) S. umgeniensis
Retronecine 9-sarracinate 7- senecioate S. caudatus Bohlmann et a l 1986b
(152) S. triangularis
S. variabilis
0 7-Senecioylretronecine (153) S. caudatus Bohlmann et al., 1986b
S. variabilis
7-Hydroxy-1-methylenepyrrolizidine; S. chrysocoma Grue and Liddell, 1993
(7/?,7aR)-form, Angeloyl (154)
7-Hydroxy-1-methylenepyrrolizidine: S. chrysocoma Liddell et al., 1993
(7S,7aR)-fonn, Angeloyl (155)
7-Hydroxy-1-methylenepyrrolizidine; S. chrysocoma Benn et a l, 1995
Angeloyl, Ar-oxide (156)
Neosarracine (157) S. chrysocoma Stelljes et a l, 1991
S. hydrophyllus
S. kaschkarovii
S. mikanioides
Rivularine. 7-Angeloylheliotridine (158) S. crisp at is Bohlmann et al., 1986b
S. rivularis
2-Hydroxy-1- S. deferens 1lirschmann et al., 1988
hydroxymcthylpyrrolizidine;
( \ R,2R,7 aS)-i'ovm. O 1-Angeloyl, A-oxide
(159)
Doriasenine (160) S. doria Roder et a l, 1988
Doronenine (161) S. doronicum Romo l)e Vivar et a l, 2007
Riddelline (162) S. eremophilus Adams et a l, 1957
S. longiflorus
S. riddellii
O-Acetylerucifoline (163) S. erucifolius Witte et a l, 1992a
S. jacobacea
Integerrimine A-oxide (164) S. erucifolius Barrero et al., 1991
S. nebrodensis
S. vulgaris
Sarracine (165) S. franc hetii Kramov, 1967
S. mikanoides
S. rhombifolius
S. sarracenius
S. sylvaticus
Sarracine A-oxide (166) S. chrysocoma Christov et a l, 2002b
S. mikanoides
S. sarracenius
FuchsiSenecionine (167) S. fuchsii Roeder et al., 1977
8-Ethoxy-3-oxo-1,2-dehydroretrorsine S. grisebachii Hirschmann et a l, 1985
(168)
Hadiensine (169) S. hadie ns is Were et al., 1991
12-O-Acetylhadiensine (170) S. hadie ns is Were et al., 1991
Petitianine (171) S. hadiensis Were et al., 1991
12-O-Acetylneohadiensine (172) S. hadiensis Wercet a l, 1991
Neorosmarinine (173) S. hadiensis Were et al., 1991
12.S-Hydroxyretroisosenine (174) S. helodes Perez-Castorena et al.,
54
Table 2.6: Pyrrolizidine alkaloids from Senecio species
Alkaloid Source Reference
S. rose us 1997b
Hygrophylline (175) S. hygrophyllus Schlosscr et al., 1965
Triangularicine (176) S. hydrophyllus Stelljes et al., 1991
S. mikanoides
Neosarranicine (177) S. hydrophyllus Stelljes et al., 1991
S. mikanoides
S. serra
Sarranicine (178) S. hydrophyllus Stelljes et al., 1991
S. mikanoides
S. serra
1.7-Dihydroxy-1- S. integrifolius Roedcr et al., 1991
hydroxymethylpyrrolizidine;
( 1R,7R,7 aR)-form, A-Me, 0 \ 0 V-
diangeloyl (179)
Rivularine N-oxide (180) S. integrifolius Bohlmann et al., 1986b
var. fauriri
Aucherine (181) S. integrifolius Sener et al., 1988
subsp. aucheri
Jacoline (182) S. jacobaea Bradbury et al., 1954
Jaconine (183) S. jacobaea Bradbury et al., 1954
Sceleratine N-oxide (184) S. latifolius Bredenkamp et al., 1985b
Merenskine N-oxide (185) S. latifolius Bredenkamp et al., 1985b
Merenskine (186) S. latifolius Gordon Gray et al., 1967
Sceleratine (187) S. latifolius Bredenkamp et al., 1985b
S. sceleratus
Diangeloylplatynecine (188) S. macedonicus Christov et al., 2002a
8-Episarracine (189) »S', macedonicus Trendafilova et al., 1995
8-Episarracine A^-oxide (190) S. macedonicus Trendafilova et al., 1995
Macrophylline (191) S. macrophyllus Danilova et al., 1955
(^-Seneciphylline epoxide (192) S. megaphyllus Bohlmann et al., 1986b
Seneciphylline epoxide (193) S. megaphyllus Bohlmann et al., 1986b
S. usgorensis
Oxyretroisosenine (194) S. mulgediifolius Klasek et al.. 1973
Mulgediifoline (195) S. mulgediifolius Klasek et al.. 1973
l,2-Dihydroxy-7- S. nemorensis Christov et al., 2005
hydroxymethylpyrrolizidine;
{\ R,2R,7R,7 aR)-form, 2-Angeloyl (196)
Nemorensine (197) S. nemorensis Klasek et al., 1973
(several varieties)
1.2-Dehydrofuchsisenecionine (198) S. nemorensis var. Bohlmann et al., 1986b
Jitchsii
S. variabilis
Oxynemorensine (199) S. nemorensis var. Klasek et al., 1973
subdecurrens
Retroisosenine (200) S. nemorensis Klasek et al., 1973
S. mulgediifolius
Bisline (201) S. othonniformis Susag et al., 2000
S. petasis Coucourakis et al., 1970,
S. ruwenzoriensis
Erucifoline N-Oxide (202) S. persoonii Roedcr et al., 1993
55
Table 2.6: Pyrrolizidine alkaloids from Senecio species
Alkaloid Source Reference
Seneciphylline N-oxide (203) S. persoonii Roeder et a l ., 1993
Neoplatyphylline (204) S. platyphyllus Jiang et a l. , 2006
S. rhom bifolius Cai and Wang, 1983
Isorosmarinine (205) S. pterophorus Liddell et a l 1993
Seneciphyllinine (206) S. pterophorus Liddell et al., 1993
Racemocine (207) S. racem osus Ahmed et al., 1991c
Racemonine (208) S. racem osus Roeder et al., 1977
Racemodine (209) S. racem osus Ahmed et al., 1993
13/?-Hydroxyretroisosenine (210) S. rose us Perez-Castorena et al.,
1997b
7-Hydroxy-1-methylenepyrrolizidine; S. schwe infurthii Benn et al., 1995
(7R J a R ) - form, A-oxide (211)
18-Hydroxyjaconine (212) S. selloi Krebs et al., 1996
Spartioidine (213) S. spartio ides Adams et al., 1957
Dihydroretrorsine (214) S. subulatus var. Pestchanker et al., 1985a
erectus
Swazine (215) S. swaziensis Gordon-Gray et al., 1974
Triangularine (216) S. triangularis Roeder et a l , 1984
Neotriangularine (217) S. triangularis Roeder et a l , 1984
Usaramoensine (218) S. usaram oensis Adams et al., 1953
Uspallatine (219) S. uspallatensis Pestchanker et a l, /985b
Senecivernine (220) S. vernal is Topuriya et al., 1982
Spartioidine jV-oxide (221) S. vulgaris Roeder et al., 1993
Retrorsine (222) S. spp Roitman, 1985
Retrorsine N-Oxide (223) S. spp Lock dc Ugaz et al., 1990
Senaetnine (224) S. spp Bohlmann et al., 1977;
1978c; 1979a
Senecionine (225) ■V. spp Barger et al., 1936
Integerrimine (226) S. spp Adams et al., 1953
Seneciphylline (227) S. spp Villarrocl et al., 1985

R, R, ‘*3

125 H II II Me

131 OH II H Me

169 H OH II Me

170 11 OH Ac Me

171 II OH II c h 2o h

172 H OH Ac M e (1 5£-isom er)

173 OH H II M e (1 5£-isom er)

204 H H II M e (15£-isom er)

214 II H II C H 2O H

56
57
 R, *3
K,
132 Ang* II OH

142 H Osene H
143 Sene* OH II

157 2-Hydroxyme-2-buten* II 1igl (2'E,2"Z)-isomer

or3
—-OR, 165 2-Hydroxyme-2-buten* H Tigl
Jt\ 167 Sene* >1 OH
/ 7 |8 \
r r2 177 2-Hydroxyme-2-buten II 1igl ;2'£,2"£>isomer
5 3 178 2-Hydroxy me-2-buten II Tigl(2'Z,2"£)-isomer

188 Tigl* 11 Tigl

189 2-Hydroxyme-2-buten tl 1igl (7a-epimer)
191 Tigl Oil II

196 H Tigl OH
207 Tigl II OH

* Abbreviations given on pages xvi-xvii

OR3 R, k2
r - or,
159 Tigl OH 11
Y r c
166 2-Hvdroxyme-2-buten H Tigl
/ R'
* 190 2-Hvdroxyme-2-buten H A ng*
O

* Abbreviations given on pages xvi-xvii

R2 *3 *4

Me H CH2OII
Me II Me (15/i-isomer, 12-epimer)
Me H Me
Me Me H

CH2OH II Me
225 ll Me II Me
226 || Me H Me (15/V-isomer)

58
 C)

134 Ac (14,15-trans)
224 Ac

137 Tigl* 3-Hydroxyme-2-buten

144 Sene 2,3-Dihydroxy-2-hydroxymebu

146 Sene 3-Aeetoxy 2-hydroxy-2-mebu

152 Sene 2-Hyroxymethyl-2-buten

153 H Sene
158 H 2-Me-2-buten
160 2-Hyroxymethyl-2-buten 3-Hyroxymethyl-2-buten

176 2-Hyroxymethyl-2-butcn Tigl (2"/>-isomer)

198 Sene H

216 2-Hyroxymethyl-2-buten Tigl

217 2-Hyroxymethyl-2-buten rigl(27:-isom er)

* Abbreviations in pages xvi- xvii

59
 R, 1*2

145 Sene 2,3-Dihydroxy-2-methylbu

147 Sene 3-Acetoxy-2hydroxy-2-mebu

151 Sene 3-Acetoxy-2,2dihydroxy-bu

180 H Tigl

R, r2

138 H Ae

141 CH2OA c H

60
61
 ()

R, r2
R;
181 OH H H Me Me

182 Me H OH OH Me

183 Me H OH Cl Me

186 Me Me OH H Cl

201 Me FI OH H Me

R,

R,
184 OH

185 Cl

62
 195 (11,14-diepimer)

63
2.4.2 2 SESQUITERPENOIDS FROM S E N E C IO SPECIES
Sesquiterpenoids comprise a large and diverse class o f isoprenoids found in plants, fungi,

select bacteria, and insects [Loomis & Croteau, 1980; Cane, 1981]. In plants,

sesquiterpenoids are often associated with essential oils [Loomis & Croteau, 1980], and

except for a limited number of cases, such as the growth regulator abscisic acid [Wareing,

1978], There is a large number o f sesquiterpenoid carbon skeletons, which all. however, arise

from the common precursor (farnesyl pyrophosphate) by various modes of cyclisations

followed, in many cases, by skeletal rearrangement [Cane, 1981].

Different classes of sesquiterpenoids [Torres et al., 19981, have been characterized from

Senecio species [Ndom et al., 2006] including eremophi lanes (228-293),

64
furanoeremophilanes (299-351), cacalol (352-376), bisabolanes (377-406), seco and

abeoeremophilanes (407-426), simple germacranes (427-454), oplapane (455-463) and

simple eudesmanes (464-472) and benzofuranoids (473-485).

2.4.2.2.1 EREMOPHILANES SESQUITERPENOIDS

Eremophilanolides are sesquiterpenes biogenetically described as rearrangement products

derived from farnesy Ipyrophosphate cyclization [Mann el al., 1994], They are derived from

eudesmanes by migration of the methyl group at C-10 to C-5 [Pindcr, 1977], There is

confusion in the literature about the numbering o f carbons 14 and 15. The biogenetic

numbering given below should be used. The normal stereochemistry is shown, although there

are several exceptions to this [Pinder, 1977]. The configuration and numbering of eudesmane

(decahydro-1,4a-dimethyl-7-( 1-methylethyl)-naphthalene) and eremophi lane (decahydro-

l.8a-dimethyl-7-(l-methylethyl)-naphthalene ) is shown below.

14 11

12
12

Eudasmane skeleton Eremophi lane skeleton

As with the other larger categories, eremophilanes can be classified further into simple

eremophi lanes, eremophi lanol ides and furanoeremophi lanes, seco- and abeoeremophi lanes

and noreremophilanes [Pinder, 1977].

These secondary metabolites, along with pyrrolizidine alkaloids, are the most common

natural products isolated from Senecio species [Bohlmann et a/., 1977: Rizk, 1991. Table

2.10 below lists some of eremophilanolides sesquiterpenoids isolated from this genus.

65
Table 2.7: Eremophilanoides of Sertecio species
Erem ophilane Source References
1,8-Dihydroxy-7(l 1),9-eremophiladien-12,8- .S', aegyptius Ciarduno-Ramirez et
olide; (1 p,8cx0//)-form (228) var. disc aide us a l ,2001

1.8-Dihydroxy-7( 11 ),9-eremophiladien-12,8- S. aegyptius (iai-duno-Ramirez et

olide; (lp,8a(7//)-form , var. discoideus al., 2001
8-Me ether (229)
l-Hydroxy-7( 11 ),9-eremophiladien-12,8-olide; S. aegyptius (iarduno-Ramirez et
(lp,8p)-form (230) var. discoideus al.. 2001
8-Hydroxy-1(10),7( 11 )-eremophiladien-12.8- .S’. aegypticus (iarduno-Ramirez et
olide; 8aO//-form , 1p, 1OP-Epoxide (231) var. discoideus al.. 2001

8-Hydroxy-1(1 ()),7( 11 )-eremophiladien-12.8- S. aegypticus (iarduno-Ramirez et

olide; 8aO//-form , Me ether, ip,lOp-epoxide var. discoideus al.. 2001
(232)
l.8-Dihydroxy-7( 11 )-eremophilen-12,8-olide; .S’. almeydae Dupre et al., 1991
(1 a,8pO//, 10P)-form,
i-Tigloyl (233)
3,9-Dihydroxy-7( 11 )-eremophilen-8-one; S. almeydae Dupre et al., 1991
(3a,9a, 10a)-form (234)
3.9-Dihydroxy-7( 11 )-eremophilen-8-one; S. almeydae Bohlmann et al.,
(3a,9a,10p)-form, 3-Angeloyl (235) S. syl vatic us 1977; 1978j; 1985
3,9-Dihydroxy-7( 11 )-eremophilen-8-one; S. almeydae Bohlmann et al.,
(3a,9a,10P)-form, 1977; 1978j,; 1985
3-Tigloyl (236)
1l-Eremophilene-3,8,9-triol; S. almeydae Dupre et al., 1991
(3a,7a//,8a,9a. 10p)-form. 9-Ketone (237)
1l-Eremophilene-3.8,9-triol; .S’. almeydae Dupre . et al., 1991
(3a,7p//,8a,9a. 10a)-form (238)
11-Eremophilene-3.8,9-triol; S. almeydae Dupre et al., 1991
(3a,7p//.8a,9a, 10a)-form. 8-Ketone (239)
11-Eremophilene-3.8,9-triol; .S’. almeydae Dupre etal., 1991
(3a,7p//,8a,9a. 10a)-form, 8-Ketone, 3-angeloyl S. syl vatic us
(240)
11-Eremophilene-3,8.9-triol; S. almeydae Dupre et al., 1991
(3a,7p//,8a,9a. 10a)-form, 8-Ketone, 3-tigloyl .S’, syl vatic
(241)
11 -Eremophilene-3.8,9-triol; S. almeydae Dupree/ al., 1991
(3a,7p/7.8a,9a, 10a)-form, 8-Ketone, 3-(3- S. syl vatic us
methyl-2-butenoyl) (242)
1,10.,7,8-Diepoxy-3,6-dihydroxy-12, S. atratus Bohlmann et al.,
eremophilanolide; (1 p,3p.6p,7a,8a, I0p, 11 p//)- 1986a
form. 6-(2-Methylbutanoyl (243)
1,10.,7,8-Diepoxy-3,6-dihydroxy-12,8 S. atratus Bohlmann et al,
eremophilanolide; (ip,3p,6p,7a.8a,10p, 11 p//)- 1986a
form, 6-(3-Methylbutanoyl) (244)
1,10-Epoxy-3.6.8-trihydroxy-7( 11 )-eremophilen- S atratus Zhao etal., 1992
12,8-olide; (lp,3p,6p,8aO//,10p)-form. 6-(2-
Methylbutanoyl) (245)

66
I able 2.7: Eremophilanoides o fS e n e c io species
Eremophilane Source References
l,10-Epoxy-3,6,8-trihydroxy-7( 11 )-eremophilen- .S’ atratus Zhao e l al., 1992
12,8-olide: (1 p.3p.6p,8a<7//. 10p)-form. 6-(3-
Methylbutanoyl) (246)
1. IO-Epoxy-3.6.8-trihydroxy-7( 1 1)-eremophilen- S. at m tu s Zhao e ta l., 1992
12.8-olide: (1 p.3p,6p.8aO//.10P)-form. 6- l
Angeloyl (247)
7(1 l)-Eremophilen-12,8-olide; (8(3,10a//)-form S. aureus Cioto el al.. 2001
(248;
8-Hydroxy-7( 11 )-eremophilen-l 2,8-olide; S. aureus Zalkow el al., 1979
(8/?.10a7/)-form, 8-Et ether (249)
1.8-Dihydroxy-7( 1 l)-eremophilen-12.8-olide; S. bracteolatus Bohlmann el al.,
(4a.5a,8aO//,10a)-form , 1986a
1-Ketone (250)
l,8-Dihydroxy-7( 11 )-eremophilen-12.8-olide; S. cachinalensis Rcina el al., 2006
( i a.8\V)H, 10p)-form, S. p o e p ig ii
8-Me ether. 1-angeloyl (251)
1l-Eremophi!ene-3,8,9-triol; S. erubescens Bohlmann el al.,
(3a.?pA/,8a.9a, 10a)-form, 3-(4-Hex-2Z- var. 1977; 1978d: !982g;
enoyloxy-2Z-hexenoyl) (252) crepidifolius 1985

1!-Ercmophilene-3,8.9-triol; S. erubescens Bohlmann et al.,

(3a,7p//,8a,9a.! 0a)-form, 3-(4-Angeloyloxy-2Z- var. 1977; 1978d: 1982g;
hexenoyl) (253) crepidifolius 1985

1l-Eremophilen-9-one; lOp-form (254) S. fila g in o id e s Bohlmann et al.,

1986a
8J 2-Epoxy-8.12-dihydroxy-1,7(11 )- S. flavus Torres et a!., 1999
ercmophiladien-3-one: {%a()H,\ 2a)-form. Di-Me
ether (255)
8.12-Epoxy-8.12-dihydroxy-1.7(1 1)- S. flavus Torres et al., 1999
eremophiladien-3-one; (SaOH, 12P)-form, Di-Me
ether (256)
8-Hydroxy-7( 11 ),9-eremophiladien-12.8-olide; S. hieracioides Zhao et al., 2002
8pO//-form (257) S. tsoongianus
1lrEremophilene-3,8,9-triol; S. gerardii Bohlmann el a l,
1(3a,7a7/,8p,9p,10p)-form, 8-Ketone. 3-angeloyl, 1977; 1978d,g,j:
9-(3-methyl-2-butenoyl) (258). 19 8 2 g ;1985

j 11 -Eresnophilene-3,8,9-triol; S. gerardii Bohlmann et al.,

! (3a,7aM8p.9p,10P)-form, 8-Ketone, 3,9-bis(3- 1977; 1978d,g,j;
methyl-2-butenoyl) (259) 1982g;1985

i 1l-Eremophilcne-3,8,9-triol; S gerardii Bohlmann et al.,

(3a,7o7/,8p,9p,10P)-form, 8-Ketone, 3-tigloyl, 9- 19/7; I978d,g,j;
(3-methyl-2-butenoyl) (260) 1982g;1985
1
' o-Hvdroxy-1(10),7(1 l)-eremophiladien-12,8- S. g /aber Dupre et a l .,, 1991
[ v)lide; (6P,8a)-form (261)
| 6-Hydroxy-l(10),7(l l)-ercmophiladien-12,8- S. glaber Perez et al., 1991

67
Table 2.7: E remophilanoides o f Senecio species
E re m o p h ila n e Source References
olide; (6a,8f3)-form (262) S. toluccanus
6-Hydroxy-1(10),7-eremophi ladien-12.8-olide; S. glaber Dupre et al. , , 1991
(6(3J I (3//)-form (263)
6,8-Dihydroxy-1 (10),7( 11 )-eremophiladien-12.8- S. glaber Perez et al., 1991
olide; (6p,8a)-form (264) S. toluccanus
1l-Eremophilene-3,8,9-triol; S. glanduloso- Bohlmann et al
(3a,7a//.8a,9p,10P)-form, 8-Ketone, 3-(4- p ilo su s 1977; 1978d,gj;
angeloyloxy-2Z-hexenoyl) (265) 1982g; 1985
1l-Eremopliilene-3,8,9-triol; S. glanduloso- Bohlmann et al
(3a,7a//,8a,9p,10P)-form, 8-Ketone, 3-(5- p ilosus 1977; 1978d,g,j;
angeloyloxy-2Z-hexenoyl) (266) 1982g; 1985
l,10-Epoxy-6.8-dihydroxy-7(l 1)-eremophilen- S. isal idetts Torres et al., 1999
12,8-olide; (1 p,6p,8p, 1Op)-form, 6-(2-
Methylpropenoyl) (267)
l,10-Epoxy-6.8-dihydroxy-7(l 1)-eremophilen- S. is at idens Zhao et al., 1992
12,8-olide; (ip,6p,8p,10p)-form, 6-(2-
Methylpropenoyl) (268)
l,10-Epoxy-6.8-dihydroxy-7( 11 )-eremophilen- S. isatideus Zhao et al., 1992
12,8-olide; (1 p.6p,8p, 1Op)-form, 6-(3-Methyl-2-
butenoyl) (269)
1,10-Epoxy-6,8-dihydroxy-7( 1 1)-eremophilen- S. isatideus Zhao et al., 1992
12,8-olide; (ip.6p,8p, 10P)-form, 6-Angeloyl
(270)
1,10-Epoxy-3,6,8-trihydroxy-7( 11 )-eremophilen- S. m auricei Bohlmann et al.,
12,8-olide; (ip,3p,6p,8aO//,IOp)-form, 6-(2- 1978f
Methylpropanoyl), 1-Ac (271)
l,10-Epoxy-3.6.8-trihydroxy-7( 11 )-eremophilen- S. m auricei Bohlmann et al.,
12,8-olide; (ip.3p,6p,8aD//,10P)-form, 6-(2- 19 78f
Methylpropenoyl), 1-Ac (272)
1.8-Dihydroxy-7( 11 )-eremophilen-12,8-olide; S. m iser Reina et al., 2001
(1 a,8pO //, 10P)-form,
8-Me ether, 1-Ac (273)
3,9-Dihydroxy-7( 11 )-eremophilen-8-one; S. ochoanus Bohlmann et al., 1983
(3a,9p, 10P)-form, 3-Tigloyl (274)
9,10-Epoxy-7( 1 l)-eremophilen-8-one; (9a«10a)- S. oldham ianus Yang et al., 2001
form (275)
8,12-Epoxy-1(10),7( 11 ).8-eremophilatriene-6,12- S. pachyphyllos Ahmed et a l, 1991 a,b
diol; (6p, 12£,)-form, 6-(2-Methylpropanoyl), 12-
Me ether (276)
8,12-Epoxy-1(10),7( 11 ),8-eremophilatriene-6,12- S. pachyphyllos Ahmed et a l, 1991a,b
diol; (6p,12^)-form, 6-Propanoyl, 12-Me ether
(277)
1,8-Dihydroxy-11 -eremophilen-9-one; S. portalesianus Jakupovic et a l , 1991
(la.8 a, 10a)-form, 8-Angeloyl, 1-Ac (278)
1,8-Dihydroxy-11 -eremophilen-9-one; S. portalesianus Reina et a l , 2001
(la.8 a, 10a)-form, 8-Tigloyl, 1-Ac (279)
1,10-Epoxy-1l-eremophilen-8-ol ; (lp.8a, 10p)- S. portalesianus Jakupovic et a l, 1991
form, Angeloyl (280)
1,10-Epoxy-1 l-ereinophilen-8-ol ; (lp.8a,10P)- S. portalesianus Jakupovic et a l, 1991
68
Table 2.7: E reinop h iIano ides o f Senecio spec ies
Eremophilane Source References
form, Tigloyl (281)
11-Eremophilene-3,8.9-triol; S. Bohlmann et al
(3cc,7a//.8p.9p,10P)-form, 8-Ketone (282) rhyncholaenus 1977; I978d,g,j;
19 8 2 g ;1985
11-Eremophilene-3,8.9-triol; S. Bohlmann et al.,
(3a,7a//.8[3,9p, IOP)-form. 8-Ketone, 3-(3- rhyncholaenus 1977; 1978d,g,j;
methyl-2-butenoyl), 9-tigloyl (283) 19 8 2 g ;1985

l,10-Epoxy-4,6-dihydroxyfuranoeremophilan-9- S. salignus Bohlmann et al.,

one; (lp,4a,6p,10p)-form, 6-(2-Methylpropanoyl) 1976a
(284)
12-Hydroxy-7( 11 ),9-eremophiladien-8-one; S. serratifolius Dupre et al., 1991
7(1 l)£-form, Ac (285)
12-Hydroxy-7( 11 ),9-eremophiladien-8-one; S. serratifolius Dupre et al., 1991
7(11)Z-form, Ac (286)
4'-Angeloyloxyisosenspeciosone (287) S. speciosus Bohlmann et al.,
1977; 1978j
5'-Angeloyloxyisosenspeciosone (288) S. speciosus Bohlmann et a l,
1977; 1978j
1l-Eremophilene-3,8,9-triol; S. speciosus Bohlmann et a l,
(3a,7p//,8a,9a,10a)-form , 8-Ketone, 3-(3- 1977; 1978j; I982g;
methyl-2-butenoyl), 9-(2-methylbutanoyl) (289) 1985

3.6.8- Trihydroxy-1( 10),7( 11 )-eremophiladien- S. toluccanus Perez et al., 1991

12.8- olide; (3a,6p,8aO//)-form , 3-Ac (290)
Toluccanolide D
6.8-Dihydroxy-1(10),7( 11 )-eremophiladien-12,8- S. toluccanus Morales et al., 2000
olide; (6p,8a)-form, 8-Et ether (291)
1,6,10-T rihydroxy-7( 11 ),8-eremophiladien-12,8- S. tricephalus Bohlmann et al.,
olide; (lp,6p, 10a)-form, 6-Ac (292) 1986a
8-Hydroxy-1( 10),7(1 1)-eremophiladien-l 2,8- S. tsoongianus Zhang et al., 2005
olide; 8pO//-form, 1p,lOP-Epoxide (293)
8-Hydroxy-7( 11 ),9-eremophiladien-12,8-olide; S. tsoongianus Zhao et al., 2002
8aO//-form (294)
10-Hydroxy-7( 11 ).8-eremophiladien-12.8-olide; S. tsoongianus Zhao et a l , 2002
lOa-form (295)
l-Hydroxy-7( 11 )-eremophilen-12,8-olide; S. viravira Bohlmann et al.,
(la,8a)-form , Angeloyl (296) 1986a
1-Hydroxy-7( 11 )-eremophilen-12,8-olide; S. viravira Bohlmann et al.,
(la,8P)-form; Angeloyl (297) 1986a
6-Hydroxy-1(10),7( 11 ),8-eremophilatrien-12,8- S. spp Bohlmann e ta l., 1977
olide; 6P-form (298)

69
 R, r2

231 H H OMc

232 H H OMe

245 OH 3-Mebu OH

246 OH OAng OH

267 H 2-Me-2-propen OH

268 H OSene OH

269 H OTigl OH

270 H OAng OH
293 H H OH

Ri

234 H
235 Ang
236 Tig
287 4-Tigloyloxy-4-mcthyI-2Z-hexenoyl
288 5-Tigloyloxy-2Z-hexenoyl

238 II
252 4-llcx-2Z-enoyloxy-2Z-hexenoyI
253 4-Angeloyloxy-2Z-hexenoyl

70
 R, R 2

239 H H
240 H Ang
241 H Tigl
242 H Sene
258 Sene Ang
259 Sene Sene
260 Sene Tigl
265 H 4-Angeloyloxy-2Z-hexenoyl
266 H 5-Angeloyloxy-2Z-hexenoyl
282 H H
283 Tigl Sene
289 H Sene

R,
R, r2
248 H H
249 H OEt

251 OAng OMe

273 Ac OMc
296 OAng H (la, 8a)-form
297 OAng R (la, 8P)-forni

R, r2
254 H H
278 Ac Ang
279 Ac Tigl

71
72
 ()

298

2 .4 .2 .2 .2 F U R A N O E R E M O P H IL A N E S F R O M SENECIO S P E C I E S
Furanoeremophilanes are eremophilanes with a furan ring at the 7(8) position in the

eremophilane ring, while the furanoeremophilanoides are furanoeremophi lanes with one or

more ester functionality.

Furanoremophilane skeleton

Table 2.8: Furanoeremophilanoides from Senecio species.

F u ra n o e re m o p h ila n o id e s S o u rc e R e fe re n c e
Alloeophyllin (299) S. alloeophyllus Garrido e ta l., 1995.
Sendarwin (300) S. alloeophyllus Garrido et al., 1995
S. d a n v in ii
S. m edley-w oodii
3.6-Di hydroxy furanoeremoph i lan-9-one; S. andreuxii Bohlmann et al.,
(3a,6p,10f3)-form, 3-Angeloyl, 6-(3-methyl-2- 1978a
butenoyl) (301)
Furanoeremophi lane-1,6-diol; (1 a,6p, 1Op)- S. auricula Torres et al., 1998
form, 1-Ketone, 6-(2-methylpropanoyl) (302)
Furanoeremophilane-1,6-diol; (1 a,6p, 1OP)- S. auricula l orres et al., 1998
form, 1-Ketone, 6-(2-methylbutanoyl) (303)
Furanoeremophilane-1,6-diol; (1 p,6p, 10a)- S. auricula Torres e ta l., 1998
form, 1-Ketone, 6-(2-methylbutanoyl) (304)
1,10-Epoxyfuranoeremophilane-6,9-diol; S. hehnii Bohlmann et al
(1 p,6p,9p, 10P)-form, 9-(2-Methylpropanoyl) 198Ig ; Dupre et al.,
(305) 1991
Furanoeremophilane-1,6-diol; (1 a,6p, 10p)- S. bergii Torres e ta l., 1998
form, 1-Ketone. 6-propanoyl (306)
Furanoeremophilane-1,6-diol; (1 a,6p, 1OP)- S. bergii Torres et al., 1998
form, 1-Ketone, 6-tigloyl (307) S. bracteolatus
6,9-Dihydroxyfuranoeremophilan-l-one; S. bracteolatus Bohlmann et al.,
(6p,9p, 10p//)-form (308) 1986a
6.9-Dihydroxy furanoeremophi Ian-1-one; S. bracteolatus Bohlmann et al.,
(6p,9p,10p//)-form, 6-Tigloyl (309) 1986a
73
Table 2 .8: Furanoeremophilanoides from Senecio species.
Furanoeremophilanoides Source Reference
6,9-Dihydroxyfuranoeremophilan-1-one; S. bracteolatus Bohlmann et a i ,
(6p,9p, 10p//)-form, 1986a
6-Cinnamoyl (310)
1,10-Epoxyfuranoeremophilan-6-ol; S. doria Bohlmann et al .,
(1 p,6p, 1OP)-, 0(2-M ethylbutanoyl) (311) 1974; 1976a
6,9-Dihydroxytliranoeremophilan-1-one; S. fila g in o id es Bohlmann et a l .,
(6p,9p,l0p//)-form, 6-Pentanoyl (312) 1986a
Furanoeremophilane-1,6-diol; (1 a,6p. 10p)- S. filaginoides Forres et al., 1998
form.l-Ketone. 6-(3-methylbutanoyl) (313)
Furanoeremophilane-1,6-diol; (1 a,6p, 10a)- S. heliopsis Forres et a i, 1998
form. 1-Ketone, 6-(2-methylpropanoyl) (314)
Furanoeremophilane-1,6-diol; (1 p,6p, 10a)- S. heliopsis Torres et al., 1998
form, 1-Ketone. 6-(2-methylpropanoyl) (315)
l,6-Dihydroxyfuranoeremophilan-9-one; S. hypochoerideus Bohlmann et a i ,
(1 a,6p, 10a//)-form . 6-(3-Methylbutanoyl) 1978a
(316)
l,6-Dihydroxyfuranoeremophilan-9-one; S. hypochoerideus Bohlmann et al.,
(1 a,6p, 10a//)-form , 1978a
6-(3-Methyl-2-butenoyl), 1-Ac (317)
l,6-Dihydroxyfuranoeremophilan-9-one; S. hypochoerideus Bohlmann et al.,
(la,6p, 10p//)-form (318) 1978a

1,6-Dihydroxyfuranoeremophi lan-9-one; S. hualtaranensis Pestchanker et al.,

(1 a,6p, 10a//)-form , 1996
6-Propanoyl (319)
1,10-Epoxy-6-hydroxy-2-furanoeremophi len- S. m auricei Bohlmann et al.,
9-one; (1 p.6p. 10P)-form, 6-Methylpropanoyl) 19 78f
(320)
1,10-Epoxy-6-hydroxy-2-furanoeremophilen- S. m auricei Bohlmann et al.,
9-one; (1 p,6p, 10P)-form, 6-Methylpropenoyl) 1978f
(321)
6,10-Dihydroxy-2-furanoeremophilene-1,9- S. m auricei Bohlmann et al.,
dione; (6p, 10P)-form. 6-(2-Methylpropanoyl) 1978f
(322)
1,10-Epoxyfuranoeremophilane-3,6-diol: S. nem orensis ssp. Novotny et al., 1973
(1 p,3p,6p. 10P)-form. 6-(2-Methylpropanoyl) fu c h s ii
(323)
1.10-Epoxyfuranoeremophilane-3,6-diol; S. nem orensis ssp. Novotny et al., 1973
(1 P,3p,6p, 10P)-form, 6-(2-Methylpropanoyl), fu c h s ii
3-Ac (324)
1,10-Epoxyfuranoeremophi lane-3,6-diol; S. nem orensis ssp. Novotny et al., 1973
(ip,3p,6p, 10P)-form, 6-(2-Methylbutanoyl) fu c h s ii
(325)
1.10-Epoxyfuranoeremophi lane-3,6-diol; S. nem orensis ssp. Novotny et al., 1973
(1 P,3p,6p,10P)-form. 6-Angeloyl (326) fu c h s ii
1,10-Epoxyfuranoeremophilane-3,6-diol; S. nem orensis ssp. Ji/ba et al., 1981
(1 p,3p,6p,10p)-form, 6-(3-Methylbutanoyl) subdecurens
(327) _ ...
1,10-Epoxy-4.6-dihydroxyfuranoeremophi lan- S. salignus Bohlmann et a l,

74
Table 2.8: Furanoeremophilanoides from Senecio species.
F u ra n o e re m o p h ila n o id e s S o u rc e R e fe re n c e
9-one; (4a,6(3,10p)-form, 6-(2- 1976a
Methylpropanoyl) (3 2 8 )
1,10-Epoxyfuranoeremophilane-6,9-diol; S. smithii Bohlmann et al.,
(ip,6p,9p, 10P)-form, Diketone (329) 1981 g
Furanoeremophilane-1,3-diol; (1 a,3 a, 1OaH)- S. sm ithii Bohlmann et al.,
form, 3-(3-Methyl-2-butenoyl) (3 3 0 ) 1981 g
Furanoeremophilane-1,3-diol; (1 a,3 a, 10 a H)- S. sm ithii Bohlmann et al.,
form, 3-Angeloyl (3 3 1 ) 198 lg
Furanoeremophilane-1,3-diol; ( 1a,3 a, 10 a / / ) - S. sm ithii Bohlmann et al.,
form, 3-(2-Methylpropanoyl) (332) 1981 g
Furanoeremophilane-3,9-diol; (3a,9a, 10a)- S. speciosus Bohlmann et al.,
form, 3-(5-Methyl-2£,4£,6£-dodecatrienoyl) 1978j
(3 3 3 )
l,6-Dihydroxyfuranoeremophilan-9-one; S. um bellatus Pestchanker et al.,
(la,6p, 10a//)-form, 1996
6-(Methylpropanoyl) (334)
6,9-Dihydroxyfuranoeremophilan-l-one; S. viravira Bohlmann et al.,
(6p,9p,10p//)-form, 6-Angeloyl, 9-(2- 1986a
Methylpropanoyl) (335)
Furanoeremophilan-1-ol; ( 1a , 10P)-form. S. viravira Bohlmann et al.,
Angeloyl (336) 1986a
Furanoeremophilane-1,6-diol; ( 1a,6p, 10p)- S. viravira Torres et al., 1998.
form, 1-Angeloyl (337)
1,10-Epoxyfuranoeremophilan-6-ol; S. spp. Bohlmann et al.,
(1 p,6p,10p)-form, 6-Angeloyl (338) 19 7 4 ;1976a
6,10-Dihydroxyfuranoeremophil-l-en-3-one; S. spp. Bohlmann et al., 1977
(6p,10p)-form, 6-(2-Methylpropanoyl) (339)
6,10-Dihydroxyfuranoeremophi 1-1 -en-3-one; S. spp. Bohlmann et al., 1977
(6p,10P)-form, 6-(2-Methylbutanoyl) (3 4 0 )
6.10-Dihydroxyfuranoeremophi 1-1-en-3-one; S. spp Bohlmann et al., 1977
(6p,10p)-form, 6-Angeloyl (3 4 1 )
1.10-Epoxyfuranoeremophilane-3,6-diol; S. spp. Bohlmann et al., 1977.
( 1p,3a,6p, 10p)-form, 6-<9-(3-Methyl-2-
butenoyl) (342)
1.10-Epoxyfuranoeremophilane-3,6-diol; S. spp. Bohlmann et al., 1977
( 1p,3a,6p, 10P)-form, 6-0-(3-Methyl-2-
butenoyl), 3-Ac (343)
1.6-Dihydroxyfuranoeremophilan-9-one; S. spp. Bohlmann et al.,
(la,6 p , 10a//)-form, 1979b
6-Ac ( 3 4 4 )
1.10-Epoxyfuranoeremophilane-6,9-diol; S. spp. Bohlmann et al.,
(ip,6p,9p, 10P)-form, 6-Angeloyl (345) 198 lg
1.10-Epoxyfuranoeremophilane-6,9-diol; S. spp. Bohlmann et al.,
(1 p,6p.9p, 10p)-form, 6-(3-Methyl-2-butenoyl) 1981 g
(346)
6.9-Dihydroxyfuranoeremophi lan-1-one; S. spp. Bohlmann et al.,
(6p,9p,10p//)-form. 1986a
6-Angeloyl (347)
6.9-Dihydroxyfuranoeremophilan-1-one; S. spp. _ Bohlmann et al.,
75
Table 2.8: F uranoeremop hi Iano ides From Senecio species.
Furanoeremophilanoides Sou rcc Reference
(6p,9p, 1Op//)-form, 1986a
6-(3-Methyl-2-butenoyl) (348)
Furanoeremophilane-1,6-diol; (1 ot,6p, 1OP)- S. spp. Torres et al., 1998
form, 1-Ketone, 6-Ac (349)
Furanoeremophilane-1,6-diol; (1 a,6p, 1Op)- S. spp. l orres et al., 1998
form, 1-Ketone. 6-(3-methyl-2-butenoyl) (350)
Furanoeremophilane-1,6-diol; (1 a,6p, 10P)- S. spp. Forres et al., 1998
form, 1-Ketone. 6-angeloyl (351)

76
 R

M e p ro p
M e b u -la , f>p, 1Op - form
M e b u -I P, 6p, 10a - form
Pro p
T ig l
313 3 -M e b u
314 2 -M e p ro p la, 6p, 1 0 a - form
315 2-IMeprop ip, 6p, 1 0 a - form

O OH

OR.

