Control of African Striga Species by Natural Products from Native Plants. Joseph Kipronoh Rugutt Louisiana State University and Agricultural & Mechanical College

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LSU Historical Dissertations and Theses Graduate School

1996 Control of African Striga Species by Natural Products From Native Plants. Joseph Kipronoh Rugutt Louisiana State University and Agricultural & Mechanical College

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CONTROL OF AFRICAN STRIGA SPECIES BY NATURAL PRODUCTS FROM NATIVE PLANTS

A Dissertation

Submitted to the Graduate Faculty o f the Louisiana State University and Agricultural and Mechanical College In the Partial fulfillm ent of the requirements for the degree of Doctor of Philosophy

The Department o f Chemistry

Joseph Kipronoh Rugutt BS., Moi University, Eldoret, Kenya, 1989 December, 1996

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UMI 300 North Zeeb Road Ann Arbor, MI 48103

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The Lord is good to all:

and his tender mercies are over a ll his works.

PSALMS 145:9

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS

In order to successfully accomplish research o f this nature, there are a number of

people who make invaluable contribution, both large and small. It is with great

pleasure that I faithfully thank them.

This research has been accomplished under the direction of Dr. Nikolaus H.

Fischer, to whom I would like to express my utmost appreciation for his endless moral

as well as material support Specifically, his endless academic support guidance and

encouragement has been a springboard of joy throughout the realization o f this work.

Dr. Fischer and Mrs. Helga Fischer are very pleasant responsible and nice people to

work with. You can always count on them in any situation. My association with the

Fischers has meant more than words can describe. In short, their support o f me and my

fam ily has been unfailing. I consider them second to none and rank them as W orld’s

heroes. Also, I wish to sincerely acknowledge the remarkable assistance provided by

Prof. Day and Mrs. Day. They constantly provided great personal encouragement and

ascertained that everything kept going smoothly. Their long time friendship has been

and w ill continue to be a never-ending source o f pride and sustenance. I am also

grateful to my wife Janny and my daughter Elizabeth who spent many lonely nights

waiting for me to complete this research. Finally, I would like to thank Dr. Frank

Fronczek for the determination of all the crystal structures; to Marcus Nauman for his

invaluable help on the use of the NMR instrumentation; to Mr. Jeff Corkem for

determination of GCTMS; Ms. Juliette Navratilova and M r. Rostem Irani for isolation of

pure compounds; Messrs. Charles Cantrell and Steven Robbs for their useful

suggestions on structure elucidation; Dr. Berner and his technical staff for doing all

bioassays; M r. John Rugutt for statistical analyses; Dr. José Castafieda-Acosta for his

help in the preparation of this manuscript; the committee members (Profs: Fischer,

Cartledge, Watkins, Bhacca, Urbatsch, Moore and Berner) and the Rockefeller

Foundation fo r financial support through African dissertation internship award.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

ACKNOWLEDGMENTS...... iü

LIST OF TABLES...... vü

LIST OF FIGURES...... ix

LIST OF SCHEMES...... xü

ABSTRACT...... x iü

CHAPTER I. INTRODUCTION ...... 1 1.1. PEST M AN AG EM EN T...... 2 1.2. PAR ASITIC PLANTS...... 2 1.3. B IO LO G Y OF S7R/GA ...... 3 1.4. D IS TR IB U TIO N OF 577Î/GA ...... 5 1.5. TH E STRIGA PROBLEM...... 6 1.6. PRACTICES TO CONTROL STRIGA SPECIES...... 7 1.6.1. Hand or Hoe Weeding ...... 8 1.6.2. Land Preparation ...... 8 1.6.3. Rotation of Land into Fallow ...... 9 1.6.4. Breeding for Resistance/Tolerance ...... 9 1.6.5. Fertilizers ...... 9 1.6.6. Herbicides ...... 10 1.6.7. False-host or "Trap" Crops...... 10 1.6.8. Germination Stimulants ...... 11 1.7. QUANTITATIVE STRUCTURE-ACTIVITY (QSAR) ...... 16 1.7.1. Basic Concepts of QSAR ...... 17 1.7.2. Bioassays ...... 18 1.8. THE RESEARCH OBJECTIVES ...... 19

n. GC/MS EVALUATION OF COMPOUNDS IN DRY AND CONDITIONED STRIGA SEEDS...... 24 n .l. INTRODUCTION ...... 25 H2. MATERIALS AND METHODS...... 26 n.2 .1 . Seed C ollection ...... 26 n.2.2. Seed Surface Disinfestation and Conditioning ...... 26 n.2.3. Preparation of Extracts of Striga Seeds ...... 27 n.2.4. GC/MS Analysis ...... 27 n.3. RESULTS ...... 28 n.4. DISCUSSION ...... 29 n.5. CONCLUSIONS ...... 36

m . ACTIVITY OF EXTRACTS FROM NONHOST LEGUMES ON THE GERMINATION OF STRIGA HERMONTHICA SEEDS...... 42 m .l. INTRODUCTION ...... 43 m.2. MATERIALS AND METHODS...... 44 m.2.1. Collection of Plant Materials ...... 44 m.2.2. Preparation of Solutions o f GR 24 ( 8 ) and GR 7 (9) ...... 45 m.2.3. Dichloromethane Extraction ...... 45 m.2.4. Water Extraction ...... 45 m.2.5. Isolation of Sterols from Leaves ...... 46

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in.2.6. Thin-layer Chromatography (TLC) ...... 46 m.2.7. Bioassays ...... 47 in.2.7.1. Crude Extracts ...... 47 m.2.7.2. Sterols ...... 48 m.2.8. Statistical Analysis...... 49 ffl.3. RESULTS...... 49 m.4. DISCUSSION...... 51 m.5. CONCLUSIONS...... 54

IV. IN VITRO GERMINATION OF STRIGA HERMONTHICA AN D S.ASPERA SEEDS BY 1-AMINOCYCLOPROPANE-1-ACID (ACC)...... 72 IV .l. INTRODUCTION ...... 73 IV.2. MATERIALS AND METHODS...... 75 rv.2.1. General ...... 75 IV.2.2. Viability TesL ...... 75 IV.2.3. Preparation of ACC (16) and GR 24 ( 8 )...... 75 IV.3. RESULTS...... 75 rv.4. DISCUSSION...... 76 IV .4.1. Mechanisms of Formation of Ethylene from ACC (16) ...... 77 rv.4.2. Hypochlorite Chemistry ...... 78 rv.4.3. Possible Basic Mechanisms o f Action of Ethylene on Germination of Striga Species Seeds ...... 80 rv.5. CONCLUSION...... 84

V. STIMULATION OF STRIGA HERMONTHICA GERMINATION BY llpH.13-DIHYDR0PARTHEN0LIDE (DHP) ...... 93 V .l. INTRODUCTION ...... 94 V.2. MATERIALS AND METHODS...... 95 V.2.1. General ...... 95 V.2.2. Seed Conditioning ...... 95 V.2.3. Measurement of Germination ...... 96 V.2.4. Molecular Modeling ...... 97 V.3. RESULTS...... 97 V.3.1. Germination Data...... 97 V.3.2. Molecular Mechanics Calculations ...... 98 V.4. DISCUSSION ...... 98 V.4.1. Conformational Analyses ...... 100 V.4.2. The Stimulant-Receptor Interaction Model ...... 101 V.4.3. Solvents ...... 103 V.4.4. Plausible Mechanism of Stimulation of Germination of Striga hermonthica Seeds by DHP (11) ...... 104 V.5. CONCLUSIONS...... 105

VI. ISOLATION OF DIHYDROELEPHANTOPIN, ISODEOXYELEP- HANTOPIN AND GRINDELIC ACID FROM THE FAMILY ASTERACEAE...... 110 V I. 1. IN TR O D U C TIO N ...... I l l V I. 1.1. Germacranes ...... I l l V I. 1.2. Elephantopus tomentosus L...... 112 V I. 1.3. Elephantopus carolinianus V/iUd...... 112 V I. 1.4. Grindelia maritima L...... 112 VI.2. MATERIALS AND METHODS...... 113 VI.2.1. Isolation of Dihydroelephantopin (10)...... 113

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VI.2.2. Isolation of Isodeoxyelephantopin (12) ...... 113 VI.2.3. Isolation of Giindelic Acid (14)...... 113 VI.3. RESULTS AND DISCUSSION...... 114

V n. RESPONSE OF TWO DIFFERENT ISOLATES OF STRIGA HERMONTHICA SEEDS TO VARYING CONCENTRATIONS OF STRIGOL ANALOG GR 7 ...... 127 V n.l. INTRODUCTION ...... 128 V n . 2. RESULTS...... 129 Vn.2.1. Effect o f Several Concentrations of GR 7 (9) on the Induction of Germination of Conditioned Striga hermonthica Seeds ...... 129 Vn.3. DISCUSSION ...... 129 Vn.3.1. Selection Criteria for Germination Stimulants ...... 129 vn.3.2. Factors which affect Germinability of Striga hermonthica Seeds ...... 130 Vn.4. CONCLUSION ...... 132

Vm . CADINANES ISOLATED FROM HETEROTHECA SUBAXILLARIS LAM...... 135 V m .1. INTRODUCTION ...... 136 V m .2. EXPER IM EN TAL...... 137 Vm.2.1. Plant Materials ...... 137 Vm.2.2. Extraction and Isolation ...... 137 Vni.2.3. NMR ...... 138 Vm.3. RESULTS AND DISCUSSION...... 139 Vm.3.1. Oc NMR Spectral Assignments in 2-hydroxy-8a- angeloyloxycalamenene (13) ...... 139 V m .3.2. 0(2 NMR Spectral Assignments in 2-hydroxy-8a- hydroxycalamenene (42) ...... 141

DC. COMPOUNDS ISOLATED FROM THE WEST AFRICAN HARRIS- ONIAABYSSINICA OLIV. (SIMARUBACEAE)...... 161 DC. IN TR O D U C TIO N ...... 162 DC.1. Lim onoids ...... 162 DC.2. M ATERIALS A N D M ETH O D S...... 165 DC.2.1. Structure Elucidation ...... 165 DC.2.2. Isolation o f Obacunone (45) ...... 166 DC.2.3. Isolation of Pedonin (46), Harrisonin (47) and 12P- acetoxyharrisonin (48) ...... 167 DC.2.4. Isolation o f (+)-ononitol (49) ...... 167 DC.2.5. Bioassays...... 168 DC.3. RESULTS AN D DISCUSSION...... 168 DC.3.1. Effects of (+)-ononitol (49) and Limonoids on Germina tion o f Striga hermonthica Seeds...... 173

X. FUTURE RESEARCH...... 201

REFERENCES...... 204

APPENDIX I: Letter of Permission to use Published Materials...... 225

VITA ...... 226

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

L I . Striga species, their economic hosts and distribution ...... 14

XL 1. Relative area (%) of components in dichloromethane extracts of Striga seeds ...... 37

m . 1. Species and common names o f legumes studied ...... 56

m .2. Percentage germination o f conditioned Striga hermonthica seeds treated with aqueous solutions o f strigol analogs, GR 24 ( 8 ) a n d O R 7 (9 ) ...... 57

m .3. Mean percentage germination of Striga hermonthica seeds treated with dichloromethane (DCM) or water extracts from leaves, stems and roots of legume cultivars ...... 58

m .4.1. Effect of dichloromethane (DCM) extracts from leaves o f legume cultivars on germination percentages o f Striga hermonthica seeds ...... 59

m .4.2. Effect of dichloromethane (DCM) extracts from stems o f legume cultivars on germination percentages o f Striga hermonthica seeds ...... 60

m .4.3. Effect of dichloromethane (DCM) extracts from roots of legume cultivars on germination percentages of Striga hermonthica seeds ...... 61

m .5.1. Effect of aqueous extracts from leaves of legumes on germination percentages o f Striga hermonthica seeds ...... 62

m .5.2. Effect of aqueous extracts from stems o f legumes on germination percentages of Striga hermonthica seeds ...... 63

m .5.3. Effect of aqueous extracts from roots of legumes on germination percentages o f Striga hermonthica seeds ...... 64

m .6. Source of variation in percentage germination of Striga hermonthica seeds as explained by the correlations (r^) ...... 65

m .7. Thin-layer chromatography (TLC) mobilities of compounds in dichloromethane (DCM) extracts ...... 66

m .8 . Composition (%) of sterols isolated from leaves ...... 68

V .l. Calculated conformational energies (kcal/mol) of strigol (2), GR 24 (8 ) and DHP (11) ...... 108

vm . 1. correlation of 2-hydroxy-8a-angeIoyloxycalamenene (13) (400 MHz, CDCI3) ...... 145

Vm.2. 1H- and NMR spectral data ( 6) for 2-hydroxy-8a-hydroxycalamenene (42) (4(10 M H z, CDCI 3X...... 146

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DC.1. correlation o f obacunone (45) (400 M Hz, CDCI 3)...... 174

DC.2. Coordinates and equivalent isotropic thermal parameters ...... 177

DC.3.1. Selected bond lengths (Â) ...... 178

DC.3.2. Selected bond angles (deg.) ...... 178

DC.3.3. Selected torsion angles (deg.) ...... 179

DC.4. Percentage germination o f conditioned Stnga hermonthica seeds treated w ith aqueous solutions o f obacunone (45), pedonin (46), harrisonin (47) and (+)-ononitol (49) ...... 180

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

LI. Compounds investigated for Striga seed gennination ...... 21

U. 1 A and B. Total ion chromatogram profiles of compounds detected in dichloromethane extracts of di^ (A) and conditioned (B) Striga hermonthica seeds ...... 39

H.2 A and B. Total ion chromatogram profiles of compounds detected in dichloromethane extracts of dry (A) and conditioned (B) Striga gesnerioides seeds ...... 40

n.3 A and B. Total ion chromatogram profiles of compounds detected in dichloromethane extracts of dry (A) and conditioned (B) Striga asperas&tàs...... 41

in. 1. Effect of chondiillasterol (15) on germination o f conditioned Striga hermonthica seeds occurring in Bida (A), Kano (B) and Abuja (C and D) locations in Nigeria ...... 69

ni.2. Effect of chondrillasterol (15) on germination o f conditioned Striga aspera seeds occurring in Kano (E) location in Nigeria ...... 71

IV. 1. Effect of ACC (16) on germination of isolates of conditioned Striga hermonthica seeds occurring in Bida (A ), Kano (B) and Abuja (C and D) locations in Nigeria ...... 90

IV .2 . E ffect o f ACC (16) on germination o f isolate of conditioned Striga aspera seeds collected in Kano location (E) in Nigeria ...... 92

V .l. Effect of dihydroelephantopin (DHP) (11) on germination of conditioned Striga hermonthica seedk ...... 107

VI. 1. Effect of dihydroelephantopin (10) and isodeoxyelephantopin (12) on germination o f isolate of conditioned Striga hermonthica seeds occurring in Bida (A), Kano (B) and Abuja (C and D) locations in N ig e ria ...... 119

VI.2. Effect of dihydroelephantopin (10) and isodeoxyelephantopin (12) on germination of isolate of conditioned Striga aspera seeds collected in Kano location (E) in Nigeria ...... 123

VI.3.1. Effect of grindelic acid (14) on germination of conditioned Striga hermonthica seeds occurring in Bida (A ), Kano (B) and Abuja (C and D) locations in Nigeria ...... 124

VI.3.2. Effect of grindelic acid (14) on germination of isolate of conditioned Striga aspera seeds collected in Kano location (E) in N igeria ...... 126

v n .l. Effect of GR 7 (9) on germination of conditioned Striga hermonth ica seeds collected in Abuja (A and B) and Bida (C) locations in N igeria...... 133

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vm .1. DEPT 90,135 and BB NMR spectra of 2-hydroxy-8a-angeloyloxycalamenene (13) ...... 147

VnL2. 2D NMR COSY spectrum of 2-hydroxy-8a-angeloyloxycalamenene (13) ...... 148

Vm.3. 2D NMR NOESY spectrum of 2-hydroxy-8a-angeloyloxycalamenene (13) ...... 149

Vm.4.1. 2D HETCOR spectrum of 2-hydroxy-8a-angeloyloxycalamenene (13) ...... 150

vm .4.2. Upfield region of HETCOR spectrum of 2-hydroxy-8a-angeloyloxycalamenene (13) ...... 151

Vm.5. Downfield region of COLOC spectrum of 2-hydroxy-8a-angeloyloxycalamenene (13) ...... 152

V m .6. 2D NMR COSY spectrum of 2-Hydroxy-8a-hydroxycalamenene (42)...... 153

Vffl.7. 2D NMR NOESY spectrum of 2-Hydroxy-8a-hydroxycalamenene (42)...... 154

V m .8 . DEPT 90,135 and BB ^^C NMR of 2-Hydroxy-8a-hydroxycalamenene (42)...... 155

Vm.9.1. The 2D HETCOR spectrum of 2-Hydroxy-8a-hydroxycalamenene (42)...... 156

Vm.9.2. Upfield region of HETCOR spectrum of 2-Hydroxy-8a-hydroxycalamenene (42)...... 157

Vm.9.3. Effect of 2-Hydroxy-8a-angeloyloxycalamenene (13) on germination o f conditioned S. hermonthica seeds occurring in Bida (A), Kano (B) and Abuja (C and D) locations in N igeria ...... 159

Vm.9.4. Effect of 2-Hydroxy-8a-angeloyIoxycalamenene (13) on germin ation o f conditioned S. aspera seeds occurring in Kano (E) locat ion in N igeria ...... 160

DC.1. DEPT 90,135 and BB ^^C NMR spectra o f obacunone (45) ...... 181

DC.2. 2D ^H NM R COSY o f obacunone (45 )l...... 182

DC.3. 2D ^H NMR NOESY spectrum of obacunone (45) ...... 183

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DC.4. 2D HETCOR spectrum of obacunone (45) ...... 184

DC.5. COLOC spectrum of obacunone (45) ...... 185

DC.6 . NMR spectrum of pedonin (46) . 186

DC.7. 2D iH NMR COSY spectrum of pedonin (46) ...... 187

DC.8 . 2D NMR NOESY spectrum of pedonin (46) ...... 188

DC.9. 13c NMR spectrum of pedonin (46) ...... 189

DC. 10. DEPT 90,135 and BB NMR spectra of pedonin (46) ...... 190

DC. 11. 2D 13c -1h HETCOR spectrum o f pedonin (4 6 ) ...... 191

DC. 12. 2D 130-lH COLOC spectrum of pedonin (46) ...... 192

DC. 13. iH NMR spectrum of harrisonin (47) ...... 193

DC. 14. Downfield region of COSY spectrum o f harrisonin (47) ...... 194

DC. 15. 2D iH NMR NOESY spectrum of harrisonin (47) ...... 195

DC. 16. iH NMR spectrum of 12|3-acetoxyharrisonin (48) ...... 196

DC. 17. 2D iH NMR COSY spectrum of 12P-acetoxyharrisonin (48) ...... 197

DC. 18. 2D iH NMR NOESY spectrum of 12P-acetoxyharrisonin (4 8) ...... 198

DC. 19. BB 13c NMR spectrum of 12P-acetoxyharrisonin (48)...... 199

DC.20. DEPT 90,135 and BB 13c NMR spectrum of 12P-acetoxyharrisonin (48) ...... 200

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF SCHEMES

LI. Life cycle of Striga...... 4

1.2. Bemer et al. (1995) scheme o f integrated 5mga-controI (modified to include chemical control) ...... 8

1.3. Oxidation of dihydrosorgoleone (3) to quinone (4)...... 15

1.4. Reduction of methylene blue (5) with hydroquinone (3) to yield the transparent reduced form ( 6) ...... 15

1.5. Extraction and bioassays of germination stimulants and/or inhibitors ...... 23

IV .l. Mechanism of Adams and Yang (1979) for the biosynthesis of ethylene ...... 85

IV.2. Formation o f ethylene by the chemical oxidation of 1 -phenyIcyclopro- pylamine (Hiyama et at., 1975) ...... 86

IV.3. Pirrung and McGheehan (1983) mechanism of formation o f ethylene ...... 87

rv.4. Proposed mechanism of Lizada and Yang (1979) for the oxidation of ACC with hypochlorite ...... 88

IV.5. A possible mechanism of action of ethylene ...... 89

V .l. Proposed mechanism for biosynthesis of ethylene from DHP (11) ...... 109

Xll

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

Obligate root-parasitic flowering plants of the genus Striga (fa m ily

Scrophulariaceae) cause considerable yield reductions o f various crops in tropical and

semi-tropical countries. Mature Striga species (witchweeds) plants produce copious

quantities of minute seeds which remain dormant in the soil for many years until

exudates from roots o f various host plants induce germination. Economically effective

means of control of Striga species are not yet available for small-scale farmers in

developing countries. In the present study data is presented which indicate that the two

most effective Striga control measures are crop rotation and suicidal germination.

For the first time we have analyzed the dichloromethane extracts of dry and

conditioned Striga species seeds by means of gas chromatography coupled to mass

spectrometry. Sixteen compoimds were identified on the basis o f their mass spectra and

their retention indices. A ll Striga species extracts contained tetradecanoic acid, cis,cis-

9,12-octadecadienoic acid, c/j-9-octadecenoic acid and sitosteroL Also, 2,6-dimethoxy-

p-benzoquinone (2,6-DMB(3) and several long chain aldehydes and n-hydrocarbons

were detected in some of the extracts. The nature of the chemical changes induced by

seed conditioning are discussed.

Dichloromethane and water extracts of various parts of legume cultivars were

tested for stimulation of germination of Striga species seeds as described in the

experimental sections. Also, several pure compounds isolated were assayed. Dilution

methods, which are very sensitive and better suited for quantifying germination

stimulant activity, were employed in identification of high and low stimulant producing

legume cultivars. New and interesting relationships between stimulatory activity and

concentration emerged. Most pure compounds tested induced significant germination

o f isolates o f Striga species seeds across a broad concentration range, from 10*3 to 10'

20 M. The mechanism of Striga germination proposed may foster synthesis of more

effective germination stimulants and/or inhibitors.

xui

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I

INTRODUCTION

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1. PEST MANAGEMENT. Producing enough food for an ever-growing

population is the biggest problem facing the human race. Efficient use of diminishing

farmland and maximization of crop returns is becoming ever more prevalent The difficulty o f this is not eased by the fact that worldwide, a large proportion of crops are

lost to insects, diseases, weeds, and parasitic plants. In the developing world where

farmers and governments struggle to feed hungry mouths, the cost o f the damage caused

by these pests on agriculture is o f utmost concern.

For many years, the main weapons against pests were chemical. From the early

sprays of tar and copper sulfate to the sophisticated modem cocktails, chemical pest

control has had much success. Traditionally, the agricultural community has relied

extensively on synthetic pesticides such as organo-chlorines, -phosphates, and

dinitrophenols (Ley et al., 1993 and references therein). Chemicals, however, undergo

biological amplification in the food chain and can be harmful to w ildlife, the environment,

and humans. Furthermore, they are expensive, and farmers may have to use more and

more because the pests develop resistance to chemicals. For these and other reasons,

there is an urgent need to develop alternative and ecologically acceptable methods of

controlling the pests.

1.2. PARASITIC PLANTS. Parasitic plant classification is based on several

criteria. Holoparasites lack chlorophyll and depend entirely on the host for nutrients and

minerals; examples include Cuscuta (Cuscutaceae) and Orobanche spp. (Orobanchaceae)

(Calder and Eager, 1979). Hemiparasites have chlorophyll and thus can fix carbon

dioxide but depend on the host for water and minerals; examples include Striga spp.

(Scrophulariaceae) and misletoes (Loranthaceae). Some parasites attack the shoot, others

the root. Parasites that require attachment to the host to complete their life cycle are

obligate parasites whereas facultative parasites can complete their life cycles without the

h o st

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. From an evolutionary standpoint, parasitic plants have lost many organs and they

have also evolved a unique set of characteristics essential to their mode of life. The

haustorium is the most important o f these. Early anatomical evidence suggested that the

haustorium o f parasitic plants served only as a bridge, transporting sap from host to

parasite. More recent studies show that the organ has a complex structure (Musselman,

1987). The haustorium is prim arily involved not only with transport but also with the

metabolism o f nutrients received from the host. The parasite and its host contain different

sugars and amino acids, and the haustorium cells may be responsible for converting

nutrients from the host into a form more suitable to the parasite. The genesis o f haustoria

is s till a subject of speculation. It is envisioned that the roots o f the parasite penetrate

those o f the host by both mechanical and enzymatic action.

1.3. BIOLOGY OF STRIGA. The genus Striga (Scheme I.l), which belongs to

the fam ily Scrophulariaceae, is among the most agronomically important parasites

particularly in Africa. A ll Striga species are obligate root parasites that strongly affect the

food resources of Africa and Asia. Botanically, the genus is characterized by opposite

leaves, irregular flowers with a corolla divided into a tube and spreading lobes,

herbaceous habit, small seeds, and parasitism (Musselman, 1987). In the field, Striga, or

witchweed as it is commonly known, looks like an ordinary green weed and has very

attractive flowers. It earned its common name “ witchweed” because although farmers

realized that the parasite's appearance in a crop meant a large loss o f yield, the farmers did

not understand the weed's parasitic nature. As far as the farmer was concerned, the crop

had been bewitched (Nour et al., 1986). In fact the word "Striga" in Latin means witch,

and describes the condition o f the host plant, which (when the parasite is s till invisible in

the soil) expresses apparently inexplicable disease symptoms.

Striga's unique parasitic lifestyle and has allowed its adaptation to the humid and

semi-arid Savannas and Sahel regions in the tropics. Parasitism is a biological attempt to

gain something for nothing, and Striga is evolutionarily adapted to this mode of life.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dispersal Dormancy (After-ripening)

Exposure to moisture, etc. (Conditioning) Seed shed

Exposure to exogenous Flowering stim ulant

Emergence Germination

Underground growth

Successful! attachment Haustoria formation

Scheme L I. L ife cycle o f Striga.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Surprisingly, the green photosynthetically active plants o f Striga rely on the host for most

o f their carbohydrates. Research (Nour et al., 1986) has shown that mature Striga plants

have low rates o f photosynthesis, even though the structure of their chloroplast is normal.

One reason is that the key enzyme o f photosynthesis, ribulose bisphosphate carboxylase,

is present only in small amounts. Thus the overall biosynthetic activity o f Striga is low .

A t the metabolic level, Striga differs from its host in a number of ways. One notable

difference is that mannitol, a sugar alcohol, is an end product o f photosynthesis in Striga,

but is absent from hosts such as sorghum and m illet. This difference raises an attractive

possibility that a substance targeted against the metabolism of mannitol might be an

effective herbicide against photosynthesizing Striga. spp

1.4. DISTRIBUTION OF STRIGA. As a whole, the genus Striga comprises

about 35 species in the Old World tropics. Striga spp. did not exist in the New Continent

but were recently been introduced (Musselman, 1987). The diversification of the genus

occurred first on the Old Continent (Table I.l). A secondary diversification center may

be Australia. Considering diversity and endemicity, the main diversification center o f the

genus seems to be located in the West- and Central Africa. It is probable that the

differentiation of Striga species arose after geographic separation o f the two continents.

The majority of Striga species are o f no agronomic importance, but those that

parasitize tropical cereals and legumes can be extremely damaging. These economically

important species include S. asiatica (L.) Kuntze, S. aspera (W illd.) Benth, S.

hermonthica (Del.) Benth and S. gesnerioides (W illd.) Vatke. S. hermonthica, is without

doubt one of the most important parasitic weeds of the world. It is the most damaging

species in sub-Saharan Africa, affecting staple cereals such as maize, sorghum, m illet,

rice and sugarcane. It is worth noting that maize, sorghum and m illet are among the

world’s most important food crop, and for the inhabitants of the sub-Saharan Africa, they

are the main sources of protein and energy (Wendorf et al., 1992). W ith the possible

exception of birds, S. hermonthica may be the single most important cause of yield

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reduction in sorghum and m illet in the whole of Africa (Parker and Riches, 1993). It is

estimated that Striga species threaten the lives o f over 100 m illion people in Africa and

infest two-thirds o f the 73 m illion hectares devoted to cereal crop production in African

savannaas alone (Lagoke et al, 1991). The greatest damage occurs in the savanna areas

stretching from Cape Verde on the west coast through west, central, east and southern

Africa. These zones also constitute the major food legume producing areas in most o f the

countries affected (Robson and Broad, 1989). For example, in Nigeria alone, the most

populous country in Africa, S. hermonthica causes crop losses of 10 to 90 percent in

maize and sorghum.

In 1956, the parasitic weed S. asiatica was found in the USA (Wunderlin et al.,

1979). This parasite has a wide geographic distribution and broad range of hosts

including many important crops (Saldhanha and Nicolson, 1976). In January 1979, an

infestation o f S. gesnerioides was reported in central Florida. This species is parasitic on

a number of broad-leaved plants in the family Leguminosae (Cowpea, Soybean,

Tephrosia pedicellata Bak.), including Indigofera, and members o f various other plant

families, including Solanaceae, Convolvulaceae (Jpomea, e.g. Sweet potatoes;

Jacquemontia tamnifolia (L.) Griseb. and Merremia tridentata L.), Euphorbiaceae

{Euphorbia), Liliaceae (Sansevieria), Acanthaceae {Lepidagathis) (Wunderlin et al., 1979)

and also some Gramineae. This parasite causes leaf chlorosis, die-back and malformation

o f parasitized cowpea plants. Yield losses due to S. gesnerioides infestation range from

26 to 50% (Aggarwal, 1987).

1.5. T H E STRIGA P R O B LE M . Striga spp. thrive in areas of low soil fe rtility and

low rainfall, causing problem for small-scale resource-poor farmers, who are often

confined to these agronomically poor areas. Control of Striga is more difficu lt than the

control o f non-parasitic weeds (Ogbom, 1987). The Striga spp. life cycle (Scheme I.l)

is closely coupled to their environment and to their host. Striga species produce large

quantities of very tiny seeds which can remain dormant in the soil up to 14 years. The

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. seeds w ill only germinate on exposure to both favorable environmental conditions and to

a germination stimulant, usually a host-root exudate. A single Striga plant can produce up to 500,000 seeds each as small as S^ig (200,000 seeds per gram). These seeds have

complex dormancy and germinate at different times. Therefore they can parasitize crops

over an extended period. As many as 50 Striga plants can parasitize a single host plant.

The control problem of Striga is compounded by the fact the very tiny seeds are spread

predominantly by activities o f man (Bemer et al, 1995).

Striga species exhibit great genetic variability and form haustoria as a common

feature. Through this special organ, the parasites attach themselves to the roots o f the

host plants and do considerable damage before they emerge above the ground. Striga

damages food production in three ways. First, there is a direct loss in yield because of

parasitism. The high-yielding hybrids of sorghum developed recently by Nour et al.

(1986) are particularly vulnerable. Secondly, the small-scale African farmers lose by

switching to low-yielding crops, such as m illet, which are less susceptible but have lower

yields. Thirdly, farmers abandon land as a result o f heavy infestation. In some areas,

whole villages move to escape the parasite infestations.

1.6. PRACTICES TO CONTROL STRIGA SPECIES. The widespread and

severe problem o f parasitism by Striga spp. in crop production differs from that caused

by other weed species. Striga spp. damage their hosts through parasitism and induction

o f disease as opposed to weeds which cause damage by competition. Striga spp. seeds

possess complex dormancy and not all the seeds conditioned for germination at the same

time. This compounds the problem of devising a persistent and adequate control method.

Among the measures employed to combat the parasite are hand-weeding, "trap" and

"catch" cropping, use of nitrogen fertilizers, herbicides, resistant crop varieties, and

germination stimulants (Musselman, 1980; Ayensu e ta l, 1984; Lagoke etal, 1991). The

control measures vary in effectiveness, cost, and knowledge required by the farmer.

None of these measures by itself seems to provide effective control. An integrated

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. package involving various methods w ill be the most effective approach to control. The article by Bemer et al. (1995) gives a complete coverage o f Striga research in Africa.

Although our attention is focused on the use of germination stimulants in Striga spp.

control (Scheme 1.2), an overview of Striga control measures implementable by small-

scale African farmers is presented below.