R 2

R
311 H 2 -M c b u
308 H 323 OH 2 -M e p ro p
309 T ig l 324 Ac 2 -M e p ro p
310 C in n 325 OH 2 -M e b u
312 Pent 326 OH A ng
347 A ng 327 OH 3 -M e b u
348 3 - M e - 2 - b u te i 338 H A ng
342 OH 3 - M c 2 - b u te n
343 OAc 3 - M e - 2 - b u te n

Ri K;

316 H 3 -M e b u
317 Ac 3 - M e - 2 - b u te n
318 H H
319 H P ro p
334 H 2 -M e p ro p
344 H Ac

77
 () R

320 Meprop
321 M ep ro p en

K,

336 A ng H
337 A ng OH

78
 2.4 2.2.3 CACALOL SESQUITERPENES FROM S E N E C IO SPECIES
Cacalolides arc biogenetic Wagner-Meerwein rearrangement products | Burgueno-Tapia et

al., 2001J derived from eremophilanes and furoeremophilanes [Burgueno Tapia et al. , 2001],

typically with an aromatic ring B, in which carbon-14 has further migrated to C-6.

Cacalol skeletons

They derive their name from cacalol. a sesquiterpenoid that was isolated from the

anlihyperglycemic species C a ca lia decom posita (Romo & Joseph-Nathan, 1964). Table 2.12

below lists some of cacalol sesquiterpenoids isolated from this genus.

Table 2.9: Cacalol sesquiterpenes from Senecio species

Cacalol Source Reference
14(5—►OVAbeo-S^-furanoeremophiladiene- S. fu e rte sii Bohlmann et a l ., 1977;
O.M-diol; 4-form, 14-Aldehyde, 9-Me ether S. p ica rd a e 1978e; 1990
(352)

14(5—»6)-Abeo-5,9-furanoeremophiladiene- S. fu e rte sii Bohlmann eta l., 1990

2,9-diol; 2a-form, 9-Me ether, 2-Ac (353)
13-Hydroxydehydrocacalohastin-15-al (354) S. heliopsis Bohlmann et al., 1985
14-Hydroxycacalol propionate (355) S. inornatus Bohlmann e ta l., 1977;
1978c; 1990
S. inornatus Bohlmann et al., 1977;
14-Acetoxycacalol propionate(356) 1978e; 1990
14(5—»6)-Abeo-5,9-furanoeremophiladiene- S. lydenburgensis Bohlmann et al.,
2,9,14-triol; 2a-form, 9-Propanoyl, 14-Ac 1982b; 1985
(357)
14(5—»6)-Abeo-5,9-furanoeremophiladiene- S. lydenburgensis Bohlmann et al.,
2,9.14-triol; 2a-form, 9-Propanoyl, 2,14-di- 1982b;1985
Ac (358)
14(5—»6)-Abeo-5,9-furanoeremophiladiene- S. lydenburgensis Bohlmann et al.,
2,9,14-triol; 2a-form, 9 .14-Dipropanovl 1982b;1985
. (359)
14(5—»6)-Abeo-5,9-furanoeremophiladiene- S. lydenburgensis Bohlmann et a l,
2,9,14-triol; 2a-form, 14-(2- 1982b;1985
Methylpropanoyl), 9-propanoyl (360)
14(5—>6)-Abeo-5,9-furanoeremophiladiene- S. lydenburgensis Bohlmann et al.,
2,9,14-triol; 2a-form. 2-(3-Methylbutanoyl), 1982b;1985
9-propanoyl, 14-Ac (361)
79
Table 2.9: Cacalol sesquiterpenes from Senecio species
Cacalol Source Reference
14(5—»6)-Abeo-5,9-furanoeremophiladiene- S. lydenburgensis Bohlmann et al.,
2.9,14-triol; 2a-form, 14-(2- 1982b; 1985
Methylbutanoyl), 9-propanoyl (362)
14(5—>6)-Abeo-5.9-furanoeremophiIadiene- S. lydenburgensis Bohlmann et al .,
2,9,14-triol; 2a-form. 14-(3- 1987b; 1985
Methylbutanoyl), 9-propanoyl (363)
14(5—»6)-Abeo-5,9-furanoeremophiladiene- S. lydenburgensis Bohlmann et al.,
2,9,14-triol; 2a-form. !4-(3-Methyl-2- 1987b;1985
butenoyl), 9-propanoyl (364)
14(5—»6)-Abeo-5,9-furanoeremophiladiene- S. lydenburgensis Bohlmann et al.,
2.9,14-triol; 2a-form, 14-Angeloyl, 9- 1982b;1985
propanoyl (365)
14(5—»6)-Abeo-5,9-furanoeremophiladiene- S. lydenburgensis Bohlmann et al.,
2.9,14-triol; 2p-form, 9-Propanoyl, 2,14-di- 1982b;1985
Ac (366)
14(5-»6)-Abeo-5,9-furanoeremophiladiene- S. lydenburgensis Bohlmann et al., 1985;
9,13,14-triol; 9-Propanoyl, 13,14-di-Ac 1990
(367)
14(5^6)-Abeo-1,2-dihydroxy-1,3,5(10)- S. m adagascariensis Burgueno-Tapia et al.,
furanoeremophilatrien-9-one; 2-Me ether 2001.
(368)
14(5—»6)-Abeo-1,2-dihydroxy-1,3,5(10)- S. m adagascariensis Burgueno-Tapia et a l,
furanoeremophilatrien-9-one; Di-Me ether 2001
(369)
I3-Acetoxycacalol methyl ether (370) S. p ica rd a e Bohlmann et al., 1978e.
14(5—»6)-Abeo-5.9-furanoeremophiladiene- S. p ica rd a e Bohlmann et al., 1985;
9,13,14-triol; 9-Me ether, 14-angeloyl, 13- 1990
Ac (371)
14(5—>6)-Abeo-5,9-furanoeremophiladiene- S. picardae Bohlmann et a l , 1985;
9,13,14-triol; 14-Aldehyde, 9-Me ether, 13- 1990
Ac (372)
14(5-»6)-Abeo-5,9-furanoeremophiladiene- S. spp Bohlmann et al., 1977;
9,14-diol; 4p-form, 9-Me ether(373) 1978c; 1990
14(5-»6)-Abeo-2,9-dihydroxy-2,5,9- S. spp. Bohlmann et a l, 1977
furanoeremophilatrien-l-one; 2-Me ether
(374)
14(5-»6)-Abeo-3,9-dihydroxy-2,5,9- S. spp. Bohlmann et al., 1977
furanoeremophilatrien-1-one; 3-Me ether
(375)
i-----------
Oj

14(5->6)-Abeo-3,9-dihydroxy-2,5,9- Bohlmann et al., 1977

o.o
w
cL

furanoeremophilatrien-l-one; Di-Me ether

(376)

80
 R, r 2 Rj

355 H P ro p OH
356 H P ro p Ac
357 OH P ro p Ac
373 H Me H

()
R, r 2

358 Ac Ac (2 a -fo rm )
359 Ac P ro p
OR.
360 H 2 -M e p ro p
361 3 -M c h u Ac
362 H 2 -M e b u

363 H 3 -M c b u
364 H 3 - M e - 2 - b u te n
365 H A ng
366 Ac (2 p -fo rm )

R, *2 R;

367 P ro p Ac Ac
371 Me Ac A ng

81
2 4 .2 .2 .4 B I S A B O L O L S E S Q U I T E R P E N E S F R O M SENECIO S P E C I E S
The bisabolanes are a fairly large group mainly found as constituents o f higher plants. The

monocyclic aromatic sesquiterpenes of the bisabolane family are constituents of a large

number of essential oils. Most of these compounds are characterized by a benzylic chiral

center often carrying a methyl group at this position [Zhang & Rajabab, 2004] and have been

isolated from natural sources in enantiomerically pure form [Fuganti el al., 1999]. Diverse

biological activities [Mayer et al., 1998] exhibited by these compounds include anti­

inflammatory, anti-viral, and anti-mycobacterial properties, and they have attracted

considerable attention from synthetic chemists. Despite their rather simple structures, the

stereocenter at the benzylic position [Cesati et al., 20041 poses a significant challenge in the

asymmetric synthesis of even the simplest of these molecules.

The numbering system used for bisabolanes is the same as the farnesane system. Since the

cyclohexane ring has a plane of symmetry, substituents in this ring should be numbered

where possible avoiding the compound locant, 1(6), for a double bond and keeping the

82
numbers for the substituents in the cyclohexane ring as low as possible. The configuration

and numbering of bisabolane, l-(l,5-Dimethylhexyl)-4-methylcyclohexanc is shown below.

14

Bisabolane skeleton

Table 2.10: Bisabolol sesquiterpenes from Senecio species

Bisabolol Source Reference
3,4., 10,11 -Diepoxy-1,8-dihydroxy-7( 14)- S. abrotanifolius Bohlmann et al ., 1981c
bisabolen-2-one; (1 a,3(3,4(3)-form. S. fu lg en s
Diangeloyl (377)
3,4-Epoxy-7( 14), 10-bisaboladiene-1,2,8- S. erubescens Bohlmann et al., 1981c;
triol; (1 p,3a,4a.6£,,8^)-form, 2-Ketone, 8- 1985
(2Z-hexenoyl), 1-Ac (378)
3.4-Epoxy-7( 14), 1O-bisaboladiene-1,2,8- S. fu lg cn s Bohlmann et al., 1981c;
triol; (1 (3,2(3,3(3.4(3,64,8^)-form, 1,2- 1985
Diangeloyl, 8-Ac (379)
1,3,5,7( 14), 10-Bisabolapentaene-1,2,4,8- S. longifolius Bohlmann et al., 1978i;
tetrol; 1,4-Quinone, 8-angeloyl (380) 1985
3,7( 14), 10-Bisabolatrien-2-one (381) S. m acroglossus Bohlmann et al., 1985
1,3.5,7( 14), 10-Bisabolapentaene-1,2,4,8- S. oxyodontus Gutierrez et al., 1988
tetrol; 8-Angeloyl (382)
3-D-Angeloylsenecioodontol(383) S. oxyodontus Gutierrez et al., 1988
1,3,5,7( 14), 10-Bisabolapentaene-1,2,4,8- S. oxyodontus Bohlmann et al., 1978h,i;
tetrol; 2,4-Di-Me ether, 8-angeloyl (384) 1985
1,3,5,7( 14), 10-Bisabolapentaene-1,2,4,8- S. oxyodontus Bohlmann et al., 1978h,i;
tetrol; 1,4-Quinone, 2-Me ether, 8-angeloyl 1985
(385)
3,4-Epoxy-7( 14), 10-bisaboladiene-1,2,8- S. p am peanus Bohlmann et al., 1986a
triol; (la,3p,4p,65)-form, 2,8-Diketone, I-
0-(3-methylbutanoyl) (386)
3,4-Epoxy-7( 14), 10-bisaboladiene-1,2,8- S. pam peanus Bohlmann et a l, 1986a
triol; (la,3P,4p,65)-form, 2,8-Diketone, 1-
tigloyl (387)
Cyclopampeanone; (9-(3-Methylbutanoyl) S. pam peanus Bohlmann et al., 1986a
(388)
Cyclopampeanone; 7,10-Diepimer, O- S. pam peanus Bohlmann et al., 1986a
tigloyl(389)
Pampeanone D-Tigloyl (390) S. pam peanus Bohlmann el al., 1986a
Pampeanone isovalerate_(391) S. pam peanus Bohlmann et al., 1986a
Pampeanone : D-(3-Methylbutanoyl), 10- S. pam peanus Bohlmann et al., 1986a
83
Table 2.10: Bisabolol sesquiterpenes from Senecio species
B is a b o lo l S o u rc e R e fe re n c e
epimer (392)
2,10-Bisaboladien-1-one; (6iS',7iS)-form S. p a l me ns is Reina et al ., 2002
(393)
3.4-Epoxy-1,11 -dihydroxy-7( 14),9- S. pom peanus Bohlmann et al., 1986a
bisaboladiene-2,8-dione; (1 a,3p,4p,9£)-
form, l-(3-Methylbutanoyl) (3 9 4 )
3.4-Epoxy-1,11 -dihydroxy-7( 14),9- S. pom peanus Bohlmann et al., 1986a
bisaboladiene-2,8-dione; (1 a,3p,4p,9£)-
form, 1-Tigloyl (395)
3.4-Epoxy-1,11 -dihydroxy-7( 14),9- S. pom peanus Bohlmann et al., 1986a
bisaboladiene-2,8-dione; (la,3p,4p,9£)-
form, 11-Hydroperoxide, l-(3-
methylbutanoyl) (396)
3,4-Epoxy-1,11 -dihydroxy-7( 14),9- S. pom peanus Bohlmann et a l, 1986a
bisaboladiene-2,8-dione; ( 1a,3p,4p,9£>
form, 11 -Hydroperoxide, 1-tigloyl (397)
3,4-Epoxy-1,11 -dihydroxy-7( 14),9- S. pom peanus Bohlmann e t al., 1986a
bisaboladiene-2,8-dione; (la,3p,4p,9£)-
form, 7a,l4-Epoxide, l-(3-methylbutanoyl)
(398)
3,4-Epoxy-1,11 -dihydroxy-7( 14),9- S. pom peanus Bohlmann et al., 1986a
bisaboladiene-2,8-dione; ( 1a,3p.4p,9£)-
form, 7p, 14-Epoxide, 1-tigloyl (3 9 9 )
3,4-Epoxy-1,11 -dihydroxy-7( 14),9- S. pom peanus Bohlmann et al., 1986a
bisaboladiene-2,8-dione; (1 a,3p,4p,9£)-
form, 7a. 14-Epoxide. 11-hydroperoxide, 1-
tigloyl (400)
1,3,5,7( 14), 10-Bisabolapentaene-1,2,4,8- S. pubigerus Bohlmann et al., 1978i;
tetrol; 2-Me ether, 8-angeloyl, 4-Ac (401) 1985
1,3,5,7( 14), 10-Bisabolapentaene- 1,2,4,8- S. pubigerus Bohlmann et al., 1978i;
tetrol; 2-Me ether, 8-angeloyl, 1,4-di-Ac 1985
(4 0 2 )
1,3,5,7( 14), 10-Bisabolapentaene-1,2,4,8- S. pubigerus Bohlmann et al., 1978i;
tetrol; 1,4-Quinone, 2-angeloyl, 8-0-(2,3- 1985
epoxy-2-methylbutanoyl) (4 0 3 )
2,10-Bisaboladiene-l,12-diol; 1-Ketone, 12- S. sm ithii Bohlmann et a l, 1983
Ac (404)
Puliglutone (405) S. sm ithii Bohlmann et al., 1983
Sessquicineol (4 0 6 ) S. subrubriflorus Bohlmann et al., 1982c

84
()

85
 o R,
394 3 -M e b u II
395 Tigl II

OR, 396 3 -M e b u OH
397 Tigl on

2 .4 .2 2 .5 S E C O A N D A B E O E R E M O P H I L A N E S

Secoeremophilanes are derivatives of eremophilanes, formed by cleavage of the C-8, C-9 or

C-9, C-10 bond followed by oxidation, while abeoeremophianes are formed by migration of

one or more bonds in eremophilane or furanoeremophilane skeleton. The numbering of the

eremophilane or furanoeremophilane skeleton is retained in the new structure. The migration

86
of the C-4, C-5 bond to C-10 in the eremophiane skeleton results to abeoeremophilane as

shown below.

Table 2.11: Seco and Abeoeremophilanes sesquiterpenes from Senecio species

Seco a n d A b e o e r e m o p h i l a n e s S o u rc e R efe re n c e
Spirosenbergiolide (407) S. bergii Bohlmann et al., 1986a.
Secomacrotolide 6(3-Tigloyloxy, 1,10- S. bergii Trendafilova et al., 1995
dihydro, l-oxo(408)
5,6-Secofuranoeremophi la-1(10),4-dien- S. elegans Bredenkamp et al., 1985a
6-ol; 0-(3-Methyl-2-butenoyl) (409)
Senglutinosin (410) S. glutinosus Bredcnkamp et al., 1985a
Norsecoglutinosone (411) S. glutinosus Zdero et al., 1989a
8-Hydroxy-6-mcthoxy-3,4,5- S. linifolius Torres et al., 1989
trimethylnaphtho[2,3-/>]furan-9(4//)-one
(412)
8-Hydroxy-6-methoxy-3,4,5- S. linifolius Torres et al., 1989
trimethylnaphtho[2,3-/?]furan-9(4//)-one,
4-Hydroxy (413)
Secomacrotolide 6-(2- S. macedonicus Trendafilova et al., 1995
Methylpropenoyloxy) (414)
6-Hydroxysecomacrolide; 6p-form, 6-0- S. macrotis Bohlmann et al., 1981 e
Angeloyl (415)
6-Hydroxysecomacrolide; 6p-form, 8- S. macrotis Bohlmann et a!., 1981 e
Epimer, 6-O-angeloyl (416)
6-Hydroxysecomacrolide; 6(3-form, 6-0- S. macrotis Bohlmann et al., 1981 e
Tigloyl (417)
6-Hydroxysecomacrolide; 6p-form, 8- S. macrotis Bohlmann et al., 1981 e
Epimer, 6-O-tigloyl (418)
6-Hydroxysecomacrolide; 6p-form, 6-(3- S. macrotis Bohlmann et a l, 1981 e
Methylbutanoyl) (419)
6-Hydroxysecomacrolide; 6p-form, 8- S. macrotis Bohlmann et a l, 1981 e
Epimer, 6-(3-methylbutanoyl) (420)
Secomacrotolide 6P-(3- S. macrotis Bohlmann et a l, 1981 e
Methylbutanoyloxy) (421)
Secomacrotolide 6p-Angeloyloxy (422) S. macrotis Bohlmann et a l, 1981 e.

Secomacrotolide 6p-Tigloyloxy (423) S. macrotis Bohlmann et al., 1981 e

—

87
fable 2.1 l : Seco and Abeoeremophilanes sesquiterpenes from Senecio species
Seco and Abeoeremophilanes Source Reference
Serratifolide A (424) S. serratifolius l)upre; et al., 1991
Serratifolide B (425) S. serratifolius Dupre et al., 1991

6-Acetoxysecomacrotolide (426) s spp - Bredenkamp et al., 1985a

414 2-Mepropen
421 3-Mebu
422 Ang
423 Tigl
426 Ac

88
 R

__ OH 415 Ang
416 Ang (8-epimer)
417 Tigl
418 Tigl (8-epimer)
O 419 3-Mebu
420 3-Mebu (8-epimer)

2 4 .2 .2 .6 S I M P L E G E R M A C R A N E S E S Q U I T E R P E N O I D S

The numbering of the germacrane skeleton poses a problem since there is plane of symmetry

through carbons 2 and 7. Germacranes are normally drawn in a conventional way as shown

below with H-7 in the a-configuration. Care should be taken with the small number of

germacranes with a double bond at C- 7 as the ring can be numbered in either direction.

Germacranes frequently have double bonds in the 1(10) and 4 positions. There have been

proposals to give different names to the skeletons with (1(10)ZAE) (melampolides) and

(1(10)£,4Z) (heliangolides) configurations. However this is confusing and all compounds are

named as germacranes.

The large germacrane group is divided into simple germacranes, that is those without a

lactone or furan ring, 12,6-germacranolides, 12,8-germacranolides, furanogermacranes, nor­

and homogermacranes, secogermacranes, and cyclogermacranes [Fischer, 1990]. The

configuration and numbering o f germacrane (1, 7-Dimethyl-4-( 1-methylethyl)-cyclodecane)

is shown below.

89
 12

Germacrane

Fable 2.12: Simple Germacrane sesquiterpenoids from Senecio species

Simple Germacrane Source Reference
1( 10),5-Germacradiene-3,4-diol; S. adenophyllus 1)upre et al., 1991.
(l(10)£,3p,4p,5£)-form (427)
1(10),5-Germacradiene-3,4-dioI; S. adenophyllus Dupre et al., 1991.
(l(10)£,3p,4p,5£)-form, 3-Ac (428)
5,10( 14)-Germacradiene-1,4-diol; (1 p,4pO//,5£)- S. adenophyllus Dupre et al., 1991.
form (429)
5,10( 14)-Germacradiene-1,3,4-triol; S. adenophyllus Dupre et al., 1991.
(lp,3p,4p,5£)-form (430)
5,10( 14)-Germacradiene-1,3,4-triol; S. adenophyllus Dupre et al., 1991.
(ip,3p,4p,5£)-form , 3-Ac (431)
5,10( 14)-Germacradiene-1,3,4-triol; S. adenophyllus Dupre et al., 1991.
(lp,3p,4p,5£)-form , 1-Hydroperoxide (432)
4,5-Epoxy-1(10)-germacrene-3,6-diol; S. crassissim us Bohlmann et al.,
(l(10)£,3p,4a,5p,6p)-form, 3-Angeloyl (433) S. cylindricus 1977; 1979d; 1980b.
S. vitalis
4,5-Epoxy-1(10)-germacrene-3,6-diol; S. crassissim us Bohlmann et a l,
(1(10)£,3P,4a,5P,6p)-form,3-Tigloyl (434) S. vitalis 1977; 1979d; 1980b.
4,5-Epoxy-1(10)-germacrene-3,6-diol; S. crassissim us Bohlmann et a l,
(1( 10)£,3p,4a,5p,6p)-form,3- (3-Methvl-2- S. cylindricus 1977; 1979d; 1980b.
hutenoyl) (435) S. vitalis
4,5-Epoxy-1(10)-germacrene-3,6-diol; S. ftco ides Bohlmann et al.,
(l(10)£,3p,4a,5p,6p)-form,3-Tigloyl, 6-Ac (436) 1977; 1979d; 1980b,
1,10:4,5-Diepoxy-11 (13)-germacrene-3.8,9-triol; S. galpinii Bohlmann et a l,
(1 p,3p,4a,5p,8p,9p,10a)-form, 3-Angeloyl, 8-(3- 1978f; 1981a; 1982g.
methyl-2-butenoyl) (437)
1,10:4,5-Diepoxy-11(13)-germacrene-3.8,9-triol; S. galpinii Bohlmann et a l,
(1 p,3p,4a,5p,8p,9p, 10a)-form, 1978f; 1981a; 1982g.
3-Angeloyl, 8-(3-methyl-2-butenoyl). 9-Ac (438)
1,10:4,5-Diepoxy-11(13)-germacrene-3.8.9-triol; S. galpinii Bohlmann et a l,
(1 p,3p,4a,5p.8p,9p, 10a)-form, 3,8-Diangeloyl 1978f; 1981a;
(439)
1,10:4,5-Diepoxy-11(13)-germacrene-3.8,9-triol; S. galpinii Bohlmann et al.,
(1 p,3p,4a,5p,8p,9p, 10a)-form, 3,9-Diangeloyl, 8- S. rhom hoideus 1978f; 1981a; 1982g.
Ac (440)
1,10-Epoxy-11 -germacrene-3,4,5,8,9-pentol; S. galpinii Bohlmann et a l,
(1 p,3p,4a,5a.8(3,9p, 10a)-form, 3-Angeloyl. 8-(3- 1982g.
methyl-2-butenoyl), 9-Ac (441)
1,10-Epoxy-11 -germacrene-3,4,5,8,9-pentol; S. galpinii Bohlmann et al.,
(1 p,3p,4a,5a.8p,9p, 10a)-form, 3-Angeloyl, 8-(3- 1982g.
methyl-2-butenoyl), 5,9-di-Ac (442)
1,10-Epoxy-11 -germacrene-3,4,5,8,9-pentol; S. galpinii Bohlmann et al.,
90
Table 2.12: Simple Gcrmacrane sesquiterpenoids from Senecio species
Simple Germacrane Source Reference
(lp,3p,4a,5a,8p,9p,10a)-form , 3,8-Diangeloyl, 9- 1982g.
Ac (443)
1,10-Epoxy-11-germacrene-3,4,5,8,9-pentol; S. galpinii Bohlmann et al.,
(1 p,3p,4a,5a,8p,9p, 1Oa)-form, 3.8-Diangeloyl, 1982g.
5,9-di-Ac (444)
4( 15),5,9-Germacratrien-l-ol; (1 p,5£,9Z)-form S. philippicus Jakupovic et al.,
(445) 1991.
1.10:4,5-Diepoxy-11(13)-germacrene-3,8,9-triol; S. rhom boideus Ilohlmann et al.,
(1 p,3p.4a,5p.8p,9p, 10a)-form, 3,9-Diangeloyl 1978f,g; 1981a;
(446) 1982f
4,5-Epoxy-1(10)-germacrene-3,6-diol: S. vital is llohlmann et al.,
(1(10)£,3p,4a,5p,6P)-form,3-(3-Methyl-2- 1977; 1979d; 1980b.
butenoyl), 6-Ac (447)
4,5-Epoxy-1(10), 11 -germacradiene-3,8.9-triol; S. spp llohlmann et al.,
(!(10)£,3p,4a,5a,8p,9p)-form, 3-Angeloyl, 8.9- 1977.
di-Ac (448)
4,5-Epoxy-1(10)-germacrene-3,6-diol; S. sp. llohlmann et a l,
(l(10)£,3p,4a,5a,6P)-form, 3-Tigloyl (449) 1977; 1979d; 1980b.
4,5-Epoxy-1(10)-germacrene-3,6-diol; S. spp llohlmann et al.,
(1(10)£,3p,4a,5p,6P)-form,3-Angeloyl, 6-Ac 1977; 1979b; 1980b.
(450)
1(10),4-Germacradien-6-ol; (1(10)£,4£,6P)-form, S. spp. llohlmann et al.,
Tigloyl (451) 1977.
1(10),4-Germacradien-6-ol; (1(10)£.4£,6P)-form, S. spp. llohlmann et al.,
0-(3-Methyl-2-pentenoyl) (452) 1977.
1(10),4-Germacradien-6-ol; (1(1 0)£,4E,6P)-form, S- spp- llohlmann et al.,
Angeloyl (453) 1977.
1(10),4-Germacradiene-3,6-diol; S. spp llohlmann et a l,
(l(10)£,3p,4£,6P)-lbrm, 3-Angeloyl, 6-Ac (454) 1977.

14

R, r 2

427 OH OH

428 OAc OH

91
 R, k2

429 H H
430 H OH
431 H OAc
432 OH OH

433 Ang H
434 Tigl H (1(10)E,3(3, 4a, 5p, 6p)-form
435 Sene H
436 Tigl Ac
447 Sene Ac

R k2

449 Tigl H (1(10)E,3P, 4a, 5a, 6P)-form.

450 Tigl Ac

R, r2 R>
437 Ang Sene If
438 Ang Sene Ac
439 Ang Ang II
OR 1
440 Ang Ac Ang
446 Ang FI Ang

O R, r2 r2

441 Ang H Sene

442 Ang Ac Sene
443 Ang II Ang
444 Ang Ac Ang
454 Ang Ac Tigl

92
2 .4 .2 .2 .7 O P L O P A N E S E S Q U IT E R P E N O ID S F R O M SENECIO S P E C I E S .
Very few oplopanesesquiterpenes have been isolated from Senecio species. Oplopane

derivatives from Senecio species usally possess a terminal double bond at C-10, an a, p-

unsaturated ketone at C-3 or an hydroxyl group at the same position. Some compounds have

an epoxy group at the 11(12) position, ester groups at C-8, C-9 or C-2 positions. Oplopanes,

from higher plants, are 3(4 —> 5)-abeocadinanes and the numbering system used here is

biogenetic. The configuration and numbering of oplopane (l-ethyloetahydro-4-methyl-7-(l-

methylethyl)-1 H-indene) is shown below, fable 2.13 below gives a summary of all

theoplopane sesquiterpenoids isolated from different Senecio species

14 14

Cadinane skeleton Oplopane skeleton

93
Table 2.13: Oplopane sesquiterpenoids from Senecio species
Oplopane Source Reference
Abrotanifolone (455) S. abrotcm ifolius Bohlmann e t a l , 1976b,c.
Implexin (4£)-form (456) S. im plexus Boh 1man n et al., 1981a.
Implexin (4Z)-form (457) S. im plexus Bohlmann e t al., 1981a.
10,14-Dihydroxy-4-oplopanone; 14- S. m exicanus Joseph-Nathan et al., 1989b.
Angeloyl (458)
4-Hydroxy-10( 14)-oplopen-3-one (459) S. m exicanus Joseph-Nathan et al., 1989b.
4,10(14)-Oplopadien-3-ol (460) S. m exicanus Joseph-Nathan et al., 1989a,
b.
4.10(14)-Oplopadien-3-ol; 3-Ketone (461) S. m exicanus Joseph-Nathan et al.,
1989a,b.
4,10( 14)-Oplopadien-3-ol; 3-Ketone, S. m exicanus Joseph-Nathan et al.,
10( 14)£-isomer (462) 1989a,b.
10( 14)-Oplopen-3-one (463) S. m exicanus Joseph-Nathan e ta l., 1990.

94
2 .4 .2 .2 .8 E U D E S M A N E S E S Q U I T E R P E N O I D

Eudesmanes are called selinanes in the older literature. The eudesmanes found in higher

plants generally have the stereochemistry shown below. E«/-eudesmanes are found in some

species o f liverworts. As with the germacrane group, the eudesmanes arc divided into groups

comprising simple eudesmanes, eudesman-12,6-olides, eudesman-12,8-olides and

furanoeudesmanes, secoeudesmanes, and noreudesmanes. There is also a large group of

esters based on the dihydro-J3- agarofuran skeleton which are grouped separately. Within the

eudesmane group, particularly with dihydro-p-agarofuran derivatives, there is some

confusion concerning the numbering of carbons 14 and 15.

14

Dihydro-P-agarofuran (2,2,5,9-Tetramethyl-2H-3, 9oc-methano-l -benzoxepin)

95
 Asteraceae family is a rich source of sesquiterpenoid natural products, especially those with

the eudesmane framework. The eudesmanoids are biosynthesised from farnesyl

pyrophosphate [Quan-Xiang et al., 2006J and approximately 1000 natural eudesmanoids have

been identified from the Asteraceae family, with many different oxygenation and cleavage

patterns [Quan-Xiang et al., 2006]. The configuration and numbering o f eudesmane (1 ,2 -

isopropyl-4a, 8 dimethyldecahydronaphthalene) is shown below.

14

12 Eudesmane skeleton

Table 2.14: Simp Ie Eudesmane from Senecio species.

Eudesmane Source Reference
l-Hydroxy-4.1 l-eudesmadien-3-one; 1p-form(464). S. bracteo/atus. Sanz et al., 1990.
1,3-Dihydroxy-4( 15), 11(13)-eudesmadien- 12.6-olide; S. chrysanthem oides Mengi el al., 1991
(la,3a,6a)-form , 11(3,13-Dihydro, 1-Ac (465)
1.4-Dihydroxy-2-eudesmen-12,6-ol ide; S. chrysanthem oides Mengi et a l , 1991
(1 a,4 a,6 a, 11 p//)-form, 1-Ac (466)
1,2-Dihydroxy-3,11(13)-eudesmadien-12,8-olid-l 4-oic S. fJam m eus Hu e ta l., 1999
acid; (la,2p,8P)-form, Di-Ac, 4,5-dimethyl-3-oxo-2-
heptyl ester (467)
4( 15)-Eudesmene-l,6-diol; (ip,5a,6a,10P)-form (468) S. m icroglossus Bohlmann et a l ,
1980c; 1983b
4( 15)-Eudesmen-5-ol; 5a-form (469) S. rhyncholaenus Bohlmann et a l ,
1978]
4( 15)-Eudesmen-6-ol; (5a,6a,7p, 10P)-form, Tigloyl S. rhyncholaenus Bohlmann et al.,
(470) 1978j; 198 If;
1982d
7-Eudesmene-l,4-diol; (1 p,4p)-form, 1-Angeloyl (471) S. spp Bohlmann et a l ,
1977
7-Eudesmene-l,4-diol; (ip,4p)-form, l-(3-Methyl-2- S. spp Bohlmann et a l ,
pentenoyl) (472) 1977

r,
R, r2 Rj
464 OH H H
469 H OH H
470 H H Tigl

96
2 A 2 . 2 .9 B E N Z O F U R A N O I D S F R O M SENECJO S P E C I E S

Benzofurans are prominent natural products of many genera o f the Asteraceae and are

especially common in the tribes Astereae, Eupatorieae, Heliantheae, Inuleae and Senecioneae

fProksch & Rodriguez, 1983]

Benzofuran derivatives are an important class of heterocyclic compounds that are known to

possess important biological properties. Substituted benzofurans find application as anti­

oxidants and anti-fungal, anti-tumor a variety of drugs and in other fields of chemistry and

agriculture [Gundogdu-Karaburun et al., 2006]. In addition, benzofurans are used in cosmetic

formulations [Sharifi et al., 2008] and have the application as synthetic precursors for optical
97
brighteners [Elvers et a l. , 1999], Simple benzofurans with 2. 3 and 5. 6 substituents have

been isolated from Senecio species. The configuration and numbering of benzofuran (benzo

[P] furan) is shown below.

2 Benzofuran skeleton

fable 2.15: Benzofuranoids from Senecio species.

Benzofuran Source Reference
5-Acetyl-2,3-dihydro-3,6-dihydroxy-2- S. d esfontainei De Guttierez et al., 1995.
isopropenylbenzofuran; (2S,3S)-form (473)
5-Acetyl-2,3-dihydro-3,6-dihydroxy-2- S. desfo n ta in ei De Guttierez et al., 1995.
isopropenylbenzofuran; (2S,3‘S)"form (474)
5-Acetyl-2,3-dihydro-3,6-dihydroxy-2- S. spp Bohlmann et al., 1977;
isopropenylbenzofuran; (2/?,3/?)-form, 3-0- 1978b; 1979c; 1980d,e,f;
Angeloyl (475) 1981b; 1982a,e.
5-Acetyl-2,3-dihydro-3,6-dihydroxy-2- S. spp Bohlmann e t al., 1977;
isopropenylbcnzofuran; (2R,3R)-form , 3-0- 1978b; 1979c; 1980d,e,f;
[2-(Acetoxymethyl)-2-butenoyl](Z-) (476) 1981b; 1982a,e.
5-Acetyl-2,3-d ihydro-3,6-dihydroxy-2- S. spp Bohlmann et al., 1977;
isopropenylbcnzofuran: (2/?,3«S)-form, 3-0- 1978b; 1979c; 1980d,e,f;
Angeloyl (477) 1981b; 1982a,e.
Euparin (478) s - spp- Ghazy et al., 1986a.
5-Acetyl-6-hydroxy-2-isopropenyl-7- S. spp. Bohlmann et al., 1977;
methoxybenzofuran; 6-Me ether (479) 1981b.
6-Hydroxytremetone S. spp. Anthonsen et al., 1970.
(iS)-form (480)
6-Hydroxytremetone S. spp. Anthonsen e t al., 1970.
(S')-form; 3’-Hydroxy (481)
6-Hydroxytremetone S. spp. Anthonsen et al., 1970.
(S)-form, 3'-Acetoxy (482)
5-Acetyl-2,3-dihydro-3,6-dihydroxy-2- S. spp Bohlmann et al., 1977;
isopropenylbenzofuran; (2/?,3/?)-form, 3-0- 1978b,k.l; 1979c; 1980;
Angeloyl (483) 1981b, d; 1982a,e.
5-Acetyl-2,3-dihydro-3.6-dihydroxy-2- S. spp Bohlmann et al., 1977;
isopropenylbenzofuran; (2R,3R)-form, 3-0- 1978b,k.l; 1979c; 1980d,e,f;
[2-(Acetoxymethyl)-2-butenoyl](Z-) (484) 1981 b,d; 1982a,e.
5-Acetyl-2,3-dihydro-3,6-dihydroxy-2- S. spp Bohlmann et al., 1977;
isopropenylbenzofuran; (2/?,3.S)-form, 3-0- 1978b.k.l; 1979c; !980d,e,f;
Angeloyl (485) 1981 b.d; 1982a,e.

98
 R.

473 OH (25,35}-form
474 OH (2/?,2/?)-form
475 OAng (2/?,3/?)-form
476 0-2-(Acetoxymethyl)-2-buten) (Z)
477 OAnj» (2A,3^)-form
480 H
483 OAn« (2/?,3/f)-form
485 OAng (2 J?,35)-form

R, r 2

478 OH H

479 OMe OMe

99
 CHAPTER THREE

RESULTS AND DISCUSSION

3.0 P R E L I M I N A R Y B I O A S S A Y T E S T R E S U L T S

The surface exudates of the fresh leaves of D. august ifol ia-Ngong, D. august ifolia-Voi and

Senecio roseiflorus were extracted by successive dipping into fresh portions of acetone for

short periods (15 seconds). The crude extracts were screened for anti-plasmodial activities

against chloroquine-senstive (D6) strain of Plasmodium falciparum, fable 3.1 summarizes

the results of these tests.

Table 3.1: Anti-plasmodial activity of plant extracts against D6 strain of Plasmodium

falciparum.
Species P la n t p a r t I C 50 v a l u e s in p g /m l

D6
Dodouaea august ifolia-Ngong forest. Fresh leaves 44.5 ±3.5
Dodouaea august ifolia-Voi Fresh leaves 56.0 ±4.2
Seuecio roseiflorus Fresh leaves 90.0 ± 9.8

The crude extract of fresh leaves of Dodonaea august ifolza-Ngong forest, Dodouaea

angustifolia-Voi and Seuecio roseiflorus were also screened for larvicidal activity against the

larvae of Aedes aegypti. The surface exudates showed minimal activity (LC50) with values

greater than 60 pg/ml after 24 hours. The extract of fresh leaves of Dodonaea angustifolia-

Ngong forest, Dodouaea august ifol ia-Vo\ and Seuecio roseiflorus were further tested for

radical scavenging activity to determine their potential as anti-oxidants. 1he results showed

that poor radical scavenging activities even at 20 pg/ml.

The extract o f fresh leaves of Dodouaea august ifol ia- N gon g forest, Dodonaea augustifolia-

Voi and Seuecio roseiflorus were also tested for anti-bacterial activity against three strains of

bacteria: Staphylococcus aureus (ATCC 29737), Escherichia coli (ATCC 25922) and

Bacillus pimilus (local strain); and anti-fungal activity against one local strain of

100
Saccharamyces cerevisiae fungus. The three crude extracts showed activity against bacteria

and fungi.

Table 3.12: Anti-microbial activities of the crude extracts o f D. angustifolia from different
geographical locations, Senecio roseiflorus and some pure compounds againgt
3 bacteria and one fungus species.
Sample pg/disc 1 |2 3 4
Crude extracts
Surface exudate of D. angustifolia (leaves)- 2500 18.863 20.05a 19.42a 10.79a
Ngong forest
Surface exudate of D. angustifolia (leaves)- 2500 17.58 19.21 18.85 12.45
Voi
Surface exudate of D. angustifolia (leaves)- 2500 19.06 18.89 18.60 14.40
Kilifi.
Surface exudate of D. angustifolia (leaves)- 2500 16.33 18.94 16.25 12.18
Garborone (Botswana)
Surface exudate of D. angustifolia (leaves)- 2500 17.87 21.68 18.43 11.45
Madagascar
Surface exudate of Senecio roseiflorus 2500 18.66 19.15 18.95 11.80
(leaves)
M icroorganism s : 1=E scherichia coli (ATCC 25922), 2= Staphylococcus aureus (ATCC
29737). 3 =B a cillu sp u m ilu s (local strain), 4 -S a cc h a ro tn yc e s cerevisiae (local strain).
Not active.
J Inhibition zone in mm.

In this study, therefore, the chromatographic separation and structural elucidation of the crude

extracts was undertaken to characterize the bioactive principles present in these extracts and

to confirm their in vitro bioactivity.

TLC analyses of the extracts obtained from fresh leaves D odonaea angustifolia-Slgong,

D odonaea angustifolia-W o\ and Senecio roseiflorus showed the presence of UV (254 nm) and

iodine active compounds. From chemotaxonomic data most o f these compounds were

assumed to be flavonoids and terpenoids (Dawson et a l. , 1966; Payne & Jefferies, 1973;

Jefferies, 1979; Jefferies & Payne, 1967; Jefferies et al. , 1973, 1974, 1981). The TLC

analyses o f extracts obtained from fresh leaves D odonaea angustifolia-N gong, Dodonaea

angustifolia-W oi were compared and the flavonoid and diterpenoid profiles of the material

collected from Ngong, representing the upland region, found not to completely match those

101
from in Taita Hills near Voi town, which is more of a coastal location (Figure 3.1). The TLC

profile below shows variation in the D. angustifolia exudates components collected from Voi,

Kilifi (MO), Mt. Kenya (Mt.K), Gaborone (Bot), Madagascar (MA). The flavonoids and

terpenoids do not completely match any of the local ones under study. From the above

overview, different Dodonaea angustifolia populations elaborate different flavonoids and

terpenoids.

Dodonaea angustifolia collections from:

1. Taita Hills near Voi. Kenya.

2. Kilifi, Kenya.

3. Ngong Forest. Kenya.

4. Madagascar, Madagascar.

5. Garborone, Botswana.

Figure 3.1: TLC profiles of extracts of D. angustifolia from different populations

The extracts of Dodonaea angustifolia from Ngong Forest and Voi were subjected to

chromatographic separation to give twenty-eight compounds. Three of these compounds have

not been reported before. The isolated compounds were characterized using UV, 'H-NMR,

nC-NMR, MS. Some of these compounds were tested for anti-plasmodial, larvicidal, anti­

oxidant and anti-microbial activity. In the following sections the isolation, structural

elucidation and biological activity of the isolated compounds will be discussed.

102
3.1 COMPOUNDS FROM D O D O N A E A A N G U S T I F O L IA - NGONG FOREST.
The extraction of the surface exudates on the aerial parts (mainly leaves) was done after

plucking out the flowers. The surface exudates of the leaves was extracted by two successive

dipping into fresh portions of acetone for short periods (15 seconds) to yield the crude

extract, thus avoiding the extraction of the internal tissue components. The extract was tested

for anti-plasmodial activity against chloroquine-senstive (1)6) strain of Plasmodium

falciparum and had an IC50 value of 44.5 ± 3.5 jig/ml. The crude extract was subjected to a

combination of chromatography methods resulting in the isolation o f eleven compounds. The

structures o f these compounds were confirmed by comparison o f their spectroscopic data

with literature and in some cases by direct comparison (co-TIX) with authentic samples. All

compounds except santin (4) have been reported here for the first time from this plant and in

the following sections the structural elucidation of these compounds will be discussed.

There is great geographical variability in the composition for this substance in Dodonaea

species, therefore the interest in the study of Kenyan species.

3.1.1. 5-HYDROXY-3,7,4-TRIMETHOXYFLAVONE (1)

Compound 1 was isolated as yellow crystals with melting point of 145-147 °C. It appears as

yellow spot (in Rf 0.3 [20% n-C6Hi2 CH2CI2]) which intensified on exposure to ammonia

vapour indicating it is phenolic. The mass spectrum showed molecular ion peak at m/z 328

[M4] corresponding to the formula CigHuOe, and also an intense peaks at 285 [M+ - 43]

(Scheme 3.1) due to the standard C-ring collapse [Harborne, 19941 for 3-methyl ether

flavone.

103
 +

OMe

la
ni/z 167

+
OMe

OMe
MeO

+
M -43
m/z 285

Scheme 3.1: MS fragmentation pattern of li-h y d ro x y ^ .T ^ '-trim cth o x y flav o n e (1)

The UV peak at [A.max 268.5 and 346.5 nm] [Mabry et al., 1970], l l-NMK signal at (8 12.63

(chelated OH)) plus the ,3C-NMR at 5 157.1 (C-2), 139.4 (C-3) and 179.0 (C-4) NMR are

consistent with a 5-hydroxyflavonol derivative [Agrawal, 1989].

The 'H-NMR (Table 3.2) indicated the presence of two meta coupled aromatic protons at 8

6.42 and 6.33 (1H, d , J = 2.0 Hz) due to ring A and an AA'BB' spin system at 8 8.05 (2H, d,./

= 9.0 Hz) and 7.00 (2H, c/, ./ = 9.0 Hz) assigned to H-2' and H-3' in ring B. With

oxygenations at C-5 and C-7 (biogenetically expected), the meta coupled protons at 8 6.33 (d,

J - 2 Hz) and 6.42 { d , J =2 Hz) are assigned to H-6 and H-8 respectively. HMBC correlation

between the chelated hydroxyl proton (8 12.63) with C-6 (8 98.1) and IIMQC correlation

104
between the proton at 8 6.33 and C-6 (5 98.1) confirmed the assignment o f the signals at 5

6.33 (d) to H-6. Similarly. HMBC correlation between the peak at 6 6.33 (cf) and C-8 (5 92.4)

allowed the assignment of the signal at 8 6.42 to H-8. 'H-NMR data further revealed peaks

for three methoxyl groups at 5 3.87 (3H, s), 8 3.85 (3H, s), and 3.84 (311. v).

In the NOESY experiment the signals at 6.33 (<7, H-6) and 6.42 (d, 11-8) were observed to

interact with the methoxyl at 8 3.87 (Figure 3.2) thus confirming this methoxyl group at C-7,

while NOESY interaction between H-37H-5' (8 7.00) and the methoxyl at 8 3.85 (Figure 3.2)

indicates the presence of a methoxyl group at C-4\

Figure 3.2: NOESY interactions in 5-hydroxy-3,7,4'-trimethoxyllavone (1)

1*3
The C-NMR of the third methoxyl group (8 60.4) is consistent with di-ortho arrangement

which suggests the presence of this group at C-3.

Based on the spectroscopic data and comparison with literature information compound 1 was

identified as 5-hydroxy-3,7,4'-trimethoxyflavone (1) | Rossi el <://., 19971. This compound has

previously been reported from Dodonaea viscosa [Wollenweber el al., 2004], but is being

reported the first time from Dodonaea angustifolia.

105
Table 3.2: ID (CDCI3: 300, 75.5 MHz) and 2D NMR data for 5-Hydroxy 3,7,4'-
trimethoxyflavone (1)
c <$1H d\3C HMBC HMBC NOESY
(m, J (Hz)) 2J 3J
2 157.1
3 139.4
4 179.0
5 162.3
6 6.33 (d, 2.0) 98.10 C-5, C-7 C-8,C-I0 H-6. OMe-7
7 165.7
8 6.42 (d, 2.0) 92.4 C-7, C-9 C-10, C-6 H-8, OMe-7
9 156.2
10 106.3
r 123.1
2' 8.05 ( d, 9) 130.4 C-6',C-2,
C4'
3' 7.00 (<7, 9) 114.3 C-4' c-5 ', c - r , 11-3', OMe-4'
4' 161.9
5' 7.03 ( d, 9) 114.3 C-4' c-3 ', c - r , 11-5', OMe-4'
6' 8.08 ( d, 9) 130.4 C-2\C-2,
C4'
OMe 3.87 (sj * 55.7 * C-7 *
OMe 3.85 (sj * 56.0 * C-4'*
OMe 3.84 (s) 60.4 C-3
OH 12.63 (s) C-5 C-6, C-10
* Chemical shift values in the same column interchangeable

3.1.2. 3,5-DIHYDROXY-7,4\-DIMETHOXYFLAVONE (2)

Compound 2 was isolated as yellow needle-like crystals with melting point of 182-184 °C. It

appears as yellow spot (Rt 0.3 [20% n-C6Hi2 - CIHCh]) which intensified on exposure to

ammonia vapour indicating it is phenolic. The E1MS showed a molecular ion peak at m/z 314

corresponding to the formula C17H14O6.

The UV peaks at [Xmax 268.5 and 346.5 nm] [Mabry et al., 1970], I l-NMR signal at 5 I 1.74

(chelated OH) and l3C-NMR peak at 6 145.7 (C-2), 135.7 (C-3) and 175.2 (C-4) [Agrawal

1989] indicates that this compound is a 5-hydroxyflavonol derivative

The 'H-NMR (Table 3.3) indicated the presence of two meta coupled aromatic protons at 8

6.50 and 6.38 (IH, d ,J = 2.1 Hz) due to ring A and an AA'BB' spin system at 5 8.19 and 7.05

106
(2H, d, J — 9.3 Hz) assigned to H-2', H-6', H-3', H-5' respectively, of a para substituted ring

B. With oxygenations at C-5 and C-7 (biogenetically expected), the met a coupled protons are

assigned to H-6 and H-8. HMBC correlation between the chelated hydroxyl proton (8 11.74)

with C-6 (8 97.9) and HMQC correlation between the proton at 8 6.38 and C-6 (8 97.9) led to

the assignment of the peak at 8 6.38 (d) to H-6. Similarly. HMBC correlation between the

signal at 8 6.38 (d) and C-8 (8 92.2) allowed the placement of the proton resonating at 8 6.50

to H-8.

Table 3.3: ID (CDCI3: 300, 75.5 MHz) and 2D NMR data for 3,5-dihydroxy-7,4'-
dimethoxyflavone (2)
c S\H d\3C HMBC HMBC
(m, J (Hz)) V V
2 145.7
3 135.7
4 175.2
5 160.9
6 6.38 (d, 2.4) 97.9 C-5, C-7 C-8,C-10
7 165.7
8 6.50 (d, 2.4; 92.2 C-7, C-9 C-10, C-6
9 156.9
10 104.0
T 123.2
2' 8.18 (d, 9.3; 129.4 C-6',C-2, C4'
3' 7.06 (d, 9.3) 114.1 C-4' C-5', C-T,
4' 161.2
5' 7.05 (d, 9.9) 114.1 C-4' C-3', C-T,
6' 8.19 (d, 9.9) 129.4 C-2',C-2,C4'
OMe 3.90 ( s ) 55.4 C-7
OMe 3.89 (s) 55.8 C-4'
OH 11.74 (s) C-5 C-6, C-10
OH 6.57 (s) C-4'

Furthermore, 'H-NMR revealed peaks for two methoxyl groups at 8 3.90 and 3.89 (3H, s).

One of the methoxyl groups was placed at C-7 and the other at C-4' but not at C-3 since they

resonate at 8 55.4 and 55.8 which is typical for isolated methoxyl groups. If either was at

position C-3 then it would have resonated ca. 8 60 in the l3C-NMR. The positions of the

methoxyl groups were confirmed by HMBC correlations of the peak at 8 3.90 to C-7

(8 165.7) and 83.89 to C -4 '(8 161.2).

107
 Figure 3.3: HMBC and HMQC of 3,5-dihydroxy-7.4'-dimethoxyllavone (2).

Based on these spectroscopic data, compound 2 was identified as 3.5-dihydroxy-7,4'-

dimethoxyflavone (2). This compound has been previously isolated from Dodonaea viscosa

[Wollenweber et al., 2004], but is being reported for the first time from Dodonaea

august ifolia.

3.1.3. KUMATAKENIN (3)

Compound 3 was isolated as yellow needle- like crystals with melting point of 253-254 °C. It

appears as yellow spot (R( 0.5 [2% MeOH - CH2CI2]) which intensified on exposure to

ammonia vapour indicating it is phenolic. The mass spectrum showed molecular ion peak at

mJz 314 [M+| corresponding to the formula C17H14O6 and an intense peaks at 271 [IVT - 43]

(Scheme 3.2) due to the standard C-ring collapse for 3-methyl ether llavone [Harborne,

1994].

108
 +

OH

M +- 43
m/z 271

Scheme 3.2: Fragmentation pattern o f kumatakenin (3)

I he UV peak [A.max 269.0 and 348.0 nm] 'H-NMR signal at (6 12.76 (chelated OH) and l3C-

NMR peak 5 148.0 (C-2), 137.6 (C-3) and 177.4 (C-4) is consistent with a 5-hydroxyflavonol

derivative [Mabry el al., 1970;Agrawal 1989].

1he 'H-NMR ( fable 3.4) indicated the presence of two meta coupled aromatic protons at 6

6.70 and 6.33 (1H, d, J — 2.1 Hz) which due to ring A and an AA'BIV spin system at 6 8.19

(2H, dd, J - 2.1, 9.9 Hz) and 5 7.03 (2H, dd, ./= 2.1, 9.9 Hz) assigned to C-4’ substituted ring

13 protons. With oxygenations at C-5 and C-7 (biogenetically expected), the meta coupled

protons are assigned to H-6 and H-8. HMBC correlation between the chelated hydroxyl

proton (6 12.76) with C-6 (8 99.1) and HMQC correlation between the proton at 5 6.33 and

C-6 (8 99.1) (Figure 3.4) led to the assignment of the doublet at 8 6.33 to H-6. Similarly,
109
HM BC correlation between the signal at 8 6.33 (d) and C - 8 (5 93.4) allow ed the confirmation

of the peak at 5 6.70 to H-8.

Table 3.4: ID (CDCI3: 300. 75.5 MHz) and 2D NMR data for 5,4'-dihydroxy-3,7-
dimethoxyflavone (3)
c £ih £|3C HM BC HMBC
(m, J (Hz)) 2J 5J
2 148.0
3 137.6
4 177.4
5 162.5
6 6.33 (d, 2.1) 99.1 C-8
7 167.4
8 6.70 {d, 2.1) 93.4 C-6, C-10
9 158.4
10 105.6
r 124.0
2' 8.19 (dd, 2.2, 9.9) 131.2 C-6', C-2, C4'
3' 7.03 (dd, 2.2, 9.9) 117.1 C-4' C-5’, c - r
4' 161.0
5' 7.03 (dd, 2.2, 9.9) 117.1 C-4' c-3', c -r,
6' 8.19 (dd, 2.2, 9.9) 131.2 C-2', C-2, C4'
OMe 3.95 (s) 57.1 C-7
OMe 3.88 (s) 60.9 C-3
OH 12.76 (s) C-5 C-6

The 'H-NMR also displayed peaks for two methoxyl groups at 5 3.95 and 3.88 (3H, s).

HMBC correlation of the methoxy at 8 3.95 with 167.4 confirmed the methoxyl at C-7 in ring

A.

The second methoxyl was confirmed at C-3 and not at C-4' due to the fact that it resonates at

8 60.9 which is typical for a d\-ortho substituted methoxyl groups.

Thus based on this and correlation with literature [Vieira el al., 19971. compound 3 was

identified as 5,4'-dihydroxy-3,7-dimethoxyflavone (3) (kumatakenin). I his compound has

been previously isolated from several plant species including the aerial parts of Achillea

ageratum [Vieira et al., 1997] and Solarium paludosum [Sarmento et al. 2002], blit is being

reported for the first time from the genus Dodonaea.

110
 OMe

Figure 3.4: HMBC and HMQC correlations for kumatakenin (3).

3.1.4. SANTIN (4)

Compound 4 was isolated as yellow needle like crystals with a melting point 159-161 °C. It

appears as yellow spot (Rf 0.4 [2% MeOH - CH2CI2]) which intensified on exposure to

ammonia vapour indicating it is phenolic.The UV peak [X,max (MeOH) 272.0 and 337.0 nm],

'H-NMR signal 6 11.76 (chelated OH) and 13C-NMR peak 5 156.0 (C-2), 138.3 (C-3) and

179.2 for (C-4) is consistent with a 5-hydroxyflavonol derivative | Mabry et al., 1970;

Agrawal, 1989].

The 'H-NMR (Table 3.5) displayed an AA'BB' system resonating at 6 8.1 I and 7.12 (2H, d, J

= 8.8 Hz) corresponding to a para substituted ring B and a singlet at 6 6.60 (1H) assigned to a

tri- substituted ring A.

The 'H-NMR (Table 3.5) further displayed signals for three mcthoxyls at 5 3.91 3.87 and

3.86, two of which are di-ortho substituted (8 60.0 and 59.6 in 1 C NMR). The location of

one of the methoxyl group was assigned to C-4' due to NOE interaction between the aromatic

protons at C-3 VC-5' (8 7.12) with the methoxyl group at 8 3.91. This led to two possible

structures 4a and 4b.

Table 3.5: ID (CDCb: 300, 75.5 MHz) and 2D NMR data for 5,7-dihydroxy-3, 6, 4'-
trimethoxyflavone (4)
c S\H ^I3C Of 8\ 3c of Santin (4a) 8\3c of 4b (Roitman
(w, J (Hz)) compound 4 (Barbera et al., 1986) & James., 1985).
5 5 6
2 156.0 156.2 155.4
3 138.3 138.7 137.5
4 179.2 179.6 178.0
5 152.5 151.8 155.9
6 131.3 130.1 98.8
7 157.4 152.2 156.9
8 6.36 (d, 2.0) 93.9 93.1 127.5
9 153.0 155.9 148.5
10 105.6 106.2 104.0
r 123.0 123.1 120.7
2' 8.11 (d, 8.8) 130.4 130.2 129.9
3' 7.12 {d, 9.0) 114.3 114.1 115.7
4' 162.1 160.2 160.2
5' 7.12 (d, 9.0) 114.3 114.1 1 15.7
6' 8.11 (d, 8.8) 130.4 130.2 129.9
OMe-3 3.91 (s) 60.0 60.1
OMe-6 3.87 (s) 59.6 61.8
OMe-4' 3.86 (s) 55.2 55.4
OH 11.76. (s)
OH 3.08 (brs)

However, the spectral data (Table 3.5) of this compound 4 is in close agreement with that

reported in literature [Barbera et al., 1986] (Table 3.5) for santin (4a) and not for 5, 7-

dihydroxy-3, 8, 4'-trimethoxyflavone [Roitman & James, 1985] ( fable 3.5) (4b) especially

the ljC-NMR for C-6, C-8 and C-9. Based on this the compound was identified as 5, 7-

dihydroxy-3, 6, 4'-trimethoxyflavone (santin) (4a) and not 5, 7-dihydroxy-3, 8, 4'-

trimethoxyflavone (4b). This compound has been previously isolated from the leaf extract of

three D odonaea species including Dodonaea attenuate var. linearis |Jefferies & Payne,

1973], D odonaea viscosa [Sachdev & Kulshreshtha, 1982; Wollenweber et al. , 1986; Abdel-

Mogib et a l ., 2001] and D odonaea angustifolia [Sachdev & Kulshreshtha.. 1984].

112
 NOE NOE

Figure 3.5: NOE correlation o f santin (4)

3.1.5. RHAMNOCITRIN (5)

Compound 5 was isolated as yellow crystals with melting point o f 221-223 °C. It appears as

yellow (Rf 0.4 (2% MeOH in CH2CI2) which intensified on exposure to ammonia vapour

indicating that it is phenolic.

The UV (?tmax 266.0 and 364.0 nm) [Mabry et al., 1970], 1H (5 12.18 (for chelated hydroxyl

group)) and l3C (8 147.2.2 for C-2, 136.3 for C-3 and 176.5 for C-4) NMR spectra [Agrawal,

1989] is consistent with the tlavonol derivative. EI-MS analysis of compound 5 showed a

molecular ion peak m/z 300 corresponding to C |6Hi20(V

fhe 'H-NMR (Table 3.6) indicated the presence of two meta coupled aromatic protons at 5

6.41 and 6.20 (1H, d, J = 2.1 Hz) which were assigned to a di-substituted ring A and an

AA'BB' spin system centered at 5 6.92 and 8.10 (./ = 9.0 Hz) which were assigned to 4'-

substituted ring B protons. The assignment of two sets of doublets to C-6 and C-8 was

confirmed from HMBC correlation of the proton at 8 6.20 with C-5 (8 161.7), C-7 (8 164.7),

C-8 (8 93.6), C-10 (8 103.7) and the proton at 8 6.41 with C-6 (8 98.4). C-7 (8 164.7), C-9

(8 157.4), and C-10 (8 103.7). C-5 (8 161.7

113
The 1 C-NMR spectrum (Table 3.6) showed signals for C-6 and C-8 of ring A at 8 98.4 and 5

93.6 respectively, which shows HMQC correlations with the corresponding protons (H-6 and

H-8).

The 'H-NMR further displayed one singlet for a methoxyl group at 8 3.89 (3H, s). The

placement of the methoxyl group at C-7 was confirmed through HMBC correlation (Table

3.6) of the protons at 5 3.89 with C-7 (8 164.7). The location o f the methoxyl group was

confirmed by NOE experiment which exhibited NOE interactions between the methoxyl at

8 3.89 (C-7) and the of meta coupled aromatic protons at 8 6.41 (C-8) and 8 6.20 (C-6).

Thus based on these spectroscopic data compound 5 was identified as 5,4'-dihydroxy-7-

dimethoxyflavonol (5) (rhamnocitrin). The data is in close agreement with that for

rhamnocitrin (5) previously isolated from the aerial parts o f Dodonaea viscosa [Ghisalberti,

1998].

Figure 3.6: HMBC correlations of 5,4'-dihydroxy-7-dimethoxyllavonol (5)

114
 NOE

NOE

Figure 3.7: NOE correlations o f the methoxy group of 5.4'-dihydroxy-7-dimethoxyflavonol

(5 )

Table 3.6: ID (CD3OD: 300, 75.5 MHz) and 21) NMR data for 5,4' dihydroxy-7-
dimethoxyflavonol (5 )
c £>ih ^I3C HMBC HMBC
(m ,J ( Hz)) 2J V
2 147.2
3 136.3
4 176.5
5 161.7
6 6.20 (d, 2 A ) 98.4 C-5, C-7 C-8, C-10
7 164.7
8 6.41 (d, 2.1) 93.6 C-9, C-7 C-6, C-10
9 157.4
10 103.7
r 122.9
2' 8.10 (d, 9.0) 129.8 C -6X -2, C4'
3' 6.92 (d , 9.0) 115.5 C-4' c - 5 '. c-r
4' 159.7
5' 6.92 (d, 9.0) 115.5 C-4' c-3', c - r ,
6' 8.10 (z/, 9.0) 115.5 C-2'.C-2, C4'
OMe 3.89 (5) 55.6 C-7
OH 12.18 (s) C-5 C-6, C-10
OH 9.2 (brs)

3 .1 .6 . I S O K A E M P F E R I D E (6 ).

Compound 6 was isolated as yellow crystals with melting point o f 299-302 °C. It appears as

yellow (R| 0.3 (2% MeOH in CH2CI2) which intensifies on exposure to ammonia vapour

indicating that it is phenolic. The L’C (5 147.6 for C-2, 136.5 for C-3 and 176.7 for C-4)

NMR is consistent with a flavonol derivative [Agrawal 1989], The mass spectrum showed

molecular ion peak at m/z 300 [M+] corresponding to C |6H |20 6 and also an intense peak at
257 [!Vf- 43] (Scheme 3.3) corresponding to the C-ring collapse for 3-methyl ether flavone

[Harborne, 1994].

M + - 43
m/z 257

Scheme 3.3: Fragmentation pattern o f isokaempferide (6)

The ’H-NMR (Table 3.7) indicated the presence of two meta coupled aromatic protons at 5

6.41 and 6.20 (1H, J = 2.0 Hz) which were assigned to a di-ortho substituted ring A and an

AA'BB' spin system centered at 5 6.92 and 8.10 (.7 = 9.0 Hz)which were assigned to 4'-

substituted ring B protons. The assignment of two sets of doublets to C-6 and C-8 was

confirmed from HMBC correlation of 8 6.20 with C-7 (8 165), C-8 (8 93.6), C-10 (8 104.0),

C-5 (8161.4) and 8 6.41 with C-6 (8 98.5), C-7 (8 165), C-9 (8 157.7). and C -10 (8 104.9).

116
The 'H-NMR displayed one singlet for a methoxyl group at 8 3.80 (311. v). The placement of

the methoxyl group at C-3 was confirmed through HMQC correlation (Figure 3.8) of the

protons at 8 3.80 with C-3 (8 136.5) and due to the fact that it resonates at 8 59.0 which is

typical for a d\-ortho substituted methoxyl.

Thus based on this and correlation of the data with literature compound 6 was identified as

5,7,4'-trihydroxy-3-dimethoxyflavonol (6) (Isokaempferide). Isokaempferide (6) has been

previously isolated from the aerial parts of Dodonaeci viscosa [Dreyer, 1978].

Figure 3.8: HMBC correlation of H-6 and H-8 of isokaempferide (6).

Table 3.7: ID (CDC13: 300, 75.5 MFIz) and 2D NMR data for isokaempferide (6)
c £ ih 3\1C HMBC HMBC
(m. ./tHzYl 3J
2 147.6
3 136.5
4 176.7
5 161.4
6 6.20 (d, 2.0) 98.5 C-5, C-7 C-8, C-10
7 164.9
8 6.41 (d, 2.0) 93.6 C-9, C-7 C-6, C-10
9 157.7
10 104.0
r 122.0
2' 8.12 (d, 9.0) 129.9 C-6',C-2, C4'
3' 6.92 (d, 9.0) 115.7 C-4' C-5', c - r
4' 159.8
5' 6.92 (d , 9.0) 115.7 C-4' c-3', c - r ,
6' 8.12 (d, 9.0) 129.9 C-2',C-2, C-4'
bo

OMe 59.0 C-3

Oj
o

117
3.1.7. 6-METHOXYKAEMPFEROL (7).
Compound 7 was isolated as yellow needle like crystals with melting point of 253-254 °C. It

appears as yellow spot (Rf 0.5 (4% MeOH in CFbCF), which intensified on exposure to

ammonia, vapour indicating it is phenolic. The mass spectrum showed molecular ion peak at

m/z 316 [M ] corresponding to C i6H |20 7 and an intense peak at 301 | M -15] (Scheme 3.4)

due to the 6-OCH3 flavonol fragmentation [Markham, 19821.

OH

7a
m/z 183

M + - 15
m/z 301

Scheme 3.4: Fragmentation pattern of 6-methoxykaempferol (7)

The 'H-NMR peak at 5 12.34 (chelated hydroxyl group) and a 1’C-NMR peak at 8 148.0 (C-

2), 137.0 (C-3) and 177.6 (C-4) NMR is consistent with a 5-hydroxyflavonol derivative

[Agrawal, 1989].

118
The 'H-NMR (Table 3.8) displayed the presence o f a singlet at 8 6.63 (IH , s) which was

assigned to a tri- substituted ring A and an AA'BB' spin system centered at 5 7.02 and 8.17

(dd J= 2.1,9.0 Hz) which were assigned to 4'-substituted ring B protons. The assignment of

the singlet to C-8 was confirmed from HMBC correlation between the chelated hydroxyl

proton at 8 12.34 with the C-6 at 8 132.4 and not with the carbon at 8 95.2 which has an

HMQC correlation with the singlet at 8 6.63.

The 'H-NMR displayed one singlet for a methoxyl group at 8 3.96 (311, .y). The placement of

the methoxyl group at C-3 was confirmed through HMBC correlation (Table 3.8) of the

protons at 8 3.96 with C-6 (132.4) and due to the fact that it resonates at 8 61.4 which is

typical for a d\-ortho substituted methoxyl.

Thus based on the 'H, 13C-NMR, COSY. NOESY, HMQC and HMBC data compound 7 was

identified as 3, 5, 7, 4'-tetrahydroxy-6-methoxyflavonol (6-methoxykaempferol) (7). 6-

methoxykaempferol (7) has not been previously reported from the Dodonaea species.

However, this compound has been isolated from the aerial parts of a number of plant species

including Heteranthemis viscidihirta [Valant-Vetschcra et al., 2003], Centaurea inca [Akkal

et al., 1997] and Carthamus tinctorius [Hattori et al., 19921.

119
Table 3.8: II) (CD3OD: 300, 75.5 MHz) and 2D NMR data for 6-methoxykaempferol (7)
c £ih due HMBC HMBC
(m, J (Hz)) 2J V
2 148.0
3 137.0
4 177.6
5 153.3
6 132.4
7 158.6
8 6.63 (.v) 95.2 C-9, C-7 C-6, C-10
9 153.8
10 105.3
r 123.0
2' 8.17 (dd, 2.1,9.0) 131.2 C-6',C-2, C4'
3' 7.02 {dd, 2.1, 9.0) 117.0 C-4' c-5', c - r
4' 160.9
5' 7.02 (dd, 2.1, 9.0) 117.0 C-4' c-3', c - r ,
6' 8.17 (dd, 2.1, 9.0) 131.2 C-2',C-2, C4'
OMe 3.96 (s) 61.4 C-6
OH 12.34 (.s) C-5 C-10. C-6
OH 9.09 (brs)

3.1.8. PINOCEMBRIN (8).

Compound 8 was isolated as white crystals with a melting point 192-I93°C. It appears as

yellow spot (Rf 0.5 (1% MeOH in CH2CI2), which intensified on exposure to ammonia,

vapour indicating it is phenolic. The UV peak at X,max (MeOH) 289.0 | Mabry et al., 1970],

'll-N M R at 5 12.14 (chelated hydroxyl), an ABX spin system at 8 5.55 {dd, J= 3.0, 13.6 Hz

for H-2) assigned to a methine proton attached to an oxygen , 8 2.77 {dd,./ = 3.4, 13.6 Hz for

H-3) , 6 3.16 (dd, J = 13.0, 17.40 Hz for H-3) and 13C-NMR signals at 8 79.9 (C-2), 8 43.6

(C-3) and 8 196.5 (C-4) is consistent with a 5-hydroxyflavanone derivative | Agrawal, 1989],

The 'll-N M R exhibited meta coupled protons resonating at 8 5.96 and 8 5.95 (d, J= 2.0 Hz)

which were assigned to ring A protons and another two sets of peaks at 8 7.53 (2H) and

8 7.43 (3H) which were assigned to an unsubstituted ring B protons. Using the above

spectroscopic data and comparison with literature [Sachdev & Kulshreshtha, 1983]

120
compound 8 was identified as 5,7-dihydroxyflavanone (pinocembrin). Pinocembrin (8) has

been previously isolated from Dodonaea viscosa [Sachdev & Kulshreshtha, 1983].

Pinocembrin (8)

Table 3.9: 'H ((CD3)2CO, 200 MHz) and l3C (200 MHz) NMR for pinocembrin (8)
c <?1H ^13C
(m, J (Hz))
2 79.91
3 43.64
4 196.53
5 163.91
6 5.95 ( d , J = 2.0) 96.86
7 167.32
8 5.94 ( d , J = 2.0) 95.84
9 165.04
10 103.05
V 139.87
T 129.33
y 7.53 m 127.18
4' 7.43 m 129.27
5' 7.53 m 127.18
6' 8.20 ( d , J = 8.8) 129.33
OH-5 12.14 5

3 .1 .9 . 2 p - H Y D R O X Y H A R D W I C K 1 I C A C I D (9 ).

Compound 9 was obtained as white crystals from CH2CI2/McOH. Rt 0.4 (3% MeOH in

CH2CI2) and was UV (254 nm) active.

121
The EI-MS of compound 9 provided the M at m/z 332 indicating the molecular formula

C20H28O4. The peaks at m/z 95 and 81 as shown in the fragments 9a and 9b respectively

suggested the presence of furan ring with an alkyl chain in 9 [Spanevello & Vila, 1994].

+CH

Typical downfield shifted broad singlets in the 'H-NMR spectrum of compound 9 (Table

3.10) at 5 6.37, 7.37, and 7.44 were attributed to the 11-14, H-16 and H-15 protons,

respectively, suggesting the presence of a (3-substituted furan ring. The 'H-NMR further

showed, a broad singlet at 5 6.62 which was assigned to a p olefinic proton conjugated to a

carboxyl group, a three proton doublet at 8 0.85 (.7 = 6.6 Hz) attributed to the secondary

methyl, a three proton singlet at 5 0.80 and 8 1.32 attributed to the tertiary methyl groups

typical o f clerodane-type diterpenes [Givovich et al., 1986; Luteijn el a/., 1982; San-Martin

el al., 1986; Sharma et al., 1984]. The 'H-NMR spectrum of 9 showed a signal at 8 4.35 (m, 1

H) indicating that one of the two protons at C-2 had been substituted by an OH group [Fang

elal., 1988],

The 'H -'H COSY experiment showed coupling between the methyl at 8 0.85 and the H-8

proton at 8 1.52. It also showed coupling between the proton at 8 6.37 with the proton

at 8 2.40 assigned to FI-12 and between the proton at 8 7.37 and the 11-12 methylene protons

at 8 2.40 and 8 2.30. In addition there were coupling between the protons at 8 6.37, 8 7.37

and 8 7.44.