[ Striga-ftQ& Planting M aterial)

[ Chemical C ontrol BiocontrolJ

Crop Rotation

( Host Seed Treatments Transplanting

[ Host-Plant Resistanc^

Scheme 1.2. Bemer et aL (1995) scheme of integrated .Srriga-control (modified to include chemical control).

1.6.1. Hand or Hoe Weeding. This is the most common practice used by small-

scale farmers. It is worth noting that Striga does most of its damage to the host crop

before they emerge. Hand pulling could be effective in controlling a light infestation and

especially if done before or during the flowering of Striga (Pieterse, 1985). However,

hand pulling is a very tedious, labor-intensive and expensive operation.

1.6.2. Land Preparation. This practice has been tested by the United Nations (UN)

Food and Agriculture Organization (FAQ) in West Africa. It was believed that if tillage

could be avoided, Striga seeds would remain on the surface o f the soil where conditions

would eliminate their chances of attacking the crop (Parker and Reid, 1981). This

method did not work well in most of the trials. Deep cultivation to bury the Striga seeds

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. has been used in some parts of Africa for quite some time. The effectiveness of this

method is lim ited since the seeds remain viable and can resurface over time.

Moreover, deep cultivation is not only tedious and expensive but may not be very

practical to the A&ican farmers especially considering their rudimentary farm machinery.

1.6.3. Rotation of Land into Fallow. This was once the traditional farming

system o f most o f the affected areas. Land was allowed to return to bush fallow for 10 to

20 years during which time the Striga seed bank in the soil would become depleted and

crops could be grown again. Where practiced, the rotation periods have declined such

that there is little effect on the Striga seed population. This method is limited by

increasing human population.

1.6.4. Breeding fo r Resistance/Tolerance. The development of resistant/tolerant

crop cultivars is one reliable approach to controlling Striga. Much effort is taking place

w ithin crop breeding departments o f national research programs and at the International

Institute o f Tropical Agriculture (Parker and Riches, 1993). Considerable progress has

been made, but there are difficulties. Resistant or tolerant crop cultivars can have

lim itations such as low quality grain. Also, new Striga strains occur or may emerge that

thwart resistance (Musselman, 1987). In effect, all resistant or tolerant crop varieties

allow infection by the parasite to varying degrees and this provides sources of re

infestation. Furthermore, breeding for resistance to Striga requires effective screening

techniques. The development o f reliable methods for screening large numbers of host

genotypes in the field has been effective through establishment of uniform levels of

infestation for reliable and reproducible results (Kim, 1991).

1.6.5. Fertilizers. Striga spp. are most abundant on cereals and very damaging on

soils o f low fertility. There are many conflicting reports on the control of Striga through

management o f soil fertility (Pieterse and Pesch, 1983). The application o f nitrogenous

fertilizers may reduce infestation (Pesch et al., 1983), but these fertilizers remain

unavailable in many parts of Africa and are often too expensive for many farmers.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10

1.6.6. Herbicides. Direct application of foliar herbicides after the emergence of

parasitic weeds has been observed to give effective reduction o f the parasites, but the cost

and the need to take precautions to avoid health hazards make this practice d iffic u lt It

should be noted that the damage by Striga spp. is done before emergence.

1.6.7. False-host or "Trap" Crops. "Trap" crops are nonhosts which stimulate

germination o f the Striga seeds without being attacked by the seedlings. When seedlings

do not find a suitable host they use up their endosperm and die within 3-7 days. Trap

crops can be cultivated alone or they can be rotated with a susceptible host crop, thus

eliminating the parasitic weed seeds that would otherwise germinate and attack the

susceptible host crop. This control approach is a promising method and is economically

advantageous for the farmer (Musselman, 1987). Since the false-host is a crop, it is

economically viable for the farmer. Also, unlike many other inexpensive control

methods, the false-host crop reduces the parasitic weed seed numbers in a field, thus

reducing the probability of more severe future crop damage. However, little is known

about false-host crops and the compounds responsible for stimulation (Lagoke et al.,

1991). More research is needed to identify the most effective plant species and cash and

food crop cultivars that can serve as false-hosts. Also, false-hosts must be identified that

address the problem o f the diversity of Striga spp. in the same field.

Although control of Striga through use of non-host crops in rotation appears to be

an attractive measure, several complicating factors hinder successful implementation.

One of these is the diversity o f Striga spp. that can be found in farmer's fields. A control

practice for one may be ineffective for the otherfs). O f particular complexity for control is

the common admixture of S. gesnerioides, a parasite of cowpea (Vigna unguiculata (L .)

Walp.), and S. hermonthica, a parasite of most cereal crops in Africa. Cowpea is a

common and important food source particularly in West Africa, but can be severely

damaged by S. gesnerioides. Because cowpea is not a host o f S. hermonthica b u t can

stim ulate S. hermonthica seed germination, certain cowpea cultivars can be used in

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11

rotation with cereals to provide S. hermonthica control. However, few non-hosts have

been identified to provide complementary control of S. gesnerioides on cowpea. Use of

rotation crops such as soybean and forage legumes, which are nonhosts to either parasite,

present a solution to this problem. Variability among cultivars of nonhosts in their ability

to stimulate parasite seed germination can be utilized and the most effective cultivars

/germplasm selected. Literature indicate that little work has been done on identifying

efficacious cultivars of nonhosts for S. hermonthica and S. gesnerioides control (Ariga et

a/., 1993).

1.6.8. Germination Stimulants. In addition to the normal germination

requirements common in many angiosperms, dormant Striga seeds germinate only in

response to chemical signals emitted by the roots o f suitable plants (Chang et al., 1986;

N etzly et al., 1988; Worsham, 1988). In the absence o f a stimulant-producing plant,

ethylene (1), strigol (2) and strigol analogues can, in the immediate vicinity (4 mm) of the

Striga spp. seeds, induce suicidal (“abortive”) germination o f Striga seeds in the soil

(Eplee, 1975; 1984). This leads to a reduction in the Striga seed bank in the soil before a

crop is planted. In the United States, S. asiatica is effectively eradicated using ethylene

(1), a natural, ubiquitous compound, is injected into the soil (Eplee, 1992; Sand and

Manley, 1990). When properly used, this method can be highly effective (Eplee, 1983).

In developing countries, this method is of lim ited potential use because ethylene (1) is a

gas under relatively high pressure and its use for large-scale control requires a high level

of skill as well as transport and application equipment that are not available or are

prohibitively expensive for the farmers in developing countries (Ogbom, 1987; Sand and

Manley, 1990). Moreover, drier soil conditions (Bebawi and Eplee, 1986) reduces the

efficacy of ethylene (1) on stimulation of Striga spp. seeds in Africa. For these reasons

the search for other naturally derived inexpensive, and easily applied germination

stimulants for control of Striga seems continues.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12

Root exudates o f natural host plants such as cereals and legumes produce, in

infinitesimal levels, a mixture o f very active stimulatory compounds w ith one compound

being predominant over the others (Worsham, 1987; Weerasuriya et al., 1993). From the

natural host sorghum, host-specific signals, collectively called sorgoleones, were recently

isolated (Chang et al., 1986; Netzly et al., 1988). The highly unstable sorgoleone 2-

hydroxy-5-methoxy-3-[(8’Z, irZ )-8 ’, ir i4 ’-pentadecatriene]-p-hydroquinone (3) is

rapidly oxidized to non-stimulatory 2-hydroxy-5-raethoxy-3-[(8’Z, irZ )-8 M ri4 ’-

pentadecatriene]-p-quinone (4) (Scheme 1.3). Since the hydroquinone (3) appears to be

the essential component o f the exudate in the induction o f Striga germination, methods

have been designed to specifically localize hydroquinone production along the sorghum

root surface (Chang, 1986 and Chang et al., 1986). Methylene blue (5), a commercially

available heterocyclic pigment, can be reduce electrochemically (E°' = + 20 mV at pH

7.6) (Clark, 1960) or with chemical reductants like sodium dithionite. The

hydroquinone, structure (3), reacts stochiometrically with methylene blue (5) to give the

visibly transparent 2e" reduced form. In the presence o f O 2, the reduced dye autoxidized

back to the blue form ( 6 ) (Scheme 1.4) and structure 3 has been isolated intact.

Therefore, in the presence o f O 2, this pigment can be used to investigate the persistence

of the production of structure 3. The oxidative lability of the crude exudate from

sorghum has already been documented (Chang et a i, 1986), and the t l /2 for biological

activity of a 10’^ M solution in H 2O at room temperature range from 12 to 24 h. The rate

o f autoxidation o f the pure hydroquinone (3) as quantified by UV and HPLC has been

found to be at least two orders o f magnitude faster than the exudate. In general, the root

exudates of grasses have been shown many years ago to be very complex mixtures

containing sugars, amino acids, organic acids, nucleotides, phenolics, and enzymes

(Rovira, 1965). Specifically, hydroquinone (3) is a general feature o f Striga's hosts. It

is possible that such a metabolically expensive process could be defensive in nature. In

that regard, related quinones have been shown to be metabolic toxins (Wong et al., 1985)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13

serving as phytoalexins (Bundenburg et aL, 1949). The autoxidation of the

hydroquinone (3) results in the generation o f reactive and toxic oxygen intermediates.

The bis-allylic methylenes o f the side chain are also known to react with oxygen radicals.

Related compounds containing both catechol (7) and olefinic (C=C) functionalities

polymerize (Takada et al., 1988) and, in fact, formed the basis o f the original lacquer

industry. Such a polymerization may generate other structures o f relevance to the

rhizosphere or a polymerization may even function to stabilize the soil around the roots.

Any o f these mechanisms could provide general protection for the sorghum roots. A

more appealing possibility would involve a specific role for these compounds.

Azospirillium spp. and nitrogen fixing bacteria colonize many o f the same species that

Striga parasitizes (Dobereiner and Pedrosa, 1987). These exudates could induce specific

root colonization by reducing the oxygen tension around the host root (most of these

bacteria can m ultiply and fix only at reduced O 2 pressures), serving as a specific

reductant or terminal electron acceptor for bacterial respiration, or even as a specific

bacterial chemoattractant (Egley and Dale, 1970). Any specific role that these compounds

might play awaits further investigations.

" x " H H

Ethylene (1)

,0 H (+) - strigol ( 2 ) '

Catechol (7)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14

Table 1.1. Striga species, their economic* hosts and distribution

Striga species Host Crop Countries

S. hermonthica sorghum, millet, maize. Africa, Yemen upland rice, fonio, tef, Saudi Arabia sugarcane, fînger millet, lowland rice S. aspera maize, millet, sorghum Africa upland rice, sugarcane, fonio S. asiatica maize, millet, sorghum. Africa, India upland rice, tef. U.S.A., China, sugarcane, finger millet Philippines S. gesnerioides cowpea, tobacco. Afnca, U.S.A, sweet potato, tomato. Saudi Arabia, Oman, Yemen, India S. forbesii sugarcane, lowland rice, Africa maize, sorghum. S. densiflora cereals Oman, India, Indonesia S. angustifolia cereals Africa, India, Indonesia S. passargei maize, sorghum Africa S. klingii sorghum, m illet Africa S. latericea sugarcane Africa S. brachicalyx sorghum, m illet Africa S. aldelii sorghum, m illet Africa S. baunumnii sorghum, m illet Africa S. bibabiata sorghum, m illet Africa

* Most species also parasitize wild monocots or dicots.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15

Oxidation H3C O ' R

HsCO* 0

OH Qninone (4) Dihydrosorgoleone (3) Does not Stimulate

Striga germination R =

- Secreted by sorghum roots

- Stimulate Striga germination - Rapidly oxidized to the quinone form - Germination occur in the immediate vicinity o f roots

Scheme 1.3. Oxidation o f dihydrosorgoleone (3) to quinone (4).

H I

I \

Methylene blue (5) Methylene blue ( 6); reduced form

Scheme 1.4. Reduction of methylene blue (5) with hydroquinone structure 3 to yield the transparent reduced form ( 6). Autoxidation occurs on exposure to air.

Strigol (2) was the first naturally occurring germination stimulant to be isolated

from root exudate of cotton (Gossypium hirsutum L.), a non-host plant (Cook et al.,

1972). This sesquiterpene is hitherto the most potent Striga germination stimulant

known, demonstrating activity at concentrations lower than 10"^^ M. This potent

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16

behavior is suggestive of a hormonal mode of action (Cook et aL, 1972; Siame et aL,

1993). However, strigol (2) is produced in low concentrations, cannot be isolated in

pure form and its synthetic procedures are lengthy, requiring about 2 0 steps, and thus

uneconomical to be o f value in Striga control In contrast to sorgoleones, Strigol (2) and

its synthetic analogs (GR-compounds), are relatively stable in soils (Babiker et aL, 1988;

Hsaio et aL, 1983 and Eplee, 1984). A number o f synthetic strigol analogs have

demonstrated success in vitro germination of Striga seeds (Johnson et aL, 1981 ; Babiker

et aL, 988). Other compounds which have been found to promote germination o f Striga

seeds include the growth hormones; kinetins, zeatin, scopletin, inositol, and inorganic

compounds such as sodium hypochlorite (Worsham, 1987).

There are attendant problems as far as Striga bioassays are concerned. Notably,

the germination stimulants are hydrophobic molecules and exhibit poor solubility in

organic solvents. Also, Striga seeds have inherent physiological differences making it

very difficult to reproduce germination data. Hitherto, the mechanism(s) o f stimulation of

Striga seeds by germination stimulants remains to be understood (Worsham, 1987).

1.7. QUANTITATIVE STRUCTURE-ACTIVITY (QSAR) ANALYSIS.

The wide diversity o f secondary metabolites known to exist throughout the plant kingdom

has been attributed to host-parasite coevolution (Erlich and Raven, 1964). The elaborate

design o f chemical defences in plants can involve mixtures o f related structures in one

biosynthetic class or multiple lines o f chemical defence in a single plant species

(Berenbaum, 1985). Some host cultivars (cereals and legumes) have developed means of

defence against attachment by Striga spp.

To date, the reasons for differences in stimulation o f germination o f Striga spp.

seeds by closely related compounds are not understood. To answer the question “Why is

ethylene ( 1) or strigol ( 2) more potent than their congener?” , the relationship between the

chemical structures and stimulatory activity o f a series o f naturally occurring compounds

(Fig. 1.1) or synthetic derivatives o f strigol (2) was studied.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17

Quantitative structurc-activity relationship (QSAR) is a complementary approach based on statistical methods fo r correlation analysis. QSAR lends its e lf to the

development o f models predictive o f the level o f stimulatory activity and even the degree

of specificity of the compound under investigation. Because of the difficulty in isolating

a series of related natural products large enough to allow detailed structural factors

contributing to activity, QSAR studies are rare. A t least five compounds are needed for

each factor to be analyzed (Martin, 1978). The alternative approach o f synthesizing

derivatives of a prototype bioactive natural germination stimulant is hampered by the

complexity o f many natural products.

Chromatographic fractionation o f dichloromethane (DCM) extracts (Scheme 1.5),

guided by assays of fractions in stimulating Striga spp. seed germination, revealed that

the active principles were widely distributed, depending on the plant material. Our

approach was both qualitative (in which the stimulatory activity was enhanced or

diminished by the different functionalities in parent molecules) and quantitative (several

concentrations were tested). Detailed structure-activity-relationships (SAR) were derived

from the analysis o f tabulated germination data which recorded the responses o f Striga

spp. seeds to a set o f concentrations o f a series o f compounds. Because the number of

concentrations (10"^ to lO'^O M; each concentration replicated twelve times) and

compounds were large, the table was complicated and therefore we had to resort to

statistical methods to achieve significant data reduction and to reformat the germination

data in easy-to-read forms. Germination data were normalized by transforming to arc

sine and curves developed by plotting log[concentration (in Moles/L)] against arc sine

transformed percentage germination.

1.7.1. Basic Concepts of QSAR. In an equation similar to that of Hansch (1989),

the father of QSAR (Fig. 1.2), we propose the following:

Stimulatory activity =7(Hydrophobic + Steric + Electronic effects + inhibitor effects).

Where stimulatory activity is considered to be a function if) of physicochemical properties

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o f the compound. QSAR analysis involving Striga seeds were expressed in terms o f the

molar concentration of germination stimulant. Hydrophobicity is a primary factor

influencing passive transport processes for germination stimulant from its source to its

site of action. Moreover, hydrophobicity has been shown to be very important in

regulating the interaction o f bioactive compound with their bioreceptors (Compadie et al,

1990). The role of hydrophobicity in QSAR analysis has been extensively reviewed

(Kabunyi, 1979; Leo et aL, 1971). Steric (Compadre et at, 1990; Hansch and Klein,

1986) and electronic (Dunn, 1977) parameters have also been described. We believe that

the inhibitor effects which were detected in dichloromethane extracts o f dry Striga seeds

(see Chapter II) affect the stimulatory capability of a given compound. The relationship

between inhibitory and stimulatory activity remain to be understood. The Hansch et at.

(1989) QSAR paradign is depicted below:

Physiological Properties Biological Activity

Statistics

V QSAR

Mechanism o f Action‘d Prediction of Action

1.7.2. Bioassays. Bioassays are an important research method for testing hypotheses

concerning plant-parasite interactions. “ Bioassays” is defined in broad terms as: any

assay in which a living organism is permitted to declare whether a specific variable (e.g.,

presence or absence o f an allelochemical, age o f the plant tissue, concentration o f a

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19

germination stimulant, presence of a plant pathogen) “ makes a difference” as to how the

organism fares; Le., does the variable have an impact on the plant’ s life history? (Leather

and Einhellig, 1988). Currently, there are no guidelines on the best way to design Striga

bioassay in any particular instance. The complexity o f a Striga system often prohibits

controlling all relevant factors; it is rare to have the total control over the genetics o f Striga

seeds, the environment to which these organisms have been exposed, or a complete

understanding o f how the testing environment interacts with the test Such uncontrolled

sources of variation often enter into an experimental design and cause errors. Since our

experiment was intended to determine the stimulatory activiQr o f different concentrations

o f various crude extracts and pure compounds of known structures, using a laboratory

population of Striga spp. seeds was appropriate.

1.8. THE RESEARCH OBJECTIVES. Since Striga spp. seed germination

activity may not necessarily be lim ited to any single class or even to a few classes o f

compounds, and since many compounds of unusual structure are known to reside in

plants, this natural source appear to offer a good opportunity for obtaining new active

compounds. Thus it is important to randomly collect and test several plants. Any plants

whose extracts show reproducible stimulatory or inhibitory activity in the Striga

germination could be re-collected in quantity and fractionated fo r the active agent(s). The

active agents could then be evaluated like “ synthetics” . I f these active agents satisfy

certain criteria for stimulatory or inhibitory activity and chemical structural novelty they

could be tested in the greenhouse; i f again, they pass certain other requirements they

could be tested in the field. The isolation of germination stimulants/inhibitors illustrates

the usefulness of fractionation guidance by means o f Striga spp. bioassays as contrasted

with the classic type phytochemical procedure of isolating the constituents first and

studying the biological properties later. Many of the compounds isolated in this study

would have remained unknown i f their presence in crude plant extracts had not been first

indicated by their stimulatory/inhibitory activities. It was the original belief that the mass

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fractionation effort would turn up a large number of gem Jnation stimulants/inhibitors of

novel chemical structure. This hope was realized.

This study focused on improving the knowledge of the relative efficacy of known

nonhost plants o f Striga spp. which were o f promising economic importance, particularly

crops which were of interest to the resource-poor African farmers. Further research

could then lead to devising universal and improved methods o f Striga spp. control. This

information could lead to a better understanding of what false-host crop chemical

compoimds are responsible for Striga spp. seed germination. The residues, crude

extracts or purified compounds from the selected plants could possibly be used directly in

Striga infested fields. Information obtained could also give clues about how the efficacy

of other false-host crops can be predetermined through analysis of their chemical

compounds. Future research could use the chemical compound information to focus

either on the direct use of these compounds or on efficient breeding programs using the

presence and quantity of the compound(s) as selection criteria. This study could improve

the understanding of how false-host crops could be better used alone or in conjunction

with other parasitic plant control practices. Finally, and most importantly, the

reproducible bioassays of structure-activity studies o f the pure compounds isolated at

LSU could enable construction of plausible molecular mechanism(s) for germination

and/or inhibition o f .J/nga seeds.

The Research Objectives included:

(i). Evaluation of compounds in dry and conditioned Striga seeds by GC/MS.

(ii). Extraction o f the plant materials from known host (for one species o f Striga, but a

false-host for another species) and suspected nonhost legume cultivars using

dichloromethane (DCM) and water as solvents. The differences in the amount o f

germination stimulants in the extracts from leaves, stems and roots on stimulation

of germination of Striga species seeds (S. hermonthica, S. aspera and S.

gesnerioides was investigated.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21

(iii). Isolation o f pure compounds from the West African Harrisonia abyssinica. Oliv.

(Simarubaceae) and the Louisiana plants o f the Asteraceae family. Detailed

analyses o f structure- and concentration-activiQf relationships o f the compounds

isolated (Figure L I) are discussed.

Ohs d V'

GR 24 (8 ) G R 7(9) ' S O

y \0 ‘

Dihydroparthenolide (11) Dihydroelephantopin (10)

Figure 1.1. Compounds investigated for Striga seed germination (fig. con'd).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22

HQ ,x\0 O

2-hydroxy-8a-angeloyloxycalamenene (13)

Isodeoxyelephantopin ( 12)

.COOH

.%%( O

Grindelic acid (14) Chondrillasterol (15)

HzNy. " COOH

(ACC) 16

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23

[ Plant Material (Air-dried; 16.5 g) ]

15g

1.5 g/L—

(Bioassays]^ 0.15 g/L CH2CI2 extract 0.015 g/L 0.15 g/L g

C TLC: Hexane-Methanol] Thin-layer chromatography (TLC)

Column chromatography

silica gel elution gradient: Hexane Ethyl acetate-Hexane Ethyl acetate Methanol Acetone

Fractions (10 mL) Mass Spectrometry (MS) L p u r e } "(structure Determination Infra Red ( Nuclear Magnetic Resonance (NMR)] m

Scheme 1.5. Extraction and bioassays o f germination stimulants and/or inhibitors.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER n

GC/MS EVALUATION OF COMPOUNDS IN DRY AND CONDITIONED STRIGA SEEDS

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25

n.1. INTRODUCTION. Striga (Scrophulariaceae) is one of the most important

genera o f parasitic plants, infecting a range o f major crops (e.g. sorghum, millet, maize,

rice sugar cane and cowpea) in West and East Afnca and Asia (Cechin and Press, 1993).

The control of Striga spp. presents serious problems, because economically effective

means o f control that have been developed in the United States are either not yet available

or are prohibitively expensive for the resource-poor small-scale farmers in Third World

countries.

Striga spp. seeds have a complex germination biology which includes the need for

an exogenous stimulant, which in nature, is provided by root exudates o f various plants.

These stimulants may not necessarily be a single substance, but may be a complex of

various factors acting individually or synergistically and constituting what Schopfer

(1943) called a constellation. However, the exact mechanism through which exudates or

active substances from external sources induce Striga germination is still largely

unknown.

In the absence of, or insufficiency o f “ conditioning” , which involves exposure to

moisture in a warm environment, Striga spp. seeds respond either not at all or poorly to

germination stimulants. Striga spp. seeds are sensitized to the germination stimulants by

conditioning (Vallance, 1950). Several plausible explanations have been put forward to

explain what happens during conditioning (Brown and Edwards, 1946; Hsiao et aL,

1979; Schopfer, 1943).

Brown and Edwards (1946) hypothesized that during conditioning Striga seeds

synthesize a substance which is the same as or similar to the stimulating substance

released from plant roots. This hypothesis implies that during conditioning the amount of

the substance formed in the seed itself gradually increases. The particularly interesting

explanation by Schopfer (1943) states that the mere fact that Striga seeds require

germination stimulants from an external source to initiate germination, suggests the loss

o f the capability of synthesizing a specific substance, which is essential for germination.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26

Such a loss o f the capability to synthesize essential active substances is by no means

uncommon in plants. Schopfer cites many examples o f lower plants in which it occurs

and also points out that it is met in higher plants as welL A more feasible explanation was

offered by Hsiao and his collaborators (1979) who suggested that some factor(s)

beneficial to the conditioning process(es) are leached out o f the Striga seeds during

conditioning. These germination inhibitor(s) are leached out into the conditioning liquid,

without leaching out the possible germination promoters such as metabolic substrates

(Vallance, 1951) or stimulants (Brown and Edwards, 1946), which had accumulated or

were synthesized during conditioning.

To the best o f our knowledge, no study concerning the chemical composition of

dry and conditioned S. hermonthica, S. gesnerioides and S. aspera seeds has been

previously done. The object o f the present investigation was to shed lig h t on the

qualitative differences in CH 2CI2 extracts of dry and conditioned Striga spp. seeds.

Preliminary identification and quantification of some of the components leached out,

synthesized or retained during conditioning are reported.

n .2 . MATERIALS AND METHODS

n .2 .1 . Seed Collection. In the United States, field and laboratory studies with

Striga are done under strict quarantine regulations approved by the US Department of

Agriculture. Therefore, studies involving Striga were performed at the International

Institute of Tropical Agriculture, Ibadan, Nigeria. S. aspera and S. gesnerioides seeds

were collected from host crops in Kano, Nigeria during the 1992 and 1993 harvesting

seasons, respectively. Similarly, S. hermonthica seeds were collected from sorghum in

Abuja (Nigeria) in 1993.

n.2.2. Seed Surface Disinfestation and Conditioning. The method of

conditioning was adapted from Igbinnosa and Okwonko (1991) and Vasudeva Rao

(1985), with minor modifications. For each experiment, 2.0 g of Striga seeds were

surface disinfested by completely immersing them in 200 mL of 1 % (w/v) sodium

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 27

hypochlorite (NaOCI). Ten drops of the detergent "Tween 80" [polyoxyethylene (20)

sorbitan mono-oleate] were added. After shaking, the mixture was allowed to stand for 4

min. Floating seeds were decanted and discarded. The remaining seeds were then

transferred to a Buchner funnel lined with Whatman No. 1 filte r paper and rinsed several

times w ith sterile deionized water until the chlorine smell had disappeared. The filte r

paper containing the surface sterilized seeds was removed and the seeds air dried. The

dried seeds were then conditioned on two sterile 9.0 cm diameter Whatman No. 1 filte r

papers, saturated with 3.0 mL sterile double-distilled deionized water, in 9.5 cm diameter

petri dishes. 100 discs of Whatman GF/A) glass microfiber filte r paper o f 5.0 mm

diameter were arranged on top o f the filte r paper. Seeds were carefully sprinkled on the

filte r disks (25-40 seeds per disc or 0.13-0.20 mg o f Striga seeds per disk; approximately

200,000 seeds per gram o f Striga seeds). The petri dishes were sealed with parafilm and

incubated at 28°C in the dark for 10 days. Throughout the conditioning period the filte r

papers were kept saturated with sterile double-distilled deionized water.

n .2 .3 . Preparation of Extracts of Striga Seeds. Dichloromethane (C H 2CI2)

used for sample preparation was obtained from Ibadan Chemical Co. (Oyo State, Nigeria)

and double-distilled before use. Two grams o f dry and conditioned seeds were extracted

six times using 20 mL of CH 2CI2 for 24 h. The solutions from each o f the six

extractions were combined and evaporated under vacuum. The gummy crude extracts

were shipped to the chemical laboratory at Louisiana State University and stored in the

refrigerator at 4°C until analysis.

n.2.4. GC/MS Analysis. A Hewlett Packard 5971A GC/MS was used with a 5% phenyl-95% methylpolysiloxane capillary column (DB-5 ms, 12 m X 0.20 mm I.D.; 0.33

pm film thickness, J & W Scientific, Folsom, CA, USA) to separate components of

extracts and obtain their electron ionization (70 eV) mass spectra. Standard stock

solutions o f the crude seed extracts (0.5 g) were dissolved in analytical grade CH 2CI2

(concentration 20 mg/mL; CH 2CI2 was obtained from Aldrich Chemical Company Inc.,

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28

Milwaukee, U.S.A.). Samples (1.0 p,L) from the standard stock solutions were

introduced via split-injection mode (50:1) into a GC inlet at 250°C. The MS detector

temperature was 250°C. The oven temperature, in itia lly set at 40°C for 3 rain, was then

raised to 280°C at a rate o f 20°C /min and the interface temperature was set at 28Q0C.

Helium was used as the carrier gas and the head pressure was approximately 5 kPa. The

mass range scanned was m/z 50-531 and spectra were acquired at a rate o f 1.5 scans/sec.

Assignments o f peak identities were made by comparing their retention times with pure

authentic samples isolated in our laboratory and/or by matching their mass spectra with

on-line library o f standard compounds (Stenhagen et a l, 1974; Stenhagen et a l, 1969;

Sidow et a l, 1970). The relative area percentages o f peaks were calculated using their

abundances.

n .3. RESULTS. Figures II. 1-3A and B show typical chromatographic profiles of

components detected in CH 2CI2 extracts o f dry and conditioned Striga seeds, as separated

on a DB-5 ms column, respectively. Several peaks were detected and their mass spectra

and retention times recorded (Table n.l). The following sixteen types o f compounds

were identified: 1 quinone, 1 sterol, 4 fatty acids, 5 aldehydes and 5 long-chain

hydrocarbons. The relative area percentages o f peaks o f these and the unidentified

compounds gave an indication of their concentrations in Striga extracts.

The constituents common to all dry and conditioned Striga seeds were

tetradecanoic acid, cis,cis-9,12-octadecadienoic acid, cis-9-octadecenoic acid and hexanal

as well as an unidentified component with a retention time o f 2.8 min. A comparison o f

dry and conditioned Striga seed extracts for the relative concentrations revealed that

tetradecanoic acid, c/s-9-octadecenoic acid and pentatriacontane all decreased during

conditioning (Table n.l). A very dramatic increase in the relative concentration of

cfs,cfs-9,12-octadecadienoic acid occurred during conditioning of S. aspera seeds.

Conditioned S. hermonthica is showed the greatest number o f compounds overall and a

larger number o f compounds in the retention time window between 5 and 10 min (Figure

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29

n.lB). Conditioned seeds o f this parasite contained trace amounts o f 2,6-DMBQ and more o f the compounds that were already present in dry seeds (Table n.l). For example,

the relative percentage areas o f ciJ-2-heptenal, rranj,/ranj-2,4-decadienal, n-pentacosane,

n-heptacosane and n-nonacosane of S. hermonthica seeds increased during conditioning.

The distribution patterns o f compounds in S. hermonthica and S. gesnerioides

seeds were similar (Figures II. 1 and n.2). Their gas chromatograms indicated that the relative concentrations o f c/j-2-decenal, hexanal and rranj, rrflns-2,4-decadienal increased

while the level o f cr crs-9,12-octadecadienoic acid remained constant before and after

conditioning. Furthermore, the seeds of both species, unlike those of S. aspera,

contained hexanoic acid and 2,6-DMBQ as well as more of several unidentified

components with retention time windows between 2.7 and 11 min.

Unique to dry S. gesnerioides extracts was the presence o f a compound with the

highest retention time of 33.4 min (Figure II.2A). 5. aspera extracts contained the

smallest number of compounds (Figures II.3A and II.3B). Compounds with retention

times 19.4 and 19.9 were detected in extracts o f dry S. aspera seeds but not in extracts o f

S. hermonthica and S. gesnerioides. It is worth noting that the concentration of 2,6-

DMBQ in S. aspera seeds decreased during conditioning, while the most abundant

component with a retention time o f 19.9 min was leached out of its seeds.

In the detected hydrocarbon series (Table ELI), the heavier odd-numbered carbon

n-alkanes, n-heptacosane (C 27 H 56), n-nonacosane (C 29H 60) and n-pentatricontane

(C35H72 ) predominated in the region above cis-9-octadecadienoic acid (Figures n.3.1-3).

Sitosterol and 2,6-DMBQ (peaks numbers 7 and 16 in Table IL l) were assigned through

comparison o f the GC/MS spectra with those o f authentic samples.

n.4. DISCUSSION. The use of CH 2CI2 for the introduction of chemicals into dry

seeds has opened up new possibilities of treating seeds with stimulators or inhibitors of

germination (Meyer and Mayer, 1971), and it has been used in this work to extract

compounds rather than introduce them. The effectiveness o f CH 2CI2 is presumably due

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30

to its ability to dissolve various polar and nonpolar compounds. A GC/MS technique was

selected for analysis of CH 2CI2 extracts because it provides identirication o f compounds

even when the chromatographic separation is not suffîcient to afford an accurate

quantification (Wittkowski et al„ 1981). To avoid changes in the elution order o f some

compounds o f CH 2CI2 extracts (Table II. 1), extraction and injection procedures as well

as GC/MS conditions were standardized.