122
The 'C-NM R spectrum (A PI) (Iable 3.10) corroborated the presence of three methyl

groups, five methylenes, seven methines and five quaternary carbon atoms. The l3C-NMR

chemical shift of methyl at C-19 was observed at 6 21.6 and the ^-positioned axial methyl

group at C-20 appeared at 8 19.5, while the P-positioned equatorial methyl group at C-17

resonated at 8 17.0. These values reveal the tram configuration at the A/B ring junction of

compound 9 | Manabe & Nishino, 1986].

The l3C-NMR showed the downfield shift of the C-2 signal at 8 69.7 due to the OH group.

The position of the OH group at C-2 was further confirmed by HMBC experiments, which

showed cross peaks between H-2 proton (8 4.35) and C -l(8 29.3*/28.6*), C-10 (8 46.8), C-3

(8 141.8) and C-4 (8 43.3). Similarly, olefinic proton at H-3 (8 6.62) showed HMBC

correlations to C-4 (8 143.6), C-2 (8 69.7), C-18 (8 169.2) and C-5 (8 40.4). The proton at 8

6.37 assigned to H-14 showed cross peak correlations to the C -13 (8 127.2), C-15 (8 144.3),

C-16 (8 140.2) and C-12 (8 19.4*/8 19.5*). The methyl protons at C-19 (8 1.32) showed

HMBC correlation with C-4 (8 143.6), C-5 (8 40.4), C-6 (8 37.2) and C-10 (8 46.8). In

addition there was HMBC cross peaks between H-10 proton (8 1.59) and C-l (8 18.1), C-8 (8

37.6), C-9 (8 40.1), C-l 1 (8 40.0) and C-4 (8 43.3).

The stereochem istry was confirmed on the basis of NOES Y cross peaks observed between H-

20/H-17 and H-20/H-I9. However, there were no cross peaks between II-20/H-17/H-19 and

H-10. Further, confirmation of the structure of 9 was provided by NOESY experiment, which

showed cross peaks between H-2 and H-10 establishing the spatial proximity of H-2 and H-

10, thus placing the hydroxyl group at C-2 to p-position. These results can be rationalized

only if C-20, C-17, C-19 and HO-C (2) are on the same face of the molecule while H-10 and

H-2 are on another face of the molecule The 'H- and l3C-NMR, HMBC, and NOESY data

were consistent with the structure of 2p-hydroxy-15,16-epoxy-5p.8p,9p,10a-cleroda-

123
3,13(16), 14-trien-18-oic acid (2f3-Hydroxyhardwickiic acid) for compound 9. 20-

Hydroxyhardwickiic acid (9) has been previously isolated from Dodonaea boroniifolia

[Jefferies el r//., 1973] and Duranta repern [Anis et a l., 2001J. However, this is the first report

of this compound from D. angustifolia.

12 14

20-Hydroxyhardwickiic acid (9)

Table 3.10: ID (CDC13: 300, 75.5 MHz) and 2D NMR data for 20-
hydroxyhardwickiic acid (9)
c £ ih £|3C
(w, (Hz))
1 29.3 2.08 (a;;), 2.00 (m)
2 69.7 4.35 (m)
3 141.8 6.63 (brs)
4 143.6 -

5 40.8 -

6 37.2 2.42 (/;/), 1.18 (m)

7 28.8 1.50 (///), 1.38 (m)
8 37.4 1.63 (m)
9 39.9 -

10 46.8 1.48 (m)

11 39.6 1.57 (m)
12 19.4* 2.40 (/?/), 2.30 (m)
13 127.2 -

14 112.6 6.37 (brs)

15 144.3 7.44 (brs)
16 140.2 7.37 (brs)
17 17.0 0.85 (J, 6.6 Hz)
18 169.2 -

19 21.6 1.32 (.v)

20 19.5* 0.80 (s)
* Interchangeable

124
3.1.10. DODONIC ACID (10)

Compound 10 was obtained as white crystals from ClBCf/M eOI I with melting point of 105-

107 °C. R| 0.5 (2% MeOH/CH2Cl2) and was UV (254 nm) active.

The 'H-NMR of 10 exhibited signals for two tertiary methyl groups at 6 0.75 and 1.28 and

one secondary methyl group at 8 0.85 (J = 6.6 Hz). The 'H-NMR further dispayed broad

singlet at 6 6.87 assigned to a [3-vinyl hydrogen in an a , (3-unsaturatcd carbonyl grouping

having a methylene group adjacent to the vinyl hydrogen. A double doublet at 6 3.60 (./ = 5.6,

10.4 Hz) was assigned to the geminal proton of a secondary hydroxyl group at C-6, which

was coupling with the two protons at C-7. Typical downlleld signals in the 'H-NMR

spectrum o f compound 10 at 8 6.35, 7.35 and 7.43 were attributed to 1114, H-16 and H-15,

respectively, suggesting the presence of a 3-substituted furan ring. The peaks between 5 1.82

and 1.55 are due to the methylene groups. The ' ’C-NMR (DEPT) corroborated the presence

of three methyl groups, five methylenes, seven methines and five quaternary carbon

atoms.The l3C-NMR chemical shift of the methyls were observed at 8 16.1, 17.1 and 18.1.

These values reveal the trans configuration at the A/B ring junction of compound 9 [Manabe

& Nishino, 1986]. From the above spectroscopic studies and comparison o f the spectral data

with that in literature [Sachdev & Kulshreshtha, 1984; Van Heerden el a/., 2000J compound

10 was deduced to be dodonic acid. Dodonic acid (10) has been isolated before from

Dodonaea angustifolia [Van Heerden et al., 20001 and Dodonaea viscosa [Sachdev &

Kulshreshtha, 1984]

10

125
3.1.11. £AT-3(3, 8a; 15,16-EPOXY-13(16), 14-LABDADIENE-3, 8-DIOL (11)

Compound 11 was isolated as white crystals from CIHCb/MeOII. Its spot on TLC had an Rt

value of 0.4 (1% MeOH in CH2CI2) and was UV (254 nm) inactive but turned brown on

exposure to iodine vapour.

The 'H-NMR o f this compound displayed signals at 8 6.29, 7.23 and 7.34 attributed to the

two a and one p-proton of a p-substituted furan ring. The 'll-NMR further displayed a

distorted quartet at 8 3.23 (J = 4.8, 10.6 Hz) which was assigned to the geminal proton of a

secondary hydroxyl group at C-3, which was coupling with the two protons at C-2. The 'H-

NMR spectrum of 11 showed the signals of four tertiary methyl groups at 8 0.76, 0.81 and

0.98 and 1.14 characteristic of a labdane skeleton. The multiplet between 8 2.47 and 1.73 was

attributed to the methylene protons.

The 13C-NMR displayed 20 signals, assigned to a diterpene skeleton. The n C-NMR (DEPT)

corroborated the presence of four methyl groups, six methylenes, three methines and three

quaternary carbon atoms. The ljC-NMR chemical shift of the methyls were observed at 8

16.4, 16.8. 21.2 and 24.8. These values reveal the fram-configuration at the A/B ring junction

of compound 11 [Manabe & Nish ino, 1986].

From the above spectroscopic studies and comparison of the spectral data with that in

literature [Mata et al., 1991] compound 11 was deduced to be ent-3[), 8a; 15, 16-Epoxy-

13(16), 14-labdadicne-3, 8-diol. ent-3p, 8a; 15, 16-epoxy-13(16), 14 labdadiene-3, 8-diol

(11) has been isolated previously from Dodonaea viscosa [Dawson et ctl.. 1966].

126
 12 14

Dodonic acid (11)

3.2 COMPOUNDS FROM DODONAEA AN G U STIF O L/A -VO \

The extraction o f the surface exudates on the aerial parts (mainly leaves) was done after

plucking out the flowers. The surface exudates of the leaves was exhaustively extracted by

successive dipping into fresh portions of acetone for short periods (less than 15 seconds) to

yield the crude extract, thus avoiding the extraction o f the internal tissue components. The

extract was tested for anti-plasmodial activity against chloroquine-senstive (D6) strain of

Plasmodium falciparum with IC50 of 56.3 ± 4.2 pg/ml. The rest of the crude extract was then

subjected to gravity column chromatography on silica gel eluting with mixtures of n-

hexane/dichloromethane and then with dichloromethane/methanol. Further purification of

compounds was done using further chromatography on silica gel and Scphadex LH 20 and

finally crystallization leading to the isolation of eleven compounds. The structures of these

compounds was confirmed by comparison of their spectroscopic data with literature and in

some cases by direct comparison (co-TLC) with authentic samples.

5-Hydroxy-3,7.4'-trimethoxyflavone (1), 3,5-dihydroxy-7,4'-dimethoxyflavone (2),

kumatakenin (3), rhamnocitrin (5) were also isolated from D. augustifolia growing in Ngong

forest and therefore their structure elucidation arc not described again. Among the

compounds isolated the flavonoids, penduletin (12), ayanin (13), 5-hydroxy-3,6,7,4'-

tetramethoxyflavone (14), kaempferol (15), the coumarin 7-hydroxy-6-methoxycoumarin

(16) and the diterpenoid hautriwaic acid (17) have not been previously described from this

127
plant species. However, most of these compounds except ayanin (13) and 7-hydroxy-6-

methoxycoumarin (16) have been reported from different Dodonaea species. This is the first

report of ^oclerodan-3,13-dien-16,15: 18,19-diolide (18), 15a-methoxy-m?oclerodan-3,13-

dien-16,15: 18.19-diolide (19), 15P-methoxy-wwclerodan-3,13-dicn-l6,15: 18,19-diolide

(20). 1he structure of these compounds was determined using a combination of spectroscopic

techniques. In this section the structural elucidation o f these compounds will be discussed

3.2.1. PENDULET1N (12).

Compound 12 was isolated as yellow crystals with melting point of 220-222 °C. It appears as

yellow spot (Rf 0.5 (4% MeOH in CH2CI2), which intensified on exposure to ammonia,

vapour indicating it is phenolic. The E1MS showed a molecular ion peak at m/z 344

corresponding to C17H14O6.

The UV (Xmax MeOH 272.0 and 341.5 nm) [Mabry et al., 1970], 'H (5 12.73 (for chelated

hydroxyl group)) and l3C (5 148.0 for C-2, 137.0 for C-3 and 177.6 for O 4) NMR [Agrawal

1989J is consistent with a 5-hydroxyflavonol derivative.

T he 'H-NMR (Table 3.11) displayed the presence of a singlet al 5 6.63 (1H, s) which was

assigned to a tri-substituted ring A and in AA'BB'spin system centered at 8 7.03 and 8.06 (dd,

J = 2.1, 9.0 Hz) which were assigned to 4'-substituted ring B protons. The assignment of the

singlet to C-8 was confirmed from HMBC correlation between the chelated hydroxyl proton

at 5 12.73 with the carbon at 5 133.8 and not with the carbon at 5 92.4 which has an HMQC

correlation with the singlet at 8 6.63.

The 'H-NMR ( fable 3.11) displayed three peaks for three methoxyl group at 8 3.98, 3.88 and

3.80.

128
The placement of the methoxyl group at C-3, C-6 and C-7 was confirmed through HMBC

correlation (Table 3.11) of the protons at 8 3.98 w ith C-6 (6 133.8), 5 3.88 with C-3 (5 139.8)

and C-7 (8 160.8).

The 1 C-NMR for the methoxyl groups at 8 60.9 and 8 61.2 requires that the methoxyl groups

are di-ortho substituted. The ,3C-NMR of the other methoxyl group was 8 57.5 which is

typical o f an isolated or mono substituted methoxyl group

Thus based on this and comparison of the data with literature compound 12 was identified as

3.5,7,4'-tetrahydroxy-6-methoxyflavonol (12) (penduletin or candirone). Penduletin (12) has

been previously isolated from the Dodonaea viscosa [Sachdev & Kulshrcshtha, 1986].

Figure 3.9: HMBC correlations in 3,5,7,4'-tetrahydroxy-6-methoxyIlavonol (12).

129
Table 3.11: ID (CDCI3: 300, 75.5 MHz) and 2D NMR data for pend u let in (12)
C d\ H <?13C HMBC HMBC
(///, (Hz)) V 3./
2 157.7
3 139.8
4 180.5
5 154.3
6 133.8
7 160.8
8 6.63 (s) 92.4 C-9, C-7 C-6. C-10
9 153.9
10 107.7
r 123.4
T 8.06 (dd, 2.1,9.0) 131.9 C-6',C-2, C4'
y 7.03 (dd, 2.1, 9.0) 117.1 C-4' c-5', c-r
4' 161.7
5' 7.03 (dd, 2.1, 9.0) 117.1 C-4'

0
i
6' 8.06 (dd, 2.1, 9.0) 131.9 C-2',C-2, C4'
OMe 3.98 (s) 57.5 C-6
OMe 3.88 (s) 60.9
OMe 3.80 (5) 61.2
OH 12.73.s- C-5 C-10, C-6

3.2.2. AYANIN (13).

Compound 13 was isolated as yellow crystals with melting point o f 173-1 74 °C. It appears as

yellow spot (Rt 0.5 (2% MeOH-CH2Cl2) which intensified on exposure to ammonia vapour

indicating it is phenolic. The EIMS showed a molecular ion peak at m/z 344 corresponding to

C |8H i60 7 and an intense peak at 301 [M - 43] corresponding to standard flavonol C-ring

collapse [Harborne, 1994].

The 'll (5 12.77 (for chelated hydroxyl group)) and 11C (5 157.6 for C-2, 140.3 for C-3 and

180.3 for C-4) NMR [Agrawal 1989] is consistent with a 5-hydroxyflavonol derivative.

The 1H-NMR (Table 3 .12) indicated the presence of two meta coupled aromatic protons at 8

6.67 and 6.32 which were assigned to ring A and an AXY spin system at 5 7.05 (lH, d , J -

8.7 Hz), 7.72 (lH, dd, ./= 2.1 and 8.7 Hz) and 7.80 ( IH, dd, ./= 2.1 Hz) assigned to C-3? and

C-4‘ substituted ring B. With oxygenations at C-5 and C-7 (biogenetically expected), the

meta coupled protons are assigned to H-6 and H-8. HMBC correlation between the chelated
hydroxyl proton (5 12.77) with C-6 (5 99.1) and HMQC correlation between the proton at 5

6.32 and C-6 (6 99.1) led to the assignment o f the doublet at 5 6.32 to 11-6. Similarly, HMBC

correlation between the doublet at 5 6.32 and C-8 (5 93.7) allowed the placement of the

doublet at 5 6.67 to H-8.

The 'H-NMR (Table 3.12) also displayed peaks for three methoxyl groups at 5 3.95, 3.92 and

3.90. One o f the methoxyl groups was placed at C-3 due to the fact that it resonates at 5 60.9

in i3C-NMR which is typical for a d\-ortho substituted methoxyl. The placement of the other

two methoxyl groups at C-7 and C-4' position was confirmed by the HMBC correlation

between the methoxyl group at 6 3.95 with C-7 (6 167.3) and that at 6 3.92 with C-4' (5

151.2). HMBC correlation of the carbon at 8 151.2 (C-4') with the three protons in the AXY

spin system was also observed.

Thus based on this and correlation of the data with literature compound 13 is identified as

5.3'-dihydroxy-3,4',7-trimethoxyflavone (13) (ayanin). Ayanin (13) has not been isolated

from Dodonaea species but from several other plant species including the aerial parts of

Bahia glandulosa [Perez-Castorena et al., 1997a] Psiadia dentate [Jakobsen et al., 2001 ].

Figure 3.10: HMBC correlations of ayanin (13)

131
 Table 3.12: ID (C D C I 3 : 300, 75.5 M H z) and 21) N M R data for ayanin (13)
c d\7,c HMBC HMBC
(Hz)) V
2 157.6
3 140.3
4 180.3
5 163.6
6 6.32 (d, 2.4) 99.1 C-5 C-8. C-10
7 167.3
8 6.67 (d, 2.4) 93.7 C-9, C-7 C-6, ( -10
9 158.4
10 107.3
r 123.5
2' 7.72 (dd, 2.1, 8.7) 124.1 c-r C-2, C4'
3' 7.05 (dd, 2.1, 8.7) 116.8 C-2' C-5'
4' 151.2
5' 148.6 C-4' c-3', c - r ,
6' 7.80 (d, 2.1) 113.4 C-2', C 2, C4'
OMe 3.90 (a) 60.9 C-3
OMe 3.95 (s) 57.1 C-7
OMe 3.92 (s) 57.1 C-4'
OH 12.77 (5) C-5 C-10, C-6
OH 8.89 (brs)

3.2.3. 5-HYDROXY-3,6,7,4-TETRAMETHOXYFLAVONE (14)

Compound 14 was isolated as yellow crystals with melting point o f 178-1 80 °C. It appears as

yellow spot Rt 0.5 (4% M eOH/CfhCh) which intensified on exposure to ammonia vapour

indicating it is phenolic. The EIMS showed a molecular ion peak at m/z 358 corresponding to

CigHisOy and an intense peak at 343 [M1 -CH3] and 315 [M - 43J corresponding to 6-OCH3

llavonol fragmentation (Markham, 1982) and standard flavonol C-ring collapse (Harborne,

1994) for 3-methyl ether flavone respectively.

The 13C (5 156.0 for C-2, 138.7 for C-3 and 178.9 for C-4) NMR |Agrawal, 1989] is

consistent with a llavonol derivative.

The 'H-NMR (Table 3.13) displayed the presence a singlet at 5 6.51 (1H, s) which was

assigned to a tri-substituted ring A and an AA'BB' spin system centered at 5 7.03 and 8.08

132
(dd, J ~ 2.1, 9.0 Hz) which were assigned to 4 '-substituted ring B protons. With the

biogenetically expected oxygenations at C-5 and C-7 the singlet at 5 6 .5 1 was assigned to

either H-6 or H-8. The peak at 8 6.51 was assigned to 11-8 due to I1MBC correlations with C-

6(5 132.3), C -7 (5 158.7), C-9 (5 152.3) and C-10(6 106.6).

The H-NMR also displayed peaks for four methoxyl groups at 5 3.96, 3.93 and 3.90 and

3.87. From the HMQC correlations, two of the methoxyl groups were placed at C-3 and C-6

due to the fact that they resonated at 5 60.1 and 8 60.9, respectively, which is typical for di-

ortho substituted methoxyl groups. The other two methoxyl groups were placed at C-7 and at

C-4\ The placement o f the methoxyl group at 5 3.96 and 3.90 to C-7 and C-4' position

respectively, was confirmed by the HMBC correlation between the methoxyl group at 5 3.96

and C-7 (8 158.7) and C-4' (8161.7), respectively.

Thus based on this and correlation of the data with literature compound 14 was identified as

5-hydroxy-3,6,7,4'-tetramethoxyflavone (14). Compound 14 has been previously isolated

from the Dodonaea viscosa [Sachdev & Kulshreshtha, 1983] and Dodonaea lobulata

[Dawson et a/., 1966]. However, this compound has never been isolated from Dodonaea

august [folia.

OH O

14

Figure 3.11: HMBC of 5-hydroxy-3,6,7,4'-tetramethoxyflavone (14)

133
 1able 3.13: ID (C D C I 3 : 300, 75.5 M H z) and 21) N M R data for 5 hydroxy-3,6,7,4'-
tetramethoxyflavone ( 1 4 )
c £ ih £l3C IIMBC HMBC
(w, (Hz)) V V
2 156.0
3 138.7
4 178.9
5 152.8
6 132.3
7 158.7
8 6.51 5 90.3 C-9, C-7 C-6, C-10
9 152.3
10 106.6
r 122.8
2' 8.08 (dd, 2.1,9.0) 130.1 C-6', C-4',C-2
3' 7.03 (dd, 2.1, 9.0) 114.1 C-4' C-5',C-1'
4' 161.7
5' 7.03 (dd, 2.1, 9.0) 114.1 C-4' c - 3 ', c - r ,
6' 8.08 (dd, 2.1, 9.0) 130.1 C-2',C 2, C-4'
OMe 3.87 s 60.1 C-3
OMe 3.96 ^ 55.3 C-7
OMe 3.93 s 60.9 C-8
OMe 3.90 5 56.0 C-4'

3.2.4. KAEMPFEROL (15).

Compound 15 was isolated as yellow crystals with melting point o f 276-278 °C. It appears as

yellow spot (Rf 0.5 (4% MeOH/CH2Cl2) which intensified on exposure to ammonia vapour

indicating it is phenolic. The EIMS showed a molecular ion peak at m/z 286 corresponding to

C 15 H 10 O 6 .

The UV (kmax MeOH 267.0.0, 324.0 and 364.5 nm) |Mabry et ah, 1970], l3C (8 148.0 for C-

2, 5 137.0 for C-3 and 8 177.3 for C-4) NMR [Agrawal. 1989] is consistent with a 5-

hydroxyflavonol derivative.

The 'H-NMR (Table 3.14) indicated the presence of two meta coupled aromatic protons at 8

6.54 and 6.27 (1H, c/, J= 2.1 Hz) which were assigned to ring A and an AA'BB' spin system at

8 8.17 and 7.02 (2H, dd, ./ = 2.1 and 9.0 Hz) assigned to C-4' substituted ring B. With

134
oxygenations at C-5 and C-7 (biogenetically expected) the meta coupled protons are assigned

to H-6 and H-8.

The assignment of two sets o f doublets to C-6 and C-8 was conllrmed from HMBC

correlation of 5 6.20 with C-7 (8 165), C-8 (8 93.6), C-l 0 (8 104.0), C-5 (8 161.4) and 8 6.41

with C-6 (8 98.5), C-7 (8 165), C-9 (8 157.7), and C-10 (8 104.9), respectively.

The ’’C-NMR (Table 3.14) further showed signals for C-6 and C-8 of ring A at 5 98.4 and

93.6, respectively, which shows HMQC correlations with the corresponding protons at H-6

and H-8.

Thus based on this and correlation of the data with literature compound 15 was identified as

5,7,4'-tetrahydroxy-flavonol (15) (kaempferol etc.). Kaempferol (15) has been previously

isolated from the Dodonaea viscosa [Khan et al., 1992].

Figure 3.12: HMBC correlations o f kaempferol (15)

135
1 able 3.14: ID (C D C I 3 : 300, 75.5 M H z) and 2D N M R data for kaempferol (15)
c 4h d\ic HMBC ' HMBC
(w, (Hz)) 2J
2 141.7
3 137.3
4 177.3
5 163.0
6 6.27 ( 4 2.1) 99.9 C-5 C-8.C-10
7 165.7
8 6.54 ( 4 2.1)

U
VO
W
95.2 C-9, C-7

l
9 158.5
10 104.9
r 124.0
2' 8.17 (dd, 2.1, 9.0) 131.2 c -r C-6', C4'
3' 7.02 (dd, 2.1, 9.0) 117.0 C-4',C-5',C-1'
4' 160.9
5' 7.02 (dd. 2.1, 9.0) 117.0 C-3', C - r , C-4'
6' 8.17 (dd, 2.1, 9.0) 131.2 c -r C-2 , C-4'
OH 9.2 (brs)

3.2.5. SCOPOLETIN (16).

Compound 16 was isolated as white needles with melting point of 203-204°C. It showed

strong blue fluorescence with UV (254 nm) and had an Rt 0.3 (CIECC). The ''C-NMR

(Table 3.15) displayed nine carbon atoms of phenylpropanoid. one of which is for an ester

carbonyl ca 8 160.7 in the 1’C-NMR and one for a methoxyl substituent at ca 5 56.0.

The 'H-NMR (Table 3.15) displayed two olefinic protons at 5 6.16 (./ 9.2 Hz) and 7.84 (./ =

9.2 Hz), two aromatic singlets at 5 7.18 and 6.79. The above data is consistent with a

coumarin derivative. The protons at 8 7.18 and 6.76 were assigned to 11-5 and H-8 on the

coumarin skeleton. The 'H-NMR further exhibited a three proton singlet at 8 3.89 attributed

to a methoxyl group. The position of the methoxyl group was confirmed by NOESY

experiment, which showed cross peaks between the methoxyl group and 11-5 (8 7.18) singlet.

From these spectral data and comparison with literature [Abyshev et al., 19801 this

compound was identified as 7-hydroxy-6-methoxycoumarin (16) (scopoletin). Scopoletin

(16) has been previously isolated from Dodonaea viscosa, and other plant species such as
136
Haplophyllum vulcanicum [Ayhan and Mehmet, 20081 but this is the first report of this

compound from Dodonaea angustifolia.

5 4
MeO

HO
l

7-hydroxy-6-methoxycoumarin (16)

7 able 3.15: H (A c e to n e ^ at 300 MHz) and 1 C (75 MHz) NMR chemical shift data for 7-
hydroxy-6-methoxycoumarin (16)
C ^1H ^>I3C
(Hz))
2 - 160.7
3 6.16 (d, 9.4) 1 12.4
4 7.84 (d, 9.2) 144.1
5 7.18(5) 109.2
6 - 150.5
7 - 151.6
8 6.79 (5) 103.0
9 - 145.4
10 - 111.2
OMe 3.89 (5) 56.0

3.2.6. HAUTRIWA1C ACID (17)

Compound 17 was obtained as white crystals from CIHCb/MeOII with melting point of 183-

184 °C. Rr 0.4 (3% MeOH/CH2Cl2) and was slightly UV (254 nm) active and turned brown

on exposure to iodine vapours.

The EI-MS of compound 17 provided the molecular ion peak at m/:: 332 indicating the

molecular formula C20H28O4. The n C-NMR spectrum (APT) corroborated the presence of

two methyl groups, seven methylenes and six methines and five quaternary carbon atoms.

The peaks at m/z 95 and 81 as shown in the fragments 17a and 17b respectively suggested the

presence of furan ring with an alkyl chain in 17 [Spanevello & Vila. 1994]. These results

indicated that compounds 17 is a diterpene with a furan ring.

137
 17a

I he 13C-NMR spectrum exhibited signals at 5 19.1 and 16.7 due to tertiary and secondary

methyl groups at C-9 and C-8, respectively, in agreement with the data of compounds having

both of these substituents as alpha on a /ram-clerodane skeleton [Givovich et al., 1986; San-

Martin et a l., 1986; Sharma et al., 1984]. In /ram-clerodanes, C-19 carbons resonates

between 8 11-19 whereas in c/T-clerodanes appears at about 5 25. Moreover, C-20 in trans-

clerodanes resonates at higher field (8 17-19) than in e/.v-clerodanes (8 21-29) [Manabe &

Nishino, 1986].

The 'H-NMR spectrum of compound 17 (Table 3.16) displayed a broad singlet at 8 6.28,

7.26 and 7.37 attributed to the H-14, H-16 and H-15 protons of the P substituted furan ring.

1H-NMR further showed an AB spin system at 8 3.74 and 4.13 (J = 12 11/.) attributed to the

H-19 hydroxyl methylene. In addition, a broad one proton singlet at 8 6.63 was assigned to a

P-olefmic proton conjugated to a carboxyl group, a three proton doublet al 8 0.87 (./= 6.6 Hz)

was attributed to the secondary methyl and a three proton singlet at 8 0.79 attributed to the

tertiary methyl group typical of clerodane-type diterpenes.

The COSY experiment showed coupling between the methyl at 8 0.87 and the H-8 proton at 8

1.63. Furthermore, the COSY experiment showed coupling between the proton at 8 6.28 with

the proton at 8 2.42 assigned to H-12 and between the proton at 8 7.26 and the H-12

methylene protons at 8 2.42 and 8 2.20. There were also coupling between the protons at

8 6.28. 7.26 and 7.34.

138
The structure of 17 was confirmed by the HMBC experiment, the olcfinic proton at 5 6.63

showed correlations to C-4 (5 138.9), C-5(43.3), C-2 (5 27.7). Similarly, the proton at 5 6.28

assigned to H-14 showed cross peak correlations to the C-13 (5 126.7), ( 15 (5 144.0), C-16

(5 139.7) and C-12 (5 18.1*/5 19.1*). In addition there was HMBC cross peaks between the

hyroxymethylene protons at 5 3.74, and C-5 (8 43.3), C-6 (5 32.9) and C -10 (8 47.8).

The stereochemistry was confirmed on the basis of NOBS Y cross peaks observed between H-

20/H-17 and H-20/H-19 (the two protons). However, there were no cross peaks between H-

20/H17/H-19 (the two protons) and H-10. These results can be rationalized only if C-20, C-

17, C-19 are on the same face o f the molecule and H-10 on another face o f the molecule. All

the data are in agreement with compound 17 being hautriwaic acid. Hautriwaic acid (17) has

been previously isolated from Dodonaea viscosa [Hsu et al., 19711 and Dodonaea attenuata

[Jefferies & Payne, 1967, 1973].

Hautriwaic acid (17)

139
Table 3.16: 'H (MeODat 300 MHz) and l3C (75 MHz) NMR chemical shift data for
hautriwaic acid (17)
c £|3C S\H
(///. (Hz))
1 19.1* 1.74 (/w), 1.69 (///)
2 27.7*
3 138.2 6.63 (bn)
4 138.9 -

5 43.3 -

6 32.9 -

7 28.1* 1.55 (/?/), 1.45 (m)

8 37.6 1.63 (m)
9 40.1 -

10 47.9 1.59 (m)

11 40.0 1.57 (m)
12 18.1* 2.40 (m ), 2.20 (/;;)
13 126.7 -

14 111.9 6.28 (bn)

15 144.0 7.37 (bn)
16 139.7 7.26 (bn)
17 16.2 0.87 (d, 6.6)
18 - -

19 66.2 4.13 (r/, 1.2), 3.74 (d, 1.2)

20 19.1 0.79 (s)

3.2.7. 7V£0CLERODAN-3,13-DIEN-16,15: 18,19-IHOLIDE (18)

Compound 18 was obtained as colourless oil from ClHCh/MeOH. Rt 0.2 (1% MeOH in

CH2CI2) and was slightly UV (254 nm) active and turned brown on exposure to iodine

vapours. The EI-MS of compound 18 provided the molecular ion peak at m/z 330 indicating

the molecular formula C20H26O4, this was also evident from the nC-NMR which showed

resonance for twenty carbons.

The clerodane skeleton of compound 18 was identified by its unique 'H and l3C-NMR signals

(Table 3.17), in which a tertiary methyl group appeared as a singlet at 8n 0.62 and 8c = 17.5

assigned to C-20 and a secondary methyl group as a doublet at 8n = 0.86 (./= 6.6 Hz) and 8C

= 15.5 assigned to C-17. The presence of two a , P-un saturated y-lactone moiety is evident in

this compound from the 'H-NMR signals at 5 6.76 (dd, J - 7.4, 2.0 Hz) and 7.14 (/, J = 1.5

Hz) for olefinic p-protons, 8 4.30 (</, J = 8.1 Hz), 3.92 (dd, 8.0. 2.0 I lx) and 8 4.79 (d,J =

140
1.8 Hz) for oxymethylenes at C-19 and C-15, respectively. The corresponding carbons in the

C-NMR for the lactone moiety appeared at 8 169.3 and 174.2 for C (): 6 71.7 and 70.2 for

oxymethylenes and the olefinic carbons resonated at 6 135.8. 138.4. 143.9 and 134.3.

The methylene protons at C-19 had an AB spin system. The pro-19S diastereotopic proton of

this group (8 3.92) was also co-coupled ( './= 2.0 Hz) with the 11-6(3 proton, indicating an a-

axial orientation for C-19 [Bruno et al., 1981; Esquivel et al., 1986a. 1986b and Stapel.,

1980],

In the H-NMR the pro-19R proton resonated at 8 4.30 which is in agreement with lack of a

substituent at C-7 position in this compound. The presence of a substituent at C-7 position

usually affects the chemical shift value of pro-R proton but has no effect on the /;ro-19S

proton. The pro-R proton is usually downfield shifted by a factor of about 0.95 ppm when an

cx-axial hydroxyl group is present at C-7 [Herz el al., 1977; Ohsaki et a/., 1986; Zdero et al.,

1989b] or by a factor of 0.50 ppm when an a-axial acetate group is bound to C-7. Change in

hybridization at C-7 also influences the chemical shift o f pro-R proton, so an average of 8 4.0

is observed [Herz et al., 1977; Esquivel el al., 1988] in oxo-dcrivatives. In the absence of the

above mentioned factors, an average of 8 4.35 is expected for the pro- 19R proton, as

observed in compound 18.

The COSY spectrum clearly indicates that the proton at 8 7.14 (/,./ 1.5 Hz), assigned to H-

14, is coupled with the oxymethylene protons at C-15. The triplet is characteristic of a proton

on the (3-carbon of an a-substituted butenoid ring.

The n C-NMR (Table 3.17) signals at 8 17.5 and 15.5 due to tertiary and secondary methyl

groups at C-9 and C-8 respectively are in agreement with the data of compounds having both

of these substituents as alpha on a /raw.v-clerodane skeleton [Givovich et al., 1986; Luteijn et

141
al., 1982; San-Martin et al., 1986; Sharma et a l 1984], This was further confirmed by the to-

coupling shown by the/?ro-19S in compound 18 (5 3.92, dd, J 8.1 and 2.0 Hz) indicating an

a-axial configuration o f the C-19 methylene group in agreement with /ram-clerodanes

having an axial methyl group at C-5 and an axial H-6 [Gambaro et al., 1986; Givovich et al.,

1986; San-Martin et al., 1986]

In /ram-clerodanes C-19 resonates between 5 11-19 whereas in c/.v-clerodanes this carbon

appears at about 5 25. Moreover, C-20 in /nmv-clerodanes resonates at higher field (8 17-19)

than in c/.v-clerodanes (8 21-29) [Manabe & Nishino 1986]. In the 1'C-NMR of compound 18

the C-20 carbon resonated at 5 17.5, confirming the tram stereochemistry.

The stereochemistry o f compound 18 was further confirmed on the basis of NOESY cross

peaks observed between H-20/H-17, H-19 (pro-S, />ro-R)/H-20 and H-6/7/H-10. However,

there were no cross peaks between H-20/H-17/H-I9 (the two protons) and H-10. These

results can be rationalized only if C-20, C -17, C-19 are on the same face o f the molecule and

H-10 on the other face.

The olefinic proton at C-3 (8 6.76) showed HMBC cross peak with the carbons at C-16 (5

169.3), C-5 (5 45.5), C-2 (5 27.6) and C-l (5 19.5). The other olefinic proton at C-14 (5 7.14)

showed HMBC cross peaks with C-16 (5 174.2), C-5 (5 70.2), C -l3 (5 134).

All the data are in agreement with compound being a //eo-clerodane skeleton. Diterpenoids

with a-substituted butenolide moieties have not been isolated from Dodonaea species before.

This is the first report of this compound in nature.

142
 Figure 3.13: HMBC of compound /7<?ocIerodan-3,13-dien-16.15: 18 .19-diolide (18).

I able 3.17: ID (CDCI3: 300, 75.5 MHz) and 2D NMR data for Areoclerodan-3,13-dien-16,15:
18,19-diolide (18).
c £|3C <?IH
(w, (Hz))
1 19.5 1.77 (m), 1.07 (m)
2 27.6 2.40 (m), 2.23 (m)
3 135.8 6.76 (</</, 7.4; 2.0 1Iz)
4 138.4 -

5 45.5 -

6 34.4 1.93 (m), 1.25 (m)

7 27.7 1.62 (w), 1.51 (m)
8 36.5 1.68 (m)
9 38.7 -

10 48.0 1.75 (m)

11 35.2 1.60 (m)
12 18.9 2.24 (/;;), 2.04 (m )
13 134.3 -

14 143.9 7.14 (/, 1.5)

15 70.2 4.79 (d, 1.8).
16 174.2 -

17 15.5 0.86 (d, 6.6)

18 169.3 -

19 71.7 pro-R 4.30 (d, 8.1), pro-S 3.92 (dd, 8.0, 2.0)
20 17.5 0.62 (s)

143
3.2.8. 15p-Af£0CLERODAN-3,13-DIEN-16,15: 18,19-DIOLIDE (19) AND 15a-
AE0CLERODAN-3,13-DIEN-16,15: 18,19-DIOLIDE (2 0 )
Compounds 19 and 20 were isolated as epimeric mixtures whose signals appear as duplicate

in the NMR. They were isolated as colourless oil which on TLC had an Rt 0.1 (1% MeOH in

CH2CI2). They were slightly UV (254 nm) active and turned brown on exposure to iodine

vapours. The EI-MS o f the epimeric mixture of compounds 19 and 20 showed a molecular

ion peak at m/z 360 indicating the molecular formula C21II28O5.

The clerodane skeleton of compounds 19/20 was identified by its unique 'H and 1’C-NMR

signals (Table 3.18), in which a tertiary methyl group appeared as a singlet at 5 h = 0.62 and

5c = 17.5 assigned to C-20 and a secondary methyl group as a doublet at 8 h = 0.85/0.86 (J =

6.6 Hz) and 5C = 15.5 to C-17. The presence of two a,p-unsaturated y-lactone moiety in the

epimeric mixtures is evident from the 'H-NMR signals at 5 6.76 (/;/) and 6.79 (m) for olefinic

p-protons; 5 4.30 (d, J = 8.1 Hz) and 3.92 (dd, J = 8.0, 2.0 Hz) for geminal oxymethylene

protons at C-19; 5 5.74 (m) for an acetal proton and 5 3.58 (.y) for a methoxy group at C-15.