In the present investigation, it has been found that plant extracts obtained from dry

and conditioned Striga seeds differ in their chemical compositions. This probably

explains the variable bioassays which have been observed in various isolates o f Striga

species (Pepperman et a/., 1982). Our results show that the relative distribution of

tetradecanoic, cis,cis-9,12-octadecadienoic, cis-9-octadecenoic and other fatty acids is an

important difference. The differences could be attributed to environmental factors which

influence their compositions in the seeds (Steams, 1970). In general, fatty acids are

biosynthesized through a series o f enzyme-mediated reactions. Acetyl-CoA carboxylase

and fatty acid synthetase are the two enzymes which catalyze the de novo synthesis o f

saturated fatty acids (Vagelos, 1974). On the other hand, desaturase enzymes, which like

fatty acid synthases are SH-enzymes (Gurr, 1974), catalyze the formation o f unsaturated

fatty acids. A substance targeted against the metabolism o f fatty acids occurring in Striga

seeds might be an effective herbicide against Striga. Specifically, these substances

(herbicides) would inhibit the function of the enzymes (Ashton et aL, 1994).

The fact that Striga spp. seeds synthesized compounds such as 2,6-DMBQ is

fully consistent with the suggestion by Brown and Edwards (1946) that during

conditioning Striga seeds synthesize a substance similar to the host-produced germination

stimulant. How this is achieved is not clear from the present results. During

conditioning, the seeds o f S. gesnerioides synthesize several unidentified compounds

w ith retention times 5.4, 7.2, 7.9 and 10.3 min while compounds w ith retention times

14.9, 17.1 and 28.5 min disappear.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31

The simple gas ethylene (C 2H4) serves as a plant hormone with profound effects

on plant growth and development (Chang and Meyerowitz, 1995). Ethylene may

enhance germination of Striga seeds, as in other seeds, by altering the promoter-inhibitor

ratio and or by promoting cell elongation during the early stages o f germination process

(Ketring and Morgan, 1972). However, Striga seed has limited capacity to convert 1-

aminocyclopropane-l-carboxylic acid (ACC), the immediate ethylene precursor, to

ethylene (Babiker et aL, 1993). It has been shown (Yu er aL, 1979) that the activity o f

ACC synthase depends on pH, the maximal activity occurs at pH 8.5 and decreases

above or below this value. We envisioned that the fatty acids detected in seed extracts

possibly lowers the pH and decrease the capacity o f ACC synthase to convert ACC to

ethylene. During the conditioning process, the relative concentrations o f tetradecanoic

and cis-9-octadecenoic acids in Striga seeds decreases (Table EE.1), and possibly relieves

the restriction on the ethylene biosynthetic pathway.

M aillard reactions are known to reduce the activities o f enzymes in dry seeds.

This results in decreased metabolic capability during seed dormancy (Feeney and

W hitaker, 1982; Njoroge and Monnier, 1989; Ebe et aL, 1983). Striga contains

significant amounts of mannose (Nour et aL, 1986), a reducing sugar, which is a driving

force o f Maillard reactions. Since dry seeds are apparently unable to exercise repair,

proteins and enzymes damaged by Maillard reaction would accumulate over time. It has

been observed that soybean seed germination decrease as Maillard products accumulate

(Wettlaufer and Leopold, 1991). In the case o f Striga seeds, the M aillard products are

leached out of the seeds during conditioning. Moreover, it should be noted that fatty

acids in Striga seeds undergo free radical oxidization to hydroperoxides, which can be

further degraded to ketones and alcohols. Alcohols can be oxidized to aldehydes. Lipid

peroxides and their secondary products can react with terminal groups o f amino acids in

proteins and enzymes (Feeney and Whitaker, 1982). The possibility that lipid

peroxidation participates in Maillard reactions suggests that free radical oxidation and

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M aillard reactions may both contribute to a common mechanism which enhances Striga seed viabiliQr.

The concentrations of aldehydes in S. hermonthica and S. gesnerioides seeds

generally increased during conditioning. Although aldehydes have been known to occur

in fruits for a long time (De Footer, 1987), the way they are synthesized is still only

solved in part Aldehydes have been reportedly formed from heat treatment of various

lipids via oxidative cleavage o f the double bond (Miyake and Shibamoto, 1995; Jennings

and Shibamoto, 1980). It is established that hexanal (peak number 1 in Table U .l) is

formed from linoleic acid. For the biosynthesis o f C 3- to Cg-aldehydes, circumstantial

evidence points to the corresponding carboxylic acids at least in part as precursors.

Aldehyde dehydrogenase is an enzyme which catalyzes the oxidation o f a number of

aldehydes to the corresponding acids (Asker and Davies, 1985). Also, a substance

targetted against the function o f aldehyde dehydrogenase might be effective in controlling

Striga.

In comparison to other plants, Striga seeds contain very low levels of 2,6-DMBQ

and sitosterol (Table II.1), the most ubiquitous compounds in plants (Vagelos, 1974;

Heftmann, 1971; Handa et aL, 1983; Gladu et aL, 1991). This indicates that Striga

species have lost the capability to synthesize these compounds as was suggested by

Schopfer (1943). The significance o f sitosterol in Striga seeds can be rationalized from

several perspectives. Sitosterol is not only one o f the biogenetic precursors o f steroid

hormones, but may have hormonal activity itself (Heftmann, 1971). Furthermore,

sitosterol may act as architectural component o f membranes (Nes et aL, 1981) and

function as a reserve supply from which plants can produce other sterols (perhaps

including cholesterol) and progesterone (Heftmann, 1971). Sitosterol is water-soluble

and might be leached out of Striga seeds in the field by rain or conditioning liquid in the

laboratory. This might be the reason why the seeds contained very low levels o f this

sterol. Squalene is the precursor for synthesis of sitosterol and several enzymes are

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involved in the conversion steps (Gaylor, 1974). A compound that enhances the activity

of squalene enzymes might be effective in inducing "suicidal" germination of Striga

seeds. Unpublished results at our laboratory indicate that exogenous application of

sitosterol induce germination of Striga seeds. The level of sitosterol might be a very

important parameter in evaluation o f germinability o f Striga seeds. Because germination

is a risky event for any plant, especially for the tiny Striga seeds, one o f its unique

strategies is to minimize the risk o f germinating by lim iting the synthesis o f endogenous

germination stimulants (e.g. sitosterol).

The exact role of 2,6-DMBQ in Striga seeds is not completely understood. This

quinone is not only an inhibitor of mitochondrial respiration (Chappel and Hansford,

1972; Redfeam and Whittaker, 1962) but also an haustoria-inducing principle. 2,6-

DMBQ has been isolated from sorghum root (a host) extracts (Chang, 1986). Using

syringaldazine, Chang and Lynn (1986) have detected laccase-type enzymes associated

with the roots of Striga asiatica (L.) Kuntze and Agilinis purpurea (L.) Raf.

(Scrophulariaceae). They proposed that such enzymes released from the parasite root

may cleave 2,6-DMBQ from the cell wall complex o f the host which then diffuses back to

Striga asiatica and trigger haustorial development They concluded that zones for both

Striga asiatica germination and haustorial induction are determined by a combination o f

factors including diffusion rates of promoters, the instability of active compounds, and

critical threshold concentrations and exposure times required for induction. In the present

study we have detected traces o f 2,6-DMBQ in dry and conditioned S. aspera seeds as

well as conditioned S. hermonthica and S. gesnerioides seeds. This is in agreement with

Brown and Edwards’ (1946) hypothesis of synthesis of host-produced substances by

Striga seeds. The fact that Striga spp. seeds usually do not develop haustoria in the

absence o f exogenous compounds suggest that the level o f 2,6-DMBQ in Striga seeds is

below the threshold concentration necessary to trigger haustorial development.

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In general, seed dormancy results from particular conditions prevailing internally

in the seed. Several causes o f dormancy have been recognized, and seeds o f numerous

species exhibit two or more o f them simultaneously (Moore, 1979). Among the many

causes o f seed dormancy that have been described are ( 1) impermeability o f seed coats to

water gases, (2) immaturity o f the embryo, (3) need for “ after-ripening” in dry storage,

(4) mechanical resistance o f seed coats, (5) presence o f inhibitors found either in the seed

coats, dry accessory structures, or, in the case o f seeds contained in fleshy fruits, in the

tissues surrounding the seeds, ( 6 ) special requirement for light or its absence, and (7)

requirement for chilling in the hydrated condition. The most common cause of seed

dormancy in woody plants native to temperate regions is a requirement for chilling o f the

hydrated seeds. Such seeds commonly germinate promptly and uniformly only after they

have become hydrated and exposed to low temperature (0-10°C) for a period o f a few to

several weeks.

It is important to note that Striga spp. seeds w ill not germinate immediately after

ripening even under conditions of moisture, temperature, and oxygen tension generally

favorable for growth. Such dormancy is o f obvious survival value to Striga plants, since

it tends to restrict germination to environmental circumstances suitable for seedling

establishment and survival. Different Striga seeds in one seed plant often vary in their

degree o f dormancy, causing germination o f the seeds to occur over an extended period

of time and thus increasing the probability that at least part o f them w ill germinate under

conditions favorable for survival.

The occurrence o f inhibitors in seeds is w ell known (Evenari, 1946) and studies

in several species indicate the physiological importance of these substances (Wareing and

Foda, 1956). There is evidence that in some tissues (e.g. dormant buds, potato tuber)

inhibitory substances play a vital role in the regulation of dormancy (Hermberg,

1949a,b). Khan (1971) hypothesized that dormancy can result from an excess of

inhibitor. However, with respect to dry Striga spp. seed dormancy, the presence and role

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of inhibitors has hitherto been obscure. The inhibitory action might possibly be brought

about by toxic concentrations of substances which are not specific germination inhibitors.

We believe that the compounds leached out o f Striga spp. seeds are germination-

inhibitors while those synthesized are germination-promotors.

These high molecular weight hydrocarbons might be responsible for the "grassy"

flavor (Body, 1977; Larick et ai, 1987) of Striga spp. extracts. The role o f these

compounds in Striga seeds warrant further investigation. It is important to point out that

all these odd-numbered n-alkanes, as well as other even-numbered hydrocarbons have

been detected in sorghum, a host plant (Kami, 1975). Generally, n-hydrocarbons

develop from reaction o f hydrogen-free radicals w ith alkyl-free radicals (hydroperoxide

decomposition products) (Selke et al, 1975). Our mass spectral data of n-hydrocarbons

and long aliphatic chain fatty acids and aldehydes detected were confirmed by the

appearance o f a large number o f fragments with a uniform difference o f 14 mass units

(Stenhagen et al., 1969; Silverstein and Easier, 1967). These compounds gave very

intensive molecular ions (M+), while the ion corresponding to (M-15)+ was lacking. The

presence o f a methyl group as side chain was confirmed by the ratio of abimdances (M-

15)+/M+ which was about 3 (Stenhagen et al., 1969; Stoinova and Hadjieva, 1969).

More intense clusters o f peaks corresponding to CnH 2n-l (m/z 83, 97, 111, 125, 139,

153, etc.) in comparison to CnH 2n+i (m/z 85, 99, 113, 127, 141, 155, etc.) further

supported acyclic olefinic nature of the unsaturated aldehydes and fatty acids detected

(Silverstein and Easier, 1967).

From the present results, it is evident that one of the compounds with retention

time of 19.9 min is the primary inhibitor in S. aspera seeds (Figure U.3A). Inhibitors

detected in S. hermonthica and S. gesnerioides included heavier n-hydrocarbons (peaks

11-16 in Figure II.2A ) in the retention time window between 14 and 30 min. Because

germination-inhibitors may be leached out of Striga seeds during conditioning (Hsiao et

a l, 1979; Went, 1957), it is not unreasonable to conclude that these inhibitors may have

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36

the potential to block stimulant-mediated germination. Furthermore, these inhibitors

m ight constitute the (external) seed structures making them impermeable to germination

stimulants. We cannot rule out the requirement for de novo synthesis of proteins

essential for both germination and ethylene biosynthesis during conditioning (Babiker et

al. , 1993). Ultimately, the process o f conditioning increases the sensitivity o f Striga

seeds to germination stimulants (Worsham 1987, Babiker etal., 1992).

n.5. CONCLUSIONS. In summary, our results indicate that the water-soluble

endogenous inhibitors are apparently washed out of Striga spp. seeds during

conditioning. Differences in relative proportions o f inhibitory substances is reflected by

gross manifestations of the seeds to respond to exogenously applied germination

stimulants. Though we cannot quantitatively substantiate, it is clear that some

compounds are also synthesized during conditioning. Future studies should emphasize

investigation of the pattern of accumulation and mechanisms by which the various

compounds are accumulated in Striga seeds. This w ill provide important information

useful in designing target specific inhibitors or germination stimulants for control of

Striga.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37

Table II. 1. Relative area (%) o f components in dichloromethane extracts o f Striga seeds

Relative area (%)^

S. hermonthica S. gesnerioides S. aspera Peak^ RTC(min) Dry Con

UnkG 2 .8 [tr]f tr tr tr tr tr

1 3.6 4 60 tr 13 tr tr

Unk 5.4 [nd]g nd nd tr nd nd

2 5.9 6 26 6 5 tr tr

3 6.1 tr 6 tr tr tr nd

Unk 7.0 tr 5 tr tr nd nd

Unk 7.2 tr 8 nd tr nd nd

Unk 7.5 tr tr tr 1 nd nd

Unk 7.9 tr tr nd 2 tr nd

Unk 8.5 9 8 tr tr 1 nd

4 8 .8 tr 37 tr 5 1 tr

5 9.1 6 36 tr tr 1 tr

6 9.3 6 52 tr 19 1 tr 7 9.7 nd tr nd I 1 tr

Unk 10.3 tr tr nd tr tr nd

Unk 11.2 nd nd nd nd 2 n

8 13.3 69 45 44 15 20 6

Unk 13.6 tr 7 nd nd nd nd

9 14.18 100 100 100 100 13 100

10 14.20 25 7 33 9 9 5 (Table con’d)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38

Relative area (%)^

S. hermonthica S. gesnerioides S. aspera Peak^ RTC(min) Dry Con^. Dry Con. Dry Con.

11 14.9 5 3 3 nd tr tr

Unk 15.8 tr 10 5 tr nd nd

12 15.9 tr 10 5 nd tr tr

Unk 17.1 nd nd tr nd nd nd

13 17.3 tr 21 tr tr 5 2

14 19.3 tr 14 9 nd tr 2

Unk 19.4 nd nd nd nd tr nd

Unk 19.9 nd nd nd nd 100 nd

15 22.4 40 11 13 tr 8 5

16 27.4 21 20 tr tr tr tr

Unk 28.5 nd nd tr nd nd nd

Unk 33.4 nd nd 12 tr nd nd

^ Calculated using relative abundance values (the area o f the most abundant peak was assigned a value o f 100%). ^ Numbering o f peaks as shown in Figures II. 1-3. Peak Identification: 1 = hexanal; 2 = cw-2-heptenal; 3 = hexanoic acid; 4 = cw-2-decenal; 5 = rra/is,frfln^-2,4-decadienal; 6 = rrnnj,clj-2,4-decadienal; 7 = 2,6-dimethoxy-p- benzoquinone; 8 = tetradecanoic acid; 9 = c f c if -9,12-octadecadienoic acid; 10 = cis-9- octadecenoic acid; 11= tricosane; 12 = pentacosane; 13 = heptacosane; 14 = nonacosane; 15 = pentatriacontane; 16 = sitosterol. RT^ (min) - retention time in minutes. Con^. - conditioned. Unk® - not identified, [tr]^-Traces. [Nd]S - Not detected.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■DCD Q.O C gQ.

Abundance ■D CD

WC/) 3o' 0 1000000 - 3 CD 8 500000 - 3" 1 3 ^—*, CD 5.00 1 0 . 00 15.00 20 . 00 25.00 30.00 35.00 c"n 3. Time (min.) 3" Abundance CD 4000000 - ■DCD Q.O C 3000 00 0 ■ a o 3 ■D O 2000000 -

CD Q. 1000000 -

■D 5 . 00 1 0 . 00 1 5 . 00 2 0 . 00 25.00 3 0 . 00 3 5 . 00 CD Time (min.) C/) C/) Figure II. 1 A and B. Total ion chromatorgram profiles of compounds detected in dichloromethane extracts of dry (A) and conditioned (B) Striga hermonthica seeds separated on DB-5ms (12m x 0.20 mm I.D.) fused silica capillary column. Numbering o f peaks is as shown in Table n .l.

w VO ■DCD O Q. C g Q.

■D CD Abundance 800000 C/) C/)

600000

8 10 400000 ■

200000 •2 ' ,.11 12 Y 14 56 15 16 „ ------13 lu _JJUL JL A- 0 1 1 r1 1------1------1— -I I I ~i i I I I I 1 1 1 1 1 1 1 , r- 5 , 00 T 3 1 0 . 00 15.00 20.00 25.00 30.00 35,00 3" Abundance (D 9 Time (min.J 8000000 m u T3 O Q. aC 6000000 3o T3 O 4000000

(D Q. 2000000 10

ik 15 16 T3 ■<—r ■-+M- r ’ r—^ n I ' I I I I I » ''i l“" [■ "I 1'^ I ■■ I ■ I I' I l'' I I I (D 5.00 10.00 15.00 20.00 25.00 30.00 35.00

(/) Time (min.) (/) Figure II.2 A and B. Total ion chromatorgram profiles of compounds detected in dichloromethane extracts o f dry (A) and conditioned (B) Striga gesnerioides seeds separated on DB-5ms (12m x 0.20 mm I.D.) fused silica capillary column. Numbering of peaks is as shown in Table n .l. ■o o Q. C g Q.

Abundance ■D CD (/) 2000000 C/)

1500000 •

1000000 8 10 ë' 500000 13 15 16 V l l l L. Lll — ' — — '— '— ' — ' — r 1 1 5.00 10.00 15.00 20.00 25.00 10.00 35.00 3. Abundance g 9 Time (min.J 3" CD 3000000 B "OCD O 2500000 Q. C a 2000000 O3 "O 1500000 O 1000000 CD Q. 500000 llj2 13 0 1— '— "O 5.00 10.00 15.00 20.00 25.00 30.00 35.00 CD Time (min.l C/) o" 3 Figure II.3 A and B. Total ion chromatorgram profiles of compounds detected in dichloromethane extracts o f dry (A) and conditioned (B) Striga aspera seeds separated on DB-5ms (12m x 0,20 mm I.D.) fused silica capillary column. Numbering o f peaks is as shown in Table II. 1. CHAPTER m

ACTIVITY OF EXTRACTS FROM NONHOST LEGUMES ON THE GERMINATION OF STRIGA HERMONTHICA SEEDS

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43

m i. INTRODUCTION. The genus Striga (Scrophulariaceae) comprises obligate

root parasites o f many graminaceous and leguminous crops. They directly affect the lives

of more than 400 million people in Africa, India, and the Middle East by severely

reducing the yields of various crops (Siame et a/., 1993, Johnson et aL, 1981). In

particular, 5. hermonthica (Del.) Benth. is a problem of enormous proportions to small-

scale African farmers who make up the majority o f rural communities in sub-Saharan

A frica. The seeds of this parasite are tiny, around 0.30 m illim eters long and 0.15

millimeters wide, and are produced in vast amounts. The parasite seeds are known to

remain dormant in the soil for as long as 14 years and w ill germinate only after exposure

to exogenous stimulating substances, usually in plant root exudates (Hauck, 1990 and

Johnson et aL, 1976).

Control methods for S. hermonthica include cultural procedures, hand-weeding,

use o f resistant crops, and biological and chemical approaches. One promising control

strategy is the use o f nonhost leguminous "trap crops" crops in rotation with host crops.

Trap crops w ill cause germination of S. hermonthica seeds, but are not parasitized

themselves. These crops induce suicidal germination and thereby reduce the S.

hermonthica seed density in the soil. However, successful implementation o f effective

nonhost rotations to control S. hermonthica has been limited because little is known about

the relative efficiency of different nonhost cultivars and the cultivar specific chemical

compounds responsible for germination (Ariga and Berner, 1993).

Various techniques have been proposed for the recovery of germination stimulants

(Herb et at., 1987). Dichloromethane (DCM) is a relatively cheap and low boiling

solvent It was used by Visser (1974) to extract germination stimulants from solutions in

which roots of various hosts and nonhost plants for S. asiatica had been growing. A

serious disadvantage of this technique of stimulant recovery is the extremely low

concentration in which these substances are produced by the host plant roots. Moreover,

several attempts have been made to identify the germination stimulants (Cook et al. 1972;

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Parker, 1983), but their relative instability have hindered isolation in pure form.

Investigation o f different plant parts o f host and nonhost legume cultivars for germination

stimulants might provide additional sources o f germination stimulants.

The advantages of including legumes in cropping systems have long been

recognized (Nutman, 1987). The prime advantage is their ability to fix nitrogen and thus

positively contribute to the nitrogen (N) balance of the cropping system. However, it

must be recognized that such contributions may be of lesser significance for grain

legumes than for forage legumes because of their high N harvest index (HI) and often

poor nodulation (Hoshikawa, 1991). Other positive effects of legumes come from their

ability to break disease cycles, improve soil physical conditions, encourage mycorrhizae,

and mobilize normally unavailable soil phosphorus sources (Hoshikawa, 1991). In

Africa at least, the ever-increasing demand for cereal grain mitigates against the use o f

grain legumes in better endowed agricultural lands and often relegates them to less

favorable, usually rainfed, environments (Saxena et al., 1993). Demand for grain

legumes is increasing but economics o f production still do not encourage their cultivation

on the more productive soils (Saxena et aL, 1993). Many o f the biotic and abiotic

stresses faced by grain legumes contribute to the large yield gap between potential yields

and realized yields as reflected in national production statistics (Johansen et aL, 1994).

As part of a program concerned with investigation of leguminous nonhost

cultivars as potential sources o f germination stimulants, we have examined the relative

efficacy in germination stimulation on S. hermonthica seeds by dichloromethane (DCM)

and aqueous extracts from ten different legume cultivars. Our goal is to develop an

effective S. hermonthica seed elimination or reduction control program compatible with

the resource-poor small-scale farmers in developing countries.

in .2 . MATERIALS AND METHODS

in.2.1. Collection of Plant Materials. All experiments were conducted at the

International Institute of Tropical Agriculture, Ibadan, Nigeria. Seeds of ten different

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legume cultivars (Table HI.1) provided by IIT A Breeding Program were planted in a

greenhouse in small pots containing river sand in June 1994. The plants were uprooted

after 21 days. Voucher specimens are deposited in the herbarium o f IIT A .

Dichloromethane (DCM) and water extracts from different plant parts were analyzed for

Striga stimulatory activities (Tables DL2-8 and Figures HI. 1-2).

n i.2.2. Preparation of Solutions of GR 24 ( 8 ) and GR 7 (9). 1.5 mg of

strigol analogues GR 24 (2-methyl-4-(2-oxo-2,3.3a,8b-tetrahydro-4H-indeno[l,2-

b]furan-3-ylidenemethoxy)but-2-en-4-olide) ( 8 ) and GR 7 (2-methyl-4-(2-oxo-

3,3a,6,6a-tetrahydro-2H-cyclopenta[b]-furan-3-ylidenemethoxy)but-2-en-4-oUde) (9)

(Johnson e ta l., 1981) were dissolved in 0.1 m L acetone and methanol, respectively.

Dilution with 9.9 mL distilled sterilized water gave standard stock solutions equivalent to

1,500 mg L "l.

in.2.3. Dichloromethane Extraction. In the laboratory, the uprooted plants of the

legume cultivars were separated into leaves, stems and roots. The plant materials were

air-dried and (15 g) exhaustively extracted by soaking in 100 mL of analytical grade

dichloromethane (DCM) (obtained from Aldrich Chemical Company Inc., Milwaukee,

U.S.A.) at room temperature for two days, six times. The extracts from each plant part

were combined, filtered by suction then evaporated below 40°C under reduced pressure

to produce a thick gummy residue (ca. 0.5 g). The dichloromethane (DCM) residue (150

mg) was dissolved in 0.1 mL of 0.1 % dimethyl sulfoxide (DMSO), a solubilizing agent,

and diluted w ith 9.9 mL sterilized distilled water to make a standard stock solutions

(equivalent to 1,500 mg L"^). All standard stock solutions were stored under

refrigeration in crimp-top vials with Teflon seals. The solutions, undiluted or after xlO or

xlOO dilution, were tested for stimulatory activity on germination o f conditioned S.

hermonthica seeds.

m .2.4. W ater Extraction. Saturated aqueous solutions were prepared by grinding

1.5 g o f fresh weight o f leaves, stems and roots in a mortar with 10 mL distilled sterilized

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water. The extracts were filtered through Whatman No. 42 filte r paper to make standard

stock solutions. Solutions o f lower concentrations were prepared by sequential dilution

then stored and assayed for S. hermonthica seed germination stimulation as with the

DCM extracts.

in .2 .S . Isolation of Sterols from Leaves. The use of leaves in screening of

legume cultivars for quantitative and qualitative differences in sterols and other

germination stimulants can be a non-destructive method for assaying efficacy o f plants in

stimulating Striga spp. seed germination. Thus five legume cultivars (Table in . 8 ) were

planted in the greenhouse and their leaves collected after fourteen days. 2 g o f dried and

powdered leaves were extracted with 50 mL DCM, three times (24 hr each). A fter

removal of the solvent by evaporation, the extracts (0.5 g each) were subjected to column

chromatography on silica gel using EtOAc-hexane (1:10-10:1, v/v; 50 mL). The EtOAc-

hexane (4:6, v/v) eluates were further rechromatographed into two fractions, 1 and 2,

using EtOAc-hexane (3:7, v/v; 50 mL). Fraction 1 gave chondrillasterol (15) while

fraction 2 yielded stigraasterol (17) (% yield calculated with respect to dried leaves).

Identification of the individual sterols were performed by GC/MS as well as by

comparing the high field and ^^C NMR with those of reference compounds.

in .2 .6 . Thin-layer Chromatography (TLC). Thin-layer chromatography (TLC)

(Felton, 1982; Fischer, 1991) was employed in this study to analyze the compounds

present in crude dichloromethane (DCM) extracts o f the different legume cultivars (Table

in.2). The best separation of the germination stimulants was obtained on 0.25 mm silica

gel 60F-254 precoated TLC (7X7 cm) plates with fluorescent indicator (EM

Laboratories, No. 5760). Using capillary tubes, dichloromethane (DCM) extracts (six

per plate) were applied as separate spots to one side of a TLC plate about 1.5 cm from the

edge (spotting line). After sample application, the plate was placed vertically into a

chamber containing developing solution of ethyl acetate/hexane (2:5) [volume ratio in

parenthesis]. The spotting line was about 0.5 cm from the developing solution. After the

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developing solution had moved 75% o f the distance from the spotting line, to the far edge

o f the TLC plate, the plate was removed from the developing chamber and dried. The

eluted spots, representing different compounds were visualized by spraying the plate with

an acidic cobalt solution prepared by dissolving 2 g cobalt chloride (CCXÜI 2.6 H2O) in 100

mL of 10% aqueous sulfuric acid (H 2SO4 ). The TLC plate was then heated on a hot plate

to 100°C for about 1 min. The different compounds present in crude dichloromethane

(DCM) extracts produced visibly colored spots whose “ ratio to fro n f ’ (Rf) values were

determined. The R f value expresses the position o f a compound on a TLC plate. In

general, R f values in the same neighborhood suggests the presence o f sim ilar or

structurally related compounds. Mathematically, Rf is a ratio of the distance that the

compound moved from the spotting line, to the distance that the solvent moved from the

spotting line. In principle, R f values are between 0 and 0.999, and without units.

in.2.7. Bioassays

in .2 .7 .1 . Crude Extracts. Seeds of one Striga hermonthica (Del.) Benth

population collected from sorghum plants by D. K. Berner in Zaria (11.07 N, 7.44 E) in

Nigeria in 1991 were used in this experiment. For each experiment, about 1440 seeds

were surface disinfested and conditioned (see Materials and Methods o f Chapter H).

Twelve 5 mm diameter filte r paper discs containing conditioned seeds (25-40 seeds per

disc) were arranged in a 9.0 cm sterile petri dish lined with two (Whatman No. 1) filte r

papers which had been moistened w ith 2.0 mL sterile deionized water. For each

bioassay, 360-480 seeds were used. Using a micropipette, aliquots of 6 (X) p.L of

dichloromethane (DCM) or aqueous extracts were uniformly applied to each petri dish

containing disks of conditioned S. hermonthica seeds. The petri dishes were sealed with

parafilm and incubated at 28°C for 24 h. In all experiments, two reference compounds,

GR 24 (8) and GR 7 (9) served as positive controls while 0.1 % (v/v) DMSO in sterile

deionized water served as negative control. Germination tests were determined 24 h.

after application o f the test solution. Three petri dishes were used as replicates for each

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test and the experiments were repeated six times over a period o f 3 weeks. Germinated and nongerminated seeds were counted under a binocular dissecting microscope at 20X

magnification. Seeds were considered to have germinated i f the radicle had emerged from

the seed coat The percentage germination was calculated for each petri dish and the

germination data expressed as overall means o f six experiments each replicated three

times.

m .2 .7 .2 . Sterols. Stock solutions (lO"^ M) of chondrillasterol (15) and

stigmasterol (17) were prepared using acetone as a solvent carrier while GR 24 (8), the

positive control, was dissolved in methanol The final solvent concentration was adjusted

to 0.1 % (v/v). Stock solutions were refrigerated at 4oC. For each experiment, fresh

solutions of lower concentrations (10*^ to lO'^O M) were prepared by serial dilution of

the stock solution and were then evaluated as germination stimulants for S. hermonthica

seeds. In order to obtain statistically meaningful information on the possible action of

different concentrations of sterols on germination of conditioned seeds, several treatments

were performed. For each treatment, approximately 420 conditioned seeds were used

and bioassayed as for crude DCM extracts. In all experiments, conditioned seeds treated

with GR 24 (8) served as positive control while those treated with sterile deionized water

containing O.I % (v/v) o f acetone or methanol were used as negative controls. Each

treatment was replicated twelve times over a period o f 3 weeks and in each test the

germination percentages were determined on three separate petri dishes. The percentage

germination data reported are the mean for twelve experiments. On the basis o f the mean

percentage germination and the percentage viability, the relative percentage germination of

S. hermonthica seeds caused by chondrillasterol (15), stigmasterol (17) and GR 24 (8)

were calculated in the following way:

i). % Absolute germination = (% Germination) - (% Germination o f negative control)

ii). % Relative germination = 100(% Absolute germination)/(% Viability of S.

hermonthica ).

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The absolute germination and viability can reach maximum values o f 100% for a

potent germination stimulant and highly viable seeds. The relative percentage germination

at each concentration were then transformed to arc sine and examined by analysis of

variance (SAS, 1989). The arc sine transformed relative percentage germination reported

are expressed as a means o f twelve experiments ± standard error (S. E.; shown only

when larger than the symbol).

ni.2.8. Statistical Analysis. Statistical comparisons were made on the amount of

germination stimulant activity for S. hermonthica seeds by the dichloromethane (DCM)

and aqueous extracts of ten legume cultivars. Germination data were transformed by

square root and compared to controls using single-degree-of-freedom contrasts w ith the

general linear models procedure o f the Statistical Analysis System (SAS) programs (SAS,

1988). A significance level o f P = 0.05 was used. Four data summaries were useful for

examining the trends in percentage germination. We first calculated the mean percent

germination o f S. hermonthica seeds by dichloromethane (DCM) and water extracts from

leaves, stems or roots o f each cultivar. We also calculated the overall amount of

germination stimulation for each cultivar using the two different extraction solvents

(Tables ni.3). To explain the differences in germination percentages, various interactions

were analyzed including combinations o f legume cultivars, legume parts, concentrations,

and treatments.

in.3. RESULTS. In all our experiments, treatment of conditioned S. hermonthica

seeds in sterile deionized water containing 0.1 % (v/v) DMSO (the negative control) did

not show observable germination under the microscope. Maximum germination for most

extracts was obtained using the undiluted or dilution xlO range. In many cases one or the

other concentration of cultivar extracts (Tables in.4.1-3) gave higher germination than

GR 24 (8) and GR 7 (9) (Tables IÏI.2 ). Treatment o f conditioned seeds w ith these

strigol analogues stimulated germination in a concentration-dependent manner. In

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particular, the strigol analogues resulted in high germination (71-72%) at 1,500 mg L ‘ l.