The corresponding carbons in the ' ’C-NMR appeared at 5 169.3 and 171.2 for C=0, 5 71.7

for the oxymethylene at C-19; 5 102.5 and 5 57.1/57.2 for an acetal and methoxy groups at C-

15, respectively; for and the four olefinic carbons resonated at 5 135.7/135.8 (C-3), 138.7 (C-

4), 5 138.4/138.5 (C-13), and 5 141.5/141.6 (C-14).

The pro-S (5 3.92) diastereotopic proton of at C-19 was co-coupled ( './ 2.0 Hz) with H-6p

proton, indicating an a-axial orientation for C-19 [Bruno et a/., 1981; Esquivel et al., 1986a,

1986b; Stapel, 1980J.

The multiplet at 5 6.79 (m) (H-14) is coupled with the doublet at 5 5.74 (///) (H-15) as evident

in the COSY spectra. The methoxy protons (5 3.58) showed HMBC correlations to C-15 (5

144
102.5). Furthermore, the substitution at this lactone ring was confirmed by HMBC cross

peaks of H-15 with the carbons at C-13 (8 138.4/138.4), C-14 (8 141.6). C-16 (8 171.2) and

the methoxy at 8 57.2/57.1 (Figure 3.19).

The placement of the methoxy at C-15 was confirmed from the COSY and 2D-NOESY

experiments which showed correlations of this group with FI-15 (8 5.74), 11-14 (8 6.79) which

in turn showed long-range allylic coupling with CH2-I2 (8 2.29 and 8 2.02).

The shielded ’C-NMR (Table 3.18) signals at 8 17.5 and 15.5 due to tertiary and secondary

methyl groups at C-9 and C-8, respectively, are in agreement w ith the data of compounds

having both o f these substituents as alpha on a /nmv-clerodane skeleton [Givovich et al.,

1986; Luteijn el a l ., 1982; San-Martin el a l., 1986; Sharma el a l ., 1984]. This tram

stereochemistry was further confirmed by the co-coupling shown by the pro- 19S in 19/20 (8

3.92, eld, J = 8.0 and 2.0 Hz) with H-6 protons, [Gambaro el al., 1986; Givovich et a l ., 1986;

San-Martin et a l ., 1986]

In the HMBC experiment of compound 19/20 (Figure 3.15), the olefinic proton at 8 6.76

showed correlations to C-4 (8 135.8/135.7), C-18 (8 169.3), C-5 (8 45.5), C-2 (8 27.7/27.6)

confirming its placemnt at C-3. Similarly, the other olefinic proton (8 6.79) showed cross

peak correlations to C-13 (8 138.5/138.4), C-16 (8 171.2), C-15 (8 102.5) and C-12 (8

18.91/18.87) which confirms its location at C-14.

The relative configuration of compounds 19/20 was confirmed by 2D-NOESY experiment

which showed cross peaks between H-20, H-17, H-19 and H-6/?. However, there were no

cross peaks between H-20/H-17/H-19 (the two protons) and H-10 and therefore the decalin

145
ring junction was deduced to be in tram configuration. This is the first report of this epimeric

mixture in nature.

Figure 3.14: HMBC correlations of 15(3-/7<?tfclerodan-3,13-dien-l 6,15: 18 .19-diolide (19)

Figure 3.15: F1MBC correlations of 15a-m,oclerodan-3.13-dien-l 6.15: 18 .19-diolide (20)

146
Table 3.18: ID (CDCI3: 300, 75.5 MHz) and 2D NMR data for the epimeric mixture of 15(3-
m?oclerodan-3,13-dien-16,15: 18.19-diolide (19) and 15a-A/eoelerodan-3,13-dien-
16,15: 18,19-diolide (20)
c £l3C 3\ H
(w, (Hz))
1 19.5 1.76 (m), 1.08 ( aw)
2 27.7 and 27.6 2.42 ( aw), 2.20 ( aw)
3 135.8 and 135.7 6.76 ( aw)
4 138.7 -

5 45.5 -

6 34.4 1.94 ( aw), 1.25 ( aw)

7 27.7 and 27.6 1.65 ( aw), 1.50 ( aw)
8 36.6 1.67 (m)
9 38.8 -

10 48.1 1.73 (m )
11 35.02 and 34.96 1.60 ( aw)
12 18.91 and 18.87 2.29 ( aw), 2.02 (w/)
13 138.5 and 138.4 -

14 141.6 and 141.5 6.79 ( aw)

15 102.5 5.74 ( aw)
16 171.2 -

17 15.5 0.86 (J, 6.6) and 0.85 (<:/, 6.6)

18 169.3 -

19 71.7 pro-R 4.30 (t/, 8.0), pro-S 3.92 (<7J, 8.0, 2.0)
20 17.5 0.62 (.y)
OMe 57.2 and 57.1 3.58 (.v)

3.2.9. PROPOSED BIOGENESIS OF COMPOUNDS 18, 19 AND 20

The precursor for the biosynthesis of diterpenes is geranylgeranyl pyrophosphate (GGPP)

which is formed by electrophilic addition reaction o f a molecule of DMAPP with three

molecules of isopentyl pyrophosphate (IPP). The biosynthesis starts by the reaction of the

double bond on GGPP with an acid which generates a carbocation. further elctrophilic

addition reactions lead to the formation of labdanyl cation. A sequence of concerted 1,2

hydride and 1.2 methyl Wagner Meerwein (WM) shifts/reaarangemcnt leads to the formation

of neoclerodiene pyrophosphate, which is a precursor for clerodane diterpenes.

147
 Neoclerodiene PP

Oxidation o f the methyl groups at C-4 and C-5 to a earboxylic acid and an alcohol

respectively, in the intermediate. The hydroxyl group in the carboxylic acid is a bad leaving

group and therefore reacts with adenosine triphosphate (ATP) to give COOP which after

reaction with coenzyme A yields the most reactive intermediate which can easily lactonize.

Further, oxidation of the methyl at C-16 to an alcohol and its subsequent reaction yields

compound 18 which is one of the new compounds isolated in this study.

148
Oxidation of 18 at C-15 and reaction with S-adenosinemcthiopnine (SAM) leads to the
epimeric mixtures 19 and 20.

149
3.3 COMPOUNDS FROM S E N E C I O R O S E IF L O R U S
The surface exudates o f the leaves was exhaustively extracted as described previousy. The

extract was tested for anti-plasmodial activity against chloroquine-senstive (D6) strains of

Plasmodium fa lcip a ru m with IC50 of 90.0 ± 9.8 f-ig/ml. which indicates that it is barely active.

The crude extract was subjected to combinations of chromatography resulting to the isolation

of eleven compounds. The structures of these compounds were confirmed by comparison of

their spectroscopic data with literature and in some cases by direct comparison (co-TLC) with

authentic samples. 5-Hydroxy-3,7,4'-trimethoxyflavone (1), 3,5-dihydroxy-7.4',-

dimethoxyflavone (2), kumatakenin (3), rhamnocitrin (5) were also isolated from the two

populations (Ngong Forest and Voi) of D. angustifolia. Isokaempferide (6 ) and 5-hydroxy-

3,6,7,4'-tetramethoxyflavone (14) were also isolated from D. angustifolia (Ngong forest) and

D. a n g u stifo lia (Voi) respectively. The compounds were characterized using combinations of

spectroscopic techniques. In this section the structure elucidation of compounds 21-27 is

discussed.

3.3.1. 5, 7-DIHYDROXY-3,4' -D1METHOXYFLAVONE (21)

Compound 21 was isolated as yellow crystals with melting point o f 233-235 °C. It appears as

yellow spot Rt 0.3 (20% CH2C12 in n-C6H6) which intensified on exposure to ammonia

vapour indicating it is phenolic.

The l3C-NMR (see Table 3.19) [Agrawal 1989] is consistent with a flavonol derivative. The

EI-MS showed a molecular ion peak at m/z 314 corresponding to C |6H| (()f,.

The 'H-NMR (Table 3.19) indicated the presence of two me la coupled aromatic protons at 8

6.43 and 6.23 (d, ./= 2.1 Hz) which were assigned to ring A and an AA'BB' spin system at 8

8.09 and 7.04 (dd, J = 2.1, 9.0 Hz) assigned to C-4' substituted ring B. With oxygenations at

C-5 and C-7 (biogenetically expected) the meta coupled protons are assigned to H-6 and H-8.

150
The H-NMR also displayed peaks for two methoxyl groups at 5 3.91 and 3.81. The NOE

interactions between H-6 and H-8 and the methoxyl at 5 3.91 places one of the methoxyl

group at C-7, while the 1’C-NMR of the other methoxyl group (5 59.7) indicates that this

group is d\-ortho substituted which is in agreement with the placement o f this group at C-3.

The placement of the methoxyl group at C-3 and C-7 was confirmed from the HMBC

correlation of the methoxyl group at 5 3.81 and 3.91 with C-3 (8 138.5) and C-7 (8 156.2),

respectively.

Thus based on this and comparison of the data with literature information compound 21 was

identified as 5,7-dihydroxy-3,4'-dimethoxyflavone (21). This compound has been previously

isolated from Dodonaea viscosa [Wollenweber et a l., 1986]. However, this is the first report

of the isolation of the compound from Senecio roseiflorus.

Table 3.19: ID (CD3OD: 300, 75.5 MHz) and 2DNM R data for 5,7-dihydroxy-3, 4'-
dimethoxyflavone ( 21)
c £ ih <5|3C HMBC HMBC
(m, (Hz)) 2./ V
2 157.6
3 138.5
4 177.0
5 162.2
6 6.23 (d, 2.1) 99.0
7 165.2
8 6.43 (d, 2.1) 93.9
9 157.5
10 102.0
r 123.0
2' 8.09 {d, 9.0) 130.4 C-6', C-4',C-2
3' 7.10 (d, 9.0) 114.3 C-4' C-5',C-1'
4' 162.3
5' 7.03 (dd, 2.1,9.0) 114.3 C-4' c-3', c - r ,
6' 8.08 (dd, 2.1, 9.0) 130.4 C -2X -2, C4'
OMe 3.81 (.?) 59.7 C-3
OMe 3.91 (.*) 55.1 C-7

151
 OMe

Figure 3.16: 5,7-dihydroxy-3,4'-dimethoxyflavone (21)

3.3.2. QUERCETIN-3, 4’-DIMETHYL ETHER (22)

Compound 22 was isolated as yellow crystals with melting point o f 235-236 °C. It appears as

yellow spot Rf0.5 (2% MeOH in CH2CI2) which intensified on exposure to ammonia vapour

indicating it is phenolic. The EIMS showed a molecular ion peak at m/z 330 corresponding to

C17H14O7. The i3C-NMR (see Table 3.20) indicated that compound 22 is a flavone (Agrawal,

1989).

The 'H-NMR (Table 3.20) indicated the presence of two meta coupled aromatic protons at 6

6.39 and 6.20 (<7, ./= 2.0 Hz) which were assigned to ring A and an AXY spin system at 8

7.06 (d, J = 8.5 Hz), 7.60 (d\ J = 2.0) and 7.80 (dd, J = 2.0 and 8.5 11/) assigned to a di-

sustituted ring B.

With oxygenations at C-5 and C-7 (biogenetically expected) the mala coupled protons are

assigned to H-6 and H-8. The 'H-NMR also displayed peaks for two melhoxyl groups at 8

3.94 and 3.80. One methoxyl group was placed at C-3 due to the fact that it resonates at 8

60.7 which is typical for a methoxyl group at this position. The other methoxy group (8 h

3.94, 8c 56.6) was placed in ring B due to its NOE interaction with ring B protons and not

ring A protons. The l3C-NMR singlet for the oxygenated aromatic carbons are shielded (at 8

151.9 and 147.9) indicating that these carbons are ortho to each other [Markham, 1982], for if

this was not the case they would have resonated at ca 160 ppm. This implies that the methoxy

group could be placed at either C-3' or C-4'. However, the NOE interactions between this

methoxyl group and the ortho-substituted aromatic proton at 8 7.06 confirms this methoxyl

group at C-4'. The HMBC data also confirms the same (see Table 3.20).

152
Thus based on this and comparison o f the data with literature information the compound is

identified as 3', 5, 7-trihydroxy-3, 4'-dimethoxyflavone (22) (trivial name quercetin-3, 4'-

dimethyl ether). Quercetin-3, 4'-dimethyl ether (22) has been previously isolated from the

aerial parts o f several plant species including Chrysothamnus viscidiflorus [Sepulveda et al.,

1994]; Psiadia dentate [Jakobsen et al., 2001].

Figure 3.17: NOE interactions of compound quercetin-3, 4'-dimcthyl ether (22)

Table 3.20: ID (CDC13: 300, 75.5 MHz) and 2D NMR data for quercetin-3, 4'-dimethyl ether
(22 )
c d\ h ^I3C HMBC HMBC
(w, (Hz)) 2J V
2 157.7
3 140.0
4 180.2
5 158.6
6 6.39 {d, 2.0) 95.0 C-5, C-7 C-8, C-10
7 166.2
8 6.20 (d, 2.0) 100.0 C-9, C-7 C-6, C-10
9 163.3
10 106.0
r 123.0
2' 7.63 {dd, 2.0 8.5) 122.3 C-6, C-4',C-2
3' 7.06 (d. 8.5) 112.5 C-4' c - r , c -5 '
4' 151.9
5' 147.9
6' 7.60 (d, 2.0) 116.3 C-5' C-2', C-4', C-2
OMe 3.80 (5) 60.7 C-3
OMe 3.94 (5) 56.6 C-4'
OH

153
3.3.3. RHAMNAZIN (23)
Compound 23 was isolated as yellow crystals with melting point of216-218°C. It appears as

yellow spot Rt 0.5 (2% MeOH in CH2CI2) which intensified on exposure to ammonia vapour

indicating it is phenolic. The EIMS showed a molecular ion peak at m/z 330 corresponding to

C17H14O7. The 'H (6 12.13 (chelated hydroxyl group)) and ’’C-NMR (see Table 3.21)

[Agrawal 1989] is consistent with a 5-hydroxyflavonol derivative.

The H-NMR (Table 3.21) indicated the presence o f two meta coupled aromatic protons at 5

6.72 and 6.33 (d, ./ = 2.0 Hz) which were assigned to ring A and an AX Y spin system at 6

7.02 ( d ,J = 8.7 Hz), 5 7.85 {dd, J = 2.1 and 8.7 Hz) and 5 7.92 {d, J= 2.0 Hz) assigned to C-

3' and C-4' substituted ring B. With oxygenations at C-5 and C-7 (biogenetically expected)

the meta coupled protons are assigned to H-6 and H-8.

The 'H-NMR also displayed peaks for two methoxy! groups at 5 3.93 and 3.90. One

methoxyl groups at C-7 while the other could be at C-3' or C-4' due to the fact that they both

resonate at 5 55.7 which is typical for isolated methoxyl groups. The placement of the

methoxyl group at C-7 was confirmed by the HMBC correlation between the methoxyl group

at 6 3.90 and C-7 (5 165.9).

The NOE correlation between the methoxy group at 6 3.93 with the meta coupled proton at 5

7.92 places the other methoxy group at C-3'.

Thus based on this and correlation of the data with literature compound 23 is identified as

3,5.4'-trihydroxy-7,3'-dimethoxyflavone (23) (trivial name rhamnazin). Rhamnazin (23) has

been isolated previously from the aerial parts Polygonum punctatum [Marin el al., 2001], and

Grindelia nana [Wollenweber et al., 1997a; 1997b].

154
 Figure 3.18: HMBC correlations o f rhamnazin (23)

Figure 3.19: NOE correlations o f rhamnazin (23)

Table 3.21: ID (CDCI3: 300, 75.5 MHz) and 2DNM R data for 3,5,4'-trihydroxy-7,3'-
dimethoxyflavone (23)
c <5ih 4l3C HMBC HMBC
(m, (Hz)) 2J V
2 148.0
3 136.0
4 178.0
5 162.0
6 6.33 (4, 2.0) 97.6 C-5, C-7 C-8, C-10
7 165.9
8 6.72 (4, 2.0) 92.1 C-9, C-7 C-6, C-10
9 158.0
10 106.0
r 123.0
2' 7.92 (</,2.1) 122.1 C-4', C-6', C-2
3' 7.02 (4 8.7) 115.3 C-2', C-4'
4' 151.0
5' 148.0
6' 7.85 (44 2.1, 111.3 C-2', C-4', C-2
8.4)
OMe 3.90 (.v) 55.7 C-7
OMe 3.93 (5) 55.7 C-5'
OH 12.13 (.v)

155
3.3.4. RETUSIN (24)
Compound 24 was isolated as yellow crystals with melting point of 235 236°C. It appears as

yellow spot on Rf value of 0.5 (2% MeOH/CHbCh) which intensified on exposure to

ammonia vapour indicating it is phenolic. The 'H (5 12.74 (chelated hydroxyl) and l3C (5

157.4.0 for C-2, 8 140.3.0 for C-3 and 8 180.3 for C-4) NMR |Agrawal 1989] is consistent

with a 5-hydroxyflavonol derivative.

The 'H-NMR (Table 3.22) indicated the presence o f two meta coupled aromatic protons at 8

6.70 and 6.34 (d, ./= 2.0 Hz) which were assigned to ring A and an AXY spin system at 8

7.16 {d, J= 8.5 Hz), 8 7.77 (d, J = 2.0 Hz) and 8 7.80 (dd, ./ = 2.0 and 8.51 Iz) assigned to a di-

substituted ring B. With oxygenations at C-5 and C-7 (biogenetically expected) the meta

coupled protons are assigned to H-6 and H-8.

The 'H-NM R also displayed peaks for four methoxyl groups at 8 3.931, 3.928, 3.918 and

3.909. The l3C-NMR (Table 3.22) further showed signals for methoxyl groups at 8 61.0,

57.0, 57.1 and 56.8 which shows HMQC correlations with the corresponding protons 8 3.928,

3.918, 3.909 and 3.931, respectively. This gives two possible structures for this compound as

24a or 24b, based on the 13C-NMR chemical shift values of these oxygenated aromatic

carbons. Oxygenated aromatic carbons with oxygenation at ortho or para position resonate at

ca 150 ppm (as in 24b), while those with no oxygenation at either position resonate at ca 160

ppm (as in 24b) [Markham, 1982].

156
Thus based on this and correlation of the data with literature compound 24 is identified as 5-

hydroxy-3,7,3',4'-tetramethoxyflavone (24) (retusin). Retusin (24) has been isolated from

Artemisia rupestris [Valant-Vetschera et al., 2003] and Mirabilis viscosa [Wollenweber &

Dorr, 1996]

OMe
/O M e
4’
5’

OH O

Retusin (24)

157
Table 3.22: ID (CDC13: 300, 75.5 MHz) and 2D NMR data for 5-hydroxy 3,7,3',4'-
tetramethoxyflavone (24)
C <S|H HMBC HMBC

n
....... ('".(H z)) 2J
2 157.4
3 140.3
4 180.3
5 163.5
6 634 (d , 2.0) 99.2
7 167.3
8 6.70 {d, 2.0) 93.6
9 158.4
10
r 124.3
2' 7.77 {d, 2.0) 123.7 C-4'
3' 7.16 {d, 8.5) 112.9 c - r , c-5'
4' 153.6
5' 150.7
6' 7.80 {dd, 2.0, 8.5) 113.2 C-4', C-2
OMe 3.931 (.v) 56.8 C-7
OMe 3.928 ( 5 ) 61.0 C-3
OMe 3.918(.y) 57.0 C-4'
OMe 3.909 (.y) 57.1 C-3'
OH 12.74 (5)

3 .3 .5 . 5 , 4 '- l) I H Y D R O X Y - 7 - M E T H O X Y F L A V A N O N E (2 5 ).

Compound 2 5 was isolated as white UV active crystals with a melting point of 152-154 °C. It

appears as yellow spot on R, value of 0.4 (1% MeOH/CH2CI2) which intensified on exposure

to ammonia vapour indicating it is phenolic. The UV (A.max (MeOH) 283.0 nm [Mabry et al.,

19701, *H (8 12.02 (for chelated hydroxyl proton), 8 5.36 {dd, J 3.0 and 13.0 Hz for H-2), a

methine proton attached to an oxygen, 8 2.79 {dd,./ 3.0 and 17 I Iz for i 1-3) and 8 3.09 {dd,

./ = 13.0 and 17. 0 Hz for 11-3) and l3C (8 79.2 for C-2, 8 43.4 for C-3 and 8 196.3 for C-4)

NMR [Agrawal, 1989] is consistent with a 5-hydroxyllavanonc derivative. The EIMS

showed a molecular ion peak at m/z 286 (Scheme 3.5) corresponding to ( kTI mO s.

The 'H-NMR (Table 3.23) indicated the presence o f two meta coupled aromatic protons at 8

6.07 and 8 6.06 {d, J - 2.0 Hz) which were assigned to a di- substituted ring A and

AA'BB'spin system centered at 8 6.89 and 7.34 {d, ./ -- 8.5 Hz) which were assigned to 4'-

158
substituted ring B protons. In the MS the presence of a fragment ion at m . 167 (2 5 a ) and m/z

120 (2 5 b ) (Scheme 3.5), resulting from a retro-Diels Alder cleavage of ring C, would place

one hydroxyl group at C-5' and one methoxyl at C-7 in ring A, and hence rings B and C

should contain the other hydroxyl group.

With oxygenations at C-5 and C-7 (biogenetically expected) the meta coupled protons are

assigned to H-6 and H-8. HMBC correlation between the chelated hydroxyl proton (8 12.02)

with C-6 (5 95.0) and IIMQC correlation between the proton at 5 6.06 and C-6 (5 95.0) led to

the assignment of the doublet at 8 6.06 to H-6. Similarly, HMBC correlation between the

doublet at 8 6.07 and C-8 (8 94.0) allowed the placement of the doublet at 8 6.07 to H-8.

The 'H-NMR displayed one singlet for a methoxyl group at 8 3.81 (311, .v). The location of

the methoxyl group at C-7 was further confirmed using HMBC (fable 3.23) which showed

correlations between the protons at 8 3.81 with C-7 (8 164.4) and due to the fact that it

resonates at 8 55.9, which is typical for an isolated substituted methoxyl group.

Thus based on this and correlation of the data with literature compound 25 is identified as 5,

4'-dihydroxy-7-dimethoxyflavanone (2 5 ). 5,4'-Dihydroxy-7-methoxyflavanone (2 5 ) has been

previously isolated from the aerial parts of Dodonaea viscosu [Mata el <//., 1991]. However, it

has not been isolated from Senecio roseiflorus.

159
 +

25b

m /z 1 2 0

Scheme 3.5: Fragmentation pattern of 5,4'-dihydroxy-7-m ethoxyi1avanone (25).

Table 3.23: ID (CDCI3: 300, 75.5 MHz) and 2D NMR data for 5,4'-dihydroxy-7-
methoxyflavanone (25)
c <5ih S\2C HMBC HMBC
(/;/, (Hz)) V V
2 5.36 { d d , 3.0,13.0) 79.2 c -r C-3’, C-4
3 3.09 { d d , 13.0, 17.0), 43.4 C-2, C-4 c -r
2.79 { d d , 3.0, 17.0) c-10
4 196.3
5 163.0
6 6.06 (</, 2.0) 95.0
7 164.4
8 6.07 {d, 2.0) 94.0 C-9, C-7 C-6, C-10
9 168.30
10 103.4
r 128.2
2' 7.34 (</, 8.5) 130.8 C-3' C-6', C-4'.C-2
3' 6.89 { d , 8.5) 115.9 C-4',C-2' C-5'
4' 156.2
5' 6.89 (</, 8.5) 115.9 C-4', 6' C-5'
6' 7.34 ( d , 8.5) 130.8 C-5' C-2',C-2, C-4'
OMe 3.81 (j ) 55.9 C-7
OH 12.02 (s) C-5 C-6, C-10
OH 5.12 (s) C-4' C-3'

160
3.3.6. METHYLPARABEN (26).
Compound 2 6 was isolated as white needles with melting point o f 127-129 °C. It appears as

yellow spot on Rf value o f 0.5 (1% M eO H /C ^C B ) which intensified on exposure to

ammonia vapour indicating it is phenolic. The 'H-NMR ( fable 3.24) exhibited an AA'BB'

spin system centred at 8 7.95 (dd, J - 9.0, 2.1 Hz) and 6.88 (2H, dd, ./ 9.0, 2.1 Hz) assigned

to aromatic protons o f a 1,4-disubstituted benzene ring. The COSY spectrum showed

correlations between two sets o f protons at 5 7.95 and 6 6.88. The 1H-NMR further showed a

broad singlet 6 6.20 attributed to the hydroxyl protons and a singlet at 5 3.90 (5, 3H) assigned

to a methoxy group.

The l3C-NMR spectrum (APT) corroborated the presence of one methoxy group, four

methines and three quaternary carbon atoms. The n C-NMR (Table 3.24) exhibited a signal at

5 167.3 for an ester group and at 8 52.0 for a carbomethoxy group.! his data is consistent with

a p-hydroxy benzoic acid skeleton for compound 26. C-2/C-6 at 8 131.9 and C-3/C-5 at 8

115.3. The HMQC experiment, showed cross peaks between the signal at 8 7.95 in the 'H-

NMR and 8 131.9 (C-2/C-6), the signal at 8 6.88 and 8 115.3 (C-3/C-5) and the signal at 8

3.90 with 8 52.0. The structure of this compound was confirmed by the 1IMBC correlations

between 8 7.95 (H-2/H-6) and the C-3 (8 115.3), C-2/C-6 (8 131.9), C-4 (8 160.3) and C-7 (8

167.3). In addition HMBC cross peaks were observed between the protons at 8 6.88 (H-3/H-

5) and C-3 (8 115.3), C-l (8 122.4) and C-4 (8 160.3). There was HMBC correlation between

the methoxy protons and the carbonyl carbon at 8 167.3. Based on these spectral data and

comparison with literature information [Yoshioka el a/., 2004 1, compound 26 was identified

as methyl-4-hydroxybenzoate (2 6 ) (trivial name methylparaben). This compound has been

isolated from the fruit and leaf of Vitex rotundifolia [Yoshioka el a!., 20041.

161
 o

Methylparaben (26)

Table 3.24: ID (CDCI3: 300, 75.5 MHz) and 2D NMR data for 4-hydroxybenzoic acid methyl
ester (26)
C #1H 3J

0
(m, (Hz))
1 122.4
2 7.95 (dd, 9.0, 2.1) 131.9 C -l, C-3 C-7, C-4
3 6.88 (dd, 9.0, 2.1) 115.3 C-2, C-4 C -l, C-5
4 160.2
5 6.88 (dd, 9.0, 2.1) 115.3 C-4, C-6 C-3, C-l
6 7.95 (dd, 9.0, 2.1) 131.9 C-5,C-l C-4, C-7
7 167.3
OH 6.20 (s)
OMe 3.90 (s) 52.0 C-7

3.3.7. 5 ,4 -DIHYDROXY-3,7,3-TRIMETHOXYFLAVONE (27)

Compound 27 was isolated as yellow crystals with melting point of 235-236 °C. It appears as

yellow spot Rt 0.3 (2% MeOH/CHzCb) which intensified on exposure to ammonia vapour

indicating it is phenolic. The EIMS showed a molecular ion peak at m/z 344 corresponding to

C i 8H i60 7. The EIMS an intense peak at 301 [M+-43] (Scheme 3.6) corresponding to standard

tlavonol C-ring contraction [Harborne, 1994] for 3-methyl ether flavone.

The 1H (5 12.64 (for chelated hydroxyl group) and l3C (8 157.0 for C-2. 139.0 for C-3 and

179.0 for C-4) NMR [Agrawal 1989] is consistent with a tlavonol derivative.

The 1H-NMR (Table 3.2) indicated the presence of two meta coupled aromatic protons at 8

6.45 and 6.36 (d, ./= 2.0 Hz) which were assigned to ring A and and an AXY spin system at

8 7.05 (d, J = 8.7 Hz), 8 7.68 (dd, J= 2.0 and 8.0 Hz) and 8 7.71 (d, ./ 2.0 Hz) assigned to

C-3' and C-4' substituted ring B. With oxygenations at C-5 and C-7 (biogenetically

expected), the meta coupled protons are assigned to 11-6 and H-8. 1IMB( correlation between

162
the chelated hydroxyl proton (8 12.64) with C-6 (8 98.10) and the HMQC correlation

between the proton at 8 6.36 and C-6 (8 98.1) led to the assignment of the peak at 8 6.32 to

H-6. Similarly, HMBC correlation between the signal at 8 6.36 (cf) and ( 8 (8 92.4) allowed

the placement of the doublet at 8 6.45 (d) to H-8.

The 'H-NMR (Table 3.25) also displayed peaks for three methoxyl groups at 8 3.99, 3.88 and

3.86. In the MS spectra the presence of a fragment ion at m/z 167 (27a) (Scheme 3.6),

resulting from a retro-Diels Alder cleavage of ring C, would place one hydroxyl group at C-5

and one methoxyl group at C-7 in ring A, and hence rings B and C should contain the other

hydroxyl and two methoxyl groups.

OMe __
OH

MeO 27a
m/z 167
+
+
27 OH

OMe

OMe

MeO

M + -43
m /z 301

Scheme 3.6: Fragmentation pattern of 5,4'-dihydroxy-3,7,3'-trimethoxyflavone (27)

163
One of the remaining methoxyl was placed at C-3 due to the fact that it resonates at 5 60.4

which is typical for a di- ortho substituted methoxyl and the other at either C-3'or C-4'.

NOESY correlations between the methoxyl at 5 3.99 with the met a proton at 8 7.71

confirmed its location at C-3' position. In addition HMBC cross peaks were observed

between the proton at 8 7.71 and C-1'(122.9*/122.7*), C-2'(122.9*/122.7*), C-3’(146.6) and

C-4'( 148.6), the proton at 8 7.68 and C-6'(112.1), C-4'(I48.6), C-2 (157.0). There was

HMBC cross peaks observed between the ortho coupled proton at 8 7.05 and C-

1'(122.9*/122.7*), C-2'(122.9*/122.7*), C-3'(146.6) and C-4'( 148.6) (Figure 3.20).

Based on these spectral data and comparison of the data with literature information

[Likhitwitayawuid et al., 2006], compound 27 was identified as 5,4'-dihydroxy-3,7,3'-

trimethoxyflavone.

Table 3.25: ID (CDC13: 300, 75.5 MHz) and 2D NMR data for 5,4'-dihydroxy-3,7,3-
trimethoxyflavone (27)
c £ ih ^13C HMBC HMBC
(nK (Hz)) 2J \J
2 157.0
3 139.0
4 179.0
5 162.7
6 6.36 (d, 2.0) 98.1 C-5, C-7 C-8, C-10
7 165.6
8 6.45 (d, 2.0) 92.4 C-9, C-7 C-6, C-10
9 157.0
10 106.3
r 122.7*/122.9*
2' 7.71 (t/, 2.0) 122.7*/122.9* C-4', C-6', C-2
3' 7.05 (d, 8.7) 114.8 C-2', C-4' c - r , c -5 '
4' 148.6
5' 146.6
6' 7.68 (dd, 2.0. 8.0) 112.1 c - r , c-5 ' C-2', C-4'. C-2
OMe 3.86 (.v) 60.4 C-3
OMe 3.88 (5) 56.0 C-7
OMe 3.99 (s) 56.3 C-5'
OH 5.98 (.y) C-4' C-3', C-5'
OH 12.6 (j ) C-5 C-6, C-10

* The 8 values in the same column can be interchangeable.

164
Figure 3.20: HMBC correlations of 5,4'-dihydroxy-3,7.3'-trimethoxyflavone (27)

3.4 CHEMOTAXONOMIC SIGNIFICANCE OF THE ISOLATED COMPOUNDS

3.4.1 D O D O N A E A A N G U S T IF O L IA (FROM NGONG FOREST AND VOI)

From this work a total of 11 compounds were isolated (7 flavones, 1 flavanone, 2 clerodane

type diterpenes and 1 labdane type diterpene) from the surface exudates of D. angustifolia

from Ngong forest. Table 3.26 below summarizes the flavonoids and terpenoids from this

population of D. angustifolia. All of these compounds except 5,7-dihydroxy-3,6,4'-

trimethoxyflavone (4) have not been previously reported from this plain but they have been

reported from other species o f the genus Dodonaea.

Table 3.26: Distribution of flavonoids, diterpenoids and shikimatcs of D.

angustifoila (Ngong Forest and Voi) and D. viscosa
TYPE D. D. viscosa
COMPOUND angustifolia
Ngong forest Voi
population population
3-Methoxyflavones
5-Hydroxy-3.7,4'-trimethoxyflavone (1) + + +
5.4'-Dihydroxy-3,7-dimethoxyflavone + +
0 )
5,7-Dihydroxy-3,6,4'-trimethoxyflavone + - +
(4)
5.7,4',-T rihydroxy-3-methoxyflavone + +
(6 )
5.4'-Dihydroxy-3,6,7-trimehtoxyflavone - + +
(12 )
5,3'-Dihydroxy-3, 4', 7- - + “
trimethoxyflavone (13)
165
Table 3.26: Distribution of flavonoids, diterpenoids and shikimates ofD.
a n g u stifoila (Ngong Forest and Voi) and D. viscosa
TYPE D. D. viscosa
COMPOUND angustifoila
Ngong forest Voi
population population
5-Hydroxy-3,6,7,4'- - + +
tetramethoxyflavone ( 1 4 )
5,7,4'-T rihydroxy-3,6- - - +
dimethoxyflavone ( 1 1 3 )
5,7-Dihydroxy-3,6,4'-trimethoxy-2'- - - +
prenylflavone ( 1 1 4 )
5, 7-Dihydroxy-3,6-dimethoxy-2'-(3- - - +
hydroxymethylbutyl) flavone ( 1 1 5 )
5,7-Dihydroxy-3,6,4'-trimethoxy-2'-(3- - - +
hydroxymethylbutyl) flavone ( 1 1 6 ) .
5,4'-Dihydroxy-3,6,7-trimethoxy-2'-(3- - - +
hydroxymethylbutyl) flavone ( 1 1 7 ) .
5-Hydroxy-3.6,7,4'-tetramethoxy-2'-(3- - - +
hydroxymethylbutyl) flavone (118).
5,6.4'-Trihydroxy-7-methoxyflavone - “ +
(119).
Flavonols
3,5-Dihydroxy-7,4',-dimethoxyflavone + + -
(2 )
3,5,4'-Trihydroxy-7-methoxyflavone + + +
(rhamnocitrin) (5)
3,5,7,4'-Tetrahydroxy-6- + - -
methoxyflavone (7)
3,5,7,4'-Tetrahydroxyflavone ( 1 5 ) - + +
5,7,2',4'-Tetrahydroxyflavonol (78) - - +
5,7,4'-Trihydroxy-2'-methoxyflavonol - - f
(1 2 0 )
Flavanone
5,7-Dihydroxyflavanone (8) + - +
5,4'-Dihydroxy-7-methoxyflavanone - - +
(79)
Coum arin
7-Hydroxy-6-methoxycoumarin (16) - + +
Clerodane type diterpenoids
2(3-hydroxzyhardwickic acid (9) + -
Dodonic acid (10) + - +
Hautriwaic acid ( 1 7 ) - + +
w oclerodan-3.13-dien-16,15 : 18,19- - + -
diolide (18)
15(3- Methoxy-/7eoclerodan-3,13-dien- - + -
16,15: 18,19-diolide (19)
15a- Methoxy-weoclerodan-3,13-dien- - + -
16,15: 18,19-diolide (20)
Methyl dodonate A (92) - - +
Methyl dodonate B (93) - - +
166
Table 3.26: Distribution of flavonoids, diterpenoids and shikimates of D.
a ngustifoila (Ngong Forest and Voi) and D. viscosa
TYPE D. D. viscosa
COMPOUND angustifolia
Ngong forest Voi
population population
Methyl dodonate C (94) - - +
Dodonolide (95) - - +
Labdane type diterpenoid
15,16-epoxy-13( 16), 14-labdadiene-3, + +
8-diol; ent-3fi, 8a form (11)

A total o f 13 compounds were isolated (8 flavones, 1 coumarin and 4 clerodane type

diterpenes) from the surface exudates of D. angustifolia population from Voi. Table 3.26

below summarizes the flavonoids and terpenoids from this population o 17). angustifolia. All

of these compounds have not been reported previously from this plant but they have been

reported from other species o f the genus Dodonaea. The new compounds from D.

angustifolia population from Voi are weaclerodan-3J3-dien-l6J5: 18 ,19-diolide (18), 15(3-

methoxy-A7eoclerodan-3,13-dien-16,15: 18,19-diolide (19) and 15a-mcthoxy-weoclerodan-

3,13-dien-16,15: 18,19-diolide (20).

The two populations of D. angustifolia are closely related in that they both elaborate the same

class o f compounds; flavonoids and terpenoids. These are the main compounds found on

plant exudates. The flavonoids are usually methylated in order to increase their solubility in

the terpenoid milieu. However, only four flavonoids (two 3-methoxyllavones and two

tlavonols) out of sixteen flavonoids isolated from the two populations are shared between

them. The terpenoids isolated from the two populations are mainly the clerodane type and

they are not shared between the two populations.