However, germination decreased with decreasing concentrations.

There was a definite difference in germination stimulation between the

dichloromethane (DCM) and water extracts o f most o f the legume cultivars studied. The

average percentage germination induced by dichloromethane (DCM) extracts o f the

various parts o f V. radiata cv. tvr28 and V. unguiculata cv. iar-48 were the same (43%)

and did not differ significantly from that due to V. radiata cv. tvr34 cultivar (44%) (Table

ni.3). Also, dichloromethane extracts of V. umbellata cv. tval and C. cajan cv. cits2

cultivars had the same stimulatory activity (48%). Dichloromethane (DCM) extracts of

the roots o f all cultivars tested contained at least a small amount o f S. hermonthica

germination stimulant activity (Tables in.4.3 and in.5.3). Of all the cultivars tested, the

dichloromethane (DCM) extracts from the roots of L. purpureas cv. tln l produced the

highest germination (93%) which was significantly greater at xlO dilution (97%)

compared to xlOO dilution (44%). The undiluted extract from the roots of the other L

purpureus cultivar, tln28, had a greater stimulatory effect on germination (98%), but

decline in activity occurred with dilution.

A ll o f Vigna cultivars tested, which are not hosts o f S. hermonthica, generally

produced large amounts of stimulant activity (Tables ni.4.1-3 and ni.5.1-3). For

example, the undiluted dichloromethane (DCM) extracts of the roots of V. unguiculata

cv. it-84d-975 and V. unguiculata cv. iar-48 gave high germination (80-89%), higher

germination (85-91%) at xlO dilution and low germination (23-54%) at x 100 dilution.

Similar trends were observed in dichloromethane (DCM) extracts o f the roots o f V.

umbellata cv. tval, L. purpureus cv. tln l and both cultivars of V. radiata and C. cajan.

The dichloromethane (DCM) extracts of the roots of V. umbellata cv. w il4 0 gave high

germination (77%), low germination (68%) and almost no response (5%) at x l, xlO and

xlOO dilutions, respectively. A similar trend was observed in all the dichloromethane

(DCM) extracts of the other cultivar, V. umbellata cv. tval.

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Aqueous extracts from all the cultivars studied either did not or only weakly

stimulated germination o f S. hermontiiica seeds at the xlO to xlOO dilution range (Tables

in.5.I-3). For example, the leaves and stems o f V. umbellata cv. w iI4 0 and V.

umbellata cv. tva l were not stimulatory at all concentrations. Also, none o f the aqueous

extracts from either C. cajan cultivars stimulated germination of S. hermonthica seeds.

This was contrary to the results with DCM. To discover the significant sources o f

variation in percentage germination of S. hermonthica seeds, we analyzed the interactions

o f legume cultivars, concentrations, treatment, and parts. A ll interactions were significant

(P < 0.05). Interactions between concentrations and treatments as well at that between

legume cultivars and treatments were the most significant sources o f variations (Table

ra.6).

Numerous spots, which appeared on developed TLC plates of crude

dichloromethane (DCM) extracts after treating with spray reagent, indicated that the

different species and cultivars produce identical and different substance(s) that could be involved in stimulatory activity (Table in.7). It is evident that four compounds (Rf 0.10,

0.60, 0.70 and 0.77) were present in most cultivars studied. However, a different

substance (R f 0.29) was detected in the roots and stems o f C. cajan cv. cits2. The

distinct separation of spots on TLC results indicate that it is possible to isolate compounds

that may have a role in S. hermonthica seed germination stimulation in acceptably high

yields. Table m.8 lists the compositions of the sterol fractions of five cultivars. The extracts contained chondrillasterol (15) and/or stigmasterol (17) as the dominant sterol

components. It is evident that the co-occurrence o f both sterols have antagonistic effect

in stimulation of Striga seeds. This prompted us to further investigate the effect of

various concentrations of chondrillasterol (15) on germination o f Striga seeds (Figures n i.l and m.2).

ni.4. DISCUSSION. The percent germination data obtained from this study

indicate that the cultivars produce chromatographically different and identical substances

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which might individually stimulate the germination o f conditioned S. hermonthica seeds.

Two types o f stimulants are produced, one which is soluble in dichloromethane (DCM)

but not in water, and a second type, soluble in both water and dichloromethane (DCM).

The stimulatory activity in dichloromethane (DCM) extracts might be due to lactone-

forming acids (Long, 1955) or related compounds. Aqueous extracts probably contain

large amounts o f water-soluble phenolic compounds (Lakshmi and Jayachandra, 1979)

which might be responsible for any inhibitory action. Any other variations in stimulatory

activity may be attributed to quantitative differences in stimulants in the dichloromethane

(DCM) or water extracts. The identification of effficaceous sources of S. hermonthica

seed germinators among nonhost cultivars is highly desirable for possible use in rotations

with host crops. The most important quality characteristics for utilization of a cultivar are

a high content of dichloromethane (DCM) and/or water-soluble germination stimulants.

Vigna unguiculata cv. it-81d-975 meets these requirements, having large amounts o f both

DCM and water extractable stimulants. Based on our germination data, it is not

unreasonable to hypothesize that the V. unguiculata cultivars and some o f the legume

cultivars studied contain large amounts o f germination stimulants highly related to alectrol

(Muller et aL, 1992). Alectrol is a highly potent germination stimulant for Alectra vogelii

and S. gesnerioides and has been isolated from the root exudates o f Vigna unguiculata

(Muller era/., 1992).

Proposed structure o f alectrol

The Rf values obtained from TLC experiments indicated a correlation between the

presence or absence o f certain compound(s) with stimulation/inhibition of germination.

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TLC can be employed as a selective and non-destructive micro-detection technique to

monitor the production o f germination stimulants by legume cultivars throughout their life

cycles. This technique could reduce the tedious S. hermonthica seed bioassays. Although the germination data (Figures HI. 1-2) are d iffic u lt to interpret, it is

evident that chondrillasterol (15) is a phytohormone (Gladu et aL, 1991; Benveniste,

1986; Heftmann, 1971). This is encouraging because chondrillasterol (15) and

stigmasterol (17) are ubiquitous in plants, and through synthetic modifications similar to

those proposed by Adam and Marquardt (1986), it may be possible to design brassinolide-like substances with potent Striga spp. germination activities. Every steroid

molecule is accessible for microbiol hydroxylation. In fact almost all the positions in a

steroid molecule have been hydroxylated by various m icrobiol strains (Mahato and

Baneqee, 1985). It is gratifying to note that several methods have been proposed for

detection of steroids (Takatsuto, et a i, 1982). Recently a new plant-growth promoting

steroid, brassinolide (18), has been isolated from rape pollens {Brassica napus L.)

(Adam and Marquardt, 1986). Brassinolide (18) is especially noteworthy because it is the first isolated natural steroid containing a seven-membered B-ring lactone and an ap

configuration at the C-22 position. Furthermore, it exhibits a broad spectrum of

biological activities (e.g. it promotes both cell elongation and cell division, resulting in

curvature, swelling and, more dramatically, splitting o f the intemode in the bean second-

intemode bioassay and shows a strong activity in the lamina inclination assay at very low

concentration) compared with known plant hormones (Yopo e ta l, 1979; Meudt e ta l.,

1979). The microanalysis o f brassinolide (18) and other brassinosteroids using GC/MS

has been investigated and the corresponding bismethaneboronates have been found to be

more suitable derivatives (Takatsuto, et aL, 1982). Since brassinolide (18) has two

vicinal diols in the side chain and the A-ring, a methaneboronate is the best derivative for

GC analysis. Bismethaneboronate (19) and its analogues exhibit sharp peaks (in GC

analysis) and the derivatives can be separated by chromatographic methods. The electron

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ion (E l) mass spectrum o f 19 shows several fragment ions. Notably, the ion at m/z 457

result from C 23-C24 fission and the strong peak at 345 result from C 17 -C20 fission. The

ion at m/z 155 correspond to the side-chain cleavage and is the base peak in the spectrum.

The characteristic ion for a B-Iactone at 332 is useful for structural determination of brassinolide analogues.

HO»,

H 0 " “

Brassinolide (18)

HL5. CONCLUSIONS. Further work on isolation and determination of the specific

structure(s) o f the germination stimulant(s) in water and dichloromethane (DCM) extracts

is necessary. Bioassays o f the purified compounds on Striga spp. seeds w ill provide

invaluable information on whether they act in additive, synergistic, or antagonistic

fashion. Control strategies which need to be investigated include direct application of

crude or purified active products, as well as a direct incorporation of plant residues

containing significant concentrations of S. hermonthica germination stimulants into

infested soils (Fischer et aL, 1990). This would also provide information about the soil

stability o f germination stimulants.

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Me— B

Bismethaneboronate (19)

Me 374

345

155

457! 71

« 332 ►

Fragments o f Bismethaneboronate (19) as observed in Electron ion-mass spectrum.

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Table E L I. Species and common names o f legumes studied

Species Common Name

Vigna radiata (L.) Wilczek cv. tvi28 Mungbean

V. ratüata (L.) Wilczek cv. tvr34 Mungbean

V. umbellata (Thumb.) Ohwi & Ohash cv. w il40 Rice bean

V. umbellata (Thumb.) Ohwi & Ohashi cv. tval Rice bean

V. unguiculata (L.) Walp. cv. iar-48 Cowpea

V. unguiculata (L.) Walp.cv. it-81d-975 Cowpea

Lablab purpureus (L.) Sweet cv. tln l Lablab

L. purpureus (L.) Sweet, cv. tln28 Lablab

Cajanus cajan (L.) Huth cv. citsl Pigeon pea

C. cajan (L.) Huth cv. cits2 Pigeon pea

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Table IIL2. Percentage germination o f conditioned Striga hermonthica seeds^ treated with aqueous solutions o f strigol analogs, GR 24 (8) and GR 7 (9)

% Germination

Concentration (mg L"l) GR 7 (9) GR 24 (8 )

1,500.0 72 (58.1)* 71 (57.2)

150.0 71 (57.7) 68 (55.8)

15.0 64(53.1) 66 (54.3)

1.5 60 (50.8) 54 (47.3)

0.15 53 (46.5) 51 (45.6)

0.015 50 (44.9) 47 (43.7)

0.0015 48 (43.5) 46 (43.2)

Mean^ 59.6 (50.7) 57.8 (49.6)

L.S.Do.05 (3.52) (2.75)

* Figures in parentheses are arc sine transformations.

^ mean percent germination (N = 42).

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Table m .3. Mean percentage germination o f Striga hermonthica seeds^ treated with dichloromethane (DCM) or water extracts from leaves, stems and roots of legume cultivars

Plant DCM Water Mean*)

V. unguiculata cv. it-84d-975 67 (56.3)* 48 (41.9) (49.1) A

V. radiata cv. tvr34 44 (39.2) 25 (23.1) (31.1) B

L. purpureus cv. tln28 54 (47.3) 8 (8.5) (49.1) D

V. radiata cv. tvr28 43 (37.6) 24 (21.4) (29.5) C

V. unguiculata cv. iar-48 43 (39.1) 17 (16.6) (27.8) D

C. cajan cv. citsl 62 (52.9) 0.01 (0.2) (26.5) E

V. umbellata cv. tval 48 (41.4) 4(4.1) (22.8) F

C. cajan cv. cits2 48 (43.3) 0.01 (0.1) (21.7) G

L purpureus cw. tln l 44 (39.1) 1 (1.8) (20.5) H V. umbellata cv. w il40 34 (30.1) 5 (6.7) (18.4) I

Mean 49 (42.6) 13 (12.4)

* Figures in parentheses are arc sine transformations.

^ Values are mean percentages o f leaves, stems and roots (N = 54; for all plants

and Water or DCM extraction, N = 540 and P = 0.0001).

^ Means associated with the same letter in a given column are not statistically different

(P =0.05).

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Table IIL4.1. Effect of dichloromethane (DCM) extracts from leaves of legume cultivars on germination percentages of Striga hermonthica seeds

% Germination

Extract dilution

Plant x l xlO XlOO Mean

V. radiata cv. tvr34 69 (56.1)* 88 (69.7) 0.01 (0.2) (42.0)

V. radiata cv. tvr28 36 (36.6) 55.3 (48.0) 0.01 (0-1) (31.2)

V. umbellata cv. w il4 0 36 (36.6) 0.01 (0.1) 0.01 (0.1) (12.3)

V. umbellata cv. tval 71 (57.6) 54 (47.4) 0.01 (0.1) (35.0)

V. unguiculata cv. it-84d-975 91 (72.8) 91 (73.3) 49 (44.5) (63.5)

V. unguiculata cv. iar-48 25 (29.6) 9 (17.9) 0.01 (O.I (15.8)

L purpureus cv. tln l 24 (29.1) 65 (53.9) 0.01 (0.1) (23.8)

L. purpureus cv. tln28 82 (66.4) 93 (74.6) 20 (26.5) (27.7)

C. cajan cv. citsl 86 (68.5) 91 (72.9) 45(42.1) (36.4)

C. cajan cv. clts2 71 (57.6) 60 (50.7) 16 (23.0) (43.7)

Figures in parentheses are arc sine transformations.

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Table m .4.2. Effect of dichloromethane (D C ^ extracts from stems of legume cultivars on germination percentages of Striga hermonthica seeds

% Germination

Extract dilution

Plant x l xlO xlOO Mean

V. radiata cv. tvr34 21 (27.3)* 31 (33.6) 5 (13.2) (24.7)

V. radiata cv. tvr28 68 (55.5) 38 (38.0) 0.01 (0.1) (31.2)

V. umbellata cv. w il4 0 85 (61.3) 37 (37.2) 0.01 (0.1) (34.8)

V. umbellata cv. tval 69 (56.4) 65 (53.6) 10 (18.5) (42.8)

V. unguiculata cv. it-84d-975 57 (48.9) 60 (50.9) 19 (25.7) (41.8)

V. unguiculata cv. iar-48 87 (69.2) 64 (53.4) 14 (21.9) (48.2)

L purpureus cv. tln l 48 (43.8) 22 (27.4) 0.01 (0.1) (27.7)

L purpureus cv. tln28 51 (45.5) 37 (37.6) 0.01 (0.1) (55.8)

C. cajan cv. citsl 50 (44.8) 55 (47.9) 8 (16.4) (61.2)

C. cajan cv. clts2 62 (52.2) 53 (46.4) 13 (21.3) (39.9)

Figures in parentheses are arc sine transformations.

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Table m .4.3. Effect of dichloromethane (DCM) extracts from roots of legume cultivars on germination percentages of Striga hermonthica seeds

% Germination

Extract dilution

Plant x l xlO xlOO Mean

V. radiata cv. tvr34 48 (43.6)* 80 (63.4) 51 (45 8) (50.9)

V. radiata cv. tvr28 46(42.7) 81 (64.0) 50 (45.0) (50.5)

V. umbellata cv. w il4 0 77 (61.3) 68 (55.3) 5 (12.6) (43.1)

V. umbellata cv. tval 69 (56.1) 80 (63.3) 11 (19.4) (46.2)

V. unguiculata cv. it-84d-975 89(71.2) 91 (72.3) 54 ( 47.2) (63.6)

V. unguiculata cv. iar-48 80 (63.5) 85 (67.3) 23 (28.6) (53.1)

L purpureus cv. tln l 93 (74.9) 97 (80.8) 44 (41.7) (65.8)

L purpureus cv. tln28 98 (81.9) 79 (62.6) 26 (30.6) (58.4)

C. cajan cv. citsl 85 (67.0) 90 (71.7) 49 (44.4) (61.0)

C. cajan cv. clts2 65 (53.7) 69 (56.4) 23 (28.5) (46.2)

Figures in parentheses are arc sine transformations.

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Table in.5.1. Effect of aqueous extracts from leaves of legumes on germination percentages of Striga hermonthica seeds

% Germination

Extract dilution

Plant x l xlO xlOO Mean

V. raSata cv. tvr34 76 (60.9)* 72 (57.9) 0.01 (0.2) (39.7)

V. radiata cv. tvr28 0.01 (0.1) 0.01 (0.1) 0.01 (0.1) (0.1)

V. umbellata cv. w il4 0 0.01 (0.1) 0.01 (0.1) 0.01 (0.1) (0.1)

V. umbellata cv. tval 0.01 (0.1) 0.01 (0.1) 0.01 (0.1) (0.1)

V. unguiculata cv. it-84d- -975 80(63.8) 75 (60.0) 11 (18.9) (47.6)

V. unguiculata cv. iar-48 0.01 (0.1) 0.01 (0.1) 0.01 (0.1) (0.1)

L purpureus cv. tln l 0.01 (0.2) 0.01 (0.1) 0.01 (0.1) (0.2)

L. purpureus cv. tln28 47 (43.5) 0.01 (0.1) 0.01 (0.1) (10.7)

C. cajan cv. citsl 0.01 (0.1) 0.01 (0.1) 0.01 (0.1) (0.1)

C. cajan cv. clts2 0.01 (0.2) 0.01 (0.2) 0.01 (0.1) (0.2)

' Figures in parentheses are arc sine transformations.

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Table in.5.2. Effect of aqueous extracts from stems of legumes on germination percentages of Striga hermonthica seeds

% Germination

Extract dilution

Plant x l xlO xlOO Mean

V. radiata cv. tvr34 21 (26.5)* 0.01 (0.2) 0.01 (0.2) (8.9)

V. radiata cv. tvr28 71 (57.3) 39 (38.5) 0.01 (0.1) (32.0)

V. umbellata cv. w il40 0.01 (0.1) 0.01 (0.1) 0.01 (0.1) (0.1)

V. umbellata cv. tval 0.01 (0.1) 0.01 (0.2) 0.01 (0.1) (0.1)

V. unguiculata cv. it-84d- -975 52 (45.9) 17 (24.1) 0.01 (0.1) (23.4)

V. unguiculata cv. iar-48 50 (45.0) 62 (52.2) 14 (21.9) (39.7)

L. purpureus cv. tlnl 7 (15.0) 0.01 (0.1) 0.01 (0.1) (5.1)

L. purpureus cv. tln28 28 (31.9) 0.01 (0.1) 0.01 (0.1) (14.6)

C. cajan cv. citsl 0.01 (0.1) 0.01 (0.1) 0.01 (0.1) (0.1)

C. cajan cv. clts2 0.01 (0.2) 0.01 (0.1) 0.01 (0.1) (0.1)

* Figures in parentheses are arc sine transformations.

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Table ni.5.3. Effect of aqueous extracts from roots of legumes on germination percentages of Striga hermonùiica seeds

% Germination

Extract dilution

Plant x l xlO XlOO Mean

V. radiata cv. tvr34 47 (43.3)* 10 (18.3) 0.01 (0.2) (20.6)

V. radiata cv. tvr28 69 (56.3) 41 (39.3) 0.01 (0.1) (32.1)

V. umbellata cv. w il4 0 13 (21.2) 25 (30.3) 2 (8.0) (19.8)

V. umbellata cv. tval 34 (35.9) 0.01 (0.2) 0.01 (0.1) (12.1)

V. unguiculata cv. it-84d-•975 77(61.6) 80 (63.2) 40 (39.2) (54.7)

V. unguiculata cv. iar-48 24 (29.2) 0.01 (0.1) 0.01 (0.1) (&9)

L purpureus cv. tln l 0.01 (0.1) 0.01 (0.1) 0.01 (0.1) (0.1)

L purpureus cv. tln28 0.01 (0.1) 0.01 (0.1) 0.01 (0.1) (0.1)

C. cajan cv. citsl 0.01 (0.1) 0.01 (0.1) 0.01 (0.1) (0.1)

C. cajan cv. clts2 0.01 (0.1) 0.01 (0.1) 0.01 (0.2) (0.1)

* Figures in parentheses are arc sine transformations.

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Table IIL6. Source o f variation in percentage germination o f Striga hermonthica seeds as explained by the correlations (r^) o f legume cultivars, legume parts, concentrations and treatments

Source o f variation % variation explained (r^ x 100

Legume cultivars (ten) 9.2*

Legume parts (Leaves, stems and roots) 35.6

Concentrations (dilutions x l, xlO and xlOO) 20.4

Treatments (DCM and water) 28.8 Legume cultivars x Legume parts 21.9

Legume cultivars x Concentrations 30.3

Legume cultivars x Treatments 47.7

Legume parts x Concentrations 22.6

Legume parts x Treatments 34.3

Concentrations x Treatments 54.7

* Figures are arc sine transformed. X interaction.

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Table in.7. Thin-layer chromatography (TLC) mobilities of compounds in dichloromethane QX]M) extracts

Crop Crop part R f values^

V. radiata cv. tvr34 Roots 0.64, 0.77, 0.88 Stem 0.10, 0.40, 0.60, 0.70, 0.88 Leaves 0.60, 0.77 V. radiata cv. tvr28 Roots 0.10, 0.77, 0.84, 0.89 Stem 0.70, 0.77, 0.84 Leaves 0.23, 0.70, 0.84 V. umbellata cv. tval Roots 0.10, 0.77, 0.86, 0.89 Stem 0.10, 0.74, 0.88 Leaves 0.10, 0.12, 0.88 V. umbellata cv. w il4 0 Roots 0.60, 0.77 Stem 0.64, 0.77 Leaves 0.10, 0.40, 0.60, 0.70, 0.88 V. unguiculata cv. it-84d-975 Roots 0.60, 0.70, 0.84, 0.90, 097 Stem 0.65, 0.70, 0.77, 0.84, 0.90 Leaves 0.77, 0.88, 0.97 K unguiculata cv. iar-48 Roots 0.10, 0.88 Stem 0.10, 0.70, 0.88 Leaves 0.77, 0.82, 0.88, 0.97 L purpureus cv. tln l Roots 0.60, 0.77, 0.88 Stem 0.10, 0.64, 0.77, 0.88 Leaves 0.10, 0.40, 0.60, 0.70, 0.88, 0.92

(Table con’d)

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Crop Crop part R f values^

L purpureus cv. tln28 Roots 0.60, 0.70, 0.77, 0.81 Stem 0.69, 0.77, 0.83 Leaves 0.10, 0.44, 0.60, 0.70, 0.78, 0.81 C. cajan cv. c its l Roots 0.60, 0.29, 0.70, 0.77, 0.93 Stem 0.70, 0.83 Leaves 0.10, 0.70, 0.81 C. cajan cv. cits2 Roots 0.29, 0.43, 0.84 Stem 0.29, 0.54, 0.86 Leaves 0.10, 0.60, 0.68, 0.70, 0.81, 0.89

^ TLC, Silica gel; EtOAc-hexane, 2:5.

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HO HO

Stigmasterol (17) Chondrillasterol (15)

0.40 TLC ;R f: 0.60

25.70 GC/MS; RT: 26.10

Table m.8. Composition (%) o f sterols isolated from leaves

Legume cultivar % Chondrillasterol (15) % Stigmasterol (17)

Vigna radiata cv. tvr34 0 52

V. umbellata cv. wil40 66 31

Lablab purpureus cv. tln l 18 15 L. purpureus cv. tln28 16 13

Cajanus cajan cv. citsl 0 0

C. cajan cv. cits2 0 40

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Control

Chondrillasterol (15) 85-1 80- 75- 70- § •a 60- .1 55- Eu 50- 0Û 45_ f 40^ g 35-| i 30- ^ 25

20 - 15-

10 -

5 T" —t— —T" n r I T I r -21 -19 -17 -15 -13 -11 -7 -5 -3

4 5 -

4 0 -

.2 3 5 -

eo 2 5 -

3I 2 0 - T%L

1 0 -

-21 -19 -17 -15 -13 11 -9 -7 -5 3 Log [concentration (in Moles/L)] Fig. E LI. Effect of chondrillasterol (15) on germination of conditioned Striga hermonthica seeds occurring in Bida (A) and Kano (B) locations in Nigeria. Data are arc sine transformed relative percentage germination expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) dissolved in methanol (fig. con'd).

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Control

Chondrillasterol (15) 4 7 -

4 2 -

O 3 7 -

22 -

1 7 -

1 2 -

-21 -19 -17 -15 -1311 9 7 5 3

50-1

4 5 -

C 4 0 - ê

•3 3 5 - 00 3 0 -

2 5 -

2 0 -

1 5 -

-21 -19 -17 -15 -13 -11 9 7 5 3 Log [concentration (in Moles/L)] Fig. in .l. Effect of chondrillasterol (15) on germination of conditioned Striga hermonthica seeds collected in Abuja location in Nigeria during the 1992 (C) and 1993 (D) harvesting seasons. Data are arc sine transformed relative percentage germination expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) dissolved in methanol.

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Control

Chondrillasterol (15)

3 5 -

O 3 0 -

2 5 -

eo 2 0 -

gI 1 5 -

10 -

5 -

0 -

-21 -19 -17 -15 -13 11 9 7 5 3 Log [concentration (in Moles/L)]

Fig. ni.2. Effect o f chondrillasterol (15) on germination of conditioned Striga aspera seeds occurring in Kano (E) location in Nigeria. Data are arc sine transformed relative percentage germination expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) dissolved in methanol.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IV

IN VITRO GERMINATION OF STRIGA HERMONTHICA AND S. ASPERA SEEDS BY 1-AMINOCYCLOPROFANE-l-CARBOXYLIC ACID (ACC)

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IV. 1. INTRODUCTION. Effects of various solutions of pure compounds on

germination of Striga spp. seeds has been the central focus o f our research. Thus it is

appropriate to define the term “seed germination” . Seed germination occurs when the

growing seed tissue pierces or breaks the seed coat, including the endosperm and/or

pericarp. Therefore, the ease of seed germination depends upon the growth potential of

the seed tissue and/or the mechanical resistance o f the seed coat Seeds display two basic

types of dormancy: (I) primary or embryo dormancy due to the failure of seed tissues to

grow, seed immaturity, or secondary dormancy due to the gradual loss of the growth

capacity of seed tissues during a period of water imbibition; (2) coat-imposed dormancy

due to the mechanical restriction of the seed coat It is possible that only the complete

loss o f the mechanical restriction of the seed envelope allows seed germination, with no

need to alter the active growth potential o f seed tissues. In the case o f Striga spp., many

researchers have tried to reduce the mechanical restraint o f the seed coat by means of

various synthetic and natural germination stimulants which include, kinetin (Williams,

1961), zeatin (Worsham et al., 1959), gibberellic acid (Cook et al., 1972, 1966)

scopoletin (Worsham et al., 1962) thiourea and allylthiourea (Brown and Edwards,

1945), sulfuric acid (Egley, 1972), sodium hypochlorite (Hsiao etal., 1981) and ethylene

(Bebawai and Eplee, 1986 and references therein). Although it has been shown that

ethylene is the most effective germination stimulant for Striga spp. (Egley and Dale,

1970), treatment of field soil with ethylene gas is not an economical option for the small-

scale African farmers.

ACC (16) was first isolated and characterized from cider apples and perry pears

by Burroughs (1957) and synthesized by Ingold etal. (1922). ACC (16) is an example

o f cyclopropane amino acids which are hypoglycemic and toxic. Several biologically

active compounds contain this moiety. For example, the chrysanthemum acids are

constituents o f the insecticidal pyrethrins, sirenin is the sex pheromone o f the water mole

Allomyces, presqualene pyrophosphate is an important intermediate in the biosynthesis of

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triterpenes and sterols, and cycloartenol may be a precursor o f phytosterols (Goad, 1970;

George and Kalyanasundaram, 1994; Law, 1971). It should he noted that in plants,

ACC (16) is present in relatively low amoimt and exposure to some types o f plant stress

can increase ACC (16) and, ultimately, ethylene synthesis (Hoffman et al., 1983).

From the structural standpoint, ACC (16) possesses no asymmetric carbon and

hence lacks enantiomers. For a clearer understanding o f its chemistry, it is instructive to

consider briefly the molecular orbital representation o f the cyclopropane ring. The carbon

atoms are believed to be hybridized in such a way that their orbitals have greater p

character than in normal sp^ bond. The two orbitals from any one carbon lie in the same

plane at an angle o f 104° to each other and, as a result, the C-C bonding orbitals ate not

directed towards each other so the bonds are described as “ bent” or “ banana” (Christie,

1970). Bonds in the cyclopropane ring, therefore, differ markedly from those in alkanes

or higher alicyclic compounds with undistorted bond angles, and cyclopropane

compounds have properties which are similar in many ways to those of alkenes. For

example, they undergo addition reactions with electrophilic reagents but with

simultaneous ring opening. The cyclopropane ring is resistant to mild oxidative

procedures including those commonly used to locate double bonds, such as ozonolysis or

permanganate-periodate fission.

To our knowledge, there are no published results on the laboratory testing o f the

Nigerian Striga hermonthica (Del.) Benth. and S. aspera (W illd.) Benth. seeds using 1-

aminocyclopropane-1 -carboxylic acid (ACC) (16). In the present study, the effect of

various concentrations of ACC (16) (ranging from 10'^ M to 10‘20 M) on germination of

these two Striga spp. seeds were examined. In addition, the various mechanisms which

have been proposed for the chemical and biological oxidation o f ACC (16) to generate

ethylene are discussed.

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rv.2. MATERIALS AND METHODS

rv.2.1. General. 5. hermonthica (Del.) Benth seeds were collected from sorghum,

in Bida (1993), Abuja (1992 and 1993), and Kano (1991) locations in Nigeria. 5. aspera

seeds were collected in Kano (1993) location. ACC (16) and the strigol analogue, GR

24 (8) were supplied by D. K. Berner.

IV.2.2. Viability Test. Viability of Striga spp. seeds was estimated using

tétrazolium red (2,3,5-triphenyltetrrazolium chloride) test (Hsiao et a i, 1979).

Approximately 400 seeds were placed on a clean sheet o f filter paper in a petri dish. The

filte r paper was then saturated with 1.0% tétrazolium red solution (pH 7). The petri dish

was sealed and incubated at 35°C for 5 days. Embryos o f viable seeds stained pink when

observed under a microscope. Very pale pink and unstained embryos indicated non-

viable seeds. V iability experiments were repeated eight times and the mean percentage

viability was determined. The total percent viability (V) was calculated on the basis o f the formula; V = (G/T) x 100, where G = the number of viable seeds and T = total number of

seeds.

rv.2.3. Preparation of ACC (16) and GR 24 (8). Because ACC (16) is very

soluble in water, stock solutions (10"^ M) were prepared using sterile deionized water.

GR 24 (8), the positive control, is hydrophobic and stock solutions (10"^ M) were

prepared using methanol as a solvent carrier. The final methanol concentration was

adjusted to 0.1 % (v/v). Solutions were refrigerated at 4oC. For each experiment, fresh

solutions of lower concentrations (10'^ to lO"^® M) were prepared by serial dilution of

the stock solution and were then evaluated as germination stimulants for Striga spp. seeds

as described in Chapter HI (see Materials and Methods).

rV.3. RESULTS. Viabilities (V) for S. hermonthica isolates from Bida, Abuja

(1992), Abuja (1993) and Kano (1991), were determined to be 87, 74, 73 and 70%

respectively. For 5. aspera isolate from Kano (1993), V was determined to be 66%. The

effect o f ACC (16) concentrations on the germination o f conditioned S. hermonthica and

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S. aspera seeds is summarized in Figures IV, 1-2. Compared to GR 24 (8), ACC (16)

did not show substantial differences in stimulation of germination. Both compounds

caused germination stimulation which did not show linear correlation with concentration.

The treatment o f conditioned Striga spp. seeds with negative control (sterile deionized

water) elicited no stimulatory activity. The isolate of S. hermonthica occurring in Bida

location showed both the least and the greatest seed germination responses to ACC (16)

and GR 24 (8), respectively. Furthermore, germination of this isolate did not

signiricantly respond to ACC (16) at concentrations lower than lO"^^ M. Conditioned

S. aspera seeds showed both the greatest and least germination response at lO'^^ M and

lO 'l^ M, respectively. It should be noted that the concentration range in our experiments

was broad and the solutions were two to five times more dilute than those employed by

Babiker gfu/. (1993).

rV.4. DISCUSSION. Babiker and coworkers (1993) observed that ACC (16) alone

did not stimulate germination or ethylene production by 5. asiatica seeds. According to

them, the inability o f 5. asiatica seeds to convert ACC (16) into ethylene was suggested

by lack o f response to ACC (16) at the concentrations used (5 to 200 mM) and is an

indication of low ethylene forming enzyme (EFE) activity. A limited ability to convert

ACC (16) into ethylene has been associated w ith dormancy o f several seeds which

require ethylene for germination (Corbineau et al., 1989; Satoh et a i, 1984). In our

experiments, the low percentage germination observed in conditioned seeds o f S.

hermonthica and S. aspera when treated with ACC (16) suggested the presence o f low

levels of EFE (Babiker et al., 1993; Jackson and Parker, 1991). Logan and Stewart

(1991) have indicated that ACC (16) at or below 100 mM did not induce germination or

ethylene production and that a very high concentration (50 mM) was needed to give

germination figures close to those attained by sorghum root exudates. This response to

ACC (16) may be due to inhibition of root elongation by the high concentration of

ethylene generated (Abeles et al, 1972; Eliasson, 1989). It is tempting to speculate that

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although some Striga spp. may have a limited capacity to convert ACC (16), the

immediate ethylene precursor, to ethylene, exogenous introduction o f 16 to conditioned

Striga spp. seeds possibly triggers the activation and/or synthesis o f EFE and consequent

conversion to ethylene (Adams and Yang, 1979).

rv.4.1. Mechanisms of Formation of Ethylene from ACC (16). The main

biological source of ethylene in plants is methionine (20) and it has been established that

ACC (16) is the immediate precursor of ethylene biosynthesis (Yang and Hoffman,

1984). The synthesis o f ethylene is regulated by two different types o f factors. One type

is promoted by internal signals, as ethylene plays an important role in some

developmental stages, e.g. seed germination (Ketring and Morgan, 1972; Logan and

Stewart, 1991; Stewart and Press, 1990), root and leaf growth (Chadwick and Burg,

1970), leaf abscission (Kao and Yang, 1983), and flower and fru it senescence (Burg,

1968). Ethylene enhanced in the developmental stage is recognized as “ auxin induced”

ethylene. Ethylene induced under stress conditions is defined as “ stress ethylene” (Yang

and Hoffman, 1984). Very pronounced stress ethylene is produced when the plant is

under environmental stresses, e.g. water stress (Hoffman et aL, 1983), chilling stress

(Field, 1984), wounding (Hoffman and Yang, 1982), the exposure to SO 2 (Meyer et ai,

1987), ozone (Langebartels etal., 1991) or other pollutants (Fuhrer, 1982).

The enhancement of ethylene production, together with the changes in

concentrations o f other kinds o f plant hormones, provides the plants with the mechanisms

in adapting or avoiding the environmental stress. Quite a few papers have been published

about the changes in the concentrations o f ACC (16) in stressed plant tissues. Many

observations demonstrate that the level o f 16 in stressed plants increases many times

more than that under normal conditions (Kende and Boiler, 1981; Elstner et aL, 1985;

Rodecap and Tingey, 1986). Treatment o f conditioned S. hermonthica and S. aspera

seeds with ACC (16) possibly relieves this stress. Although the present study was not

aimed at investigating the mechanism of action of ACC (16), the germination data

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suggested a hormonal type o f action (Lockwood and Brain, 1976). Various mechanisms

showing how ACC (16) stimulate the biosynthesis of ethylene are depicted in Schemes

IV . 1-4.

Adams and Yang (1979) postulated a mechanism to account for the formation o f

ethylene (Scheme IV . 1). The first step is the activation o f methionine (20) by adenosine

triphosphate (ATP) to produce S-adenosylmethionine (SAM) (21), which then reacts

w ith pyridoxal enzyme (in Scheme IV . 1, pyridoxal phosphate stands fo r pyridoxal

enzyme) to form a Schiffs base (22). It is well known that pyridoxal enzymes are

capable o f catalyzing y-elimination (1,3-elimination) of the proton from the a-carbon of

an amino acid yielding a carbanion (Davis and Metzler, 1972). Because the positive

sulfonium group of SAM (21) is an excellent leaving group, once the carbanion is

formed, an intramolecular nucleophilic displacement reaction can occur, resulting in the

elimination of 5' -methyIthioadenosine (MTA) and the formation of ACC (16). The

carboxyl, C-1, C-2, and C-3 of 16 are derived, respectively, from C-1, C-2, C-3, and C-

4 of methionine (20). The enzyme mediating the conversion o f ACC (16) to ethylene

has not been isolated and characterized. However, the characteristics o f the in vivo

reaction strongly suggest that the enzyme is membrane-associated, labile and disrupted by

various treatments (Yu et aL, 1979). The conversion process is oxygen-dependent and is

inhibited by Co^+, temperatures above 35°C, light and uncouplers such as dinitrophenol

(Yu cf aL, 1979). Based on the enzymic reaction catalyzed by this enzyme (ACC

synthetase ?) it can be classified as SAM-MTA-lyase (o,y-eliminating).

rv.4.2. Hypochlorite Chemistry. For satisfactory and reproducible germination

data, the use of a specific concentration of sodium hypochlorite (NaOCl) is critical.

Striga spp. seeds disinfested with solutions o f different concentrations o f NaOCl for one

to several minutes showed differences in germination response to the positive control,

GR 24 (8) (data not shown). NaOCl is effective as a disinfecting and sterilizing agent

against a broad range o f bacteria, viruses, and fungi (Mercer and Somers, 1957). It is

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commonly used in pre-treatment for seeds in Europe and North America (Guthrie, 1978),

particularly for seeds which may be excessively contaminated with saprophytic fimgi

(Knudson, 1950). The chemical is used to eradicate those surface-home organisms

which may interfere with germination. NaOCl has a strong oxidizing property which

makes it highly reactive with amino acids (Kantouch, 1971), nucleic acids, amines, and

amides (Hayatsu et al., 1971). The general reaction between amino acids and NaOCl

produces the respective aldehyde, NH 4CI and CO 2 (Kantouch, 1971).

Hiyama et al. (1975) found that substituted cyclopropylamines react with NaOCl

or other oxidants to form ethylene and other products. For example, 1-

phenylcylopropylamine (26) is oxidized via nitrenium ion intermediate (27) to yield

ethylene and benzonitrile (Scheme IV.2). Piming and McGeehan (1983) suggested ring

opening o f the nitrenium ion (27) to afford the ketimine (28) (Scheme IV.3). According to them, hydrolysis of 28 yields 2-oxo-3-butenoic acid (29) which cyclizes to a-

Ketobutyrolactone (30). Support for the processes in scheme IV.3 was obtained by

following the oxidation of [l-^^C, ^^N] ACC (16) in an NMR spectrometer. The ^^N

atom split the enriched C-1 signal in its ^^C NMR spectrum. The signal at 37.8 ppm

appears as a doublet, with a coupling constant of 8.7 Hz. Addition of NaOCl to the

solution results in a new doublet appearing at 122.8 ppm with a larger coupling constant

(18.5 Hz) (Route A). This coupling is characteristic o f cyanide groups and is assigned to

the cyanide group of the cyanoformic acid. Moreover, oxidation of labeled ACC (16)

with N-chlorosucciniraide result in appearance o f strong singlets at 207,142, and 97 ppm (Route B). These, they assigned to the 2-keto group o f 29, C-3 o f (31) (the enol o f a -

ketobutyrolactone), and C-2 of (32) (the hydrated form of (29)), respectively. It is w ell known that NaOCl reacts with a-amino acids with the formation o f N-

chloroamine as an intermediate which is ultimately degraded into aldehyde, ammonia, and

CO2 (Schonberg and Moubacher, 1952). In keeping with the results o f Hiyama et al.

(1975), Lizada and Yang (1979) developed a sensitive assay for ACC (16), in which the

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amino acid is treated with sodium hypochlorite in the presence of mercuric chloride,

resulting in 80% yield of ethylene (Scheme IV.4). Cupric ions also catalyzed the

reaction. In the absence of the divalent ions the yield o f ethylene was only 13%. The C-

1 and the carboxyl group of ACC (16) were converted to oxalic acid via its monoamide (33).

rv.4.3. Possible Basic Mechanisms of Action of Ethylene on Germinat

ion o f Striga Spp. Seeds. The biological effects o f ethylene are w ell known (Pratt

and Goeschl, 1969). There are three ways in which ethylene might stimulate germination

o f Striga spp. seeds; (1) by serving as a cofactor in some reaction; (2) by being oxidized

to some essential component and being incorporated into tissue; or (3) by binding to a

receptor, providing some essential function, and then either diffusing away or being

destroyed as is the case with other hormones. Each o f these ways has been investigated

(Beyer, 1979; Abeles et ai, 1972). However, the in itia l events that occur at the ethylene

receptor site(s) which cause these effects are entirely unknown. Likewise, the location,

chemical nature, and the type o f bonding which occurs between the receptor site(s) and

ethylene (e.g., covalent, coordinate, van der Waals) and the subsequent molecular

changes, i f any, that ethylene undergoes during receptor site activation are unknown.

Burg and Burg (1967) have proposed on the basis o f indirect evidence that ethylene binds

to a metal-containing receptor site. Deuteration, especially where the deuterium is directly

bound to an unsaturated carbon atom as in ethylene, increases the stability o f silver-ion

olefin complexes (Jones, 1968). A basic question concerning the primary mechanism o f

ethylene action is whether ethylene acts catalytically or whether it is permanently

incorporated into the tissue during mechanism of action. Conflicting results have

appeared in the literature as to the extent o f incorporation when radioactive ethylene is

applied to fru it tissues. Jansen (1963, 1964) found that mature green avocados and green

pear fruits exposed to 1 ml/L of 1^-labeled ethylene for several days incorporated 0.05%

ethylene o f the applied radioactivity. In other experiments, however, Buhler et al. (1957)

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failed to obtain any incorporation o f radioactivity into ripe oranges, limes, papayas, green

apples, tomatoes, and grapes. Abeles etal. (1972) suggested that covalent bonding is

important in ethylene action. Based on their results deuterated ethylene had the same

physiological effect as protonated ethylene suggesting that C-H bonds were not broken.

Furthermore, isotopic discrimination experiments did not make the deuterated ethylene

(C 2D4) approximately half as effective as its lighter analogue (C 2H4 ). There is no

support that hydrogen bonding might be important in ethylene action. It appears that the

forces which bind ethylene to its site of action must be of the weak van der Waals type (Abeles era/, 1972).

From the structural point of view, two o f the sp^ orbitals of each carbon atom of ethylene overlap with the Is orbitals o f the two hydrogen atoms to form two strong o C-H

bonds, while the third sp^ orbital of each carbon atom is used to form a strong

~~bond between the two carbons. The two unhybridized 2p orbitals of the two carbon~~

~~atoms are parallel to each other and can themselves overlap to form a molecular orbital (the K orbital) spreading over the two carbon atoms and is situated above and below the~~

~~plane containing the carbon and hydrogen atoms. The electron occupying this molecular~~

~~orbital are called n electrons. The carbon atoms are drawn closer together by the k bond.~~

~~The lateral overlap o f the p orbitals that occurs in forming a % bond is less effective than~~

~~the linear overlap that occurs in forming a G bond, and the 7t bond is less effective than~~

~~the a bond, as well as being different in position (Sykes, 1965).~~

~~The direct chemical reaction of ethylene with an enzyme, a structural protein,~~

~~nucleic acid or other cell components is a possible basic mechanism o f action o f ethylene.~~

~~The most reactive portion o f ethylene is, of course, the double bond, for the distribution~~

~~of the 7c electrons in two layers, above and below the molecule and extending beyond the~~

~~C-C bond axis, means that a region of negative charge exists to attract any electron- seeking (i.e. oxidizing) agents. Because the two electrons in there orbital are less firm ly~~

~~held between the carbon nuclei, they are more readily polarizable than those o f the a~~

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~~bond. Additions to ethylene can proceed through ionic mechanisms, as the tc electrons~~

~~become polarized, either by approaching agent or other causes, as follows:~~

~~K — X — X lomc Ethylene Free radical~~

~~This type o f process tends to predominate in polar solvents, while free radical~~

~~additions predominate in non-polar solvents, particularly in the presence of other radicals~~

~~and light. Ethylene is an effective free radical scavenger (Stahmann, 1968). Water, a~~

~~polar solvent, is the major constituent of living organisms, and most biochemical~~

~~reactions occur in aqueous solutions. Also, membranes o f the cell have a high proportion~~

~~o f proteins and phospholipids, both polar in nature. Neutral lipids make up a small~~

~~proportion o f the membranes, but constitute the majority o f fat deposits of o il globules in~~

~~plant cells.~~

~~Ethylene, with its high solubility in both lipids and water, could be expected to be~~

~~found in all these locales, both polar and non-polar. Because polar solvents for ethylene~~

~~predominate in the cell, one would expect ionic reactions to be quantitative and most~~

~~important, with the electrophilic reagents attacking the double bonds. However, it must~~

~~be kept in mind that a quantitatively unimportant reaction may be qualitatively significant,~~

~~because o f the chain reactions it sets in motion.~~

~~Certain metallic ions might fill the role of an electrophilic reagent in an attack on~~

~~the double bond. Suppose, for example, that a metal M is part o f an oxidoreductase or~~

~~other electron transferring agent, and that it could be reversibly oxidized and reduced~~

~~between two states, M++ and M+. For the sake o f sim plicity, we shall assume that the~~

~~electron acceptor is oxygen, although it could well be an intermediate electron carrier that~~

~~would either directly or through other electron carriers transfer its electrons to oxygen.~~

~~We envision a series of reactions (scheme IV.5) similar to that proposed by Jones (1968).~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 83~~

~~In its oxidized state the metal is assumed to attack the double bond, but not in its~~

~~reduced state (Scheme IV.5). The affinity of ethylene for R-M++ is much greater than for~~

~~R-M^. After the metal removes an electron from ethylene, the now positively charged~~

~~carbon on the ethylene can take up an electron 6om a donor (or could form a bond with a~~

~~nucleophilic reagent). For these reactions to continue, since the amount o f compound R-~~

~~M in the system is probably limited in typical biological fashion, R-M+must give up its~~

~~electrons. Ethylene is also regenerated in its initial form. For an increase in respiratory~~

~~rate in the presence of trace amounts o f ethylene, the ethylene-R-M complex, if it exists,~~

~~must complete the process o f taking up and giving up its electrons more rapidly than does~~

~~R-M alone. Large amounts o f ethylene might crowd out the oxygen ( if it were the~~

~~immediate acceptor o f electrons from R-M), preventing access to the M+ site on M-R and~~

~~thus slowing down electron transfer. This probably explain the observed decline in~~

~~respiration after the climacteric peak in both respiration and ethylene production. The~~

~~mechanism would also explain why low oxygen tensions reduce the efficacy o f ethylene~~

~~in producing some of its physiological effects. Another factor to consider is that certain~~

~~concentrations o f carbon dioxide act as competitive inhibitors o f ethylene in at least some~~

~~o f its effects. The fact that the concentrations required are high may also be linked to the~~

~~crowding concept. The affinity of CO 2 for R-M+would have to be much less than that of~~

~~ethylene, i f it were so. On the other hand, CO 2 could have its effect through an entirely~~

~~different mechanism. However, the grossly observable physiological effects result from~~

~~complex interweavings of reactions; and their interpretation in terms of action of one~~

~~substance is premature to our knowledge.~~

~~No direct evidence for this basic mechanism of action (Scheme IV.5) o f ethylene~~

~~exists at present, but perhaps the outline o f it w ill stimulate the search fo r i t In principle,~~

~~there are a number o f metal containing-compounds involved in electron transfer in living~~

~~systems, not all o f them react with ethylene with equal ease. The influence on the metal~~

~~o f the R group as well as of the properties of the metal itself are involved in determining~~

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~~the reactivity; and little information exists on this influence, where biological compounds~~

~~are concerned.~~

~~It is also important to consider the chemicals surrounding ethylene in the cell. For~~

~~example, lipids also have double bonds, and free radical reactions w ith ethylene are a~~

~~possibility. What other changes can one expect to take place in the ethylene molecule~~

~~itse lf when it accomplishes its biological functions? One thinks first, in these days of~~

~~allostery, of conformational changes, where the same group of atoms can assume~~

~~different arrangements without the breaking of any bonds. However, it is known that~~

~~there is resistance to rotation about the carbon-carbon double bonds. The reason for this is the overlap o f the 2 p atomic orbitals forming the n bond, and thus the strength of the~~

~~n bond, w ill be at maximum when the two carbons and four hydrogen atoms are exactly~~

~~coplanar. It is only in these position that the k atomic orbitals are exactly parallel to each~~

~~other and capable of the maximum overlapping. Any disturbance o f this coplanar state by~~

~~twisting about the c bond joining the two carbon atoms would lead to reduction in k~~

~~overlapping and hence a decrease in the strength o f the n bond: it w ill thus be resisted.~~

~~Even i f one does not expect the conformational changes in ethylene itself, one should~~

~~keep in mind that other substances attracted by the ethylene itself, may alter their~~

~~conformation. W ith enzymes, conformational changes often accompany changes in~~

~~reactivity.~~

~~IV.5. CONCLUSION. There are many potential mechanisms of formation of~~

~~ethylene from ACC (16) as an intermediate and also for the action of ethylene in~~

~~biological systems. To thoroughly explore these possibilities it w ill take the combined~~

~~efforts of many specialists, including chemists, physicists, horticulturalists,~~

~~physiologists, biochemists and pathologists. The knowledge gained thereby w ill also~~

~~have application in many fields, most foreseeable o f which is the control o f the noxious~~

~~Striga spp. seeds.~~

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~~.COOH œOH ATP ♦ NHa NH3 Methionine (20) SAM (21) Ade~~

~~pyridoxal phosphate OH OH~~

~~COOH H~~

~~Elimination o f a-H Iu (^ C H~~

~~H Aromatization Schiff base (22) O2 f H2O MTA- I ^COOH Hydrolysis O-OH~~

~~H2N COOH ACC (16)~~

~~NH3~~

~~H2c X~~

~~Ethylene H2n '^ COOH (25) «COOH " ^ Formic acid f H2N Formamide Scheme IV. 1. Mechanism o f Adams and Yang (1979) for the biosynthesis o f ethylene.~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 86~~

~~Pb(OAc )4 e x ; CH2CI2 K 1-phenyIcyclopropylamine (26) OAc~~

~~+ HOAc~~

~~+ OAc~~

~~Nitrenium ion (27)~~

~~H H Ph — c = N + H' Benzonitrile Ethylene~~

~~Scheme IV.2. Formation o f ethylene by the chemical oxidation of I-phenylcyclopropylamine (26) (Hiyama et al., 1975).~~

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~~[0] .COOH N—H~~

~~B 37.8 ppm NH2 Ck-\,COOH ACC (16) Nitrenium ion (27)~~

~~COOH~~

~~COOH r NH * Ketimine (28)~~

~~O 207 ppm~~

~~H H Ethylene 2-oxo-3-butenoic acid (29) HI 122.8 ppm / COOH /OH / - ■ f C c-O H • 97 ppm 1 ° ° H O - ^ 0 CN‘ + CO2 a-Ketobutyrolactonejtyrolactone (30) (30) ’ * (32)~~

~~.OH CX (31)~~

~~Scheme IV.3. Pirrung and McGheehan (1983) mechanism o f formation o f ethylene.~~

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~~NaOCl~~

~~COOH COOH ACC(6) HO~~

~~NH Ethylene HO COOH Monoamide (33)~~

~~COOH I COOH oxalic acid~~

~~Scheme IV.4. Proposed mechanism o f Lizada and Yang (1979) for the oxidation o f ACC with hypochlorite.~~

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~~Electron from biological oxidations~~

~~oxidized reduced electron carrier electron carrier~~

~~© 2 ,C-C~~

~~• f V.2 '^2~~

~~2 H+~~

~~H ,0~~

~~Scheme IV.5. A Possible mechanism of action o f ethylene.~~

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~~ACC (16)~~

~~8 5 - Control 80 — 7 5 - 7 0 - § 6 5 - 'Ü 6 0 - .2 5 5 - I 50- 00 4 5 - & 40 - 1 3 5 - p 3 0 - £ 2 5 -~~

~~2 0 - 1 5 -~~

~~10 -~~

~~-21 -19 -17 -15 -13 -11 9 7 5 3~~

~~4 5 -~~

~~4 0 - 0 ’2 3 5 - .5 1 50-~~

~~I ao.~~

~~10 -~~

~~-21 -19 -17 -15 -13 11 9 7 •5 3 Log [concentration (in Moles/L)]~~

Fig. IV . 1. Effect of ACC (16) on germination o f isolates o f conditioned Striga hermonthica seeds occurring in Bida (A) and Kano (B) locations in Nigeria. Data are arc sine transformed relative percentage germination values expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) in methanol (fig. con'd).

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 91~~

~~ACC (16)~~

~~4 5 - Control S 4 0 -~~

~~2 0 -~~

~~15-~~

~~-21 -19 -17 -15 -13 -11 -9 7 5 3 55-, 50- 4 5 - 4 0 -~~

~~& 30- & 2 5 -~~

~~2 0 - 15-~~

~~1 0 -~~

~~21 -19 -17 -15 -13 -11 -9 75 3 Log [concentration (in Moles/L)]~~

Fig. rv.l. Effect of ACC (16) on germination of isolates of conditioned Striga hermonthica seeds collected in Abuja location in Nigeria during the 1992 (C) and 1993 (D) harvesting seasons. Data are arc sine transformed relative percentage germination values expressed as means ± S. E. of twelve replicates (P = 0.05). Control is GR 24 (8) in methanol.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 92~~

~~ACC~~

~~Control 25-1~~

~~2 0 -~~

•3

~~-21 -19 -17 -15 -13 11 9 -7 5 3~~

~~Log [concentration (in Moles/L)]~~

Fig.IV.2. Effect of ACC (16) on germination of isolate of conditioned Striga aspera seeds collected in Kano location (E) in Nigeria during the 1993 harvesting season. Data are arc sine transformed relative percentage germination values expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) in methanol.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER V~~

~~STIMULATION OF STRIGA HERMONTHICA GERMINATION BY llpH,13-DIHYDROFARTHENOLIDE (DHF)~~

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~~V .l. INTRODUCTION. Plant-derived chemicals are of considerable agronomic~~

~~interest because o f their low level o f hazard to nontarget plant species (Duke et al., 1987).~~

~~Strigol (2), a natural product isolated from the root exudates of a nonhost, cotton~~

~~(Gossypium hirsutum L.), has proven to be particularly effective in stimulating the~~

~~germination of witchweed seeds (Cook et a i, 1972). It has been found that application~~

~~o f 10"D M solutions of strigol (2) result in over 50% germination of S. asiatica seeds~~

~~(Kendall et at., 1979). Thus much effort has been made to explore the potential o f~~

~~synthetic strigol analogues in inducing Striga spp. seed germination. These strigol~~

~~analogues are also being used as reference compounds in evaluation o f other strigol~~

~~related compounds. However, the d ifficu lt and expensive syntheses (Kendall et at.,~~

~~1979; Mac Alpine et at., 1976; Johnson et at., 1981) as well as the requirement o f many~~

~~different types o f chemical plant equipment (V ail et at., 1990) makes the use strigol (2)~~

~~and its analogues prohibitively expensive for control o f Striga in small-scale farming.~~

~~Sesquiterpene lactones (SLs) which are ubiquitous in nature appear to be attractive~~

~~candidates in control o f Striga spp.~~

~~Sesquiterpene lactones (SLs) constitute a large and diverse group o f secondary~~

~~metabolites, which are common constituents of several plant families. However, the~~

~~greatest number o f these bitter principles have been reported in the family Asteraceae with~~

~~over 3500 reported structures (Fischer, 1991; Pieman, 1986; Seaman, 1982). It is~~

~~generally accepted that the SLs are derived from trans,trans-famesy\ pyrophosphate (34)~~

~~by in itia l cyclization process and subsequent oxidative biomodifications (Fischer, 1991).~~

~~SLs can exhibit a broad spectrum of biological activities including antibiotic, phytotoxic,~~

~~allergenic and plant growth regulatory properties (Pieman, 1986). Previous reviews~~

~~(Fischer et al., 1979; Seaman, 1982) have discussed the chemistry and taxonomic~~

~~significance o f sesquiterpene lactones. Many other studies (Pieman, 1986; Kupchan,~~

~~1970) have shown that the biological activities o f SLs can be mainly attributed to the a-~~

~~methylene-y-lactone (35) moiety and that this structural feature is almost indispensable~~

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~~for this action (Kupchan et al., 1971). Structures containing this functional group have~~

~~been reported to alkylate by conjugate addition to biological nucleophiles (Hall et at.,~~

~~1977).~~

~~The sesquiterpene lactone dihydroparthenolide (DHP, 11), has been isolated~~

~~(Fischer et at., 1981) in multi-gram quantities from Ambrosia artemisifolia (common~~

~~world-wide ragweed) and identified (Parodi et at., 1987). Dihydroparthenolide (DHP,~~

~~11) occurs in significant concentrations (0.15% o f weight) in Louisiana populations o f~~

~~A. artemisifolia and has been previously investigated for S. asiatica germination~~

~~stimulatory activity (Fischer et al., 1989; 1990). This present study was undertaken in~~

~~order to develop improved S. hermonthica control procedures based on the concept of~~

~~suicidal germination by DHP (11). To facilitate concentration-activity comparisons,~~

~~various aqueous solutions of 11 (isolated at the LSU laboratory by Fischer et al., 1981,~~

~~1989) were prepared using acetone and methanol as carriers.~~

~~V.2. MATERIALS AND METHODS~~

~~V.2.1. General. A ll bioassays were done at the International Institute o f Tropical~~

~~Agriculture, Ibadan, Nigeria. Seeds of S. hermonthica were collected in 1993 by D. K.~~

~~Berner from sorghum plants in Bida, Nigeria. A pure sample of DHP (11) was isolated~~

~~(Fischer et al., 1981) in multi-gram quantities from Ambrosia artemisifolia (common~~

~~ragweed) and identified (Parodi et al., 1987) at the LSU laboratory. GR 24 ( 8 ) was~~

~~provided by D. K. Berner. The viability (Hsiao et al., 1979) o f S. hermonthica seeds~~

~~was determined to be 87%.~~

~~V.2.2. Seed Conditioning. For each experiment, about 1440 S. hermonthica seeds~~

~~were surface disinfested by completely immersing in 3.6 mL of 1 % (w/v) sodium~~

~~hypochlorite (NaOCl) to which three drops o f the detergent "Tween 80" [polyoxyethylene~~

~~(20) sorbitan mono-oleate] were added. After shaking, the mixture was allowed to stand~~

~~for 4 min. Floating seeds were decanted and discarded. The remaining seeds were~~

~~transferred to a Buchner funnel lined with Whatman No. 1 filte r paper and rinsed several~~

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~~times with sterile deionized water until the chlorine smell disappeared. The filte r paper~~

~~containing the surface disinfested seeds was removed and the seeds air dried. The~~

~~disinfested, dried seeds were conditioned on 1 00 glass fiber disks (o f 5.0 mm diameter)~~

~~arranged on top o f two sterile Whatman No. 1 filte r papers in 9.5 cm diameter petri~~

~~dishes. S. hermonthica seeds (25-40 seeds per disk) were carefully sprinkled on each~~

~~glass fiber disk. The petri dishes were sealed with parafilm and incubated at 28°C in the~~

~~dark for 10 days. Throughout the conditioning period the filte r papers were saturated~~

~~with sterile deionized water.~~

~~V.2.3. Measurement of Germination. Because of the low water solubility of the~~

~~DHP (11) and GR 24 ( 8 ) aqueous stock solutions were prepared using acetone and~~

~~methanol as carriers. Two sets o f stock solutions o f 10'^ M DHP (11) were prepared by~~

~~dissolving 2.5 rag in 0.1 mL acetone or methanol and diluted w ith 9.9 mL distilled water~~

~~to obtain the desired concentration. 10’ ^ M GR 24 ( 8 ) was similarly prepared by~~

~~dissolving 2.98 mg in 0.1 m L methanol before diluting with 9.9 m L distilled water. The~~

~~stock solutions were refrigerated at about 4°C. For each experiment, fresh solutions of~~

~~lower concentrations were prepared by serial dilution o f stock solutions and evaluated as~~

~~germination stimulants for conditioned S. hermonthica seeds. Twelve 5.0 mm diameter~~

~~glass fiber disks containing the conditioned seeds were arranged in a 9.0 cm sterile petri~~

~~dish lined with two (Whatman No. 1) filter papers which had been moistened with 2.0 mL sterile deionized water. Using a micropipette, aliquots of 600 pL of test solutions~~

~~were uniformly applied to each petri dish containing disks of conditioned S. hermonthica~~

~~seeds. The petri dishes were sealed with parafilm and incubated at 28°C for 24 h. In all~~

~~experiments, conditioned 5. hermonthica seeds treated with sterile deionized water~~

~~containing 0.1% (v/v) acetone or methanol served as negative controls. Germination tests~~

~~were determined 24 h after application o f the test solution. Because o f the difficulties in~~

~~reproducing Striga bioassays, it was necessary to replicate each test in three separate petri~~

~~dishes per experiment and repeat the experiments twelve times over a period o f 3 weeks.~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 97~~

~~For each test, approximately 360 conditioned seeds were used. Germinated and nongerminated seeds were counted under a binocular dissecting microscope at 20X~~

~~magnification. Seeds were considered to have germinated i f the radicle had protruded~~

~~from the seed coat. The germination data were transformed to arc sine (Gomez and~~

~~Gomez, 1984) and processed by analysis o f variance (SAS, 1989).~~

~~V.2.4. Molecular Modeling. Energy-minimized geometries and conformational~~

~~energies (enthalpies) required by the major conformers o f DHP (11), strigol (2), and GR~~

~~24 (8 ) were calculated using a PCMODEL molecular mechanics program developed by~~

~~Burkert and Allinger (1982). This program uses the M M X force field. The van der~~

~~Waals volumes (VDWs) and the dipole (DP) moments were also calculated. As there are~~

~~presently no data from which it is a priori possible to choose the conformer(s) to use as a~~

~~model for DHP (11) active in stimulating germination of conditioned S. hermonthica~~

~~seeds, the energy minimization procedure was carried out starting from every conformer~~

~~that could be formed using Dreiding models. Conformers 11a and 11b are modelled by~~

~~homologues of DHP (11). MM calculations of conformation of strigol (2) were carried~~

~~out using the coordinates of X-ray crystallographic analyses by Coggon et al. (1973). In~~

~~the case of the synthetic strigol analogue GR 24 ( 8 ), the coordinates o f strigol (2) were~~

~~used in energy minimization calculations except in the A-ring. The entropy terms have~~

~~not been explicitly considered in the present work, but differences in the entropy~~

~~contributions for the molecules in the series investigated should largely be compensated~~

~~by differences in hydrophobic binding (Liljefors et at., 1985).~~

~~V.3. RESULTS~~

~~V.3.1. Germination Data. The curves of means of arc sine transformed relative~~

~~percentage germination o f 5. hermonthica seed germination plotted against the log~~

~~[concentration] of GR 24 ( 8 ), the positive control, and DHP (11) are shown in Figure~~

~~V .l. Neither compound inhibited germination at concentrations as high as 10’ ^ M. GR~~

~~24 (8 ) in methanol as a carrier exhibited high stimulation effects (54-79%) on seed~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 98~~

~~germination at all concentrations. Solutions o f DHP (11) prepared in acetone exhibited a~~

~~broader range of seed germination stimulation (19-75%) than those in methanol (32-~~

~~64%), and paradoxically, the highest stimulation of germination (75%) at IQ-^O ^~~

~~Solutions o f DHP (11) in methanol showed two seed germination “ plateaus” between 10"~~

~~^ to 10"^ M and IG'^5 to 10"16 m concentration range, respectively. Unlike DHP (11) in~~