The two populations of Dodonaea in Kenya (Ngong I Tills and Voi) do not have the same set

of surface flavones / flavonols and diterpenoids in both quality and quantity proportions of

the exudates (Table 3.26). All flavonoids from the two populations of D. angustifolia except

5,3'-dihydroxy-3.4',7-trimethoxyflavone (ayanin) (13) are kaempferol methyl ethers. The

167
composition of the surface exudates of D. angustifolia was compared with that of D. viscosa

because o f the controversy that exists between them. Dodonaea angustifolia is variously

considered synonymous with, sub-species of or distinct genus from D. viscosa depending on

the particular authority. In Kenya D. angustifolia was declared synonymous with D. viscosa

from Australasia.

The oxygenation pattern of some of the tlavonoids isolated from both l). angustifolia and D.

viscosa is similar as shown in the flavones 1, 3-6, 12, 14, 15. 113, and 119 and the flavanones

8 and 79. However, prenylation at C-2' is evident in compounds 78. 114-118 and 120 from

Dodonaea viscosa which is lacking in compounds from D. angustifolia Prenylation at C-2'

can serve as a chemotaxonomic marker for Dodonaea viscosa. Clerodane and only one

labdane type diterpenes were isolated from the two species of Dodonaea. The diterpenoid

profile o f D. angustifolia consists of 2p-hydroxyhardwickic acid (9), dodonic acid (10),

15,16-epoxy-13(16), 14-labdadiene-3, 8-diol; ent-3p, 8a form (11) from the Ngong Forest

population and hautriwaic acid (17), weoclerodan-3,13-dicn-16.15: 18,19-diolide (18), 15p~

methoxy-w?oclerodan-3,13-dien-16,15: 18,19-diolide (19), 15a-mcthoxy-«coclerodan-3,13-

dien-16,15 :18,19-diolide (20) from Voi population. Compounds 9, 10 and 11 could serve as

chemotaxonomic markers for D. angustifolia from Ngong Forest while hautriwaic acid (17)

and the three new clerodane diterpenes from D. angustifolia could also serve as

chemotaxonomic markers for D. angustifolia species from Voi. The two D. angustifolia

populations arc also qualitatively different, in their terpenoid profile, from D. viscosa (Table

3.26) which seems to produce methyl dodonate diterpenoids |Ortega., 2001] which could be

genetically advanced structures from dodonic and hautriwaic acids observed in D.

angustifolia from Ngong Forest and Voi, respectively. These compounds could also be used

as chemotaxonomic markers for D. viscosa.

From these phytochemical work a sound conclusion can be made that different populations of

D. angustifolia and D. viscosa have different flavonoid and diterpenoid profiles in their

168
exudates. The two populations of D. angustifolia and D. viscosa present three different

chemotypes, and probably taxonomically distinct varieties or species. However, the

HPLC/HPTLC profiles of these populations and other populations in Kenya need to be

determined before a definite conclusion can be made.

3.4.2 SEN E C IO ROSEIFLORUS.

This study led to isolation o f fourteen compounds, all of which were phenolic (thirteen

tlavonoids and one carbomethoxy phenol), from the surface exudates ol'.S’. roseiflorus. Table

3.27 summarizes the compounds isolated from the exudates of S. roseiflorus and those shared

with the two populations of D. angustifolia.

Table 3.27: Distribution of tlavonoids. diterpenoids in S. roseiflorus and the two populations
of D. angustifoila from Ngong Forest and Voi
CLASS S. roseiflorus D. angustifolia
COMPOUND
Ngong Voi
Forest
3-Methoxyflavones
5-Hydroxy-3,7,4'-triinethoxyflavone (1) + + +
5.4'-Dihydroxy-3,7-dimethoxyflavone (3) + + +
5,7,4',-T rihydroxy-3-methoxy flavone (6) + +
5,3'-Dihydroxy-3,4 ',7-trimethoxyflavone (13) + +
5,7-Dihydroxy-3, 4'-dimethoxyflavone (21) +
3',5,7-Trihydroxy-3,4'-dimethoxyflavone (22) +
5-Hydroxy-3,7,3',4'-tetramethoxyflavone (24) +
Flavonols
3.5-Dihydroxy-7,4'.-dimethoxyflavone (2) -l- + +
3,5-Dihydroxy-7.4',-dimethoxyflavone (5) + + +
3.4',5-Trihydroxy-3',7-dimethoxyflavone (23) +
Flavanone +
5,4'-Dihydroxy-7-dimethoxyflavanone (25) +
Phenol -t-
3-Carbomethoxyphenol (26) +

Four tlavonoids from S. roseiflorus were similar to those isolated from the two populations o f

D. angustifolia. Terpenoids and methyl ethers o f the widespread flavonoids, apigenin,

kaempferol, quercetin occurs scattered within the Angiospertns as well as in farinose exudate

of gymnogrammoid ferns (Wollenweber et al, 1982). We sometimes find in one plant the

169
whole series of possible methyl derivative (normally with the exception of 5-methyl ethers)

of a distinct basic skeleton. The flavonoid and terpenoid profiles of plant resins overlap in

some plant species, belonging to totally different genera as is the case with D. angustifolia

and S. roseiflorus. In such cases the flavonoid and terpenoid patterns between different

genera alone cannot be used for chemotaxonomic purposes. However, the patterns of these

compounds within some genera such as Notholaena (Wollenweber, 1975) are, used to typify

species, recognized varieties, and even chemical races.

This study and previous studies have revealed that flavonoids and terpenoids are

characteristic compounds on surface exudates, that being the reason why some methylated

flavonoids were common in S. roseiflorus and the two populations of D. angustifolia studied.

The flavonoids are usually methylated or have hydrophobic side chains to increase their

solubility in the terpenoid milieu. The localization of these compounds (flavonoids and

diterpenoids) on leaf surface is specially suited for ecological protection.

3.5 BIOLOGICAL ACTIVITIES.

3.5.1 ANTIPLASMODIAL TESTS
The extract o f fresh leaves o f Dodonaea angustifolia-Ngong forest. Dodonaea angustifolia-

Voi, and Senecio roseiflorus were tested for anti-plasmodial activities against Plasmodium

falciparum. The tests were done against two different strains of Plasmodium falciparum

parasites. These strains are the chloroquine-senstive Sierra Leone I (D6) and chloroquine-

resistant Indochina I (W2) that are commonly used in drug sensitivity assays. The most

commonly used anti-malarial drugs chloroquine, quinine and mefloquine are used as positive

control. Some of the compounds isolated were also tested for anti-plasmodial activities and

most of them showed potent and dose dependent activities. In this section the bioassay results

of this study are discussed.

170
3.5.1.1 ANT1-PLASMOD1AL ACTIVITY OF D. ANG U STIFO LIA-NGONG FOREST
The acetone extract o f the fresh leaves (surface exudates) of I). august ifolia-Ngong Forest

showed mild anti-plasmodial activity (IC50 41.5 ± 3.9 pg/ml) against chloroquine-sensitive

(D6) strain of the P. falciparum. The pure compounds from the surface exudates had

moderate activity (IC50 7.60-18.40 pg/ml) as listed in Table 3.28. All the eight flavonoids

were moderately active and their activity was considerably more than that o f the crude extract

and hence the need to test the rest of the compounds from this plant. The anti-plasmodial

activities of some of the compounds isolated from this plant are summarized in Table 3.28.

Table 3.28: In vitro activity (IC50) o f compounds from D. august ifo //a-N go ng Forest against
D6 strains of Plasmodium falciparum.
I( so in pg/ml
Tested compound D6
Flavonoids
5-Hydroxy- 3,7,4'-trimethoxyflavone (I) 13.8 ±4.2
3,5-Dihydroxy-7,4'-dimethoxyflavone (2) 13.0 ±2.4
5,4'-Dihydroxy-3,7-dimethoxyflavone (Kumatakenin) (3) 7.6 ±2.3
5,7 Dihydroxy-3,6.4'-trimethoxyflavone (Santin) (4) 8.6 ± 1.7
3,5,4'-Trihydroxy-7-methoxyflavone (Rhamnocitrin) (5) 17.4 ±3.9
5,7,4'-Dihydroxy-3-methoxyflavone (Isokaempferide) (6) 1 1.1 ±4.0
3,5,7,4'-tetrahydroxy-6-methoxyflavone (6- 18.4 ±4.8
methoxykaempferol) (7)
5.7-Dihydroxyflavanone (Pinocembrin) (8) 10.7 ± 1.3
Terpenoids
2(3-Hydroxyhardwickiic acid (9) 10.8 ±2.2
Dodonic acid (10) 9.7 ±2.8
Chloroquine 0.003 ±0.001
Quinine 0.063 ±0.003
Mefloquine 0.002 ±0.001

3.5.1.2 ANTI-PLASMODIAL ACTIVITIES OF D. A N G U S T IF O L IA VOI

The acetone extract of the fresh leaves of D. angustifolia-Voi showed very mild anti-

plasmodial activity (IC50 values of 56.3 ± 4.2 pg/ml) against chloroquine-sensitive (D6) strain

of Plasmodium falciparum. Hautriwaic acid (17) showed moderate antiplasmodial activity

(IC50 23.6 ± 2.6 fig/ml and 23.0 ± 2.3 jag/ml) against chloroquine-sensitive (D6) and resistant

(W2) strains of Plasmodium falciparum, respectively. The crude extract of the two

populations of D. augustifolia did not have good anti-plasmodial activity. However, their

171
activities were comparable. The anti-plasmodial activities of other compounds isolated from

this plant were not determined.

3 .5.1.3 ANTI-PLASMODIAL ACTIVITIES OF S E N E C IO R O S E IF L O R U S

The acetone extract o f the fresh leaves of Senecio roseiflorus showed no anti-plasmodial

activity (IC50 90.0 ± 9.8 pg/ml) against chloroquine-sensitive (1)6) strain of Plasmodium

falciparum.

The anti-plasmodial activity o f some of the compounds isolated from this plant are

summarized in Table 3.29. The flavanone 5, 4'-dihydroxy-7-dimethoxyllavanone (25) is the

most potent among the flavonoids tested. The other flavonoids showed moderate activity. The

activity o f all the compounds tested against the two strains of Plasmodium falciparum were

comparable, with no significant differences. It would be of interest to screen a wide range of

flavanones to ascertain their importance as lead structures for clinically useful products.

Table 3.29: In vitro activity (IC50) from compounds o f Senecio roseiflorus against D6 and
W2 strains of Plasmodium falciparum.
ICso in pg/ml
Tested compound D6 W2
Flavone
5.4'-Dihydroxy-3,6,7-trimethoxyflavone (12) 18.2 ±3.5 28.9 ± 1.0
5.7-Dihydroxy-3,4'-dimethoxyflavone (21) 8.9 ± 1.7 8.5 ± 1.4
3',5,7-Trihydroxy-3,4'-dimethoxyflavone (quercetin-3, 18.2 ±3.5 28.9 ±2.3
4'-Dimethyl Ether)(22)
3.4'5-Trihydroxy-3',7-Dimethoxyflavone (rhamnazin) 18.6 ± 7 12.1 ±3.3
(23)
5-Hydroxy-3,7,3',4'-tetramethoxyflavone (retusin) (24). 10.7 ±5.7 9.4 ±3.7
5.4'-Dihydroxy-3,7,3'-trimethoxyflavone (27) 10.9 ±2.1 -
Flavanone
5, 4'-Dihydroxy-7-Dimethoxyflavanone (25) 3.2 ± 0.8 4.4 ±0.01
Chloroquine 0.43 ± 0.002 0.51 ±0.004
Quinine 0.069 ±0.001 0.073 ±0.002
Mefloquine 0.004 ±0.001 0.002 ±001

3.5.4 ANTI MICROBIAL ACTIVITY

The tests were carried out on the acetone extracts of D. angustifolia from five different

geographical locations for comparison and Senecio roseiflorus. Some o f the pure compounds

from D. angustifolia (Ngong Forest and Voi) and S. roseiflorus were also tested for activity.
172
Evaluation of anti-microbial activity of extracts and pure compounds was accomplished using

the agar well-diffusion method [Bauer et al., 1966]. The extracts and pure compounds were

tested for activity against three strains of bacteria; Stciphyloccocus aureus (ATCC29737),

Escherichia coli (ATCC25922) and Bacillus pumilus (local strain) and a local strain of

fungus, Saccharamyces cerevisiae. The inhibition zones were measured in millimeter and the

results obtained are presented in Table 3.34. All the extracts showed activity against the

organisms tested. The anti-microbial activity of D. angustifolia extracts from the five

different geographical locations were similar. All compounds except 3.4',5-trihydroxy-3',7-

dimethoxyflavone (quercetin-3,4'-dimethyl ether) (22) were inactive against Escherichia coli

which is a virulent strain of bacteria. The fact that compound 22 was active and 3, 4',5-

trihydroxy-7-dimethoxyflavone (5), with a similar substitution pattern except for the absence

of a methoxyl substituent at the C-3', was inactive implies that the presence of a methoxyl

group at C-3' in compound 22 appears to be important for activity against Escherichia coli.

The methylated flavonoids 5, 8, 9, 10, 17, 22 and 25 were active against Staphylococcus

aureus. Previous investigations have shown that such lipophilic flavonoids display anti­

microbial activity. It was argued that this property was due to their ability to penetrate

biological membranes [Harborne, 1983].

Although it is not possible to establish a general structure-activity relationship, some trends

can be observed. For a good biological activity against Staphylococcus aureus a 3,5,4'

trihydroxy substitution as encountered in compounds 5 and 22 seems to be important for the

flavones. It is not clear whether it is the presence of just three hydroxyl groups or the

substitution pattern of three hydroxyl groups on the flavonoid skeleton that determines

activity. The activity of the two flavanones 5,7-dihydroxyflavanone (pinocembrin) (8) and

5,4'-dihydroxy-7-dimethoxyflavanone (25) is almost identical. For activity against

Staphylococcus aureus a minimum of three hydroxyl groups in flavones and may be two in

flavanones seems to be necessary. The flavonoids, 5-hydroxy-3,7,4'-trimethoxyflavone (1)

173
5.7-dihydroxy-3,6,4'-trimethoxyflavone (santin) (4) and 5.4'-dihydroxy-3,6,7-

trimethoxyflavone (penduletin) (12), with three methoxy groups were inactive even at 500

pg/ml against this strain of bacteria. While the phenolic groups may interact with biological

structures through hydrogen bonding [McClure, 19751 a certain degree of lipophilicity is

apparently required for the biological activity of the flavonoid.

A number of compounds 4, 5, 8, 9, 10, 12, 17, 22 and 25 were found to be active against

Bacillus pumilus. 5,7-Dihydroxy-3,6,4'-trimethoxyflavone (santin) (4). 3,5,4'-trihydroxy-7-

methoxyflavone (rhamnocitrin) (5) and 3,5,-4',-trihydroxy-3',7-dimethoxyflavone (quercetin-

3,4'-dimethyl ether) (22) were active against this local strain of bacteria, with inhibition zones

of 9.89 mm at 250 pg/ml, 11.73 mm at 125 pg/ml and 8.97 mm at 31.25 fig/ml, respectively.

As was observed with Staphylococcus aureus methoxy substitution at the C-3' is important

for activity against this bacteria. The two flavanones, 5,7-dihydroxyflavanone (pinocembrin)

(8) and 5,4'-dihydroxy-7-dimethoxyflavanone (25) were active against this bacteria, but their

structure activity relationships could not be established.

Compounds 4, 8, 9, 10, 12, 17, 22 and 25 and hautriwaic acid lactone were active against the

local strain of fungus, Saccharomyces cerevisiae. Santin (4) was the most active flavone with

an inhibition zone of 11.15 mm at 31.25 pg/ml while 5,4'-dihydroxy-3,6.7-trimethoxyflavone

(penduletin) (12) was less active with an inhibition zone of 1 1.50 mm at 500 pg/ml. The

presence of 5,7-dihydroxy substitution seems to be important for activity o f flavones against

Saccharomyces cerevisiae fungus. This is evident from the observation that 4 with a 5,7-

dihydroxy substituents was more active than 2 with 5,4'-dihydroxy substituents. The

flavones, 5-hydroxy-3,7,4'-trimethoxyflavone (1) and 5,4'-dihydroxy-3, /-dimethoxyflavone

(kumatakenin) (3) were inactive even at 500 pg/ml. The flavanones, 5,7 ciihydroxyflavanone

(pinocembrin) (8) and 5,4,-dihydroxy-7-dimethoxyflavanone (25), were both active against

Saccharomyces cerevisiae. 3,5,4'-Trihydroxy-7-methoxyflavonc (rhamnocitrin) (5)with three

hydroxyl groups had no activity while 22 had minimum activity (1/ o f 10.80 mm at 500

174
pg/ml). The presence o f two polar groups (hydroxyl), but not more, and a minimum of three

methoxy groups, appears to be necessary for good activity while complete methylation except

for C-5 removes activity as encountered in 5-hydroxy-3.7.4'-trimcthoxyflavone (1).

Compared to the anti-bacterial flavones, the anti-fungal compounds tend to be more

lipophilic.

Hautriwaic acid lactone showed activity with an inhibition zone o f 10.34 mm (31.25 pg/ml):

almost as active as santin (4), the most active compound, probably due to its lipophilic nature.

The other diterpenoids, 2(3-hydroxyhardwickic acid (9), dodonic acid (10) and hautriwaic

acid (17) showed low activity (1Z of 10.80, 10.10, 9.65 mm at 125 p.g/ml, 62.5 and 500

pg/ml, respectively). The three diterpenoids 9, 10, 17 have the same carbon skeleton and one

hydroxyl substituent each, but differ in the position of the hydroxyl group on the ring. The

hydroxyl group is at C-2, C-6 and C-19 in compounds 9, 10, 17, respectively. Consequently,

the activity o f these diterpenoids is determined by the position of the hydroxyl group. The

order o f activity depends on the position of hydroxyl group: C-6 (10) > C 2 (9)> C-19 (17).

Although substituents were found to be important in determining whether or not compounds

were fungitoxic, an unambiguous relationship between structure and activity was not

revealed. Thus for both flavonoids and diterpenoids, with different structural features, the

anti-fungal activity may depend on some common physicochemical attribute, perhaps

lipophilicity and ability to penetrate fungal membranes, rather than a common structure.

Table 3.34: Anti-microbial activity of the acetone extracts of D. angustifolia from different
geographical locations, Senecio roseiflorus and some pure compounds
Sample pg/disc 1 2 3 4
Crude extracts
Surface exudate of D. angustifolia (leaves)- 2500 18.86a 20.05a 19.423 10.79a
Ngong forest
Surface exudate of D. angustifolia (leaves)- 2500 17.58 19.21 18.85 12.45
Voi
Surface exudate of D. angustifolia (leaves)- 2500 19.06 18.89 18.60 14.40
Kilifi.
175
Table 3.34: Anti-microbial activity of the acetone extracts of D. angustifolia from different
geographical locations, Senecio roseiflorus and some pure compounds
Sample ug/disc 1 2 3 4
Surface exudate o f D. angustifolia (leaves)- 2500 16.33 18.94 16.25 12.18
Garborone (Botswana)
Surface exudate of D. angustifolia (leaves)- 2500 17.87 21.68 18.43 11.45
Madagascar
Surface exudate of Senecio roseiflorus 2500 18.66 19.15 18.95 11.80
(leaves)
Compounds
5-Hydroxy-3,7,4"-trimethoxyflavone (1) 500 - - - -

5,4'-Dihydroxy-3,7-dimethoxyflavone 500 - - - -

(Kumatakenin) (3)
5,7-Dihydroxy-3,6,4’-trimethoxyflavone 500 - - 10.08 11.92
(Santin) (4)
250 - - 9.89 11.89
125 - - - 11.60
62.50 - - - 11.22
31.25 - - - 11.15
3.5.4,-Trihydroxy-7-methoxyflavone 500 - 11.95 12.68 -

(Rhamnocitrin) (5)
250 - 11.68 11.83 -

125 - 11.40 11.73 -

62.50 - 10.80:1 - -

31.25 - - - -

5.7-Dihydroxyflavanone (Pinocembrin) (8) 500 - 13.88a 13.42a I4.723

250 - 12.90 13.12 14.66
125 - 11.53 11.49 14.08
62.50 - 9.49 9.87 11.92
31.25 - - - -

2p-hydroxyhardwickic acid (9) 500 - 11.83 11.71 10.95

250 - 10.17 10.47 10.82
125 - - - 10.80
Dodonic acid (10) 500 - 13.74 12.74 10.94
250 - 11.13 10.71 10.90
125 - - - 10.40
62.50 - - - 1 0 .1 0
5.4'-Dihydroxy-3,6,7-trimethoxyflavone 500 - - 9.38 11.50
(penduletin) (12)
Hautriwaic acid (17) 500 - 11.77 12.27 9.65
250 - 9.93 11.07 -

125 - - 9.48 -

Hautriwaic acid lactone 500 - - - 10.90

250 - - - 10.89
125 - - - 10.84
62.50 - - - 10.63
31.25 - - - 10.34
3,5,4'-Trihydroxy-3’,7-dimethoxyflavone 500 12.76 11.72 10.57 1 0 .8
(quercetin-3, 4'-dimethyl ether) (22)
250 12.47 9.68 9.72 -

125 11.97 9.58 9.42 -

176
Table 3.34: Anti-microbial activity of the acetone extracts of D. angustifolia from different
geographical locations, Senecio roseiflorus and some pure compounds
Sample |ig/disc 1 2 3 4
62.50 10.66 9.37 9.20 -

31.25 9.08 - 8.97 -

5,4‘-Dihydroxy-7-methoxyflavanone (25) 500 - 13.20 10.84 12.44

250 - 12.12 10.53 12.21
125 - 12.04 10.18 12.04
62.50 - 11.32 - 11.48
31.25 - - - 10.59
Gentamicin 30 12.39 24.74 30.34 -

Nystatin 25 - - - 25.6

Microorganisms: 1=Escherichia coli (ATCC 25922), 2 -Staphylococcus aureus (ATCC

29737). 3 =Bacilluspumilus (local strain), 4=Saccharomyces cerevisiae (local strain).
Not active.
a Inhibition zone in mm.

3.5.2 LARVICIDAL ACTIVITY

3.5.2.1 LARVICIDAL ACTIVITY OF D. A N G U S T IF O U A -N G O N G FOREST AND
ITS COMPOUNDS
The acetone extract of the fresh leaves of Doclonaea angustifolia-Ngong f orest and some of

its compounds were tested against the larvae of Aedes aegypti. The objective of this study is

to identify botanical insecticides for the control o f disease vector insects. Rotenone was usd

as the standard insecticide.

The extracts, compounds 5-hydroxy-3,7,4'-trimethoxyflavone (3) and 5,7-

dihydroxyflavanone (8) did not show significant activities even at 20 pg/ml, (LC50 > 60

pg/ml after 24 hours) (Figure 3.21).

177
Figure 3.21: Larvicidal activity of the extracts of Dodonaea angustifolia-Ngong Forest on the
second instar Aedes aegypti larvae

The results of the tests are summarized in Fable 3.30. 3,5,4'-'Trihydroxy-7-methoxyflavone

(5) and 5,7 dihydroxy-3,6,4’-trimethoxyflavone ( 4 ) showed good and dose dependent activity

(LC50 1.75 pig/ml and 5.1 pg/ml respectively, after 24 hours) (Table 3.30). 3,5,4-Trihydroxy-

7-methoxyflavone ( 5 ) which was the most active, caused 100% mortality at 6.5 pg /ml

(Figure 3.22) after 24 hours.

Table 3.30: Larvicidal activity (LC50) of compounds from Dodonaea angustifolia-Ngong

Forest on second instar Aedes aegypti larvae
Com pound I X so in p g /m l
F la v o n o id s
5-Hydroxy- 3,7,4'-trimethoxyflavone (1) 75.0
3,5-Dihydroxy-7,4'-dimethoxyflavone (2) 20.0
5,4'-Dihydroxy-3,7-dimethoxyflavone (3) 17.5
5,7- Dihydroxy-3,6,4'-trimethoxyflavone (Santin) (4 ) 5.1
3,5.4'-Trihydroxy-7-methoxyflavone (5) 1.75
5,7-Dihydroxyflavanone (Pinocembrin) (8) >100
D iterp e n o id
Dodonic acid (10) >100
R o ten o n e 0.68

178
Figure 3.22: Larvicidal activity o f 3,5,4'-trihydroxy-7-methoxyflavonc (5) on the second
instar Aedes aegypti larvae

3,5-Dihydroxy-7,4’-dimethoxyflavone (2) was inactive (LC50 20 (ig/ml, after 24 hours). All

the active compounds were less potent than rotenone (LC50 0.68 pg/ml, after 24 hours). The

larvicidal activity of the rude extract of D. angustifolia (Ngong Forest) and some compounds

after 24 hours is summarized in figure 3.23.

Crude extract
Conpoind 1
Corrpoind 2
Conpoind 3
Compound 4
Conpound 5

Figure 3.23: Larvicidal activity of the extracts of D. angustifolia (Ngong Forest) and some of
its compounds after 24 hours

179
3.5.22 LARVICIDAL ACTIVITIES OF D. A U G U S T //O ilA -V O l AND ITS
COMPOUNDS.
The acetone extract o f the fresh leaves of Dodonaea angustifolia-V o\ forest and one of its

compounds hautriwaic acid (17) were tested against the larvae of Aedes aegypti. The extract

was inactive (LC50 > 100 jig/ml, fter 24 hours). However, hautriwaic acid (17) showed good

larvicidal (LC50 10.2 pg/ml, after 24 hours) (Figure 3.24).

Figure3.24: Larvicidal activity o f hautriwaic acid on the second instar Aedes aegypti larvae

The activity of hautriwaic acid (17) was compared with that of its lactone. It is interesting to

note that hautriwaic acid (17), showed activity (IC50 10.2 jig/ml, after 24 hours) while its

lactone (Figure 3.25) was inactive (LC50 > 100 pg/ml, after 24 hours), indicating the

importance of free acidic group as a structural requirement for activity.

180
 60

Figure 3.25: Larvicidal activity o f hautriwaic acid lactone on the second instar larvae of
Aedes aegypti

Flowever, hautriwaic acid (17), was less potent than rotenone (Table 3.31), suggesting that

structural modification may be required to achieve activity that is comparable to that of

rotenone.

Table 3.31: Mosquito larvicidal activities (LC50) against Aedes aegypti of the hautriwaic acid
and its lactone
Tested compound LCso in pg/ml
D iterp e n o id s
Hautriwaic acid ( 17 ) 10.2
Flautriwaic acid lactone > 100
R o ten o n e 0.68

181
3.5.23 LARVICIDAL ACTIVITIES OF S E N E C IO R O S E IF L O R U S AND ITS
COMPOUNDS.

I he acetone extract of the fresh leaves of Senecio roseiflorus and two of its compounds were

tested against the larvae o f Aedes aegypti. The crude extract and did not show good larvicidal

activity, as its (LC50 > 100 pg/ml, after 24 hours) (Figure 3.26).

Figure 3.26: Larvicidal activity o f extracts of Senecio roseiflorus on the second Aedes aegypti
instar larvae

Compounds 5,4'-dihydroxy-7-methoxyflavanone (25) and 5,7-dihydroxy-3,4'-

dimethoxyflavone (21) showed moderate and dose dependent activity (LC50 14.3 pg/ml and

15.5 pg/ml after 24 hours), respectively. The isolated compounds seem to have better activity

than the crude extracts in all the three plant species. Consequently, there is need to test all the

pure compounds isolated from this plant for their activity to establish their use to control

malaria vectors.

3.5.3 ANTI OXIDANT ACTIVITIES

3.5.3.1 ANTI OXIDANT ACTIVITIES OF D O D O N A E A AN G U ST11 O U A -N G O N G
FOREST.
Preliminary radical scavenging activities, using 1,1 -diphenyl-2-picryIhydrazyl (DPPH) free

radical as a spray reagent on TLC plates, of the acetone extract of fresh leaves of D.

angustifolia-Ngong Forest indicated that this extract contains compounds with radical

182
scavenging activities. Some o f the compounds isolated from this plant were tested for

activity. Radical scavenging activities were observed in the extract and some compounds

(Table 3.32). Using spectrophotometric method, the radical scavenging activity of the acetone

extract of D. angustifolia was tested at 11.4 pg/ml while the pure compound were tested at 50

pM. The compounds that had comparable activity to quercetin at that concentration were

tested at lower concentrations. The scavenging activities of the samples were measured as the

percent decrease in absorbance (at 517 nm) of DPPH radical after mixing the sample with

DPPH. The diterpenoids tested showed no RSA activity at all as expected but the crude

extract showed radical scavenging activity of 54.6 % at 11.4 pg/ml and all the flavonoids

tested showed some activity at 50 pM (Table 3.32). The flavonol, 3,5,4'-trihydroxy-7-

methoxyflavone (5) was found to be the most active followed by 3.5-dihydroxy-7,4'-

dimethoxyflavone (2) while the flavanone, 5,7-dihydroxyflavanone (8), was the least active

of all the flavonoids tested. The radical scavenging activity of 3,5,4'-trihydroxy-7-

methoxyflavone (5) was ascertained at lower concentrations and found to be lower than to

that of quercetin (Figure 3.27).

fable 3.32: Radical scavenging activities of flavonoids o f D. angustifolia-Ngong forest

COMPOUND TLC ASSAY % RSA (50
RESULTS pM)
Quercetin + 96.7
Flavonols
3,5-Dihydroxy-7.4'-dimethoxyflavone (2) + 25.4
3,5.4'-Trihydroxy-7-methoxyflavone (5) + 96.2
3-metlioxyflavones
5,4'--Dihydroxy-3,7- + 18.4
dimethoxyflavone(Kumatakenin) (3)
5,7- Dihydroxy-3,6,4'-trimethoxyflavone - 6.67
(Santin) (4)
5,7.4'-Dihydroxy-3-methoxyflavone (6) + 11.2
Flavanone
5,7-Dihydroxyflavanone (8) - 2.31

183
3.5 3.2 ANTI OXIDANT ACTIVITIES OF DODONAEA ANGUSTII O LIA-VO l
Some of the compounds isolated from Dodonaea angustifolia-Vo\ were tested for radical

scavenging activity. The diterpenoid tested showed no RSA activity while the flavonoids

tested were active at 50 pM. The flavonol, kaempferol (15) was found to be the most active

with RSA o f 96.8% at 50 pM. 5,4'-Dihydroxy-3,6,7-trimethoxyflavone (12) had RSA of only

2.55% at 50 pM. The activity o f kaempferol (15) is comparable to that o f quercetin at 50

pM. However, at lower concentrations the activity of quercetin is higher than that of

kaempferol (15). The activity o f 3,5,4'-trihydroxy-7-methoxy flavone (5) and kaempferol

(15) are comparable at all concentrations. Flavonols had appreciably good anti-oxidant

activity compared to the 3-methoxyflavones, indicating the importance o f the the hydroxyl

group at C-3.

3.5 3.3 ANTI-OXIDANT ACTIVITIES OF SENECIO ROSEIFLORUS.

The acetone extract and two compounds isolated from Senecio roseiflorus were tested for

radical scavenging activity. The extract had minimal activity with 9.25 % RSA at 11.4 pg/ml,

which is higher than that of 5, 4'-dihydroxy-7-dimethoxyflavanone (25) (Table 3.33). The

highest activity was observed in quercetin-3, 4'-dimethyl ether (22). However, the activity of

quercetin was higher than that o f its derivative (Table 3.33).

Table 3.33: Radical scavenging activities of quercetin and flavonoids of S. roseiflorus.

COMPOUND TLC ASSAY % RSA (50 pM)
RESULTS
Quercetin + 96.7
3-methoxy flavone
Quercetin-3,4’-dimethyl ether (22) + 77.1

Flavanone
5, 4'-Dihydroxy-7- - 1.22
methoxyflavanone (25)

184
 — Quercetin
— Conrpound 5
—a—Corrpourd 15
— Compcxnd 22

Figure 3.27: Radical scavenging activity o f the standard (quercetin), rhamnocitrin (5),
kaempferol (15) and quercetin-3, 4'-dimethyl ether (22).

185
 CHAPTER FOUR

CONCLUSIONS AND RECOMMENDATIONS

4.1 CONCLUSIONS

The phytochemical investigation of Dodonaea angustifolia from Ngong Forest and Voi and
Senecio roseijlorus was undertaken which led to the isolation and characterization of 27
compounds. All the compounds except compounds 16 and 26 were flavonoids and
diterpenoids.

From the surface exudates o f Dodonaea angustifolia-N gong Forest isolation and
characterization of eight flavonoids (7 flavones and I flavanone) and three diterpenoids (2
ert/-clerodane and one e/7/-labdane type) was achieved.

The phytochemical study on the surface exudates o f Dodonaea augustijolia-Voi led to the
isolation and characterization o f a total of 8 flavonoids, a coumarin and four diterpenoids (all
e/7/-clerodane). Three of these compounds «eo-clerodan-3,13-dien-16,15: 18 , 19-diolide (18),

15p-«eo-clerodan-3,13-dien-16,l 5: 18 , 1 9-diolide (1 9 ) and 1 S a -n e o clerodan-3,13-dien-

16,15: 1 8 , 1 9-diolide ( 2 0 ) are new.

Additional chemotaxonomic information that could help solve the taxonomical controversy
between Dodonaea angustifolia-Ngong Forest and Dodonaea augustifolia-\lox, which are
morphologically similar, was achieved. The two populations of D. angustifolia are of
different chemotypes and may not be different species.

The surface exudates o f Senecio roseiflorus led to the isolation and characterization of a total
of 10 flavonoids (seven flavones and one flavanone) and one benzene derivative. This is the
first phytochemical report of this plant.

The presence of a chelated hydroxyl group at C-5 position was identified as the main
structural feature of all flavonoids isolated from the surface exudates of Dodonaea
angustifolia and Senecio roseiflorus. The flavonoids from these plants arc mainly kaempferol
and quercetin methyl ether.

186
The anti-plasmodial activity of the crude extracts, some of the flavonoids and diterpenoids of
these plants were tested. The results showed that among the flavonoids tested the flavones
had moderate activity and the highest anti-plasmodial activity was exhibited by flavanone
(25), while the diterpenoids had the least activity. Earlier studies (Andayi. 2005) have shown
that tlavanones have good anti-plasmodial activity against both chloroquine sensitive (D6)
and chloroquine resistant strain (W2). This group of compounds can be potential candidates
for use as lead compounds in developing drugs to combat chloroquine resistant malaria.

The surface exudates o f the D. angustifolia-Ngong Forest and Senecio roseiflorus screened
for anti-oxidant activity at 11.4 fag/ml showed 54.6 and 9.25% RSA respectively. The two
plant species elaborate surface exudates consisting mainly of terpenoids and flavonoids. The
RSA o f the surface exudate is due to the flavonoids. The difference in RSA of the two plant
species could be in the qualitative and quantitative composition o f the exudate. The surface
exudates o f D. angustifolia could be richer in flavonoids that have higher radical scavenging
activity, in this case the flavonol, as compared to Senecio roseiforus. The pure compounds
from the two plant species were also tested for anti-oxidant activities and some of the
flavonoids showed RSA activity. The potential use o f the surface exudates and flavonoids
from the surface exudates of the two plants species as radical scavenger was established. The
structure-activity relationship o f the active flavonoids showed that flavonols had appreciable
activity as compared to 3-methoxyflavones isolated from the surface exudates.

The crude extracts of the plant extracts and some pure compounds were tested for larvicidal
activity against Aedes aegypti. The results indicated that flavonoids 5. 4, 25, 21 showed good
and dose dependent activity after 24 hours. Compound 5, being the most active, caused 100%
mortality at 6.5 pg /ml after 24 hours. The diterpenoid, hautriwaic acid (17) showed good
larvicidal after 24 hours. The activity of this compound (17) was compared with that of its
lactone. It is interesting to note that hautriwaic acid (17), showed activity while its lactone
was inactive (LC50 > 100 pg/ml, after 24 hours), indicating the importance of free acidic
group as a structural requirement for activity. These compounds could have potential use for
small scale control o f mosquitoes in rural communities in Fast Africa where mosquito
transmitted diseases such as malaria is endemic. The isolated compounds seem to have better
activity than the crude extracts in all the three plant species. Consequently, there is need to
test all the pure compounds isolated from this plant for their activity to establish their use to
control malaria vectors.

187
The surface exudates o f fresh leaves of Dodonaea angustifolia-'Hgong forest, Dodonaea
angustifolia-Voi and Senecio roseiflorus showed anti-bacterial activity against three strains of
bacteria Staphylococcus aureus (ATCC 29737), Escherichia coli (A I CC 25922) and Bacillus
pimilus (local strain) but minimum anti-fungal activity against one local strain of fungus
Saccharamyces cerevisiae. Compounds 1, 3, 4, 5, 8, 9, 10, 12, 17, 22 and 25 isolated from
this plant were tested and only 4, 8, 9, 10, 22 and 25 were found to be active against at least
one bacteria and fungi.

4.2 RECOMMENDATIONS.

HPLC (High Perfomance Liquid Chromatography) profiles of Dodonaea angustifol'ia-Ngong

Forest, Dodonaea angustifolia-V oi and Dodonaea angustifolia from different geographical
locations within Kenya should be compared in order to unequivocally determine their
taxonomic relationships.

The oil from the surface exudates of the plants should be studied using IIPLC to isolate all
the constituent compounds. The internal tissue compounds of the plants should also be
investigated.