~~acetone, GR 24 ( 8 ) elicited a single seed germination “ plateau” between IG'^^-IG'^'^M.~~

~~It is o f interest to note that while IG"^ M solutions o f DHP (11) in acetone and methanol~~

~~showed the lowest seed germination stimulation activities of 19 and 32%, respectively, a~~

~~IG'^ M solution o f GR 24 ( 8 ) elicited a high germination of 71%.~~

~~V.3.2. Molecular Mechanics Calculations. The results of MM calculations are~~

~~shown in Table V .l. The conformational energy, van der Waals volume and dipole~~

~~moment o f strigol (2) were calculated to be 43.71 kcal/mol, 2.35 and 6.38, respectively.~~

~~The energy of the most thermodynamically stable conformer ( 8 ) was determined to be~~

~~45.37 kcal/mol. The energy of conformer DHP (11a) with H-13 in a-orientation was~~

~~within 1.48 kcal/mol of the energy of the thermodynamically most stable conformer. The~~

~~interconversion between the two conformers may thus be described as a large amplitude~~

~~torsional motion. However, the energy barrier for the interconversion o f 11 to 11b (Me- 14 in a-orientation) was calculated to be large (104.04 kcal/mol).~~

~~V.4. DISCUSSION. The germination data indicated variable seed germination~~

~~stimulatory activities by DHP (11) and GR 24 ( 8 ) which can be attributed to structural~~

~~differences. GR 24 ( 8 ) possesses an aromatic A-ring and contains all o f the features of~~

~~strigol ( 2 ) w ith the exception of both o f the gem-dimethyl groups and the introduction of~~

~~formal double bonds in the 4,5 and 6,7 positions. In addition, GR 24 ( 8 ) does not have~~

~~a hydroxyl group at the 4-position as does strigol (2). Other stractural similarities of 8~~

~~and 2 that merit comment include the butenolide ring structure (D-ring o f strigol) joined to~~

~~another methyleneoxy bridge. The fact that butenolides undergo alkaline hydrolysis or~~

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~~degradation during storage, leading to variable results in Striga bioassays (Pepperman et~~

~~al., 1982; Hassanali, 1984) appears to be a major lim itation for strigol and its analogues.~~

~~On the other hand, the structure of DHP (11) is unique in several respects.~~

~~Firstly, it has a germacranolide skeleton (36) with a 4,5-epoxide of a 1(10), 4(5)- cyclodecadiene system. Secondly, it has C(14) and C(15) lying syn on the p-face o f the~~

~~medium ring. Thirdly, it does not contain the common alkylating a-methylene-y-lactone~~

~~moiety (35), which is generally considered to be essential for biological activities~~

~~(Pieman, 1986). However, the epoxide ring at the 4,5-position can be opened by~~

~~transannular cyclization generating a cationic center at C-10 which can be a receptor for~~

~~biological nucleophiles. It should be noted that 11 is stable at room temperature and its~~

~~lactone ring is similar to the butenolide (‘D’) ring of strigol (2), except that it lacks the~~

~~double bond (Fig. V .l). Previous studies has revealed a positive correlation between~~

~~general phytotoxicity and the presence o f the C -13 exocyclic methylene cyclopentenone~~

~~groups (Rodriguez, 1977). The absence o f these functionalities in DHP (11) makes it an~~

~~attractive nontoxic candidate in Striga control.~~

~~The germination data demonstrated that DHP (11) can promote germination at~~

~~concentrations lower than 10'^^ M. These activities are considerably higher than the~~

~~activities o f other natural compounds reported to affect seed germination (Duke, 1986),~~

~~and suggests that DHP (11) may be a representative o f a new class o f plant growth~~

~~hormones (Cook et at., 1972; Kalsi et at., 1977; Johnson etal., 1976). We hope these~~

~~results can be extrapolated to greenhouse and eventually, field experiments. It is~~

~~reasonable to assume that the rain washes can readily transport DHP (11) from the source~~

~~plant (crop residue or decomposing litter) into the soil where they can easily attain~~

~~concentration levels applied in our study. It has been demonstrated that iso-alantolactone~~

~~(37) can persist in mineral soil and organic soil for 3 months (Pieman, 1987), supporting~~

~~the assumption that DHP (11) may persist in the soil at least for 3 to 7 days, sufficient~~

~~time to stimulate Striga spp. seed germination.~~

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~~The failure of 10"® M solutions of DHP (11) to induce signiHcant germination~~

~~may be interpreted as a lack of intrinsic hormone-like activity and that the ability to~~

~~stimulate the endogenous ethylene biosynthesis probably failed at this concentration.~~

~~Alternatively, this could be viewed as the poor penetration or metabolic inactivation of~~

~~conditioned S. hermonthica seeds by the 10"® M solutions. Further work is needed to~~

~~establish the factors responsible for this behavior.~~

~~V.4.1. Conformational Analyses. While NMR studies have shown that strigol~~

~~(2), GR (8 ) and DHP (11) the stable conformations at room temperature (Cook et al.,~~

~~1972; ; Johnson et at., 1981; Fischer et al, 1989), it is not certain that they are the only~~

~~forms populated since the NMR method is unable to distinguish between a single “ fixed”~~

~~conformation or a mixture of conformations in rapid equilibrium resulting an averaged~~

~~symmetry equivalent to that of the single conformation. For the latter possibility, a much~~

~~lower temperature would be required to slow down the residual process on the NMR time~~

~~scale. To solve this enigma, the utilization o f molecular mechanics (MM) calculations on~~

~~the conformational analysis of germination stimulants is inevitable (Osawa et al, 1989;~~

~~Shimazaki etal., 1991 and references therein).~~

~~Ten-membered-ring system o f the germacrane skeleton o f DHP (11) is flexible~~

~~(Rscher et al., 1979). Furthermore, it is well established that the conformational freedom~~

~~is greatly reduced in the cyclic structures, and this is where the possibility exists in~~

~~developing method for exhaustive conformer generation (Goto and Osawa, 1989).~~

~~Analysis o f the conformers of DHP (11) indicated that there are at least two conformers~~

~~(1 1 and 1 1 a) which are candidates for the biologically active structure (w ithin the~~

~~context o f our “ stimulant-receptor interaction model” proposed below). Our interest was~~

~~to compare the minimum energies of DHP (11) with those o f the leading germination~~

~~stimulants, strigol (2) and its analogue, GR 24 ( 8 ) (Table V.l). Significantly high~~

~~stimulatory activities of compound 2 may be attributed to electrostatic effects of the~~

~~functionalities and also intra- and intermolecular hydrogen-bonding between strigol ( 2 )~~

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~~molecules and receptor (Lijefors et al., 1985). A t the moment, the only way we can~~

~~reconcile the relatively low stimulatory activity of DHP (11), as compared to GR 24 ( 8 )~~

~~is that it exists in a mixture o f conformers with large energy variations.~~

~~V.4.2. The Stimulant*Receptor Interaction Model. We propose the presence~~

~~o f receptor sites in Striga seeds in which the reactive sites o f germination stimulants attach~~

~~and induce the production o f ethylene. Plausible features o f receptor models may include:~~

~~( 1) the use of the entire strigol ( 2 ) molecule in the construction o f the models instead of~~

~~parts o f the molecule; (2) the receptor has a specific and probably flexible van der Waals~~

~~volume; (3) the use o f the complete structure o f different conformers in the calculations of~~

~~conformational energies; and (4) addition o f flexibility to the model with respect to the~~

~~required location of the actiphore o f the germination stimulants. Furthermore, we assume~~

~~the receptor sites are complementary to a wide variety of functional groups including the~~

~~double bond, lactone moiety, hydroxyl, and/or methyl groups. The natural strigol (2) is~~

~~thus very useful in defining spatial relationships in the cavity of the receptor active site~~

~~between positions of molecular parts crucial for full stimulatory activity (Liljefors etal.,~~

~~1985). The corresponding molecular arrangements in the studied conformers (Table V. 1)~~

~~may, through conformational changes, be positioned in these space locations. The~~

~~energy required for such a conformational change probably correlates with the stimulatory~~

~~activity of the conformer. A low conformational energy should correspond to a high~~

~~biological activity and vice versa. It should be noted that at the present stage of~~

~~development our proposed model is applicable to the natural strigol ( 2 ) and its analogues~~

~~as w ell as sesquiterpene lactones which have the capability to position the crucial~~

~~molecular parts, as defined above, in the required positions. Our stimulant-receptor~~

~~interaction model should be understood as a model for an “ activated complex” related to~~

~~the efficacy (intrinsic stimulatory activity) rather than as a model for the initial binding of~~

~~the substrate. In terms of stimulant-receptor binding energies, this model implies that~~

~~strigol ( 2 ) and its analogues have very similar interaction energies with the assumed~~

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~~receptor sites. It must also be borne in mind that the exact details o f the germination~~

~~mechanism are yet to be proven experimentally and that the mechanism may be a complex~~

~~multistep process as shown in Scheme V .l. It is necessary for a germination stimulant to~~

~~mimic the natural stimulant, possibly strigol (2) or its analogues (Hauck et a i, 1992),~~

~~with respect to the spatial locations of the actiphore. The high activity of strigol (2)~~

~~compared to its synthetic analogue GR 24 ( 8 ) indicate that not only the ggm-dimethyl~~

~~groups but also the hydroxyl group (OH-4) may efficiently interact with a hydrophobic~~

~~region o f the assumed receptor cavity, a region which is complementary to van der Waals~~

~~surface o f the substrate molecule. Thus, the shape o f the part o f the receptor cavity~~

~~interacting with the A, B, C, and D rings of strigol (2) may be a deep “groove” or~~

~~“ pocket,” at least fu lly circumscribing the actiphore (rings C and D).~~

~~The different stimulatory activities o f the biologically active compounds strigol (2)~~

~~and DHP (11) and GR 24 ( 8 ), are, according to our stimulant-receptor interaction model,~~

~~due to the differences in binding capabilities to the receptor, or to form an “ activated~~

~~complex” with the receptor. This is reflected in the differences in the conformational~~

~~energies (Table V .l). We believe that the differences in energies between the lowest~~

~~conformational energy structures (thermodynamically favorable) and higher energy~~

~~conformations correspond to the energies required to bring the molecules from their~~

~~preferred conformations to their biologically active conformations. The probability of a~~

~~germination stimulant binding to the receptor sites o f enzymes in Striga seeds probably~~

~~depends on the energy of the conformationally rearranged structure relative to that o f the~~

~~thermodynamically preferred one. Gratifyingly, the van der Waals volumes of the natural~~

~~strigol (2) and GR 24 ( 8 ) are comparable but differ from that o f DHP (11) (Table V .l).~~

~~The extra volume required by compound GR 24 ( 8 ) relative to strigol (2) is probably not~~

~~tolerated by the receptor site and this is reflected by their overall performances in~~

~~stimulation of S. hermonthica seeds (Figure V .l).~~

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~~V.4.3. Solvents. The relative stimulatory activity of DHP (11) and GR 24 (8) in~~

~~germination o f S. hermonthica seeds generally depended upon the concentration and~~

~~further modulated by the carrier systems used. This may account for the large differences~~

~~in the percentage germination by a wide range of concentration tested. The stimulatory~~

~~activity may also be associated with the ease o f penetration and subsequent metabolic~~

~~conversion of the germination stimulant to one active functional group. Alternatively,~~

~~intercalation o f each substance at a critical concentration directly into the membrane~~

~~systems o f conditioned S. hermonthica seeds may be the most relevant factor, as for the~~

~~alcohols (Taylorson, 1988). Organic solvents have been used for extraction and~~

~~dissolving natural stimulants (Brown et al., 1951). It was known that the compounds to~~

~~be tested as germination stimulants, DHP (11) and GR 24 ( 8 ) are hydrophobic, and~~

~~solutions of known concentrations would be difficult to obtain without the use of a carrier~~

~~system. The purpose of the carrier is to solubilize the germination stimulant prior to~~

~~dilution with water. The influence of carrier system on stimulatory activities on Striga~~

~~spp. seed germination was perceptible (Fig. V .l). As the concentrations of the~~

~~compounds and the solvents decreased, the percentage germination fluctuated. We~~

~~envisioned that the observed germination at concentrations below 10* 1“* M, was largely~~

~~due to the solvents employed. Increases in seed activity germination stimulation may~~

~~have been due to solvent polarity (e.g. acetone versus methanol) suggesting the existence~~

~~of different conformations in equilibrium (Bhatt etal., 1965). Djerassi etal. (1960) have~~

~~shown that in passing from a relatively non-polar to polar medium, the proportion o f the~~

~~conformer(s) with higher dipole moment increases. It is not unreasonable to hypothesize~~

~~that in general methanol-induced respiration and hence Striga spp. seed germination rate~~

~~is higher than acetone-induced. Moreover, methanol treatments have been reported to~~

~~greatly increase productivity and/or water requirements o f desert crops (Nonomura and~~

~~Benson, 1992). Methanol in small quantities is a natural product o f plant metabolism~~

~~(Zhang etal., 1993; Hansen etal., 1994; Cossins, 1964; McDonald and Fall, 1993), but~~

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~~the mechanism by which methanol affects growth and water use efficiency is hitherto~~

~~unknown. Methanol is known to accumulate in maturing seeds (Obendorf et a i, 1990),~~

~~probably as a product of pectin déméthylation by the enzyme pectin methylesterase~~

~~(PME), The presence o f methanol during germination has been reported (Vancura and~~

~~Stotzky, 1976; Taylorson, 1979). Non-viable seeds have low levels o f methanol due to a~~

~~decrease in pectin methylesterase activity (Obendorf et ai, 1990). In this study methanol~~

~~was used to dissolve DHP (11) and GR 24 ( 8 ) at much lower concentrations than would~~

~~naturally occur.~~

~~By soaking seeds o f a number o f plant species in acetone fo r six months,~~

~~Milborrow (1963) demonstrated that acetone is not toxic and does not reduce viability. He~~

~~further showed that acetone-soluble and not water-insoluble compounds penetrate seeds~~

~~and could be recovered from the seedlings. These findings suggests that acetone~~

~~employed in dissolving germination stimulants acted primarily as a solvent carrier and had~~

~~no adverse effects on the seeds.~~

~~V.4.4. Plausible Mechanism of Stimulation of Germination of Striga~~

~~hermonthica Seeds by DHP (11). W ith the success o f the laboratory bioassays and~~

~~the attendant promise of using DHP (11) in Striga control, it becomes important to~~

~~understand the molecular mechanistic basis o f its stimulatory activity. To the best o f our~~

~~knowledge, there have been no mechanistic proposals or even speculation regarding the~~

~~ways in which DHP (11) might stimulate the germination of Striga seeds. Several~~

~~unusual “ maxima” and “ minima” of percentage germination (Table V .l) suggest that DHP~~

~~(11) act in hormonal fashion (Abeles et a i, 1972) and indirectly stimulate the~~

~~biosynthesis of ethylene which then triggers seed germination (Logan and Stewart,~~

~~1991). The indirect role o f an enzyme in converting a chemically unreactive "DHP (11)"~~

~~to a reactive intermediate, possibly 38, seems to be a necessary, but not a sufficient~~

~~condition. A ll these can be understood in terms o f a plausible mechanism similar to that~~

~~proposed by Adams and Yang (1979).~~

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~~In considering stimulation o f germination initiated by an alkylation process, it is~~

~~important to recognize that DHP (11) contains as a reactive receptor site, the 4,5-epoxide.~~

~~In general, epoxides have alkylating properties and can react with biological nucleophiles,~~

~~in particular, thiol-containing amino acids o f enzymes w ith essential thiol functions, e.g.,~~

~~L-cysteine and the enzymes phosphofructokinase and glycogen synthetase (Hall et al.,~~

~~1977). In DHP (11) the epoxide function is not directly accessible due to steric~~

~~hindrance of the associated medium ring structure. However, the epoxide provides~~

~~increased reactivity, as well as regio- and steteo-specificity o f subsequent intramolecular~~

~~cyclizations (Rscher, 1991). The epoxide is activated by bio-protonation and undergoes~~

~~a Markovnikov-type transannular cyclization to give cation (38) as outlined in Scheme~~

~~V .l. I t is not unreasonable to hypothesize that the conditioned S. hermonthica seeds~~

~~contain methionine ( 2 0 ) (active nucleophilic site is sulfur) which is also present in many~~

~~plant tissues (Yang, 1974; Adams, 1977). It can be envisioned that, the cation (38) react~~

~~with the sulfur group of methionine ( 2 0 ), a reaction mediated by pyridoxal phosphate.~~

~~The positive charge imposed on the sulfur atom o f 39 would create a strong leaving~~

~~group, thus facilitating formation of Schiff base (22) via a concerted y-elimination (Davis~~

~~& Metzler, 1972) o f the thiol moiety (41) along with a-hydrogen. This result in the~~

~~formation cyclopropane adduct (24) which undergoes “chelotrophic” elimination of~~

~~ethylene as outlined in Scheme IV. 1. It should be noted that conditioned Striga seeds~~

~~have high oxygen (O 2) tensions (Hsiao, 1981) necessary for the formation o f hydrogen~~

~~peroxide (H 2O2) which reacts with ACC (16) leading to the formation o f ethylene.~~

~~V.5. CONCLUSIONS. Our germination data demonstrate that DHP (11) possesses~~

~~significant hormone-like activity in stimulating germination o f conditioned S. hermonthica~~

~~seeds. Thus in the future DHP (11) could prove very useful in control o f S. hermonthica~~

~~in agricultural situations where hormonal activity is desired. In addition, if DHP (11)~~

~~mimics hormones in its mechanism of action, it may prove very useful in studies~~

~~concerning the biochemistry of the actions of germination stimulants. Also, the carrier~~

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~~system used may affect solubilization and activity of germination stimulants. It is hoped~~

~~that the results obtained by MM analysis should be of great value in establishing the~~

~~groundwork for the use of computer molecular modeling in explaining structure-activity~~

~~relationships of agricultural compounds that exhibit Striga stimulatory activities. The goal~~

~~o f this tool in agriculture is to be predictive in the design of potent stimulatory substances.~~

~~OPP~~

~~trans.trans-îzmosyl pyrophosphate ( 3 4 ) a-methylene-y-lactone moiety (35)~~

~~Germacranolide (36) iso-alantolactone (37)~~

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~~Dihydroparthenolide (11) in acetone~~

~~Dihydroparthenolide (11) in methanol~~

~~O GR 24 (8) in methanol 85-1~~

~~80-~~

~~70-~~

~~65-~~

~~60 — I 55 & 50 H 45~~

~~40 - Î 35- 30-~~

~~2 0 -~~

~~15-~~

~~1 0 - T- T~ 1 — T" —|- “T -21 -19 -17 -15 -13 -11 -9 -5 -3~~

Log [Concentration (in Moles/L] Fig. V.L Effect of dihydroparthenolide (DHP) (11) on germination of conditioned Striga hermonMca seeds. Data are means ± S. E. o f twelve different experiments performed in triplicate (P = 0.05). Control is GR 24 (8) in methanol. Small vertical lines: standard error of mean value.

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~~Table V .l. Calculated conformational energies (kcal/mol) o f strigol (2), GR 24 (8) and DHP (11).~~

~~Compound # H-1 H-2 H-4’ OH-4 Energy VDW“ DP^ mom~~

~~2 3 5 63843.71~~

~~Compound # H-1 H-2 H-4’ Energy VDW“ DP** mom~~

~~45.37 1.92 5.60~~

~~Compound # Me-15 Me-14 H-5 H-6 H-7 H-13 Energy VDW* DP*’~~

~~P p a p a p 25.40 4.78 5.41~~

~~P P a p a a 26.88 5.12 5.41~~

~~lib P a a P a P 104.04 1.84 2.81~~

~~VDW*, van der Waals volume. DP**, Dipole mcment~~

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~~cyclization~~

~~M III I~~

~~Methionine (20) DHP (11)~~

~~H pyridinecarboxaldehyde~~

~~Schiff base (22)~~

~~CHO~~

~~pyridinecarboxaldehyde H2O + H3O+~~

~~..X% N CO2 HN = N ACC (16)~~

~~Scheme V .l. Proposed mechanism for biosynthesis o f ethylene from DHP (11). The substituted pyridinecarboxaldehyde stands for pyridoxal phosphate.~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER VI~~

~~ISOLATION OF DIHYDROELEPHANTOPIN, ISODEOXYELEPHANTOPIN AND GRINDELIC ACID FROM THE FAMILY ASTERACEAE~~

~~110~~

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~~VLl. INTRODUCTION~~

~~V I.1.1. Germacranes. Germacrane type sesquiterpenes are considered to be an~~

~~important intermediates in the biosynthetic pathway towards guaiane-, eudesmane-,~~

~~elemane- and other types o f sesquitepenes (Piet et al., 1995). Despite the fact that a large~~

~~number o f germacrane have been identified, the synthesis o f these compounds is not well~~

~~developed due to lack of suitable methods for construction o f 10 -membered rings with~~

~~appropriate functional groups. The sesquiterpenoids are C-15 compounds formed of~~

~~three isoprenoid units. Based on biogenetic assumptions, it is now generally accepted~~

~~that a ll sesquiterpene lactones arise from a common precursor, famesyl pyrophosphate,~~

~~by various modes o f cyclizations followed in many cases by skeletal rearrangements. In~~

~~sesquiterpene lactones, the y-lactone ring is capable of adopting two main conformations,~~

~~S and A, as shown below The A and the S conformations o f the lactone ring differ by the~~

~~sign of the torsion angle 0)4 . For the A conformation it is positive , and for the S~~

~~conformation, it is negative. A nwis-linked lactone ring has the S-conformation when the~~

~~torsion angle 0)4 ^ 120°C, and the A-conformation when 004 > 1200C.~~

~~Ra Ha 0 ms-lactone, S-type trans -lactone, A-type~~

~~0)4^120°C Q)4^120°C~~

~~Ra^ Hb « > 0 ^ y-lactone Ra^ Rb q~~

~~Ha c/j-lactone, S-type c/s-lactone, A-type~~

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~~V I.1.2. Elephantopus tomentosus L . The genus Elephantopus (Compositae) is a~~

~~member o f the tribe Veraonieae, and is composed o f approximately 32 species which are~~

~~distributed throughout the tropical areas of the world. The chemistry of the genus~~

~~Elephantopus is hitherto not well understood to clarify its phyletic position within the~~

~~tribe Vemonieae; therefore, a survey of the chemistry of members of the genera~~

~~composing this tribe is s till needed. E. tomentosus L. (common names; tobacco-weed,~~

~~devil's grandmother) is found in the United States and distributed from Eastern Texas~~

~~through the Southeastern portions o f the country. We now report the isolation and~~

~~structure elucidation of dihydroelephantopin (10) as a major constituent of E.~~

~~tomentosus.~~

~~V I. 1.3. Elephantopus carolinianus W illd . The presence o f desoxyelephantopin,~~

~~a type o f sesquiterpene lactone seems to be characteristic o f this genus (Bohlraann et at.,~~

~~1984). From E. carolinianus , deoxyelephantopin and several other sesquiterpene~~

~~lactones with marked anti-tumor activity have been isolated (Zhang et a i, 1986).~~

~~Isodeoxyelephantopin (12) a minor constituent o f this plant has been isolated previously~~

~~from India E. scaber (Govindachari et al., 1972), Sri Lanka E. scaber (De Silva et al,~~

~~1982), West Virginia E. carolinianus and North Carolina E. carolinianus (Zhang et al,~~

~~1986). 12 has shown significant cytotoxicity with ED 50 = 2.5 lig/m L against the in vitro~~

~~growth o f KB tissue culture cells.~~

~~V L1.4. Grindelia maritima L . Grindelia species are annual, biennial and perennial~~

~~shrubs and herbs o f the Asteraceae (tribe Astereae, subtribe Solidagininae ). One o f the~~

~~main problems in the tribe Astereae is the delimitation o f its subtribes (Jakupovic et al.,~~

~~1986); chemical characters therefore might prove helpful here. So far chemical~~

~~investigations have shown that labdane diterpenes (which have the e/ir-labdane absolute~~

~~configuration) and clerodane type are widespread in this subtribe (Bohlmann et al., 1982;~~

~~A d in o lfi et al. 1988). The elucidation o f the ecological roles o f the resins in Grindelia~~

~~spp. is only beginning. Resins are useful for a variety of industrial applications including~~

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~~tackifiers, adhesives, and paper sizings; their derivatives are widely used in the~~

~~production of synthetic polymers. Studies of resin biosynthesis, metabolism and~~

~~transport w ill lead to significant taxonomic advances. The marked variation in both~~

~~quantity and composition of resin produced by different populations make them attractive~~

~~models fo r study o f genetics and inheritance o f resin production (Timmermann et al.,~~

~~1987).~~

~~The North American Grindelia maritima L. (common name “ gumweed” ) is an~~

~~annual herb and like the other members in the same genus, is characterized by the~~

~~abundant production o f resinous exudates which cover the surfaces o f the leaves, the~~

~~involucre o f the flower heads and stems. The production and accumulation o f these~~

~~conspicuous hydrophobic, non-volatile resins appear to be associated with m ulticellular~~

~~resin ducts which occur in the leaf mesophyll and stem cortex and sessile resin glands~~

~~which occur in shallow pits on the surfaces o f the leaves, stems, and involucre o f the~~

~~flower heads. Heavy resinous coatings may be a phytochemical and ecological adaptation~~

~~to the arid and semi-arid environments to which these plants are exposed (Hoffmann et~~

~~a l, 1984).~~

~~VI.2. MATERIALS AND METHODS~~

~~VI.2.1. Isolation of Dihydroelephantopin (10). Aerial parts of E. tomentosus~~

~~were collected at the flowery stage on October, 19, 1992 at Lee Forest Station,~~

~~Washington Parish, Louisiana. A voucher specimen is on deposit in the Herbarium o f~~

~~LSU (Fischer collection 435. Air-dried leaf material (40 g) was ground in a Waring~~

~~blender w ith 150 mL o f dichloromethane (CH 2CI2). After filtration, the dichloromethane~~

~~extract and washings were evaporated to afford a yellow-brown syrup which was~~

~~chromatographed over a column of silica gel 60 (Merck). Elution was effected with~~

~~hexane and EtOAc (gradient elution; increasing the volume o f the more polar solvent,~~

~~EtOAc). The medium polar fraction (EtOAc-hexane, 7:20 v/v) yielded 560 mg of~~

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~~dihydroelephantopin (10) as a white crystalline solid (C 19H22O7 , Mr. 362.38; m.p.: 266-~~

~~268°C; Rf, 0.55, EtOAc-hexane, 3:7).~~

~~VI.2.2. Isolation of Isodeoxyelephantopin (12). The air-dried leaves (3.0 g)~~

~~o f £. carolinianus was extracted with dichloromethane at room temperature. The syrup~~

~~(1.0 g) was column chromatographed on silica gel by elution with EtOAc-hexane (3:7).~~

~~Fifty-three fractions of 4 mL were collected and examined by TLC. Fractions 25-27~~

~~yielded unstable crystals which were purified by prep. TLC [silica gel, C 6 H6-CH2CI2-~~

~~Et2 0 ( 1: 1: 1)] and recrystalization from CHCl 3-Et2 0 afforded isodeoxyelephantopin ( 1 2 )~~

~~[C 19H20O6 , M+ 344] (101 rag) as colorless needles: m.p. 161-1162°C.~~

~~VI.2.3. Isolation of Grindelic Acid (14). G. maritima was collected by Dr.~~

~~Fischer about 20 miles North of Santa Cruz, CA on Highway 1 between road and the~~

~~coast on July 1, 1993. A voucher specimen is deposited in the Herbarium o f LSU;~~

~~collection Fischer 464. Ground dried aerial parts (flowers (33 g), stems (46 g) and~~

~~leaves (32 g)} of the plant were exhaustively extracted using a number of different~~

~~solvent systems (methanol, ethyl acetate, hexane). Dichloromethane was the most~~

~~efficient solvent for the recovery of grindelic acid (14) (C2 0 H32O3), although the~~

~~drawback was that it also extracted pigments and chlorophyll Evaporation o f the solvent~~

~~under reduced pressure yielded 5.0 g, 4.0 g and 3.0 g o f the extractables from the~~

~~flowers, leaves and stems, respectively. The dry extracts were chromatographed over~~

~~silica gel packed in acetone ((CH 3)2CO}. Fractions eluted with EtOAc-hexane (2:5 v/v)~~

~~were rechromatographed to yield 1.6 g (32 %; calculated with respect to the crude~~

~~extract), 1.0 g (25%) and 0.4 g (13%) o f grindelic acid (14), respectively. Grindelic acid~~

~~(14) was isolated as a yellow oil (TLC; R f 0.67, EtOAc-hexane, 7:20 v/v) and its spectral~~

~~data (IR, ^H, and GC/MS) agreed with those reported in the literature (Brunn et al.,~~

~~1981).~~

~~VI.3. RESULTS AND DISCUSSION. The IR spectrum of dihydroelephantopin (10) contained absorptions typical of a,p-unsaturated y-lactone groups (endo: 1770;~~

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~~exo: 1750 cm 'l), double bonds (1660, 1640 cm"^) and a sharp absorption at 1720 c m 'l~~

~~for a saturated ester function. The ^H-NMR spectrum (CDCI 3 , 400 MHz) exhibited a~~

~~broad singlet at 7.41 ppm for H-1; two doublets at 6.30 ppm and 5.80 ppm (J = 4 Hz)~~

~~typical for the two methylene protons of the C l 1-C13 exocyclic double bond; a multiplet~~

~~at 4.20 ppm for H- 8 ; a complex multiplet between 2.60 ppm and 4.30 ppm for H-5 and~~

~~H-6 . In the high-fîeld region o f the spectrum a sharp singlet appeared at 1.32 ppm for the~~

~~C-4 methyl group; the two side chain methyl groups (o f the isobutyrate ester group) gave~~

~~doublets at 0.87 ppm and 1.15 ppm (J = 5 Hz).~~

~~Our X-ray analysis data o f dihydroelephantopin (10) tallied with those reported~~

~~by Watson (1982). The data confirmed that 10 is a germacranolide-type sesquiterpene~~

~~lactone containing a ten-membered ring which exhibits a chair-chair conformation in the~~

~~solid state (torsion-angle sequence + - + - + - + - + -) which is found in most~~

~~germacranolides. The C(4)-C(5) double bond of the germacradiene skeleton (34b) has~~

~~been transformed into a trans epoxide function. The C(15) methyl group has been~~

~~oxidized and forms an a,|3-unsaturated y-lactone by closure at C(2) with the C(l)-C(10)~~

~~double bond remaining undisturbed. The second ot,P-unsaturated lactone is /rons-fused~~

~~at C(6)-C(7) and exhibits an exocyclic double bond. The absolute configuration at C( 6 )~~

~~is the same for all germacranolides, and the 6 S configuration [i.e. H( 6 ) is |3] has been~~

~~confirmed (Kupchan and Kelsey, 1967). The two five-membered rings in~~

~~dihydroelephantopin (10) are flattened envelopes with C( 6 ) and C(2) being the flaps.~~

~~The C(13)C(11)C( 12)0(12) and O(15)C(15)C(10)C(l) torsion angles are -05° and~~

~~-178.3°, respectively.~~

~~The IR and NMR data o f isodeoxyelephantopin (12) were very similar to those o f~~

~~dihydroelephantopin (10) and indicated the presence o f an a-methylene-y-lactone [IR~~

~~1754 (y-lactone) and 1635 (C=C) cm*^; % NMR: 5.67 ppm (IH , d, Jv.iSa = 4.0 Hz, H-~~

~~13a) and 6.21 ppm (IH , d, 17,135 = 4.0 Hz, H-13b)] and a methacrylate ester group [IR;~~

~~1704 and 1636 cm"^; ^H NMR: 5.69 ppm and 6.16 ppm (each IH , br s, H-18), and 1.94~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 116~~

~~(3H, dd, Ji8,19a = Jl8,19b = 10 Hz, H-19); MS: m/e 69 (base peak,~~

~~[CH2=CH(Me)C0]+) and 258 (strong peak, [M-C4H6023+].~~

~~Unequivocal proof o f the structure and stereochemistry of isodeoxyelephantopin~~

~~(12) has been derived from single-crystal X-ray analysis o f dimethylamine adduct (12b)~~

~~by Zhang et al. (1986). However, their data departed significantly as compared to results~~

~~from X-ray diffraction studies on related germacradienes (McPhail and Sim, 1973). With an a-orientation of the oxygen substituent at C-2 in isodeoxyelephantopin (12), in~~

~~contrast to the ^-orientation at this center in dihydroelephantopin (10), formation of the~~

~~corresponding y-lactone ring incorporating C-15 requires that the latter also lies on the a-~~

~~face o f the rrans, rrans-cyclodeca-1,5-diene ring, that is anti to the ^-oriented C-14 methyl~~

~~group.~~

~~Plant derived chemicals (pesticides) that deter herbivory are of great practical~~

~~value (Elliger et at., 1976) and growing in demand, not only because o f their derivation~~

~~from a renewable source, but also because they are often highly specific, and most~~

~~importantly, cause a minimum ecological disturbance. In the last few years considerable~~

~~interest has been focused on the isolation and structure elucidation o f grindelane~~

~~diterpenoid acids, mainly due to insect feeding deterrent properties exhibited by some~~

~~members o f the genus Grindelia (Rose, 1980; Rose et al., 1981). To date, however,~~

~~most successful discoveries o f new insecticides from natural products have been from~~

~~microbial transformations. For a long time, there existed no direct chemical synthesis o f 3a-hydroxygrindelic acid (14b), a potential starting material for a novel biorationally~~

~~designed insecticides. Recently, Hoffmann and Punnapayak (1988) have successfully~~

~~produced 14b from grindelic acid (14) through microbial chemical transformation~~

~~involving Aspergillus niger. It is our hope that the naturally abundant grindelic acid (14)~~

~~w ill be used to inhibit germination o f Striga spp. seeds.~~

~~The dichloromethane extracts o f the aerial parts o f the previously uninvestigated~~

~~G. maritima showed a definitive and elaborate quantitative variation o f grindelic acid~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 117~~

~~(14), the major constituent. The leaves contained the highest amount while the stems, the~~

~~lowest. The observed quantitative differences in resin acid composition possibly~~

~~represent differences in morphological characters o f the aerial parts.~~

~~Our germination data (Figures VI. 1-2) indicated that both dihydroelephantopin~~

~~(10) and isodeoxyelephantopin (12) are promising germination stimulants for Striga spp.~~

~~seeds. Grindelic acid (14) is a weak germination stimulant (Fig. V I.3 .1-2). The~~

~~carboxylic acid group is probably responsible for this behavior. This molecule might be~~

~~used as a 5. hermonthica seed germination inhibitor. Several derivatives of~~

~~dihydroelephantopin (10), isodeoxyelephantopin (12), and grindelic acid (14) need to be~~

~~synthesized and bioassayed in order to derive meaningful information concerning~~

~~structure-activity relationships.~~

~~13 19~~

~~NMC2~~

~~Isodeoxyelephantopin a-dimethylamine adduct (12b)~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 118~~

~~COOH~~

~~3a-hydroxygrindelic acid (14&^~~

~~Germacradiene skeleton (34 b)~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 119~~

~~Dihydroelephantopin (10) Isodeoxyelephantopin (12) Control~~

~~8 5 -]~~

~~80 —~~

~~7 5 -~~

~~‘S 65 I g 60 4 u 00 5 5 4~~

~~g1 5 0 - u 0 45^~~

~~4 0 -~~

~~3 5 -~~

~~3 0 -~~

~~2 5 -~~

~~2 0 -~~

15 —r- —I— T" —t— —T— “T n r -21 -19 -17 -15 -13 -11 -7 -5 -3 Log [concentration (in Moles/L)] Fig. VI. L Effect of dihydroelephantopin (10) and isodeoxyele phantopin (12) on gemination o f isolate o f conditioned Striga hermonthica seeds occurring in Bida (A) location in Nigeria. Data are arc sine transformed relative percentage germination values expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) in methanol (fig. con'd).

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120~~

~~Dihydroelephantopin (10)~~

~~Isodeoxyelephantopin (12)~~

~~B - Control~~

~~4 0 -~~

~~I 3 5 - .5 30 & 2 5-~~

~~2 0 -~~

~~I15-~~

~~10 -~~

~~5 - 1~~

0 - —r- T- —I— —r- -T- T“ T" T" - r -21 -19 -17 -15 -13 -11 -9 -7 -5 -3 Log [concentration (in Moles/L)] Rg. VI. 1. Effect of dihydroelephantopin (10) and isodeoxyele phantopin (12) on gemination o f isolate of conditioned Striga hermonthica seeds occurring in Kano (B) location in Nigeria. Data are arc sine transformed relative percentage germination values expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) in methanol (fig. con'd).

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 121~~

~~Dihydroelephantopin (10)~~

~~Isodeoxyelephantopin (11)~~

~~Control~~

~~50-1~~

~~4 5 -~~

~~4 0 -~~

~~I 35- I~~

~~15-~~

~~-21 -19 -17 -15 -13-11 ■9 •7 ■5 •3 Log [concentration (in Moles/L)]~~

Fig. VI. L Effect o f dihydroelephantopin (10) and isodeoxyele phantopin (12) on gemination of isolate of conditioned Striga hermonthica seeds collected in Abuja location in Mgeria during the 1992 (C) harvesting season. Data are arc sine transformed relative percentage germination values expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) in methanol (fig. con’d).

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 122~~

~~Dihydroelephantopin (10) Isodeoxyelephantopin (12) D Control~~

~~46 “~~

~~4 1 -~~

~~I•3 3 6 - & t~~

~~2 6-~~

~~-21 -19 -17 -15 -13 11 ■9 7 5 3 Log [concentration (in Moles/L)]~~

Fig. V L l. Effect of dihydroelephantopin (10) and isodeoxyele phantopin (12) on gémination o f isolate of conditioned Striga hermonthica seeds collected in Abuja location in Nigeria during the 1993 (D) harvesting season. Data are arc sine transformed relative percentage germination values expressed as means ± S. E. of twelve replicates (P = 0.05). Control is GR 24 (8) in methanol.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 123~~

~~Dihydroelephantopin (10) Isodeoxyelephantopin (12)~~

~~-O-— Control~~

~~4 0 -1~~

~~3 5 -~~

~~3 0 -~~

~~■a 2 5 -~~

~~Sb 2 0 - &~~

~~1 0 - O.~~

~~-21 -19 -17 -15 -13 11 9 7 -5 3 Log [concentration (in Moles/L)]~~

Fig. VI.2. Effect o f dihydroelephantopin (10) and isodeoxyele phantopin (12) on gemination o f isolate o f conditioned Striga aspera seeds collected in Kano location (E) in Nigeria during the 1993 harvesting season. Data are arc sine transformed relative percentage germination values expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) in methanol.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 124~~

~~Grindelic acid (14)~~

~~Control 85 80~~

~~70 I " .5 60- I a) 50~~

~~45 40 Î 35 30~~

~~25 2 0 . T“ T" —I— —I------f —T- ~ r T" -T 20 -1 8 -1 6 -14 -12 -10 -8 -6 -4 30 -1~~

~~I2 0 -~~

~~0 -~~

-20 -18 -1 6 -1 4 -12 -10 8 6 4 Log [concentration (in Moles/L)] Fig. VI.3.1. Effect of grindelic acid (14) on germination of conditioned Striga hermonthica seeds occurring in Bida (A) and Kano (B) locations in Nigeria. Data are arc sine transformed relative percentage germination expressed as means ± S. E. of twelve replicates (P = 0.05). Control is GR 24 (8) dissolved in methanol (fig. con'd).

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 125~~

~~Grindelic acid (14)~~

~~Control 45 n~~

~~4 0 -~~

~~3 5-~~

~~2 5-~~

~~0 -~~

~~-20 -18 -16 -14 -12 -10 8 6 -4 50-1~~

~~4 5 -~~

~~2 0 -~~

~~15-~~

~~-20 -18 -16 -14 -12 -10 8 6 4~~

~~Log [concentration (in Moles/L)]~~

Fig. VI.3.1. Effect of grindelic acid (14) on germination o f conditioned Striga hermonthica seed s collected in Abuja location in Nigeria during the 1992 (C) and 1993 (D) harvesting seasons. Data are arc sine transformed relative percentage germination expressed as means ± S. E. of twelve replicates (P = 0.05). Control is GR 24 (8) dissolved in methanol.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 126~~

~~Grindelic acid (14)~~

~~Control 45-1~~

~~4 0 -~~

~~S ”~~

~~5 -~~

~~-20 -18 -16 -14 -12 -108 6 4 Log [concentration (in Moles/L)]~~

Fig.VI.3.2. Effect of grindelic acid (14) on gemination of isolate o f conditioned Striga aspera seeds collected in Kano location (E) in Nigeria during the 1993 harvesting season. Data are arc sine transformed relative percentage germination values expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) in methanol.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER V n~~

~~RESPONSE OF TWO DIFFERENT ISOLATES OF STRIGA HERMONTHICA SEEDS TO VARYING CONCENTRATIONS OF STRIGOL ANALOG GR 7~~

~~127~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 128~~

~~VII. 1. INTRODUCTION. Populations of Striga species from different~~

~~geographical areas and different hosts have been reported to exhibit marked differences in~~

~~virulence. Striga hermonthica (Del.) Benth. has a high level of genetic diversity~~

~~(Bharathalakshmi et al., 1990), even in comparison with other species that share its life-~~

~~history features. There are many reports o f inter-provenance differences in virulence for~~

~~S. hermonthica parasitizing a given cereal cultivar host (King and Zummo, 1977).~~

~~Several lines of evidence indicate that the control of S. hermonthica presents a serious~~

~~problem, because economically effective means of control are not yet available for small-~~

~~scale farmers in developing countries. The high application o f nitrogen fertilizer needed~~

~~to bring about a significant reduction in Striga infestation is prohibitively expensive for~~

~~small-scale farming. Besides, the paradoxical effect (Pieterse, 1991; Pieterse and~~

~~Verideij, 1991) is yet to be fully understood. The introduction of synthetic germination~~

~~stimulants to deplete Striga seed reserves in the soil is an intriguing possibility (Egley and~~

~~Dale, 1970). Various reports have indicated that the strigol analogues, such as GR 7 (9)~~

~~could induce germination o f several Striga species and strains (Hassanali, 1983; Ibrahim~~

~~et al., 1985). Johnson et al. (1981) described the preparation o f GR 7 (9) and GR 24~~

~~(8). Their results indicate that the former can readily be synthesized in large quantities~~

~~from the so-called "Corey's lactone" following the standard coupling procedure.~~

~~"Corey's lactone" is commercially available (even in optically pure forms), which makes~~

~~GR 7 (9) an attractive compound for germination stimulation of seeds of Striga species.~~

~~The objective of this work was to assess the effects of different concentrations of GR 7~~

~~(9) on germination o f isolates o f S. hermonthica seed lots collected in Bida and Abuja~~

~~locations in Nigeria (Experimental details as described in Materials and Methods of~~

~~Chapter IV).~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 129~~

~~Vn.2. RESULTS Vn.2.1. Effect of Several Concentrations of GR 7 (9) on the Induction~~

~~of Germination of Conditioned Striga hermonthica Seeds. In the laboratory,~~

~~at 28°C during the 24h period, germination of conditioned S. hermonthica seeds did not~~

~~occur in either 0.1 % methanol or sterile double-distilled deionized water, the negative~~

~~controls. Aqueous solutions of GR 7 (9) and the positive control, GR 24 (8) induced~~

~~germination. Activity of the solutions as indicated by percentage germination was~~

~~dependent on concentration of GR 7 (9) and the S. hermonthica isolate under~~

~~investigation (Figures VII.1A-C). There was not an obvious relationship between~~

~~concentration and germination o f the parasite seeds. A t 10*3 M, GR 7 (9) was less~~

~~stimulatory on germination o f both isolates. Surprisingly, 10'^^ M solution o f GR 7 (9)~~

~~showed higher germination rate than the positive control on both isolates of S.~~

~~hermonthica. The isolate o f S. hermonthica collected from Bida showed the highest~~

~~germination responses which were not reproduced by either o f the isolate from Abuja~~

~~location. Moreover, this isolate gave 52-81% germination, while those collected from~~

~~Abuja during the 1992 and 1993 harvesting seasons resulted in 21-32% and 18-42%~~

~~germination, respectively. The isolate from Abuja (1993) gave higher germination rates~~

~~than GR 24 (8) in all concentrations except at 10"^, 10"8, lO’ l^ and 1 0 "M. A low~~

~~“ double maximum” o f germination at 33% was noted for the isolate from Abuja (1992)~~

~~which occurred at 10"? and 10" M.~~

~~vn.3. DISCUSSION~~

~~VII.3.1. Selection Criteria for Germination Stimulants. To date, GR-~~

~~compounds (GR is referring to Gerry Rosebery, one o f the members o f Johnson's~~

~~group), especially GR 24 (8) and GR 7 (9) are the most widely field evaluated m ultiring~~

~~strigol analogues. These compounds, developed to control Striga spp., meet the several~~

~~criteria for germination stimulants for field use: (1) activity at low concentration; (2)~~

~~reasonable water solubility; (3) environmental safety; and (4) ability to promote~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 130~~

~~germination in a range o f species (Bradow, 1986; Egley, 1980). Specifically, GR 24 (8)~~

~~(mol. wL 298) has been shown to stimulate Alectra vogelii Benth. (Visser and Johnson,~~

~~1982) and S. hermonthica but not S. gesnerioides (W illd.) Vatke (Okonkwo and Okafor,~~

~~1988). The other strigol analogue, GR 7 (9) (moL w t 248), has been shown to exhibit a~~

~~high germination activity for S. hermonthica as well as 5. asiatica (L.) Kuntze and~~

~~Orobanche ramosa L. both in the field (Pavlista and Worsham, 1979; Johnson et al.,~~

~~1976) and in the laboratory (Johnson and Hassanali, 1981a,b). However, it has been~~

~~shown that GR 7 (9) was more active in vitro (Johnson et al., 1976) than when soil~~

~~applied (Babiker and Hamdoun, 1979; Stevens and Eplee, 1979). GR 7 (9) shows~~

~~adequate persistence (6-8 days) in acidic soils (pH 5.0-6.3), but residual activity is short~~

~~(1-3 days) in alkaline soils (Babiker, 1988). This decreased activity in soils was~~

~~attributed to the breakdown o f the chemical (Stevens and Eplee, 1979).~~

~~Vn.3.2. Factors which aHect Germinability of Striga hermonthica Seeds.~~

~~Despite the enormous seed production by weeds which help them maintain their~~

~~populations (Chandler et al., 1977), the number o f seeds reaching the soil bank is limited~~

~~by various environmental factors. A proportion of the seeds produced by Striga spp.~~

~~plants is carried a considerable distance away by activities o f man. Losses due to seed~~

~~eating animals and birds, and destructive microbes also affect the number o f germinable~~

~~seeds entering the seed pool.~~

~~Factors such as soil type, soil moisture, soil fertility, temperature, light, rainfall~~

~~pattern during the season, crop planting date, and synchronization o f the host growing~~

~~cycle with the growing season can have a significant effect on germinability o f Striga~~

~~seeds in the laboratory. These arguments find further support from Babiker et al. (1988)~~

~~who have shown that the germination o f S. hermonthica seeds in response to GR-~~

~~compounds was influenced by soil moisture and soil pH. Ogbom (1972) observed that~~

~~Striga spp. seeds thrives on intermittent dry conditions and is suppressed by continuous~~

~~soil moisture. Excessive soil moisture decreases oxygen level in the soil and induces~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 131~~

~~anaerobic conditions (Kimmerer and Kozlowski, 1982). Oxygen may be vital for an~~

~~oxidative process, which leads to removal o f an inhibitor(s), or attainment o f critical~~

~~levels o f required factors or biochemical stages needed for the stimulant to be effective.~~

~~In addition, anaerobic conditions may increase accumulation o f ethanol and acetaldehyde,~~

~~which affect seed dormancy and germination (Davis, 1980; Taylorson and Hendricks,~~

~~1981). The inhibitory effects o f these compounds are pronounced at low oxygen levels~~

~~(Holm, 1972). There is thus good reason to believe that all or a combination o f these~~

~~factors affect the survival of germinable seed lots o f the parasite recovered from the soil in~~

~~Bida and Abuja locations.~~

~~A noteworthy aspect of the present investigation is the observation that the isolates~~

~~o f S. hermonthica occurring in Bida and Abuja locations exhibited mariced differences in~~

~~germination responses to various concentrations of GR 7 (9) (Figures VILIA-C).~~

~~Generalizations about the influence o f the various concentrations on germination should~~

~~be made with caution. The germination data could be rationalized from several~~

~~perspectives. The adverse effect that application of 10-3 M GR 7 (9) had on~~

~~responsiveness o f all isolates o f S. hermonthica is in line with the findings o f Fischer et~~

~~al. (1990) for Striga asiatica. The adverse effect might be, as suggested by Pavlista et al.~~

~~(1979), due to the interference o f GR 7 (9) with leaching o f an inhibitor from the seed.~~

~~However, interactions between GR 7 (9), the inhibitor and/or other components of the~~

~~system cannot be dismissed. Prior to germination, Striga spp. seed requires a period of~~

~~conditioning, the optimum time being temperature dependent (Logan and Stewart, 1992).~~

~~Previous studies by Jackson etal. (1991) have shown that S. hermonthica, and probably~~

~~S. aspera and S. asiatica, require the jo in t action o f ethylene (produced by the seed itself)~~

~~and strigol-related substances before they w ill germinate. In our experiment, we~~

~~employed a 10-day conditioning period and 24 h incubation with the test solutions {GR 7~~

~~(9) and GR 24 (8)}. We cannot rule out the possibility that these experimental conditions~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 132~~

~~might not have been adequate for all the isolates o f S. hermonthica occurring in Bida and~~

~~Abuja locations.~~

~~Isolate of S. hermonthica from Bida gave the highest response to all~~

~~concentrations o f GR 7 (9). This isolate might be the most virulent parasite in the field.~~

~~The very low percentages o f germinable seeds particularly those collected in Abuja in~~

~~1992 was evident This may be due to the loss o f some seeds via germination before the~~

~~collection o f soil samples and also to the presence o f non-germinable seeds from the~~

~~preceding year. In addition, some of the seeds which did not germinate were probably~~

~~under induced or enforced dormancy (viable but non-germinable in the laboratory). This~~

~~is in agreement with the observation made by Hintikka (1988) that the seeds of many~~

~~plant species enter dormancy when buried in the soil or humus. However, factors that~~

~~cause the onset o f dormancy remain to be fully understood.~~

~~Vn.4. CONCLUSION. Isolates o f conditioned Striga hermonthica occurring in~~

~~Bida and Abuja locations in Nigeria exhibited different germination responses to GR 7~~

~~(9) under laboratory conditions. It is possible that the different isolates have different~~

~~seed physiologic specialization in mechanism o f dormancy. Using GR 7 (9) and other~~

~~potentially promising germination stimulants, further experimental work is needed, both~~

~~in the laboratory and in the field.~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 133~~

~~GR 7 (9) in methanol~~

~~Control 50-1~~

~~4 5 -~~

~~40 -~~

~~35-~~

~~25-~~

~~2 0 -~~

~~15-~~

~~-21 -19 -17 -15 -13 -11 9 7 ■5 3 50-1~~

~~40 - •a~~

~~0Û 30- OQ~~

-21 -19 -17 -15 -13 -11 9 7 5 3 Log [concentration (in Moles/L)] Fig. V n.l. Effect o f GR 7 (9)on germination o f conditioned Striga hermonthica seeds collected in Abuja location during the 1992 (A) and 1993 (B) harvesting seasons. Data are arc sine transformed percentage germination values expressed as means ± S.E. o f twelve replicates (P = 0.05). S.E. shown only when larger than the syrnbol. Control is GR 24 (8) in methanol (fig. con'd).

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 134~~

~~GR 7 (9) in methanol~~

~~85-1 Control~~

~~8 0 -~~

~~I”-~~

~~7 0 -~~

~~5 5 -~~

~~5 0 -~~

~~-21 -19 -17 -15 -13 -11 97 5 3 Log [concentration (in Moles/L)]~~

Fig. V n .l. Effect o f GR 7 (9) on germination o f isolate o f conditioned Striga hermonthica seWs collected in Bida (C) location in Nigeria. Data are arc sine transformed relative percentage germination values expressed as means ± SB o f twelve replicates (P = 0.05). SE shown only when larger than the symbol. Control is GR 24 (8) in methanol.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER Vni~~

CADINANES ISOLATED FROM HETEROTHECA SUBAXILLARIS LAM.*

*This chapter contains some materials which have been reprinted with permission from

~~Spectroscopy Letters.~~

~~135~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 136~~

~~V n i.l. INTRODUCTION. The polymorphic Heterotheca subaxillaris (Lam.) Britton & Rushy (family Asteraceae; tribe Astereae), commonly referred to as~~

~~camphorweed, is widespread, spaning the coastal plain o f the Southern and Eastern~~

~~United States. Although the chemistry o f many Asteraceae has been investigated,~~

~~members of the tribe Astereae have received only limited attention (Lincoln and~~

~~Lawrence, 1984). These taxa appear to be characterized by the absence o f sesquiterpene~~

~~lactones or alkaloids, but commonly accumulate mono-, sesqui- and diterpenoids. The~~

~~five species of the North American genus Heterotheca, which have so far been~~

~~investigated chemically are rich in cadinanes which exhibit diverse biological activities~~

~~w ith remarkable dependence on minor structural and stereochemical changes (Kalsi and~~

~~Talwar, 1981; Borg-Karlson and Norin, 1981). Cadinanes, which are common in higher~~

~~plants, have been further divided into four subclasses based on the nature o f the ring~~

~~fusion and the orientation of the isopropyl group at C(10) (Bordoloi et al., 1989). Due to~~

~~complex stereochemistry of these compounds, many of the previous structural and~~

~~configurational assignments appear to be uncertain (Borg-Karlson and Norin, 1981). It~~

~~is also evident that the present classification of cadinane-type compounds needs a revision~~

~~o f nomenclature (Vlahov et at., 1967).~~

~~NMR represents an important tool for structure determination, since~~

~~chemical shifts are far more sensitive to their chemical environment than proton shifts,~~

~~providing valuable structural information (Bax et a i, 1986). The normal and DEPT-~~

~~edited experiment (Doddrell et al., 1982) can be used to partially assign spectra in~~

~~terms o f the number of attached protons. Furthermore, the recent development o f two-~~

~~dimensional NMR spectroscopy has provided a number of new NMR assignment~~

~~techniques which are useful in the area of natural products chemistry (Derome, 1989;~~

~~Shoolery, 1984). In particular, the direct heteronuclear (^H-^^c) chemical shift-~~

~~correlated spectra allow simultaneous determination o f and 1% chemical shifts for~~

~~directly bonded units (Bax and Morris, 1981).~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 137~~

~~As part of an ongoing study of 2-hydroxy-8a-angeloyIoxycaIamenene (13) and~~

~~2-hydroxy-8a-hydroxycalamenene (42) as germination stimulants for Striga spp. seeds,~~

~~it was deemed important to report their complete NMR assignments, information~~

~~which is invaluable for the structure elucidation of other terpenoids in general and~~

~~calamenene-type compounds in particular. The calamenenes are A-ring-aromatized~~

~~cadinanes w ith a methyl group at the C-7 position (Bordoloi et al., 1989). It is o f some~~

~~medicinal interest to note that the skeleton o f 2-hydroxy- 8 a-angeloyloxycalamenene (13)~~

~~and 2-hydroxy-8a-hydroxycalamenene (42) is the same as that of the spermicidal~~

~~gossypol (Davila-Huerta et al., 1995). Unambiguous assignments for all skeletal carbon~~

~~resonances o f both compounds were made primarily by the application o f 2D~~

~~heteronuclear correlated spectroscopy (HETCOR) (Bax and Morris, 1981) and long-~~

~~range heteronuclear correlation experiments (COLOC) (Kessler et al., 1984).~~

~~Vni.2. EXPERIMENTAL~~

~~VlJJ.2.1. Plant Materials. The aerial parts of H. subaxillaris were collected by N.~~

~~H. Fischer, H. D. Fischer and L. Quijano on February 2,1989, in Jefferson Parish along~~

~~the beach at Grand Isle, Louisiana, U.S.A. (Voucher No. Fischer 375; voucher deposited~~

~~at the Louisiana State University Herbarium). IR spectra were recorded on a Perkin-~~

~~Elmer 1760x FT-IR spectrometer as a film on KBr plate using CHCI 3 solution. Mass~~

~~spectra were run on a Hewlett-Packard 5971 A GC/MS spectrometer. Comparison by~~

~~TLC (Fischer, 1991) (EtAOc-hexane, 2:5), GC/MS and ^H NMR of the crude extract of~~

~~stems and leaves showed that they contained the same compounds with minor quantitative~~

~~differences.~~

~~V ill.2.2. Extraction and Isolation. Powdered leaves (502 g) were extracted with~~

~~CH 2CI2 (6 X 900 mL) at room temperature for 72 h. Evaporation o f the combined~~

~~CH 2CI2 extracts under reduced pressure gave 5.6 g o f crude extract which was~~

~~chromatographed by VLC (Pelletier et al., 1986) using 100 g silica gel (M N Kieselgel G)~~

~~and ethyl acetate-hexane as eluent. The percentage o f EtOAc in hexane was gradually~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 138~~

~~increased {(EtOAcrhexane (v/v): 0:1; 1:9; 1:4; 3:7; 1:1; 7:3; 9:1; 1:0, 50 mL each)}.~~

~~Forty fractions of 10 mL each were collected and monitored by TLC performed on~~

~~precoated M N Sil-G 25 UV 254 plates having a layer thickness o f 0.25 mm. Fraction 12~~

~~provided 14 mg of 2-hydroxy-8a-angeloyloxycalamenene ( 1) [R f 0.86 (SiC> 2 , EtAOc-~~

~~hexane, 2:5)] as a yellow o il The medium polar fractions 20-25 eluted with 25% EtAOc-~~

~~hexane were combined and evaporated to afford 120 mg o f 2 -hydroxy- 8 a -~~

~~hydroxycalamenene (42) [R f 0.45 (SiC> 2. EtOAc-hexane, 2:5)] as a yellow oil.~~

~~V in .2 .3 . NM R . One and two-dimensional NMR spectra were obtained in a Bruker~~

~~AM X 400 NMR spectrometer at room temperature. Samples of 6 mg of 1 or 2 were~~

~~dissolved in 0.7 mL of CDCI 3 (99.8% isotopically pure in deuterium, from Aldrich~~

~~Chemical Company Inc., Milwaukee, U.S.A) in 5 mm diameter NMR tubes. The~~

~~and 1% chemical shifts are reported in 8 -values (ppm) and referenced to the signal of~~

~~CDCI3 at 7.26 and 77.00 ppm, respectively. To make unambiguous assignments of~~

~~carbon signals additional COSY (Aue et al., 1976), NOESY (Bax and Morris,~~

~~1981), DEPT (DoddreU et al., 1982), correlation (HETCOR) (Bax and Morris,~~

~~1981) and COLOC (Kessler et al., 1984) experiments were performed. The 2 D~~

~~experiments were acquired and processed with the software provided by Bruker on AM X~~

~~400. Typical acquisition time and processing conditions for COSY and NOESY experiments were: relaxation delay o f 1 and 2 seconds, 512 t i increments; 1024 to 2048~~

~~t2 points; sweep width o f 2 ppm. Sine bell squared and shifted (îc/4, tc /6 and tc/8)~~

~~apodization functions were used for processing. The mixing time in NOESY~~

~~experiments, generally set at 1.2-1.5 seconds, was also varied between 0.8 and 2~~

~~seconds, w ithout substantial change in the results. For ( l^ c detected)~~

~~correlations, the same relaxation delays were used, 256 to 512 t i increments, 1024 to~~

~~2048 t 2 points, the sweep width being 60 ppm for Lorentz and Gaussian~~

~~deconvolution were generally used in the processing. The number of scans was set for an~~

~~overall acquisition time of about 12 to 16 hours. HETCOR was carried out at a ^^C~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 139~~

~~frequency o f 100.62 MHz using 4K data points in the t 2 dimension and 256 increments on ti. A Gaussian window function was applied to the t; dimension and a sinebell~~

~~window function was applied to the t i dimension. Zero fillin g to IK was also applied in~~

~~the dimension. Three-bond long-range shift correlations (COLOC)~~

~~experiment were performed using a pulse sequence reported by Kessler et al. (1984).~~

~~The data were acquired in 12 hours using 256 experiments, each with a block size o f 4K.~~

~~The follow ing parameters were used; D1 = 2 sec, D f = 3 msec, D3 = 0.071 sec, D4 =~~

~~0.036 sec. The D3 and D4 values were computed using the observed three-bond coupling~~

~~constant, = ?Hz. The data were processed using sinebell multiplication in both~~

~~dimensions (SSB1 = SSB2 = 0) and Gaussian m ultiplication in the second dimension~~

~~(WDW2 = G; LB2 = 2.0) before Fourier transformation. The contour plot was plotted at~~

~~the 256 K level~~

~~Vin.3. RESULTS AND DISCUSSION. The NMR spectrum of the crude~~

~~dichloromethane extract of H. subaxillaris showed absorptions both in the aromatic~~

~~region as well as aliphatic methyl signals. From fractions of the medium polarity fractions, the cadinanes, 2-hydroxy-8a-angeIoyIoxycalamenene (13) and 2-hydroxy-8a-~~

~~hydroxycalamenene (42) were isolated. Both compounds had previously been isolated~~

~~from H. subaxillaris o f an unspecified collection site and their structures were determined~~

~~by spectroscopic methods (Bohlmann and Zdero, 1979). VIII.3.1. i^C NMR Spectral Assignments in 2-hydroxy-8a-~~

~~angeloyloxycalamenene (13). The spectral data (% NMR, IR, MS) of 2-hydroxy- 8a-angeloyloxycalamenene (13) were in full agreement with the reported values~~

~~(Bohlmann and Zdero, 1979). Only the homonuclear coupling constants in the NMR~~

~~spectrum showed marginal differences. The ^^C NMR spectrum of 13 indicated the~~

~~presence of 20 carbon signals in the molecule. Its relative stereochemistry was~~

~~determined from NOESY (Bax and Morris, 1981) experiment and proton coupling data~~

~~which allowed the unambiguous assignment of carbon and proton resonances (Table~~

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~~V in.I). The DEPT spectrum of 13 (Fig. V III. 1) exhibited six methyls, one methylene and seven methine signals, while the remaining 6 signals in the broad-band spectrum~~

~~were due to the quaternary carbon atoms. The methyl protons were assigned by analyses~~

~~of COSY (Aue et al., 1976), NOESY (Bax and Morris, 1981), and 2D one-bond~~

~~heteronuclear correlation (HETCOR) (Bax and Morris, 1981) contour diagrams shown in~~

~~Figures VHI.2, VHI.3, and VHI.4, respectively. The aromatic methine protons H-1 and H-4 at 6.58 and 6.92 ppm showed cross peaks with carbon resonances at 113.6 and~~

~~130.3 ppm, respectively (Fig. Vm.4.1). The upfield region of the HETCOR spectrum~~

~~(Fig. Vm .4.2) showed cross peaks o f the methyl protons H-12, H-13, H-14, H-15, H-~~

~~4’ and H-5’ at 17.2, 21.1, 17.3, 15.6, 15.5, and 20.5 ppm, respectively. The signal at~~

~~72.3 ppm was due to the oxygen-bearing carbon, C-8 (Silverstein et al., 1991). The ^^C~~

~~NMR resonances o f one methylene carbon (C-9) and the remaining 5 methine carbons~~

~~were sim ilarly assigned from the correlation diagram (Fig. Vni.4.1) and are depicted in~~

~~Table V U L l. The only difficulty in assignment o f the NM R resonances came from~~

~~the non-protonated carbons, C-2, C-3, C-5, C-6, C-1’ , and C-2’ which do not have~~

~~cross peaks in the HETCOR diagram. To unambiguously assign these NMR~~

~~signals, a three-bond long-range heteronuclear correlation experiment (COLOC) was~~