The in vitro activity o f all the flavonoids isolated should be tested against the two strains of
P. falciparum and then other strains as well. The in vivo activity of the llavonoids isolated
should be tested against the two strains of P. falciparum. The bioactivy of the new
compounds against different bacteria strains, the two strains o i Plasmodium falciparum (D6
and W2) and larvicidal activity against the 2nd instar stage of Aedes aegypti should be done.

Comprehensive structure-activity relationship studies should be carried out on the active

compounds to determine the properties responsible for anli-plasmodial. mosquito larvicidal
and anti-microbial activities.

Methylation of 5-OH of the isolated flavonoids should be done and the 5-methyl ethers
flavonoids subjected to anti-fungal activities to establish their activities because previous
investigations have shown that methylation of 5-011 is the structural Feature essential for
good anti-fungal activity [Tomas-Barberan et «/., 1988], The anti-viral activity of different 3-
methoxyl flavonoids should be established, as these compounds have been reported to exhibit
anti-viral activity [Van Hoofe/ al., 1984].
188
Structural modifications of the isolated compounds should be carried out and screened for
bioactivity in order to determine the functional groups that are necessary for activity.

DNA or genetical profiling of Dodonaea species for taxonomic identification should be

included, besides essential oil (GC) and polar compound profiles (I IPL(').

189
 CHAPTER FIVE

EXPERIMENTAL

5.0 GENERAL

The NMR spectra were recorded on a Varian-Mercury 200MHz and Brucker 300, 500 and

600 MHz instruments. Chemical shifts were measured in ppm in 8 values relative to the

internal standard tetramethylsilane (TMS). COSY, NOESY, DEPT, AP I', 11MQC and HMBC

spectra were acquired using the standard Bruker software. El MS spectra were recorded on 70

eV SSq 710 Finnigan MAT spectrometer. UV values were obtained using SP8 150 UV/VIS

spectrophotometer. Melting points were recorded using a Gallenkamp melting point

apparatus with capillary tubes.

Column chromatography was carried out using silica gel 40 (Merck, 70-230 mesh) and

Sephadex LH 20. Analytical thin layer chromatography and preparative thin layer

chromatography (PTLC) were done using Merck pre-coated 60 F254 and Merck 60 PF254-

5.1 PLANT MATERIALS

5.1.1 DODONAEA ANGUSTIFOLIA (NGONG FOREST)
The fresh leaves of Dodonaea angustifolia were collected from Ngong Forest, on 20th

November 2001. The plant was identified by Mr. S.G. Mathenge of the University of Nairobi

Herbarium, School of Biological Sciences (SBS), where a voucher specimen is kept.

5.1.2 DODONAEA ANGUSTIFOLIA (VOI)

The fresh leaves of Dodonaea angustifolia were collected from Voi on 30th August 2002. It

was identified by Mr. S. G. Mathenge of the School of Biological Sciences (SBS),

Herbarium, University of Nairobi, where a voucher specimen is deposited.

5.1.3 SENECIO ROSEIFLORUS

The fresh leaves of Senecio roseiflorus were collected from Mt. Kenya Forest, Meru, at about

1300-1500ft, on 30th August 2002 with the assistance of Mr. S.G. Mathenge of the School of

190
Biological Sciences (SBS), Herbarium. University o f Nairobi, where the specimen is

deposited.

5.2 EXTRACTION AND ISOLATION OF COMPOUNDS

5.2.1 DODONAE ANG U STIFO LIA (NGONG FOREST)
5.2.1.1 EXTRACTION AND ISOLATION OF COMPOUNDS FROM FRESH
LEAVES (AERIAL PARTS)

The leaves of this plant were shinny and gummy indicating that they were coated with resin.

Extraction of the surface exudates on the aerial parts (mainly leaves) was done after plucking

out the flowers. The surface exudates of the leaves. 450 g, was washed out into solvent by

successive dipping into fresh portions of acetone for short periods (< 15 seconds) to yield 52

g of extract, thus avoiding the extraction of the internal tissue components.

A portion o f the extract (7 g) was kept aside for bioassays. The rest of the extract, 45 g, was

adsorbed on silica gel (45 g) and subjected to column chromatography silica gel (450 g)

under C H 2Cl2->?-C6H|4 in the ratio of 1:1. Separation was carried out by stepwise gradient

elution with mixtures of CH2C12-h-C6H|4 and MeOH/CH2CI2 in increasing polarities. Yellow

armophous solids of 3,5-dihydroxy-7,4'-dimethoxyflavone (2) (30 mg) [Drayer, 1978]

precipitated out of the fraction eluted in 50% CH2CI2-/7-C6ll|4. The fraction eluted in 60 %

CH2Cl2-f?-C6H|4 afforded yellow needles o f 5-hydroxy-3, 7,4'-trimethoxyflavone (1) (204

mg) [Dreyer, 1978]. 5,7-dihydro-3,6,4'-trimethoxyflavone (santin) (4) (350 mg) [Sachdev &

Kulshreshtha, 1982; Wollenweber el al., 1986; Abdel-Mogib el al., 2001 | precipitated out of

the fraction eluted with 90% CH2C12-t7-C6H]4 and neat CH2CI2 Purification of the mother

liquor o f the fraction eluted in 90% CH2C12-A2-C6H|4 and neat CH2CI2 using PTLC (Si02,

CH2C12 multiple development) afforded ent-3(3, 8 a - 15.16-epoxy-13(16). 14-labdadiene-3, 8-

diol (11) (10 mg) [Dawson el al., 1966]. The fraction eluting in 1 % MeOH-CH2Cl2 after

column chromatography on sephadex LH 20 (MeOH-CH2Cl2 (1:1)) gave dodonic acid (10)

(500 mg) [Van Heerden et al., 2000; Sachdev & Kulshreshtha, 1984|. while that eluting in 2

% MeOH-CH2CI2 after column chromatography on sephadex Ell 20 (MeOH-CH2Cl2 (1:1))

191
gave 5,7-dihydroxyflavanone (pinocembrin) (8) (120 mg) [Sachdev & Kulshreshtha, 1983J.

The fraction eluted in 3 % MeOH-CH2CI2 afforded 5,4'-dihydroxy-3.7 dimethoxy flavone

(kumatakenin) (3) (200 mg) [Vieira et al., 1997; Sarmento et al., 2002 1and 3,5.4'-trihydroxy-

7-methoxyflavone (rhamnocitrin) (7) (184 mg) [Valant-Vetschera et a/.. 2003; Akkal et al.,

1997; Hattori et al., 1992]. The fraction eluted in 4% MeOH-CH2Cl2 alter purification with

column chromatography on sephadex LH 20 (MeOH-CH2Cl2 (1:1)) afforded 2(3-

hydroxyhardwickiic acid (9) (778 mg) [Jefferies et al., 1973; Anis et al., 2001], 3,5,4'-

trihydroxy-7-methoxyflavone (rhamnocitrin) (5) (60 mg) |Wollenwebcr et al., 1986] and

5.7,4'-trihydroxy-3-methoxyflavone (isokaempferidc) (6) (40 mg) | Dreyer, 1978]. The

fraction eluted in 5% MeOH/CH2Cl2 yieded 3,5,7,4'-tetrahydroxy-6-mcthoxyflavone (7. 60

mg) [Valant-Vetschera et al., 2003; Akkal et al., 1997; Hattori et al., 19921.

5.2.1.2 PHYSICAL AND SPECTROSCOPIC PROPER TIESOF COMPOUNDS OF D.

ANGUSTIFOLIA FROM NGONG FOREST.
5-Hydroxy-3,7,4 '-trimethoxyflavone (1)
Yellow crystals (CH2Cl2-n-C6H |4), mp 145-147 °C lit. mp 145-147 °C |I)NP]; R, 0.3 [20% n-
C6H|2 C H 2C12]; molecular formular CigH^Of,; % yield 0.45; UV: A.maN (MeOH) 268.5 and
346.5 nm; 'H (see Table 3.2); 13C-NMR (see Table 3.2); E1MS m/z (rel. int.): 328 [M]+(49),
327 [M -H]+ (47), 285 [M -43]+ (26), 167(14).

3,5-Dihydroxy-7,4',-dimethoxyflavone (2)
Yellow crystals (CH2Cl2-«-C6H |4), mp 180-181 °C lit. mp 178-180 °C |I)NP]; R, 0.4 [20% n-
C6H 12 C H 2C12]; molecular formular C17H14O6; % yield 0.07; IJV: X.max (McOH) 269 and 348
nm;'H (Table 3.3); ,3C-NMR (see Table 3.3); FJMS m/z (rel. int.): 314 [M]+ (18.6),
167(12.9).

5,4 '-Dihydroxy-3,7-dimethoxyflavone (kumatakenin) (3)

Yellow crystals (CH2CI2-«-C6H 14), mp 253-255 °C lit. mp 253-254 °C |DNP]; R, 0.5 [2%
MeOH - CH2C12]; molecular formular C17H14O6; % yield 0.44; IJV: I maN (MeOH) 268.5 and
346.5 nm; 'll (Table 3.4); l3C-NMR (see Table 3.4); EIMS m/z (rel. int.): 314 [M]+(35), 271
[M - O M e]+ (25), 167(10).

5, 7-Dihydroxy-3,6,4 '-trimethoxyflavone (santin) (4).

Yellow needle like crystals (CH2Cl2-/7-C6Hi4), 159-161 °C lit. mp 159-161 °C [DNP]; Rt 0.4
[2% MeOH - CH2C12]; molecular formular CigH^O?; % yield 0.78; UV: X.max (MeOH) 272.0
and 337.0 nm; 'H (Table 3.5); ,3C-NMR (see Table 3.5).

192
3,5,4'-T rih yd ro xy- 7 -m eth o xyfla vo n e (rham nocitrin) (5)
Yellow crystals (MeOH-CH2Cl2), mp 221-222 °C lit. mp 22I-223°C |I)NP]; R, 0.4 [2%
MeOH - CH2C12]; molecular formular C)6H i206; % yield 0.13; UV X,max (MeOH) 266.0 and
364.0 nm; 'H (Table 3.6); ,3C-NMR (Table 3.6); E1MS m/z (rel. int.): 300 |M ]+(10), 167 (4).

5,7,4',-Trihydroxy-3-methoxy flay one (isokaempferide) (6).

Yellow crystals (MeOH-CH2Cl2), mp >300 °C lit. mp 299-302 °C [DNP|; Rt 0.3 [2% MeOH
in CH2C12]; molecular formular Ci6H|20 6; % yield 0.09; 'll (fable 3.7); ' ’C-NMR (Table
3.7); EIMS m/z (rel. int.): 300 [M]+ (100), 257 [M - O M e f ( 12.3). 150 (31.1).

3,5,7,4 '-tetrahydroxy-6-methoxyflavone (6-methoxykaempferol) (7).

Yellow armophous solid (MeOH-CH2Cl2), 265-266 °C lit. mp 268-270 °C |DNP]; Rt 0.5 [4%
MeOH in CH2C12]; molecular formular C|6H)207; % yield 0.41; 'll (Table 3.8); ' ’C-NMR
(Table 3.8); EIMS m/z (rel. int.): 316 [M]+(87.0), 301 | M - Me]‘ (14.0).

5,7-Dihydroxyflavanone (pinocembrin) (8).

White crystals (CH2Cl2-«-C6H|4), mp 192-193 °C lit. mp 192-193 °C |DNP]; Rf 0.5 [1%
MeOH in CH2C12]; molecular formular Ci7H]204; % yield 0.27; UV X.maN (MeOH) 289 nm;
'H (Table 3.9); ,3C-NMR (Table 3.9).

2(3-Hydroxy-15,16-epoxy-5ft,8[3,9(3,10a-cleroda-3,13(lb),14-trien-18-oic acid. 2(3-

hydroxyhardwickiic acid (9).
White needles (CH2Cl2-«-C6H 14). mp**: Rt 0.4 [3% MeOH in CH2C12]; molecular formular
C2oH280 4; % yield 1.73; 'll (Table 3.10); 13C-NMR (Table 3.10); EIMS m/z (rel. int.): 332
[M]+ (7), 95 [C6H70 ] + (15), 81 [C5H5OJ+ (23), 54 (41). 46 (100).

15,16-epoxy-6-hydroxy-3,13(16),14-clerodatrien-l8-oic acid. (Dodonic acid) (10)

Colourless crystals (MeOH-CH2Cl2), mp 105-107 °C lit. mp 105-107 °C [DNP]; Rt 0.5 [2%
MeOH/CH2Cl2]; molecular formular C2oH2804; % yield 1.11; 'll-NMR (200 MHz, CDCI3):
5 7.39 (cl, J = 16.6 Hz) (H-15), 5 6.86 (distorted t, J -4.8 Hz, H-16) and 6.35 (s, H-14), 3.60
(dd, J = 5.6, 10.4 Hz, H-6), 1.28 (s, Me-19), 0.85 (d, J = 6.6 Hz, Me-17), 0.75 (s, Me-20);
13C-NMR (50 MHz, CDCI3): 5 143.5 (C-4), 142.2 (C-15), 140.9 (C-3), 139.3 (C-16), 126.3
(C-13), 1 11.6 (C-14), 75.0 (C-6), 18.1 (Me-20), 17.1 (Me-17), 16.1 (Me-19).

ent-3/3, 8 a -l5,16-Epoxy-13(16), 14-labdadiene-3, 8-diol (11)

White crystals (MeOH-CH2Cl2), mp**; Rr 0.4 [1% MeOH in CH2C12|; molecular formular
C20H32O 3; % yield 0.02; 'H-NMR (200MHz, CDCI3): 5 7.34 (s, 11-15). 7.23 (s, H-16) and
6.29 (.v, H-14). 3.23 (dd, J = 4.8, 10.6 Hz, H-3), 1.14 (.v, Me-17). 0.98 (s, Me-20), 0.81 (s,
Me-18) and 5 0.76 (s, Me-19). I3C-NMR (50 MHz, CDC13): 8 143.5 (C 14), 139.5 (C-16),
126.2 (C-13), 111.8 (C-14), 79.6 (3), 74.8 (8), 61.9 (C-9). 55.8 (C-5), 28.9/29.1 (Me-17), 24.8
(Me), 16.6 (Me), 16.4 (Me).

193
5.2.2 D O D O N A E A N G U S T IF O L IA ( \ O l )

5.2.2.1 EXTRACTION AND ISOLATION OF COMPOUNDS FROM FRESH

LEAVES (AERIAL PARTS)

The leaves of this plant were shinny and gummy indication that they were coated with resin.

Extraction o f the surface exudates on the aerial parts (leaves) was done after plucking out the

flowers. The surface exudates o f the leaves (143 g) was extracted by successive dipping into

fresh portions of ethyl acetate for short periods (< 15 seconds) to yield 143 g of crude extract,

thus avoiding the extraction of the internal tissue component.

5.2.2 2 ISOLATION AND PURIFICATION OF SURFACE COMPOUNDS FROM

D O D O N A E A A N G U S T IF O L IA (VOI)

A portion of the extract (5 g) was kept aside for various bioassays. The rest of the extract

(138 g) was subjected to column chromatography on silica gel (1.4 kg) eluting with different

mixtures of CH2CI2-rt-C6H|4 followed by MeOH-CH2Cl2 in increasing polarities. Yellow

crystals o f 5-hydroxy-3,7,4'-trimethoxyflavone (1) (206 mg) [Dreyer, 1978] precipitated out

of the fraction eluted in 50% CH2CI2-«-C6H|4 mixture. The mother liquor was purified by

column chromatography on Sephadex LH- 20 (eluting with McOH/Cl LCI2; 1:1) to yield 5-

hydroxy-3,6,7,4'-tetramethoxyflavone (14) (46 mg) [Wollenwcber et c//., 1986] and 3,5-

dihydroxy-7,4',-dimethoxyflavone (2) (1 g) (Dreyer, 1978]. The fraction eluted in 60 %

CH2C l2/n-hexane afforded crystals of 5,4'-dihydroxy-3,7-dimethoxyflavone (kumatakenin)

(3) (1.5 g) | Vieira et a!., 1997] [Sarmento el ai, 20021. The mother liquor was separated by

PTLC (S i0 2) (CH2Cl2-/7-C6Hi4; 1:1) severally times to give yellow crystals of 5,4'-

dihydroxy-3,6,7-trimethoxyflavone (penduletin) (12) (108 mg) [Sachdev& Kulshreshtha.,

1986]. Colourless crystals o f hautriwaic acid (17, 2.53 g) [Jefferies & Payne, 1967; 1973;

Hsu et cil., 1971] precipitated out of the fraction eluted in neat CH2C12. The mother liquor of

this fraction was concentrated in vacuo and subjected to recrystallization to yield a second

crop o f hautriwaic acid (17) (277 mg). The fraction eluted in I % MeOH-CH2Cl2 after

194
purification by PTLC (S i0 2) 20 % EtOAc-^-CftHu with multiple development, afforded 5,3'-

dihydroxy-3, 4', 7-trimethoxytlavone (ayanin) (13) (36 mg) [Perez-Castorena et al., 1997a;

Jakobsen et al., 2001]. The mother liquor was purified by PTLC developing severally in

(Si02. CH2CI2) to yield 7-hydroxy-6-inethoxycoumarin (16) (22 mg) | Andrianova et al.,

1975] as the major band.

The fraction eluted in 2 % MeOH-CFLCL after purification by column chromatography

(sephadex LH 20, MeOH-CH2Cl2; 1:1) and preparative TLC (1 % MeOH-CH2Cl2 x 2)

afforded /7<?o-clerodan-3,13-dien-16,15:18.19-diolide (18) (60 mg), 15a-methoxy-«eo-

clerodan-3.13-dien-16,15:18,19-diolide (19) (78 mg), and 15(3-mcthoxy /7eo-clerodan-3,13-

dien-16,15:18,19-diolide (20) (78 mg). The fraction eluted in 3 % MeOH-CH2Cl2 after

purification by column chromatography (sephadex LH 20, MeOH/Cl 12C12; 1:1) yielded

3,5.4'-trihydroxy-7-methoxyflavone (rhamnocitrin) (5) (33 mg) [Wollenweber et al., 1986]

and 3,5.7,4'-tetrahydroxy-flavone (kaempferol) (15) (57 mg) [Khan et a l ., 1992].

5.2.2.3 PHYSICAL AND SPECTROSCOPIC PROPERTIES OF COMPOUNDS

FROM D. ANGUSTIFOLIA FROM VOI.

5,4'-Dihydroxy-3,6,7-trimehtoxyflavone (penduletin) (12)

Yellow armophous (CH2Cl2-«-C6H,4). mp 220-222 °C lit. mp 221-222 °C |DNP]; R, 0.5 [4%
MeOH in CH2C12]; molecular formular CisH^O?; % yield 0.08; UV: X.maN (MeOH) 272.0 and
341.5 nm; 'll (Table 3.11); l3C-NMR (Table 3.11); EIMS m/z (rel. int.): 344 [M]+(55.0), 329
[M-Me]+ (31.5), 301 [M-]+ (12.0).

5.5 '-Dihydroxy-3, 4 \ 7-trimethoxyflavone (ayanin) (13)

Yellow crystals (MeOH-CH2Cl2), mp 173-174 °C lit. mp 173-I74°C [DNP]; 0.5 (2% MeOH-
CH2C12]; molecular formular C^Hi^O?; % yield 0.03; 'll (fable 3.12); ' ’C-NMR (Table
3.12); EIMS m/z (rel. int.): 344 [M]+(66.0), 329 [M-Me]+ (35.0), 301 [M-()Me]+ (35.0).

S-Hydroxy-3,6,7,4 '-tetramethoxyflavone (14)

Yellow crystals (MeOH-CH2Cl2), mp 178-180 °C lit. mp 178-180 °C | DNP]; Rf 0.5 [4%
MeOH/CH2Cl2]; molecular formular C19H18O7; % yield 0.03; 'll (see fable 3.13); 13C-NMR
(see Table 3.13); EIMS m/z (rel. int.): 358 [M]+(62.0), 343 [M-Me]+ (32.0), 315 [M-OMe] +
(14.0).

195
3,5,7,4'- T etra h yd ro xy-fla vo n e (kaem pferol) (15)
Yellow armophous (MeOH-CH2Cl2), mp 276-278 °C lit. mp 276-278°( [ D N P ] ; Rf 0.5 [4%
M eOH/C^CH]; molecular formular C15H10O6; % yield 0.04; UV: X.max (MeOH) 267.0, 324.0
and 364.5 nm; 'H (Table 3.14); 13C-NMR (Table 3.14); EIMS m/z (rel. int.): 286 [M]+
(100.0), 329 [M-Me] + (31.5), 301 [M-OMe]+ (12.0).

7 -H yd ro xy-6 -m eth o xyco u m a rin (16)

Colourless crystals (CH2C12-a?-C6H,4), mp 201-202 °C lit. mp 203-204 °C [DNP]; R, 0.3
(CH2C12); molecular formular C]oH8Q4; % yield 0.2; 'll (Table 3.15); 1 C NMR (Table 3.15).

ent-15 ,1 6 -E p o x y -1 9 -h y d ro x y -3 ,l3(16),14-clerodatrien-18-oic acid (hautriw aic acid) (17)

Colourless needles (MeOH-CH2Cl2), mp 179-180 °C lit. mp 183-184°C |DNP]; Rf 0.4 [3%
MeOH/CH2Cl2]; molecular formular C20H28O4; % yield 2.03; 'll (fable 3.16); 'C-NM R
(Table 3.16); EIMS m /z (rel. int.): 333 [MH]+(32), 332 [M]+(23), 284 |C ,9H240 2]+ (45), 220
[C,4H2o0 2]+ (23), 219 [Ci4Hi902]+ (13), 189 [C,3H,70 | + (27), 95 [C6H7OJ+ (95), 81 [C6H70 ]
+(99).

Neoclerodan-3,13-dien-16,15: 18,19-diolide (18)

Colourless oil (CH2CI2-H-C6H14), mp**; Rf 0.2 [1% MeOH in CH2C12]; molecular formular
C20H26O4; % yield 0.04; 'H (Table 3.17); 13C-NMR ( fable 3.17); HR-E1MS: m/z = 331.1894
[MH]+: EIMS m/z (rel. int.); 330 [M] (13), 300 |M -CH20 ] (13), 219 |C 1 iH|g02]+ (53), 189
(100), 161 (15), 105 (20), 91 (30), 84 (28).

15/3-!Weoclerodan-3,l3-dien-l 6,15:18,19-diolide (19) and 15a-neoclerodan-3,13-dien-

16,15: 18,19-diolide (20)
Colourless oil (CH2CI2-/7-C6H14), mp**; Rt 0.1 [1% MeOH in CH2C12]; molecular formular
C21H28O5; % yield 0.06; 'H (Table 3.18); ,3C-NMR (Table 3.18). IIR-EIMS: m/z = 360.8609
[M f; EIMS m/z (rel. int.); 361 [M H ]\ 342 [M-H20 ] + (18), 330 [CH20 ] f (36), 329 (54), 310
(52), 283 (52), 189 (32), 91 (43), 86(59), 84 (100).

2.3 S E N E C IO R O S E IF L O R U S

5.2.3.1 EXTRACTION AND ISOLATION FROM FRESH LEAVES (AERIAL PARTS)

Extraction of the surface exudates on the aerial parts (mainly leaves) o f Senecio roseiflorus

was carried out on fresh plant material, by successive dipping into fresh portions of ethyl

acetate after short periods (< 15 seconds). Whenever the colour of the solvent become intense

yellow', it was changed. The extracts were combined, filtered and concentrated in vacuo. In

the process of removing the solvent, a white precipitate was formed which was determined to

be an inorganic material, NaCl.

196
This was filtered out to give 15 g of material. Further removal of solvent of the filtrate

resulted in a pale brown gummy solid (223 g). The remaining aerial parts of the plant were

spread on a bench to dry out.

5 2.3.2 ISOLATION AND PURIFICATION OF SURFACE COMPOUNDS FROM

S E N E CIO ROSEIFL OR US.

A portion o f the extract (15 g) was kept aside for various bioassays. I lie rest of the extract

(50 g) was adsorbed on silica gel 50 g and subjected to column chromatography (Si02, 500 g

CH2C12- a7-C6H,4 1:1. Separation was carried out by stepwise gradient elution using mixtures

of CH2C l2-w-C6H|4 followed by MeOH-CH2Cl2 in increasing polarities. The fraction eluted in

60 % C H 2Cl2-rt-C6H,4 was purified by CC (sephadex-LH 20, MeOI l-CI 12C12; 1:1) to give 5-

hydroxy-3,7,4'-trimethoxyflavone (1) (33 mg) |I)reyer, 1978], 3,5-dihydroxy-7,4'-

dimethoxyflavone (2) (54 mg) [Dreyer, 1978], 5,4'-dihydroxy-3,7,3'-trimethoxyflavone (27)

(103 mg) and 3,5-dihydoxy-3',4',7-trimethoxyflavone (28) (21 mg). Yellow crystals of 3,4',5-

trihydroxy-3',7-dimethoxyflavone (rhamnazin) (23) (39 mg) [Marin et a/., 2001] precipitated

out from the fraction eluted in 80 % CH2Cl2/n-hexanc. The mother liquor was separated by

CC (sephadex-LH 20; MeOH- CH2C12; 1:1) to yield yellow' crystals of 3,5,4'-trihydroxy-7-

methoxyflavone (rhamnocitrin) (5) (24 mg) [Wollenweber et al., 19861. White crystals of

5.4'-dihydroxy-7-dimethoxyflavanone (25, 1 .480 g) |Mata el al.. 19911 precipitated out of the

fraction eluted in neat CH2C12. The mother liquor was purified by CC (sephadex-LH 20;

MeOH- CH2CI2; 1:1) coupled with PTLC (Si()2), 90 % CH2( h-/7-C6Hi4 multiple

development to yield 20 mg o f 5-hydroxy-3,6,7,4'-tetramethoxyfiavone (14) [Wollenweber et

a/., 1986] and 5-hydroxy-3,7,3',4-tetramethoxyflavone (retusin) (24) (28 mg) [Valant-

Vetschera et al., 2003; Wollenweber & Dorr., 1996].

The fraction eluted in 2 % CH2Cl2-MeOH after purification by CC (Si(K 1% MeOH-CH2Cl2

) followed by PTLC (S i0 2, CH2C12) multiple development yielded 3,5,4'-trihydroxy-7-
methoxyflavone (rhamnocitrin) (5) (15 mg) [Wollenweber et al., 1986] and 5,7,4',-

197
r

trihydroxy-3-methoxy flavone (isokaempferide) (6) (24 mg) [Dreyer., 1978]. The fraction
eluted with 3 % CH2Cl2-MeOH afforded 104 mg o f 5, 7-dihydroxy-3.4'-dimethoxyflavone
(21) [Wollenweber et al., 1986] and 53 mg of 3\5,7-trihydroxy-3,4'-dimethoxyflavone
(quercetin-3,4'-dimethyl ether) (22) [Sepulveda et al., 1994; Jakobsen et al., 2001]. The
mother liquor was purified by preparative TLC (S i02, CH2CI2 multiple development) to yield
3.4',5-trihydroxy-3',7-dimethoxyflavone (rhamnazin) (23) (35 mg) | Marin et al., 2001] and 4-
hydroxy-methylbenzoate (26) (22 mg) [Yoshioka et al., 2004].

5 2.3.3 PHYSICAL AND SPECTROSCOPIC PROPERTIES OF COMPOUNDS

FROM S E N E C IO R O S E IF L O R U S .

5,7-D ih y d r o x y -3,4 f-d im eth o x yfla vo n e (21)

Yellow armophous (CH2C12-a?-C6H|4), mp 233-235 °C; Rt 0.3 [20% ( H2CI2 in n-C6Hh];
molecular formular C17H14O6; % yield 0.21; !H (Table 3.19); ' ’C-NMR ( fable 3.19); EIMS
m/z (rel. int.): 314 [M]+(100.0), 271 [M-OMe]+ (75.0).

3',5,7-T r ih y d r o x y -3 ,4 f-d im eth o xyfla vo n e (quercetin-3,4 '-d im eth y le th er) (22)

Yellow armophous (CH2Cl2-/7-C6Hi4), mp 232-234 °C lit. mp 235-236°C |DNP]; Rf 0.5 [2%
MeOH in CH2CI2]; molecular formular C17H14O7; % yield 0.11; 'll ( fable 3.20); |1C-NMR
(Table 3.20). EIMS m/z (rel. int.): 330 [M]+(l00.0), 287 [M-OMe]+ ( 12.3), 167 (18.5).

3 ,4 \5 - T rih yd ro xy-3 \ 7-d im eth o xyfla vo n e (rham nazin) (23)

Yellow armophous (CH2Cl2-/7-C(sH|4), mp 216-218 °C lit. mp 2 16-218 °C |DNP]; Rf 0.5 [2%
MeOH in CH2CI2]; molecular formular C17H14O7; % yield 0.07; 'll ( fable 3.21); i3C-NMR
(Table 3.21); EIMS m/z (rel. int.): 330 [M]+(l00.0), 287 [M-OMe]+ (12.2), 167 (16.7).

5 -H y d ro x y -3 ,7,3 ',4 '-tetram ethoxyflavone (retusin) (24)

Yellow armophous (M eO H -C ^C h), mp 160-162 °C lit. mp 159-160°C |I)NP]; R( 0.5 [2%
M eOH/CfhCh]: molecular formular Cjyl118O7; % yield 0.06; 'll (Table 3.22) and n C-NMR
(Table 3.22).

5,4 '-D ih yd ro xy-7 -m eth o xyfla va n o n e (25)

White crystals (CH2Cl2-/7-C6Hi4), mp 151-153°C lit. mp 152-154 °C [DNP|; R, 0.4 [1%
MeOH in CH2CI2]; molecular formular C16H14O5; % yield 2.96; UV >tmax (MeOH) 283.0 nm);
'H (Table 3.23) and ,3C-NMR (Table 3.23). EIMS m/z (rel. int.): 286 [M ] (43.7), 167
(100.0), 120(34.4).

4-H ydroxy benzoic acid m e th y l ester (26)

Colourless oil, mp 127-129 °C literature 127-129 °C [DNP]; R, 0.5 11% MeOH/CH2Cl2];
molecular formular C rHsO s; % yield 0.04; 'll (Table 3.24); l3C-NMR ( fable 3.24).

198
5.4 BIOLOGICAL ACTIVITY STUDIES

5.4.1 IN VITRO ANTI-PLASMODIAL TEST

The extracts and the pure compounds were assayed using an automated micro-dilution

technique to determine 50% growth inhibition of cultured parasites |('hulay et al., 1983;

Desjardins et al., 1979]. Two strains of Plasmodium falciparum parasites, from the Walter

Reed Army Institute o f Research, that are commonly used in drug sensitivity assays were

cultured. The chloroquine sensitive Sierra Leone I (D6) and chloroquinc- resistant Indo-china

I (W2) strains were grown in continuous culture supplemented with mixed gas (90%

nitrogen, 5% oxygen, 5% carbon dioxide), 10 % human serum, and 6% hematocrit of A+ red

blood cells. Once cultures reach a parasitemia level of 3% with at least a 70% ring stage

development, parasites were transferred to a 96 well microtiter plate with wells pre-coated

with sample. The samples were serially diluted across the plate to provide a range of

concentration used to accurately determine IC50 values. Plates were incubated in a mixed gas

incubator for 24 hours. Following the specified incubation time, | 11|-hypoxanthine was

added and parasites allowed to grow for an additional 18 hours. Cells were processed with a

plate harvester (Tom Tec) onto a filter paper and washed to eliminate unincorporated [ H|-

hypoxanthine. Filters were measured for activity in a microtiter plate scintillation counter

(Wallac). Data from the counter was imported into a Microsoft Excel spreadsheet which

isand subsequently imported into an Oracle database/program to determine (IC50) values.

5.4.2 LARVICIDAL ACTIVITY ASSAY

The eggs of Aedes aegypti L. (Diptera: Culicidae) were obtained from the Department of

Zoology, University of Nairobi. The eggs were flooded w ith 0.08% NaCI solution and left to

hatch at 28 °C. Twenty second instar larvae were transferred into a petri-dish containing 10ml

of 0.08% NaCI solution. The larvae were treated with the test extracts and pure compounds

according to Mwangi and Rembold [1998]. Twenty milligrams o f test samples were

dissolved in 2 ml of DMSO. From the stock solution different concentrations were prepared

199
by serial dilution and the larvae were tested for mortality at 20, 10, 5, 2.5, 1.25 and 0.75

pg/ml o f sample solutions. Control larvae in all cases received 50pl o f DMSO as in test

larvae. Each experiment was run in triplicate.Mortality were checked after 24. 48 and 96

hours. LC50 values were calculated from the average of three observations for each

concentration using Finney's probit analysis for quantal data [Mwangi et al., 1988,

McLaughlin et al., 1991].

5.4.3 RADICAL SCAVENGING TEST USING DPPH

To a MeOH solution (3 ml) o f DPPH (100 uM), 0.5 ml of test compounds at 50 pM (10

pg/'ml for crude extract) were added and the mixture was shaken and left to stand for 30 min.

The Radical Scavenging Activities were estimated as the percentage decrease of absorbance

of DPPH (100 pM) at 517 nm [Ohinishi et al., 1994]. For 3,5,4’-trihydroxy-7-

methoxyflavone (rhamnocitrin) (5), 5,7,4\-trihydroxy-3-mcthoxy flavonc (isokaempferide)

(6), and quercetin etc, tests were done at six different concentrations ( 50.25. 12.5, 3.13 and

1.56 pM ). In all cases the mean values were used from triplicate experiments. EC50 values

were calculated using Finney's probit analysis for quantal data [McLaughlin et al., 1991].

5.4.4 ANTI MICROBIAL TEST

Evaluation of anti-microbial activity of extracts and pure compounds was accomplished using

the agar well-diffusion method [Bauer et al., 1966]. The extracts and pure compounds were

tested for activity against three strains of bacteria: Staphyloccocus aureus (ATCC29737),

Escherichia coli (ATCC25922) and Bacillus pumilus (local strain) and a local strain of a

fungus, Saccharamyces cerevisiae. The bacterial test organisms were cultured on tryptone

soya agar while the fungus was cultured in Saboraud’s dextrose agar.

Seven cylindrical plugs were removed from the solidified nutrient agar plates at equidistant

points, using a sterile cork borer, to produce wells (5 mm diameter, 2 mm depth). The seven

wells were used for six different plant extract at 2500 pg/ml and a control.

200
The extracts 50 mg and pure compounds (10 mg) were dissolved in 1 ml o f solvent (DMSO)

to give the stock solution of 50 mg/ml for extract and 10 mg/ml for pure compounds which

were 31.25, 62.5, 125, 250 and 500 pg/disc subjected to anti-microbial test for the crude plant

extracts. However, for the pure compounds, different concentrations were prepared by serial

dilution o f the stock solution. Only 50 pi of the solution of extract, pure compound, standard

drug (Gentamicin 0.3 mg/ml, nystatin 0.25 mg/ml) or solvent (DMSO) were filled in each

well. All experiments were done in triplicate. The inoculated petri dishes prepared as

described above were left for 30 minutes for diffusion and then incubated overnight (18

hours) at 37 °C and 25 °C for bacteria and fungi, respectively. The anti-microbial activity was

recorded as the w idth (mm) o f the clear zone of inhibition surrounding the agar well after 18

hrs of incubation for the bacteria and fungi.

The minimum inhibitory concentration (MIC) was determined by the agar well-ditfusion

method. MIC of a compound is defined as the lowest concentrations of the compounds that

visually inhibits growth compared with growth in control wells.

201
 CHAPTER SIX

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Zdero, C., Bohlmann, F. Niemeyer, H. M. (1990): Seco-Labdanes and other constituents
from Ophryosporus floribundus. Phytochemistry' 29. p. 3247-3253.
Zdero, C., Bohlmann, F., Liddell, J. R. (1989a): .seco-Eremophilanes and other constituents
from South African Senecio species. Phytochemistry 28. p. 3532-3534.
Zdero, C., Bohlmann. F., Solomon, J. C., King, R. M., Robinson, H. (1989b): e«/-Clerodanes
and other constituents from Bolivian Baccharis species. Phytochemistry 28, p. 531 -
542.
Zhang, A. and RajanBabu, T. V. (2004): Chiral Benzyl Centers through Asymmetric
Catalysis. A Three-Step Synthesis of (/?)-(-)-a-Curcumene via Asymmetric
Hydrovinylation. Organic Letters 6, p. 3159-3161.
Zhao, Y., Wang, P., Hao, X. J., Parsons, S., Rankin, D. W. H., Shi, 1L, Yu, H., Sevenet, T.,
Gueritte, F. (2002): Enantiomeric sesquiterpene lactones from Senecio tsoongianus.
Chin. Chem. Lett. 13, p. 754-757.
Zhao, Y., Jia, Z. J., Tan, R. X. and Yang, L. (1992): Eremophilanolides from the roots of
Ligularia veitchiana. Phytochemistry 31, p. 2785-2787.
Z hdanovich, E. S., Men'shikov, G. P. (1941): The alkaloids o f Senecio othonnae. J. Gen.
Chem (USSR) 11, P. 835-838.

http://www.mediainquiries@who. int retrieved on 2nd September. 2009

http://en.wikipedia.org/wiki/Asteraceae retrieved on 28th September, 2009
http://www.plantatlas.usf.edu/. retrieved on 28,h September, 2009

224
APPENDIX I: SPECTRA FOR COMPOUND 1

225
ppm 'H-NMR spec tr u m fo r COMPOUND 1

226
 IJC-NMR SPECTRUM FOR COMPOUND 1

o
(-*
00
o 179.035

-J
o
165.649
162.283
CTv
O 161.919
157.096
156.233
LJ1
O
.£*
o
LO
O 1 3 0 . 4C7

123.069

114.299

106.296
CD
CD
98.049

92.398
o

oo
O 77.499
77.246
76.992
-J
o

CTv
CD ------------ 6 0 . 3 8 1
____----- 5 6 . 0 2 9
------- 5 5 . 6 7 0
oi
o

O
o
J
29.933

M
o

n
TJ
3

227
 DEPT SPECTRUM FOR COMPOUND 1

M
<T\
rs)

' I .......................... I ............................1 ............................I ' ’ r"Tr"' rrT r I ^ ...........■’■ ’T .................

130 120 110 100 90 80 70 60 50 40 30 20 pprr

COSY SPECTRUM FOR COMPOUND 1

228
NOESY SPECTRUM FOR COMPOUND 1

HMBC SPECTRUM FOR COMPOUND 1

229
 HSQC SPECTRUM FOR COMPOUND 1

s'.

MASS SPECTRUM FOR COMPOUND 1

230
APPENDIX II: SPECTRA FOR COMPOUND 2

231
 'h - n m r spec tr u m for COMPOUND 2

0.963 11.736

8.203
8.194
8.187
8.170
8.163
8.153
2.000 7.606
7.327
7.261
7.066
2.148 7.057
7.050
2.912 7.033
7.026
6.910
6.567
6.503
6.495
6.385
6.378

6.130 3.897
3.892

3.079 1.549

232
 UC-NMR SPECTRUM FOR COMPOUND 2

17 b .2 C 0
170

------------- 1 6 b . / 4 4

--- 1 6 1 .1 7 7
160

160.863
---------- 1 5 6 . 8 6 7
150

1 4 5 . 7C2
140

135.667
130

129.397

123.223
120

114.112
110

103.956
100

97.893

92. 238
90
80

77.424
77.204
77.001
76.578
75.555
70
ppm

bb.834
55.419

233
COSY SPECTRUM FOR COMPOUND 2

HMBC SPECTRUM FOR COMPOUND 2

234
HMQC SPECTRUM FOR COMPOUND 2

MASS SPECTRUM FOR COMPOUND 2

235
APPENDIX III: SPECTRA FOR COMPOUND 3

236
 ‘h - n m r spectrum FOR COMPOUND 3

12.768

--- -
CD 2.082 -----8 . 0 4 7

^ — 7.035
-vl - 2.000
— 6.677
: 0.953 ^Z 6.672
----6 . 331
: 0.957 ^ ----6 . 3 2 7
CT> -

3. 930
3.881
cn 3.854
3.586
3.576
3. 572
3. 562
3.535
7.213 3.526
3.034
"374T9 2.994
2.98/
2.954
CO -
2.947
2.921
2.907
2.095
2.080
ro - 2.076
2.068
2.063
31,505 2.059
2.055
8.959
ppm

2. C50
9.191 2.043

237
 IJC-NMR SPECTRUM FOR COMPOUND 3

167. 219
166. 533
163. 481

158.410

131. 882

117. 100

99. 143

93. 502

60. 868
58. 369
57. 099
33. 280
31. 904
31. 328
31. 290
31. 245
31. 107
31. 059
30. 963
30. 905
30. 810
30. 752
3 0. 656
30. 602
30. 503
ppm

30. 349
30. 195

238
HMBC SPECTRUM FOR COMPOUND 3

239
HMQC SPECTRUM FOR COMPOUND 3

8 7 6 5 4 3 2 1 ppm

MASS SPECTRUM FOR COMPOUND 3

240
APPENDIX IV: SPECTRA FOR COMPOUND 4

241
 'h - n m r spectrum FOR COMPOUND 4
Z*Dt
an
BIZI

242
i3C-NMR s p e c t r u m FOR COMPOUND 4

243
DEPT SPECTRUM FOR COMPOUND 4

244
APPENDIX V: SPECTRA FOR COMPOUND 5

245
 'h - n m r spectrum FOR COMPOUND 5

VO
ui
VO

8.114
8 . C84

o 6. 934
6. 904

/ 6.412
— 6.406
CP> - 6. 200
^ 6.193
O

c
n

c
n
o
4.874

Ui

o .894
3.812
CjO . 365
cn s. 337
3.331
CaJ 3. 326
3.321
O 3.315

fo
c
n

NJ
O

C
n

O
ppm

246
190
180 i3c - n m r s p e c t r u m FOR COMPOUND 5

----- 1 7 6 . 5 3 3
170

---------- 164.741
160

— ------- 161.678
---------- 159.713
------ 157.419
150

--------- 1 4 7 . 2 0 5
14C

----- 1 3 6 . 2 9 5
130

_ — — 130.570
-— 129.838

122.893
120

116.742
115.723
115.462
110

---------- 103.704
100

---------- 100.538
------ 98. 421
— ----- 93 . 6 1 8
— ----- 92. 261
90
80
70
60

55.625
49.012
48.728
50

48.444
48.161
47.877
‘■ 1 . 593
40

47. 309
30
20
1C ppm

247
 DEPT SPECTRUM FOR COMPOUND 5

1........ ' ........ ' I' ......... I......... T...................... ^ ■■----- |.......... | ....... •| -— ' "I■
140 130 120 110 100 90 80 00 60 50 40

'H, H-COSY SPECTRUM FOR COMPOUND 5

248
NOESY SPECTRUM FOR COMPOUND 5

i
--------p — ----1---
1 1
JL J .1
- -1“ “
1
. - -i —
1_ _ _ _ JJL
r -
ppm
1 1 i 1 i 1 i 1
1 1 i 1 1 *
1 1 i 1 i 1 I \S
1 7 1 i 1 i \
1 1 i 1 i 1 i i 1
1 1 I 1 l 1 i 1
1 1 i 1 i 1 i I 1
1 1 i 1 I 1 i o I 1
■4.5
1
1 1 i 1 i 1 1
1 1 i 1 i 1 oJ 1
:4
1
1 ~ -.0
1 i 1 i 1 l 1
1 1 i 1 i 1 1 l 1
• 5. 5
1 1 i 1 i 1 • ? 1 1
1 1 i 1 i 1 1 1
1 1 i 1 1 1 l 1
- 6.0
1
1 i 4 | 1 1 1
a 1
1
i
i
1
\*
* 1 1
1
1
1
1 1
• 1 i 1 1 1 1 1 ■6.5
1 1 i 1 1 1 l
1 1 1 1 1 1 1 1
*
--------1-------
1 1 1 1 •7.0
1 1 1 1
1 1 i 1 1 1 1 1
1 i 1 1 1 1 1 1
1 i 1 1 1 1 l 1
■7.5
1 * 1 i 1 1 1 1 i 1
1 * 1 i 1 1 1 1 1 1
1 i» 1 1 1 1 1 1 ■ 8.0
a •
i 1 i 1 1 1 1 1 1
i 1 i 1 1 1 1 1 1
...1__ __ |__
J ‘ 1’ * J 1 VU ■ ■ l ■ ■
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4 . r: ppm

HMBC SPECTRUM FOR COMPOUND 5

249
HSQC-DEPT SPECTRUM FOR COMPOUND 5

MASS SPECTRUM FOR COMPOUND 5

250
APPENDIX VI: SPECTRA FOR COMPOUND 6

251
'H-NMR spectrum FOR COMPOUND 6

3.898
3.889
3.883
3.804
3.798
3. 331
3.328
3.325
3.322
3.319

1. 320
1.295
0.925
0.912
0.898
0.885
0.878

252
i3C-NMR s p e c t r u m FOR COMPOUND 6

253
 DEPT SPECTRUM FOR COMPOUND 6

1 ...... '........ I.....1 ■ •■I......... |... Il--- |......... I......... |....... " I '■ ■*.... I......... I - ’ -r-r,*-' •!•••■• '■ ■■|........ ,.........
140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

'h , h -c o s y s p e c t r u m FOR COMPOUND 6

jL _____ i L u i i ________ . ppm

254
 NOESY SPECTRUM FOR COMPOUND 6

HMBC SPECTRUM FOR COMPOUND 6

b. 5 8. 0 7.5 7.0 6.3 fc.O 5. 5 5. 0 4.5 4. 0 j .5 3.0 - .5 1. 0

255
HSQC-DEPT SPECTRUM FOR COMPOUND 6

MASS SPECTRUM FOR COMPOUND 6

jtouncUruTt*
28000 -

40 «0 80 100 120 140 160 180 200 220 2 4 0 260 2 6 0

256
APPENDIX VII: SPECTRA FOR COMPOUND 7

257
 'h -n m r spectrum FOR COMPOUND 7

7.009
6. 999
6. 973
6. 917
6.91 0
6. 894
6 .8 8 8
6. 625
6. 558
6. 276
5. 959
5. 955
3. 955
3. 945
3. 889
3. 822
3. 769 «
3. 757
3. 314
2. 865
2. 768
2. 757
2. 711
2.7C0
2. 262
2. 195
2. 188
2. 128
2.121
2. 114
2. 090
2. 069
2. 062
2. 054
2. 047
2. 040
2. 003
1 .995
1.988
1.981
1. 973
1 .934
1. 929
1.922
1. 850
ppm

1 .843
1. 835

258
 i3C-NMR s p e c t r u m FOR COMPOUND 7

------- 177.602
160

------- 160.90b
------- 158.555

_____ 153.804
150

-— 153.285

--------- 147. 959

140

----------- 139.531
------- 137. 037

-------- 132.421
130

------- 131. 206

124.082
120

------- 117.040
110

------- 105.304
100

------- 95.233
90
80

61.449
61.044
70

61.449
61.044
ppm

259
 APT SPECTRUM FOR COMPOUND 7

'H, H-COSY SPECTRUM FOR COMPOUND 7

260
HMBC SPECTRUM FOR COMPOUND 7

HMQC SPECTRUM FOR COMPOUND 7

261
MASS SPECTRUM FOR COMPOUND 7

R tljtw * Abundance

262
'h -n m r spectrum FOR COMPOUND 8
12 t l

264
r

UC-NMR SPECTRUM FOR COMPOUND 8

265
APPENDIX IX: SPECTRA FOR COMPOUND 9

266
 'h - n m r spectrum FOR COMPOUND 9

Tsl 7. 1 3 8
in 0.994
0.991
■Nj
b

p> 1.000
In
0.989

o>
b

Ul 0.841
cn

cn
b

4^
cn
1.098

co
cn

co
b

ro
cn
3.384

ro
b
y~
8.028
cn
3.224,
1.700 1 .202
1.0 ppm

1.188
3 1 3 2 ‘; 1.159
1.145
3.511._ 0.862
0.840
0 . 7 95

267
I3c - n m r spectrum FOR COMPOUND 9

- ---- 169. 197

144. 329
143. 644
141. 750
140. 170

---- 127. 165

--- 69.731

46. 806
40. 358
39. 901
39. 569
37. 429
37. 197
31. 271
31 . 014
30. 758
30. 245
29. 988
29. 732
29. 290
28. 575

268
 APT SPECTRUM FOR COMPOUND 9

' " ! .................r T" " ' T ,| ..............T .r ,~ T r r T T T ,T ..,.,. „ . , f T 1

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 ppm

' h , h -c o s y spectrum FOR COMPOUND 9

ll I l ppm

269
HMBC SPECTRUM FOR COMPOUND 9

HMQC SPECTRUM FOR COMPOUND 9

270
MASS SPECTRUM FOR COMPOUND 9

Relative Abundance

271
APPENDIX X: SPECTRA FOR COMPOUND 10

272
 ' h - n m r SPECTRUM FOR COMPOUND 10

1
x .«l
1.27
1.00

o»
D.14
25.34

-
I
4.79
2.^1
19.22
4.13
6 . OB

273
FOR COMPOUND 10

274
spectrum
'H-NMR .77
1. 2 9
1. 0 0
0. 11
24.68
5.07
7 . CO
10. 70
4. 24
B . 07
180
160
140
120
100
80
60
40
20
ppa UC-NMR SPECTRUM FOR COMPOUND 10

275
APPENDIX XI: SPECTRA FOR COMPOUND 11

277
 'H-NMR spectrum FOR COMPOUND 11
2. 36
1.00
0.18
1.34
2 . BO
45.12

278
I3C-NMR spectrum FOR COMPOUND 11

279
DEPT SPECTRUM FOR COMPOUND 11

iwwxwo

280
APPENDIX XII: SPECTRA FOR COMPOUND 12

281
 'H-NMR spectrum fo r COMPOUND 12

GO
0.761 12.72b

ro

0.802
CD

8.070
CO 2.030 3.063
8. 0- 17
8 . 0 -10

7.040
1.951 7.033
7.017
1.000 7.010
6.803

CD

CD -

3.981
f 3.882
9.175 3. 873
V 3.816
3. 803

w-
2.868

ro

~o
x>
3

282
 i3C-NMR SPECTRUM FOR COMPOUND 12
180

— 180.481
170

--- —--- 161.650

160

----- "— 160.790

— — 157.741
—— 154.28/
~~~— 153.873
150
140

-— 139.843

--- — 133.873
130

------------- I V . 9 0 0

---- — 123.358
120

— — 117.104
110

---- — 107.733
100

----— 92.3 64
90
80
70

61.227
60.877
ppm

--- — 57.4 91

283
 APT SPECTRUM FOR COMPOUND 12

180 170 160 150 140 130 120 110 100 90 80 70

' h , h -c o s y spec tr u m FOR COMPOUND 12

ppm

284
HMBC SPECTRUM FOR COMPOUND 12

ppm

-105

-no
-115

120

-125

-130

-135

140

-145

-150

-155

-160

-165

-170

ppm

HMQC SPECTRUM FOR COMPOUND 12

285
MASS SPECTRUM FOR COMPOUND 12

Relative Abundance

286
APPENDIX XIII: SPECTRA FOR COMPOUND 13

287
 'h - n m r spectrum FOR COMPOUND 13

S i ____
0.825 12 . 75C

" 1

CO -
j
7. 906
0.759 7. 876
] 0 .2 3 6 V 7. 804
00 -
0.451^ 7. 797
7. 731
JL037^ 7. 724
1.242/ 7. 702
7. 695
1.431/ Z 7. 0 2 9
7. 000
1.000/~~ 6. 6/4
6. 670
0 .9 5 0 V 6.666
CT> 6. 3 2 6
6.32?
6. 3 1 9

cn
3. 970
3. 951
3. 931
■u 3. 920
10 0 8 9 V
3. 903
3. 8 8 8
3. 882
2. 8 3 7
Co - 2.090
2.077
2.068
2. 0 6 1
2. 0 5 3
ro - 2.046
2.039
1. 371
1. 3 5 6
10.571 1. 351
T3 3TJ39 1. 3 0 6
T3 1. 290
3

288
I3C-NMR SPECTRUM FOR COMPOUND 13

180. 284
180. 214
------ 176. 305

------ 167. 274

------ 163. 560

------- 158. 414

— ---- 157. 585

------ 151. 231

------ 148. 978

140. 292
140. 167

------- 134. 044

------ 132. 693
127. 875
126. 675
125. 272
124. 109
123. 479
_____ 117. 508
—----- 116. 777
----- 113. 418

------ 107. 251

_____ 99. 147

----- 97.76C
93.661
93. 610

------ 84. 677

----- 60. 925

57. 146
57. 115

289
APT SPECTRUM FOR COMPOUND 13

290
HMBC SPECTRUM FOR COMPOUND 13

HMQC SPECTRUM FOR COMPOUND 13

291
 MASS SPECTRUM FOR COMPOUND 13

Relative Abundance

100
100
150
200
250
m fz
300

292
APPENDIX XIV: SPECTRA FOR COMPOUND 14

293
 'H-NMR SPECTRUM FOR COMPOUND 14

8.094
8.087
8.070
8 . 0 63
7 . 2 63
7.C42
7.035
7.019
7.011
6.505

3.975
3.969
3.959
3.925
3.902
3.683
3 . 8 65
2.374
2.350
2.324
1.636
1.612
1.396
1.390
1 .304
1.255
1.188
1 .1 65
pprr

1.146
1. 122

294
i3C-NMR SPECTRUM FOR COMPOUND 14

----- 178. 936

----- 161. 709

-------- 1 5 8 . 7 4 7
------ 155. 993
_____ 1 5 2. 798
152. 342

----- 1 38. 738

----- 132. 312

------ 130. 137

----- 122. 830

----- 1 14. 079

----- 106. 624

----- 90. 316

----- 77. 204

60. 861
60. 128
56.296
55. 997
55.429

33. 675
31 . 9 0 9
2.9.679
29.499
29. 417
29 . 3 3 8
29. 221
29 . 0 5 7
24.703
22.669
14. 083

295
 APT SPECTRUM FOR COMPOUND 14

V i V ffii i ii W W W

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 ppm

' h , H-COSY SPECTRUM FOR COMPOUND 14

296
HMBC SPECTRUM FOR COMPOUND 14

HMQC SPECTRUM FOR COMPOUND 14

297
MASS SPECTRUM FOR COMPOUND 14

Relative Abundance

298
APPENDIX XV: SPECTRA FOR COMPOUND 15

299
'h - n m r spectrum FOR COMPOUND 15

12. 804

12. 176

3. 872

-------- 2. 9 6 8

2. 071
2. 0 6 3
2. 0 5 6
2.049
2. 0 4 2
1. 990

300
I3c - n m r spectrum FOR COMPOUND 15

1 7 7 . 3C4

-------- 165. 660

------- 16.3.040
------- 160. 860
------- 1 58. 506

147. 732

--------- 1 3 7 . 3 3 1

------- 131. 161

124. 044

117. 030

------- 104. 873

99. 865

95. 195

301
 APT SPECTRUM FOR COMPOUND 15

180 170 160 150 140 130 120 110 100 ppm

'h , h -c o s y SPECTRUM FOR COMPOUND 15

302
HMBC SPECTRUM FOR COMPOUND 15

HMQC SPECTRUM FOR COMPOUND 15

303
 MASS SPECTRUM FOR COMPOUND 15

Relative Abundance
o o i o o i o u J 8 o h 8 & S $ o o i o o i o <->i O
■ I I ■ . I I . ■ ■ ■ I ■ ■ ■ I I ■ ■ ■ ■ I . ■ ■ I I ■ I ■ I I ...........I I I . 1 I . ■ I I ■ 1 . 1 I ■ I . 1 I I I I 11 LJ.I 1 1 1 I I 1 1 1 1 1 1 1 1 1 1 1 1111-1 1 1

__s

8-b

8 -u ^ -

8 --

28C

(O
ft
CO
w

304
APPENDIX XVI: SPECTRA FOR COMPOUND 16

305
1.01 ' h - n m r SPECTRUM FOR COMPOUND 16

306
,3C-NMR SPECTRUM FOR COMPOUND 16

307
NOESY SPECTRUM FOR COMPOUND 16

11 t ■ - . .................................................................
u it i * ? t i i } : t «i
f i (p p « )

308
APPENDIX XVII: SPECTRA FOR COMPOUND 17

309
 'H-NMR SPECTRUM FOR COMPOUND 17

"J
cn 1.000
1.052
-si
b

CT> 1.140
cn
1.049
CT>
b

Ol
In

Ol
o
- 2 . 2 8 il
■t* -2.234
cn -2 .2 1 5
2. i y4
1.291
* -
1.260
co
cn

CO
b

ro
cn
6.294
ro
o
r
10.621
x
2.686
1.845
1.0 ppm

y
8.842

310
 l3C-NMR SPECTRUM FOR COMPOUND 17

143.983
140

1 3 9 . 73C
138.924
130

126.731
120
110

111.854
100
90
80
70

66.177
60
50

47.865

---------- 4 3 . 3 1 1
^ --- 4 0 . 1 4 4
40

--- 3 9 . 9 5 0
---------- 3 7 . 5 5 2

32.863
30

28.047
27.649

19.122
19.055
ppm

18.099
16.200

311
 APT SPECTRUM FOR COMPOUND 17
140
130
120

------ 111. 856

110
100
90
80

Ii
1
70

------ 66. 167

60

49. 856
49. 572
49. 289
49. 005
50

48. 721
48. 437
48. 153
47. 881
43. 325
40

40. 153
39. 963
37. 565
32. 876
30

--- 28. 057

'------ 27. 649

19.131
19. 048
1 8.111
ppm

16. 197

312
'H, H-COSY SPECTRUM FOR COMPOUND 17

HMBC SPECTRUM FOR COMPOUND 17

313
HMQC SPECTRUM FOR COMPOUND 17

MASS SPECTRUM FOR COMPOUND 17

314
 ‘h - n m r spectrum FOR COMPOUND 18

^1
In

~>l 1.085
o
1.001
O)
Ln

CT)
b

Ol
In

Ol
b
2.332 . 800
. 796
4* . 791
Ln
0.983 • 1 . 748
4* 1. 704
b 0.931 • 1. 6 9 4

co
Ln
1.636
co
b

1.151

cn 1.805

4.139
3.468

316
UC-NMR SPECTRUM FOR COMPOUND 18

. 181
. 30 2

.86/
. 431
. 763
. 299

203

/O b
184

013
526
742
533
150
418
843
619
711
5 94
476
940
51 7
518

317
 APT SPECTRUM FOR COMPOUND 18

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 ppm

' h , H-COSY SPECTRUM FOR COMPOUND 18

318
NOESY SPECTRUM FOR COMPOUND 18

HMBC SPECTRUM FOR COMPOUND 18

319
HMQC SPECTRUM FOR COMPOUND 18

MASS SPECTRUM FOR COMPOUND 18

320
HIGH RESOLUTION MASS SPECTRUM FOR COMPOUND
18

331.1894

321
APPENDIX XIX: SPECTRA FOR COMPOUND 19 AND

322
 'H-NMR SPECTRUM FOR COMPOUNDS 19 AND 20

7.270
6.797
6.79-1
6. 7 92
6.770
6. 7 5 6
5. 741
4. 306
4.290
3 . 92 9
3.925
3.913
3.909
3.593
3. 585
3.580
3.573
2.195
2.187
2.182
2.173
2.046
2.022
1.954
1.948
1.942
1.928
1.922
1.747
1 . 735
1 . 724
1.690
1.670
1.663
1 . 64 6
1. 6 4 1
1.633
1. 61 3
1. 601
1.595
1.589
1.579
1.517
1.513
1.509
1.506
1. 4 9 0
1 . 486
1.483
1. 2 7 4
1.270
1. 2 6 6
1. 254
1.246
1.069
1.035
0.886
0.878
0. 8 6 4
0.859
1.0 ppm

0.851
0.846
0.8.33
0.660
0.66
0.585

323
i3C-NMR SPECTRUM FOR COMPOUNDS 19 AND 20

------- 1 7 1 . 2 0 6
-------- 1 6 9 . 2 6 5

141.575
141.533
138.652
138.468
138.440
135.765
135.700

102.542

77 . 2 0 4

71.689

--- 5 7 . 1 9 1
“ — 57.101

48.059
45.544
38.793
36.580
35.022
34.956
34.431
30.868
29.656
27.728
27. 612
19.503
ppm

18.907
'8.872

324
'H, H-COSY SPECTRUM FOR COMPOUNDS 19 AND 20

NOESY SPECTRUM FOR COMPOUNDS 19 AND 20

325
HMBC SPECTRUM FOR COMPOUNDS 19 AND 20

HMQC SPECTRUM FOR COMPOUNDS 19 AND 20

326
MASS SPECTRUM FOR COMPOUNDS 19 AND 20

327
h ig h r e s o l u t i o n m a s s s p e c t r u m f o r c o m p o u n d
19 & 20

361.1995

m/z

328
APPENDIX XX: SPECTRA FOR COMPOUND 21

329
 ' h -n m r spectrum FOR COMPOUND 21

00

00 8.101
8.071
o

Cn

7 .1 1 2
7 .1 0 6
o 7 .0 8 2

<T>
cn 6.4 3 2
6 .4 2 5
___ 6.2 2 8
6. 221
cn
O

cn
cn

cn
o
4.871

cn

3 .9 0 7
o 3 .9 0 5
3.811
3 .8 0 9
CO 3 .8 0 5
cn 3 .3 3 2
3.326
3.321
3 .3 1 6
Co

N)
cn
ppm

330
,3C-NMR spectrum for COMPOUND 21

165.225
162.259

157.619

_--- _---- 1 3 0 . 6L2

— 130.371

115.701
1 1 4 .272

98.961

93.9 0 3

59.7 1 5

55.590
55.0 6 6

331
 DEPT SPECTRUM FOR COMPOUND 21
<
T\r-j
130.369
130.610

m co
<7. O
in t

,,,-,^-.Tr,,,j" I... . l ’r•' r l " ■’I. • • ■

?5 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 .5 50 ppm

'H, H-COSY SPECTRUM FOR COMPOUND 21

JL. _ PPm
~ - F- --F-
1 1 1 1 ——i—1 i 1 1 1 1
1 1 1 1 11 i I 1 1 1
K 1 _ _ _ J _ _ __ 1__ ___ 1__ __
_L _ .J _
- - 1
11 11 11 11 11 i l
l
i
l
11 11 - i -4.0
i
1 1 1 1 i l i 1 1
1
_ r —-
1 1 1 1 i 1 i 1 1 r ••• 4, )j
. -J l 1 1 1 1 i l i 1 1
1 1 1 1 -5.0
r
1 1 i l i
11 11 11 11 i
i
i
i
l
i
11 11
i - 11" T l ‘ 1 “ 11 1 T“
l
---- J
1 r
i
-" “
1
1 i
r -5.5

- -f —
1 1 1 1 1 l i 1 i
♦--6.0
— i 1 l 1 !. . l i i 1 i
L i.. _ _ _L _ _ _ j _ ___ i__ __L _ ' i _ i • 6. 5
- 1 1 1 1 • i • 1 l 1 1 i
a 1
“ T* “
1 1 1 l 1 1 i
-7.C
11 I
11 11 l
l
l
l
l
i
11 11 i
i
1 l 1 I 1 i 1 1 i
- X- - X-7.5
11 1l 11 11 l l I 1
11 1 i
11 11
l
1l
l
11 I
l
1 11 i
i
r T
l
---- 1- - 1 i
-8.0
" T ~~ r“' i f ---- “ 1 " " i
J i i i l l i 1 i
i
-+ —
1 i i i 1 1 i 1 i !
**-8.5
■ - i ..... •~ + T - ~ H -...- i
3.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4 .0

332
 NOESY SPECTRUM FOR COMPOUND 21

HMBC SPECTRUM FOR COMPOUND 21

b.-> B.O 7.5 7. 0 6.5 6. 0 5. 5 5.5 . 4.0

333
HSQC-DEPT SPECTRUM FOR COMPOUND 21

MASS SPECTRUM FOR COMPOUND 21

$U>iiS3Sri[c*

26 000

24000

22000

20000

180 00

16 000

14 000

12000

10000

0000

6000

4000

2000

0 l~
40 60 80 100 120 140 160 180 200 220 240 260 280 300

334
APPENDIX XXI: SPECTRA FOR COMPOUND 22

335
 'h - n m r spe c t r u m for COMPOUND 22

647
645
641
628
623
603
599
071
053
898

563
396
392
315
203
199

496

869

946
933
881
802
796
326
323
320
317
313

fo -
ppm

336
FOR COMPOUND 22

337
i3C-NMR s p e c t r u m

L90 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 ppm
 DEPT SPECTRUM FOR COMPOUND 22
»r> cm O VO O
r- m csj oo o*-» r» <o«nrt h in
rn»n hv a) ri«
»rr* o r*
-
<N CM VO r* vo vo »n r* <o <r n h o>
o o vo vo ov ^ Oi o\ oo
vo vo mm *r »r <7 «r <r

YV ^

w JL v

'' T ' ' ' ' I..........." ' I ' ' ' ' I ............ I~....... I ................ I ■ ' ■ ■ | ' ■ ■ ' - ' T ' ' ' ' 1 ' ' ' ' , r- ^ - r -r - r ,...............
125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 ppm

'h , h co sy spec tr u m FOR COMPOUND 22

__Ma_____ ^ V
_1_L—
^— J_______J ' Ppm

338
 NOESY SPECTRUM FOR COMPOUND 22

8.0 7.5 7.0 6.5 6. 0 5.5 5.0 4.5 ppm

HMBC SPECTRUM FOR COMPOUND 22

IP T H T I— rTTT1T

339
HSQC-DEPT SPECTRUM FOR COMPOUND 22

MASS SPECTRUM FOR COMPOUND 22

Ste uiiidajvc*

340
APPENDIX XXII: SPECTRA FOR COMPOUND 23

341
 'h - n m r SPECTRUM FOR COMPOUI) 23

12.128

3.939
3.928
3.902
uidd

342
 l3C-NMR SPECTRUM FOR COMPOUI) 23

------ 2 0 5 . 3 6 5
------- 2 0 5 . 1 0 4

165.924

122.759
122.10b
115.302
111.332

97.600
92.081

CTi
O
55.676

o 29.836
29.579
U
o
) 29.323
29.067
28.810
NJ 28.554
o 28.298
ppm

343
 DEPT SPECTRUM FOR COMPOU1) 23

O' cr> cr> cd

NNNN

r . ’ irrn' ir,r11,irTTi......... r ''........ t........... i....... rn .......... i..........i....... ’ ~t.......... i t ..........iM’ rrr"' ’ i '"M........... r 1 1 i.......... i...........i.......... i....... .
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 ppm

'h , h -c o s y SPECTRUM FOR COMPOUD 23

13 12 11 10 9 3 7 6 S 4 i /. ppm

344
 HMBC SPECTRUM FOR COMPOUD 23

*3 12 11 F'P-

HSQC-DEPT SPECTRUM FOR COMPOUD 23

---------------- A---------------------------------------------------------
1
— A_A_l l _________ L i ............... J L J L l ppm

1 1 1
-1--------------- 1---------------- — — ; -------------- 1 -------------- r - 0
1 i i
1 1 1
i i i
1 1
------- 20
1
1 1 ! \
t 1 i i • i
1 i - - - - -
- -i - •10
1 • i
1 i i
I t < i i
1 • *1 6 i
1 > i i
00
1 1 i i
1 i i i
i
1 1
1 1 i . i 30
i *
1 1 i i
1
1 t «
—
100

1 i i
120
i
1
i i
i i 140
i t

i
U . r-r- ----------- -------------------- --------- ■ 1 ..................... 1 I ' r r p ...... .,1 ---------------- --------------1 1
12 11 10 3 8 6 ?. 4 3 2 ppm

345
MASS SPECTRUM FOR COMPOUD 23

346
APPENDIX XXIII: SPECTRA FOR COMPOUND 24

347
 ' h - n m r SPECTRUM FOR COMPOUND 24

■ 12. 739
CJ /- 7 805
0.888 - 7 801
/84
7 777
0.091 7 772
ro - 7 167
7 150
6 696
6 692
6.392
6. 338
3.931
3. 928
3. 918
3. 909
3. 889
3. 860
3. 690
3. 586
576
571
CD 562
557
599
543
3. 535
3. 523
2.143 3.041
3.001
2. 961
2.929
2. 862
2. 099
2. 096
2. 080
2 . 0/6
2. 068
2. 064
2. 060
2. 055
2.051
2. 019
2. 015
1.291
1.246
1. 163
1.137
1. 123
1 .109
0. 993
0. 957
0. 942
0. 927
0. 905
0. 902
0. 899
0. 895
0.891
0.881
0. 875
0. 867
0. 863
0. 857
ppm

0. 853
0. 846

348
i3C-NMR SPECTRUM FOR COMPOUND 24

. 283

. 431
. 290
.487
. 417
. 424
. 51 6
. 683

. 330

. 277
1. 730

1 .231
1.912
1.006
' . 230

167

625

978
372
, 123
, 925
. 817
. 280
. 493
, 413
. 255
. 197
,11C
.062
. 967
. 909
.812
.755
.656
.504
.351
. 197
.044
. 608
.1 51
.2 94

349
'h , h -c o s y SPECTRUM FOR COMPOUND 24

HMBC SPECTRUM FOR COMPOUND 24

HMQC SPECTRUM FOR COMPOUND 24

351
APPENDIX XXIV: SPECTRA FOR COMPOUND 25

352
 ' h - n m r SPECTRUM FOR COMPOUND 25

1 2 . 02!
0.911 7.607
7.354
7.326
7.261
6.910
6.902
6.896
6.880
6.873
6.080
6.072
6.050
6.042
5.386
5.376
5.343
5.333
2,210 3.809
2.000 3.144
3.101
3.087
1.791 3.044
2.821
2.810
1.088 2.763
0.658 2.753
1.572
1.333
0.156 1.321
1.255
2.858
1.089
0.992
0.968 0.959
0.952 0.945
0.927
0.920
0.895
0.880
0.852
0.828
- 15.226
1 ppm

0.800
0.783

353
 i3C-NMR SPECTRUM FOR COMPOUND 25

*95.991

CD
o
00
o

o-si ------ 1 6 8 . CIO

. ______164. 161
------- 16 2 . 6 8 7
o>
o ------ 1 56. 113

cn
o

o
00 ------1 3 0. 637
o ------ 1 2 7. 953

ro
o

103. 156
o
o 95. 127
94. 252
CO
o
78. 962
00 77 . 9 7 8
o 1 1 . 424
77.203
77.001
"s i 76. 577
o
CD J
O :
- ------55 . 6 7 7

cn J
O :
43 . 2 2 6
40
ppm

J
1 ----- 2 9 . 6 8 9

354
‘h , H-COSY SPECTRUM FOR COMPOUND 25

HMBC SPECTRUM FOR COMPOUND 25

355
HMQC SPECTRUM FOR COMPOUND 25

MASS SPECTRUM FOR COMPOUND 25

356
APPENDIX XXV: SPECTRA FOR COMPOUND 26

357
 'H-NMR SPECTRUM OF COMPOUND 26

7.976
7.967
7. 960
7.945
7.938
7.929
7.552

7.262
6. 901
6.892
6.885
6.869
6.862
6. 8 5 3

6. 1 8 3

3. 962
3.895
3.829

. 548
1. 362
1. 3 3 9
1 .336
1. 317
1. 3 0 0
1 . 296
1 .292
1 .255
C. 984
0. 971
0.962
PPm

0.949
0. 9 4 0
0. 9 1 6

358
 i3C-NMR SPECTRUM OF COMPOUND 26

VI
o
167.329

O)
o 1 6 0 . 22C

■u
o

131.931
00 131.025
o 128.847

122.358

115.267

o
o

(O
o

00
o

CT>
o

52.017
T3
■o
3

359
 167.308
APT SPECTRUM OF COMPOUND 26

52.002
T 1*****
1

" l......... i' '' r

170 160 150 140 130 120 110 100 90 80 70 60 PPm

H, H-COSY SPECTRUM OF COMPOUND 26

360
HMBC SPECTRUM OF COMPOUND 26

ppm

- 110

- 115

120

-1 2 5

-1 3 0

-1 3 5

- 140

145

-1 5 0

-1 5 5

- 160

165

170

h 175

ppm

HMQC SPECTRUM OF COMPOUND 26

361
APPENDIX XXVI: SPECTRA FOR COMPOUND 27

362
 'H-NMR SPECTRUM FOR COMPOUND 27

.870

. 901
.800

.327
.322
.317

. 297
ppm

363
 "C-NMR SPECTRUM FOR COMPOUND 27
190
180
170

166.218
163.616
160

163.324
158.669
150
140
130

___ .____ 1 3 1 . 4 2 8
130.658

------ 1 2 4 . 0 2 2
120

115.329
115. 052
110
100

100.001
94.947
90
80
70

60.768
60

56.122
50. 009
49. 725
49.441
50

49. 157
48.874
48.590
40

48.306
30

------ 3 0 . 9 1 1
20

14.595
10
ppm

364
 DEPT SPECTRUM FOR COMPOUND 27
131.421
130.6 54
OVtO

O
<7V
\

V V

• I ............................... I ................................. I .................................I ................................. I ................................ I ............................. ...................................... .............. T ' ■ . - ' ■ • ' ' T ............................. | '
130 120 110 100 90 80 70 60 50 40 30

'h , h -c o s y SPECTRUM FOR COMPOUND 27

[
u _
___
___
___
___
_
1 — JU
L
___
_j_
___ ppm
_
..... T~
_
___
___
__1 1
: 1
J_.3 . 5
1
1 1
1
- -J- 4 . 0
1
* ; 1
1
Jm_ _ __ L _ J _
1 4.5
1
: 1
-’i _ _ J _ _ __ L _ 14 J _5 . 0
1
1
1
___ _
__ __ ___ L_ • J ' 5.5
1
1
1
_ L_
__ i J 6. 0
1
«» 1
1
__j• i i 1'
1 6.5
1
1
-- flL
___ 1
1 7.0
* 1
1
1
1 7.5
t: 1
1
1
— —
f—
8.0
$ "1 i
i
i
8.5
1— ......1.......!■■■■!.
..-..H- h ~ f -....1 •
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 0 3 .5 3 . 0 ppm

365
 HMBC SPECTRUM FOR COMPOUND 27

HSQC-DEPT SPECTRUM FOR COMPOUND 27

366
MASS SPECTRUM FOR COMPOUND 27

367