~~carried o u t Figure V n i.5 shows the non-protonated carbons at the downfield region o f the COLOC contour plot for 2-hydroxy-8a-angeloyloxyca-lamenene (13). The carbonyl~~

~~resonance at 168.1 ppm showing long-range correlation w ith the H-5’ was assigned to C-~~

~~r . Based on the correlation with the H-5’ proton signal, the carbon resonance at 128.2~~

~~ppm could be assigned to C-2’ . Three carbon resonances at 130.3 (C-4), 152.0, and~~

~~121.7 ppm showed correlation with H-15, indicating either a two-bond or three-bond~~

~~coupling to H-15. The carbon resonances at 121.7 and 152.0 ppm were tentatively~~

~~assigned to C-3 and C-2, respectively. The carbon resonance at 152.0 ppm showed~~

~~correlation peaks with H-1, H-4 and H-15 could be unambiguously assigned to C-2,~~

~~whereas the carbon at 121.7 ppm was assigned to C-3, values in accord with chemical~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 141~~

~~sh ift considerations. Three correlation peaks were observed between the H-4 proton~~

~~signal at 6.92 ppm and carbon resonances at 130.4 (C-4), 152.0 (C-2) and 138.8 ppm.~~

~~The carbon signal at 138.8 ppm showed a three-bond correlation w ith C-4 and was~~

~~unambiguously assigned to C-6. The remaining 1% signal at 130.0 ppm showed a three-~~

~~bond correlation peak with the H-1 signal at 6.58 ppm and was therefore assigned to C-5. VIII.3.2. l^C NMR Spectral Assignments in 2-hydroxy*8a-~~

~~hydroxycalamenene (42). The structure of 2-hydroxy-8a-hydroxycalamenene (42)~~

~~was illustrated to be a cadinane sesquiterpenoid with a secondaiy hydroxyl group at C-8.~~

~~Its spectral data resembled those o f 13 occurring in the same plant The IR spectrum of~~

~~42 showed the presence of hydroxyl and double bond (C=C) absorptions at 3500 and~~

~~1650 c m '\ respectively. The high resolution ^H NMR spectrum provided considerable~~

~~inform ation (Table VHI.2) and the chemical shift values were in close agreement~~

~~(differences o f < 0.01 ppm) with the literature data (Bohlmann and Zdero, 1979). There~~

~~were IH singlets at 6.95 and 6.61 ppm indicating the aromatic hydrogens (H-4 and H-1,~~

~~respectively) in a para relationship. A singlet for an aromatic methyl group appeared at~~

~~2.20 ppm and an OH group signal at 6.15 ppm. The remaining two positions of the~~

~~benzene ring are occupied by benzylic MeCE< and Me2CHCH< groups (the underlined~~

~~protons being vicinal to the phenyl ring).~~

~~The 2D ^H-^H COSY and NOESY experiments revealed and clarified the~~

~~presence and connectivities o f each o f the three partial segments, A - C:~~

~~OH HX3C B A~~

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~~One oximethinic proton (H-8) located downfield at 4.12 ppm correlated with both~~

~~methylene protons at C-9 carbon and a secondary methyl (H-14) in segment A. The C-4~~

~~proton (H-4) correlated with a tertiary methyl (H-15) in segment B and H-1 and in~~

~~segment A. Two secondary isopropyl methyl groups (H-12 and H-13) were correlated~~

~~w ith the methine proton (H-11) in segment C and with H-14 methyl group in segment A.~~

~~A pair o f 3H doublets (J = 6.8 Hz) at 0.66 and 0.98 ppm, which were scalar-coupled to a~~

~~IH multiplet at 2.20 ppm in COSY spectrum (Figure Vin.6) confirmed the presence of~~

~~an isopropyl moiety (segment C). Moreover, the El-mass spectrum established a~~

~~molecular weight of 234 [M]+ (corresponding to C 15H22O2) with major fragment ions~~

~~resulting from loss of isopropyl group.~~

~~The stereochemistry o f 2-hydroxy-8a-hydroxycalamenene (42) was deduced by~~

~~the NOESY experiment. Several signifîcant NOEs observed were not in agreement with~~

~~the previously assigned stereostructures of 2-hydroxy-8a-angeloyloxycalamenene (13)~~

~~and 2-hydroxy-8a-hydroxycalamenene (42) (Bohlmann and Zdero, 1979). As~~

~~noticeable in Figures Vin.3 and V III.7, cross peaks between H-12 and H -11, H-13 and~~

~~H-14, between H-14 and H-11 strongly suggested that the C-7 methyl group and the C-~~

~~10 isopropyl group in both 13 and 42 are cw-oriented. Chemical shifts correlations,~~

~~integrals and determination o f the relevant coupling constants (e.g. Jqjq and Jy g) further~~

~~supported the relative configurations at C-7, C-8 and C-10.~~

~~The NMR spectrum of 2-hydroxy-8a-hydroxycalamenene (42) showed~~

~~signals o f fifteen carbon atoms, four o f them being quaternary. The protonated carbon~~

~~signals were assigned by the polarization transfer experiment (DEPT) (Figure Vin.8)~~

~~which indicated the presence of four methyl groups, one methylene and six methines (one~~

~~directly bonded to an oxygen function at 70.7 ppm). Two sets o f aromatic carbon signals~~

~~arose at d 113.6, 130.3 and were attributed to (-C=CH) and at d 121.8, 130.6, 138.0,~~

~~152.2 due to quaternary aromatic carbons. Two-dimensional ^^C-^H COSY spectral data~~

~~(Figures Vni.9.1 and Vin.9.2) was employed in the assignments o f chemical shifts of~~

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~~protonated carbons in the 1% NMR spectrum. Quaternary carbons (C-2, C-3, C-5 and~~

~~C-6) were assigned by comparison w ith the spectral data for 13 and for sim ilar skeletal~~

~~structures (Davila-Huerta etal., 1995).~~

~~Because 2-hydroxy-8a-angeloyloxycalamenene (13) is the major constituent of~~

~~H. subaxillaris, various concentrations were assessed for germination o f conditioned~~

~~Striga seeds. The results are depicted in Figures Vni.9.3-4. Isolates o f S. hermonthica~~

~~occurring in Bida (Fig. VIII.9.3A) and S. aspera occurring in Kano (Fig. VIII.9.4E)~~

~~showed higher germination responses to 13 than the control (GR 24 (8)). More work is~~

~~needed to establish the mechanism o f germination/inhibition of Striga seeds by 2- hydroxy-8 a-angeloyloxycalamenene (13). The presence of two antagonistic functional~~

~~groups, the OH (inhibitor of germination) as w ell as the angelate ester group (a Michael~~

~~receptor site for methionine (20) which can lead to formation of ethylene) makes this~~

~~molecule an interesting model for future studies.~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 144~~

~~2-hydroxy-8a-angeIoyIoxycaIamenene (13)~~

~~HO, *0H~~

~~2-hydroxy-8a-hydroxycalamenene (42)~~

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~~Table V IU l. correlation of 2-hydroxy-8a-angeloyloxycaIamenene (13) (400MHz,CDCl3)~~

~~513c MulL^ 5attacbed{] Assignment~~

~~113.6 CH 6.58 s 1 152.0 - 2 121.7 Cd - 3 130.4 CH 6.92 s(br) 4~~

130.0 Cd - 5 138.8 O» - 6 36.1 CH 3.05 dq 7 72.3 CH 5.37 ddd 8 25.2 CH2 1.85 ddd; 2.05 ddd 9 40.8 CH 2.80 ddd 10 32.9 CH 2.10 dqq 11 17.2b CHg 0.99 d 12 21.1C CH3 0.75 d 13 17.3 CH3 1.21 d 14 15.6 CH3 2.10 s(br) 15

~~168.1 Cd - r 128.2 C4 - 2’ 137.6 CH 6.02 3’ 15.5 CH3 1.89 4’ 20.5 CH3 1.85 5’~~

J(Hz); 7a, 8p, = 4.4; 7a, 14 = 6.8; SP, 9p = 8.5; 9a, 9P = 14; 9a; 10a = 9p, 10a = 10a, 11 = 6 ; 11, 12 = 11,13 = 6.8. ^ Carbon multiplicities determined through DEPT experiments. Assignments may be interchanged in each vertical column. ^ Assignments based on COLOC experiment.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 146~~

~~Table V in.2. and NMR spectral data (6) for 2-hydroxy-8a-hydroxycalamenene (42) (400 MHz, CDCI3)~~

~~813c MulL^ SaoacbedH Assignment~~

113.6 CH 6.61 s 1 152.2 Cb - 2 121.8 Cb - 3 130.3 CH 6.95 s(br) 4 130.6 Cb - 5 138.0 Cb - 6 37.7 CH 2.84 dq 7 70.7 CH 4.12 ddd 8 28.9 CH2 1.50 ddd; 1.95 ddd 9 39.4 CH 2.85 ddd 10 33.0 CH 2 .2 0 dqq 11 16.6C CH3 0.98 d 12 2 0 .8 C CH3 0 .6 6 d 13 16.6 CH3 1.29 d 14 15.5 CH3 2 .2 0 s(br) 15

J (Hz): 7a, 8 g, = 3.5; 7a, 14 = 7; 8 p, = 3; 9p, 9a = 13.5; 9a. 10a = 9p; 10a = 7; 10a, 11=6. & Carbon multiplicities determined through DEPT experiments, b These carbon number assignments were made by comparison with assignments for 13 and related compounds (Davila-Huerta, et a i, 1995). c Assignments may be interchanged in each vertical column.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 147~~

~~Ocu 1 . 0 \ CN- E -Sa~~

~~§ oV 1' 1 g C\& cn a 23 à Ë: o oo a CO :k Q X 2 -a >» JS cs o Cm CO 0 2~~

~~CL Q. 1 a o o cn I cnU n m o cu X3 o m ■ § CN . 'oo Cv § 'O * o cs I CN- {= cn o CO > 2 I~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 148~~

~~y u~~

~~1.0~~

~~2.0~~

~~_ 3.0~~

~~_ 4.0~~

~~- 5.0~~

~~_ 5.0~~

~~_ 7.0~~

~~PPM PPM~~

~~Figure Vm.2. 2D NMR COSY spectrum of 2-hydroxy-8a-angeloyloxycalamenene (13).~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 149~~

~~HO.~~

~~9| 0~~

~~U U l .~~

~~1.0~~

~~^ 2.0~~

~~_ 3.0~~

~~_ 4.0~~

~~_ 5.0~~

~~_ 6.0~~

~~7.0~~

~~PPM T T 6.0 PPM~~

~~Figure vm.3. 2D NMR NOESY spectrum o f 2-hydroxy-8a-angeloyIoxy- calamenene (13).~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 150~~

~~O o o CVJ 10 m~~

~~m Ov I -a K 0X 1§ OQ è oo X s JS% CN CkHO E en I 0~~

~~es 1 ffi I = m i •— — ds .z~~

~~m2 z\z\n 'oô I II iZ >o SI s T36~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 151~~

~~tn o m o in cu cu m en m~~

~~EI "E K X o 1 > v <8> 0 & 1 cu à oo :k X 2 "O >» ON (4 CL 0 E L.3 en 1 I 1 en X~~

~~c o ’ôb o 2 y ■a o u C 1 Q. D r i~~

~~> 2 3 BD iZ~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 152~~

~~o o O cu 'T (O X a. • CL m~~

~~Ei es O eu iX I.0~~

~~coo _o e 1 I~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 153~~

~~H 14~~

~~H 111~~

~~1.0~~

~~. 2.0~~

~~3.0~~

~~4.0~~

~~5.0~~

~~6.0~~

~~7.0~~

~~PPM~~

~~Figure VHI.6. 2D NMR COSY spectrum of 2-Hyclroxy-8a-hydroxycalamenene (42).~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 154~~

~~_ 1.0~~

~~_ 2.0~~

~~_ 3 .0~~

~~_ 4.0~~

~~- 5 .0~~

~~6.0~~

~~L 7 .0~~

~~PPM~~

~~Figure VHL7. 2D NMR NOESY spectrum of 2-Hydroxy-8a-hyciroxycaIam- enene (42).~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 155~~

~~S 3 o — OJ Ù~~

~~no - Z.-D oio- )i-;) N i c\ i ca o S3 co 12 00 00 Ô' I 1 12 CN W o O h o ca S 2 2 CQ o ■C\J CQ 7 3 c m CQ en~~

~~CM 'O o i 0 “ S TM oo 0 - > 2 3 OO E~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 156~~

~~o o o cu co~~

~~CL Sl-D J §3 1 6-3 11-3 :-3~~

~~► i~~

~~1ca K X 00 2 "O > » x: é CL 00 X 2 •o Z (S o E rO J i. CO O~~

~~z z mû 'ôo û O i-H rn CN g~~

~~G\ tT 'm S)~~

~~Çl-H 2 â ÎL,~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 157~~

~~i g I~~

~~l£3 O m o in o \a i cu eu en en "3 6 00 >s U) 1 I es -s o E % I~~

~~O m I X t—,0 ù 1 2 _d i o . -O X es Ov m > 2 ~rvY 7irrr)riî & B r r B g E~~

~~'en 'es EI-H~~

~~SI-H~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 158~~

~~2-Hydroxy-8a-angeloyIoxycalamenene (13)~~

~~85 q Control 8 0 - 7 5 - 7 0 - 6 5 - 60 — I 5 5 - & 5 0 - 4 5 -~~

~~Ic 4 0 - 3 5 - £ 3 0 - 2 5 -~~

~~2 0 - 1 5 -~~

~~10 T" T“ 1 1— —T- T" T r -21 -19 -17 -15 -13 -11 -9 -7 -5 -3~~

~~25-1~~

~~2 0 -~~

~~I-S 1 5 - & & 2 10 - I~~

~~-21 -19 -17 -15 -1311 ■597 3 Log [concentration (in Moles/L)]~~

Fig. Vni.9.3. Effect of 2-Hydroxy-8a-angeloyloxycalamenene (13) on germination o f conditioned S. hermonthica seeds occurring in Bida (A) and Kano (B) locations in Nigeria. Data are arc sine transformed relative percentage germination expressed as means ± S. E. o f twelve replicates ^ = 0.05). Control is GR 24 (8) dissolved in methanol (fig. con'd).

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 159~~

~~2-hydroxy-8a-angeloyloxycalamenene(13)~~

~~Control~~

~~4 0 -~~

~~3 5 -~~

~~es 3 0 -~~

~~& 2 5 -~~

~~I C 2 0 -~~

~~1 5 -~~

~~1 0 -~~

~~-21 -19 -17 -15 -13 11 9 7 5 3 50-1~~

~~4 5 - jS 4 0 - 3 5 -~~

~~2 0 -~~

~~1 5 -~~

~~-21 -19 -17 -15 -13 -119 7 5 3 Log [concentration (in Moles/L)]~~

Fig. Vin.9.3. Effect of 2-Hydroxy-8a-angeloyloxycalamenene (13) on germination o f conditioned S. hermonthica seeds collected in Abuja location in Nigeria during the 1992 (C) and 1993 (D) harvesting seasons. Data are arc sine transformed relative percentage germination expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) dissolved in methanol.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 160~~

~~2-Hydroxy-8a-angeIoyIoxycalamenene (13)~~

~~Control~~

~~45-1~~

~~4 0 -~~

~~3 5 -~~

~~2 5 - 00 op 20-~~

~~1 5 -~~

~~10 -~~

~~-21 -19 -17 -15 -13 - I I 97 5 3 I Log [concentration (in Moles/L)]~~

Fig. Vm.9.4. Effect of 2-Hydroxy-8a-angeIoyIoxycalamenene (13) on germination o f conditioned S. aspera seeds occurring in Kano (E) location in Nigeria. Data are arc sine transformed relative percentage germination expressed as means ± S. E. o f twelve replicates (P = 0.05). Control is GR 24 (8) dissolved in methanol.

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IX~~

COMPOUNDS ISOLATED FROM THE WEST AFRICAN HARRISONIA ABYSSimCA OLIV. (SIMARUBACEAE)*

~~♦This chapter contains some materials which have been reprinted with permission from Spectroscopy Letters.~~

~~161~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 162~~

~~IX. INTRODUCTION~~

~~IX.1. Limonoids. Limonoids, also referred to as tetranortriterpenoids, constitute a~~

~~group o f highly oxidized natural products known to occur in the Meliaceae, Rutaceae and~~

~~Cneoraceae. These compounds apparently are formed by enzymatic oxidation and~~

~~degradation o f the tetracyclic tiiterpenoid precursor, euphol (43) (Akisanya et al., 1960).~~

~~The limonoids have been classified on the basis of which of the four rings in the~~

~~triterpene nucleus have been oxidized. The biosynthetic evolution of the limonoids has~~

~~recently been proposed (Champagne et al., 1992). The citrus bitter principle limonin~~

~~(44) was firs t isolated in 1960 (Arigoni et al., 1960), but intensive investigation of~~

~~limonoids did not begin until the last decade (Hasegawa et al., 1984).~~

~~The separation of mixtures of limonoids by column chromatography is very~~

~~difR cult These compounds are often very similar, unstable and may interconvert during~~

~~isolation. High pressure liquid chromatography has been much used, but is not free from~~

~~problems of isomerization. Separation is further complicated by the occurrence of~~

~~mixtures o f esters. Because o f this, some compounds which have been isolated may be~~

~~artifacts. When isolated, the limonoids may be of considerable molecular complexity,~~

~~and the structure determination usually present formidable problems. Even in simple~~

~~cases, the structure determination may present unexpected pitfalls for the unwary, and~~

~~several methods o f determining stereochemistry have failed when applied to limonoids~~

~~(A rigoni et al., 1960). Moreover, the molecular conformation is not always readily~~

~~predictable, and NMR spectroscopy of limonoids use of the Karplus equation has led to~~

~~mistaken conclusions (Halsall etal., 1975).~~

~~From NMR point of view, the rigid steroid framework of limonoids contain a~~

~~relatively large number of functional groups that can exhibit diamagnetic anisotropy and~~

~~would induce fields which can change the chemical shifts o f neighboring protons in~~

~~accord w ith their individual orientation to the functional group. These secondary fields~~

~~can then be evaluated in a series o f compounds where regular changes in structure have~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 163~~

~~been made. In many cases where overlapping bands occur, they can be separated by or~~

~~shifted by taking the spectra in different solvents. Band shapes and coupling constants~~

~~could thus be established with certainty. Some higher melting limonin derivatives are not~~

~~sufficiently soluble in chloroform for good NMR structure. They would usually~~

~~dissolve in mixtures of solvents, for example deuteriochloroform-dimethyl sulfoxide,~~

~~deuteriochloroform-acetonitrile, and trifluoro acetic acid. More reliable has been the~~

~~effect of substituents on the chemical shift of the nearby methyl groups (Jibodu et al.,~~

~~1970). It has been shown that the presence o f a hydroxy substituent causes a~~

~~considerable downfield shift o f the methyl substituents in a 1,3 diaxial relationship, while~~

~~an acetoxy or other carbonyl containing substituent may or may not produce a similar~~

~~shift depending on orientation effects. This can produce good evidence of~~

~~stereochemistry, and in suitable cases, of the position of a substituent (Halsall et al.,~~

~~1975). The general features of the NMR spectra o f limonin (44) and its derivatives~~

~~starting from the high field end of the spectrum can be summarized as follows;~~

~~i). Bands due to the C-methyl resonances. ii). The absorption bands for the four protons at C-2 and C-6, a- to the carbonyl~~

~~groups. For most limonin derivatives these bands are not interpretable.~~

~~iii). A sharp singlet due to the proton at C-15, adjacent to the epoxy group. This band~~

~~sometimes overlap the band due to the proton at C-1.~~

~~ix). A broad band due to the protons on C-1 which in some cases is resolved into a~~

~~symmetrical triplet~~

~~v). An AB system from the protons on C-19, which in several derivatives collapses into~~

~~a broadened singlet.~~

~~vi). A very slightly broadened singlet caused by the proton at C-17, adjacent to the furan~~

~~ring.~~

~~vii). The structural changes in the D-ring is greatly influences the m ultiplicity o f the furan~~

~~absorption bands.~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 164~~

~~In most derivatives, structurally similar to limonin (44), the C-methyl resonance~~

~~furthest downfield is that due to the C-methyl at C-13. The downfield shift o f this methyl~~

~~group is presumably due to the anisotropic effect of the furan ring. Since the gem-~~

~~dimethyls at C-4 are attached to a carbon atom which is in turn attached to an ether~~

~~oxygen, the chemical shift o f such a group would be further downfield than that observed~~

~~for the C-methyl at C-8. Thus, the assignment o f the highfield C-methyl peak falls to the~~

~~C-methyl group at C-8 largely by elimination.~~

~~Limonin (44)~~

~~The West African shrub Harrisonia abyssinica Oliv. (Simarubaceae) (“ Msabubini”~~

~~or “Mpapura-doko” in Swahili) is widely used in various folk remedies, including~~

~~treatment for tuberculosis, fever, bubonic plague, haemorrhoid, snake-bite, etc~~

~~(Hassanali et al., 1987). Four tetranortriterpenes, obacunone (45), pedonin (46),~~

~~harrisonin (47) and 12p-acetoxyharrisonin (48), have so far been isolated from this~~

~~shrub. The first two limonoids showed mild antifeedant activities against the larvae o f the~~

~~East African monophagous crop pest Spodoptera exempta (Kubo et al., 1976) and the~~

~~third against the Southern armyworm, Spodoptera eridania (Hassanali et at., 1987). Our~~

~~recent interest in comparative activities of Striga species seed germination stimulants from~~

~~native Nigerian plants has led to an examination of the methanolic root extracts of H.~~

~~abyssinica. The complete carbon-13 assignments of obacunone (45) could not be~~

~~unambiguously assigned by previous workers (Okorie, 1982; Kubo et at., 1976), and we~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 165~~

~~now report the high field and spectral assignments of methyl groups and~~

~~quaternary carbons (C-4, C-10 and C-13).~~

~~K.2. MATERIALS AND METHODS~~

~~IX.2.1. Structure Elucidation. Mp measured on a Thomas Hoover apparatus (uncorr.). Mass spectrum were recorded on a Hewlett-Packard 5971 A GC/MS~~

~~spectrometer. The IR spectrum was recorded on a Perkin-Elmer 1760x FT-IR~~

~~spectrometer as a film on KBr plate using CHCI 3 solution.~~

~~One- or two-dimensional ^H- and ^^C-NMR spectra were recorded on a Bruker~~

~~A M 4(X) (4(X) MHz) spectrometer at room temperature. The spectrometer was locked on~~

~~the deuterium signal of CDCI 3. A ll experiments were performed on obacunone (45)~~

~~samples o f the same weight as ca. 10% CDCI3 solutions in 5-mm NMR tube (0.6 mL of~~

~~solution used). A ll chemical shifts are expressed in 5 (ppm) using TMS as internal~~

~~standard (S tm s = 0 ppm). Chemical shifts are reported in ppm on the S scale. The~~

~~following are the 2D NMR techniques employed: ‘H-^H correlation spectroscopy~~

~~(COSY) (Aue e ta l, 1976), NOESY(Odenhausen e ta l, 1984), HETCOR (Jch = 125)~~

~~(Bax and Morris, 1981) and the long-range COLOC (Jch = 7) (Kessler et a l, 1984). The DEPT experiments (Doddrell et a l, 1982) were carried out with 0 = 90° and 135°.~~

~~The quaternary carbons were determined by subtraction o f these spectra from the broad~~

~~band ^^C NMR spectra. The standard pulse programs for the 2D NMR experiments~~

~~employed were those provided by Bruker instruments, COSY.AU for ^H shift~~

~~correlation, XHCORR.AU for CH-correlation with homonuclear ^H decoupling,~~

~~COLOC.AU for long-range (3-bond) CH-correlation with polarization (D2 0.06 sec) set~~

~~fo r 8 Hz and NOESY.AU for nOe difference. The ^H-^H-COSY correlation and~~

~~NOESY maps consisted o f 256 X 1 K data points per spectrum, each composed o f 32~~

~~transients. The HETCOR and COLOC sequences used were those described by Bax and~~

~~Morris (1981) and Kessler et a l (1984), respectively. In both experiments, a 2-sec delay~~

~~was allowed between each scan, and the coupling constant was optimized for J-125 Hz~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 166~~

~~and 7 Hz, respectively. The final size o f the data matrix was 2K X 2K. The number of acquisitions at each ti was 32 with 4 dummy acquisitions also being acquired. The~~

~~HETCOR and COLOC correlation maps consisted of 256 X 4 K data points per~~

~~spectrum, each composed o f 64 transients. In all ID and 2D experiments, the sample was spun.~~

~~IX.2.2. Isolation of Obacunone (45). Roots of H. abyssinica were collected near~~

~~Moniya market, Ibadan, Nigeria on August 1,1994. Authentication was done by Mr. C.~~

~~W . Agyakwa in the Weed Science Section, International Institute o f Tropical Agriculture~~

~~(ETA). A voucher specimen is deposited in the ETA Herbarium (Voucher No. Rugutt~~

~~61). The air-dried powdered roots (50 g) were extracted with methanol (6 X 250 mL) at~~

~~room temperature for 3 days. Each crude extract was filtered in a Buchner funnel. The~~

~~extracts were combined and methanol evaporated under reduced pressure at low~~

~~temperature (40°C). The resulting crude extract was shipped to the Chemistry laboratory~~

~~at Louisiana State University (LSU) and stored in the refrigerator at 4°C until analysis.~~

~~A t LSU, the crude extract (1.1 g) was chromatographed on a VLC (Pelletier et al., 1986)~~

~~column packed with 100 g silica gel (MN Kieselgel G) using ethyl acetate-hexane as~~

~~eluent The percentage of EtOAc in hexane was gradually increased (0:50, 5:45,10:40,~~

~~15:35, 25:25, 35:15, 45:5, 50:0). Forty fractions of 10 mL each were collected and~~

~~monitored by TLC (Fischer, 1991) performed on precoated M N Sil-G 25 UV 254 plates~~

~~having a layer thickness o f 0.25 mm. The TLC plates were developed w ith 35% ethyl~~

~~acetate in hexane and made visible by spraying with a solution prepared by dissolving~~

~~C 0CI2.6H 2O (2 g) in 10% aqueous H 2SO4 (100 mL) and heating until a colored spot~~

~~appeared. The homogeneous fractions 30-39 eluted with 35% EtAOc-hexane were~~

~~combined and evaporated to afford 5.1 mg of obacunone (45) as a yellow flu ffy solid [Rf~~

~~0.25 (Si02» EtOAc:Hexane, 2:6); MS 454 [M+] and mp 227-231° {lit(Kubo etal.,~~

~~1976) 226-228°)}.~~

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~~IX.2.3. Isolation of Pedonin (46), Harrisonin (47) and 12^ -~~

~~acetoxyharrisonin (48). Air-dried roots (30 g) of the same plant was extracted~~

~~consecutively with solvents of increasing polarity (hexane, ether, dichloromethane,~~

~~methanol and water). The dichloromethane extract was concentrated under reduced~~

~~pressure to give a residue (1.2 g) which was chromatographed on 350 g silica gel with~~

~~benzene and increasing amounts o f the ether in benzene (0:50, 5:45,10:40, 15:35, 45:5~~

~~and 50:0, 50 mL each). The fraction eluted with benzene-ether (45:5), (5:45) and~~

~~(10:40) afforded (30 mg) pedonin (46) {C 27 H32O9, Mr. 500.5, mp. 258-260°C; Rf~~

~~0.53, hexane-ether, 2 : 1}, (15 mg) harrisonin (47) {C 27 H 32O 10, M f. 517, rap. 154-~~

~~155°C ; R f 0.40, hexane-ether, 2:1} and (8 mg) 12P-acetoxyharrisonin (48)~~

~~{C 29H 34O12, Mr. 575, mp 252-254°C; R f 0.42, hexane-ether, 2:1}, respectively. A ll~~

~~these limonoids were isolated as white powder.~~

~~IX.2.4. Isolation of (+) ononitol (49). The methanolic extract (0.5 g) of the~~

~~roots was chromatographed on a silica gel column. The fraction obtained by elution with~~

~~1:1 acetone-methanol afforded 90 mg of colorless prism-like crystals which were~~

~~identified by X-ray structure analysis to be (+)-ononitol (49) {4-O-methyl-myo-inositol-~~

~~hydrate, C 7 H 14O6.H2O, rap 186-188°C; Rf 0.46, hexane-methanol, 1:2}.~~

~~The crystal structure determination was carried out by Frank R, Fronczek. A~~

~~colorless crystal fragment o f dimensions 0.30 x 0.40 x 0.52 mm was used for data collection on an Enraf-Nonius CAD4 diffractometer equipped with CuKa radiation (X =~~

~~I.54184 Â), and a graphite monochromator. Crystal data are: C 7 H 14O6.H 2O M r =~~

~~212.2, triclinic space group PI, a = 5.079(1), b = 6.604(1), c = 7.758(1)Â, a =~~

~~106.00(1), p = 93.49(1), Y= 109.10(1)“ , V = 233.0(1)Â3, Z = 1, dc = 1.512 g cm-3, T~~

~~= 23°C. Intensity data were measured by O)-20 scans of variable rate. A hemisphere of~~

~~data was collected within the limits 2 < 0 < 15°. Data reduction included corrections for~~

~~background, Lorentz, polarization, and absorption effects. Absorption corrections (p. =~~

~~II.4 cm 'l) were based on scans, with minimum relative transmission coefficient~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 168~~

~~90.5%. O f 1894 unique data, 1890 had I> 0 and were used in the refinement.~~

~~Coordinates of Kailiang et al (1988) were used as a starting model and refined using the~~

~~Enraf-Nonius M olEN programs. Refinement was by full-m atrix least squares, treating~~

~~nonhydrogen atoms anisotropically and hydrogen atoms isotropically. Convergence was~~

~~achieved w ith R = 0.054 for 189 variables. Maximum residual electron density was 0.63 eÂ-3.~~

~~IX.2.5. Bioassays. Test solutions were prepared by dissolving 10 mg of~~

~~obacunone (45), pedonin (46), harrisonin (47) and (+)-ononitol (49) in 0.1 mL~~

~~dimethyl-sulfoxide (DMSO) as the initial solvent carrier followed by diluting with 9.9 mL~~

~~sterile distilled water to a final concentration o f 1,000 m gL'l. Other test solutions (i.e.~~

~~100,10 m g L 'l) were prepared by diluting with sterile distilled water. Each test solution (600 jxL) was added separately to a sterile glass petri dish lined with Whatman No. 1~~

~~filter paper. Stimulatory activity of test solutions was assayed using conditioned S.~~

~~hermonthica seeds collected in 1993 in Bida, Nigeria. The seeds were surface disinfested~~

~~by immersion in 1% NaOCl for a brief period and then rinsed several times with sterile~~

~~distilled water. Treatments consisted o f three replicates o f about 360 seeds in twelve~~

~~evenly spaced 5 mm diameter disks (per petri dish). Six replicates of each treatment were~~

~~arranged and the experiment repeated three times. Treated seeds were kept in the dark in~~

~~an incubator at 28°C. Germination (radicle protrusion through the seed coat) o f all assay~~

~~seeds were recorded after 24 h. Germination data were transformed to arc sine and~~

~~compared to controls using single-degree-of-freedom contrasts with the general linear~~

~~models procedure o f the Statistical Analysis System (SAS, 1988) programs.~~

~~IX.3. RESULTS AND DISCUSSIONS. Obacunone (45, C 26H 30O 7 ), an~~

~~optically active limonoid, has previously been isolated from the West (Okorie, 1982) and~~

~~the East (Kubo et a l, 1976) African H. abyssinica plants and also from citrus species~~

~~(Emerson, 1948) as one of their bitter principles. This compound contains two lactone~~

~~rings, which can be opened reversibly with alkali, a ^-substituted furan residue, a~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 169~~

~~ketonic oxygens present in the 6-membered ring and one epoxide (Barton and Pradhan,~~

~~1961). The assignment o f a ll proton and most carbon o f NMR signals o f obacunone~~

~~(45) have been made partly through comparison o f the chemical shifts with the published~~

~~data o f the same compound (Okorie, 1982; Kubo et al., 1976) and partly through high~~

~~field ID and 2D experiments (Table DCl).~~

~~Obacunone (45) exhibited IR bands which were typical o f a limonoid skeleton (a ^-substituted furan at 'Dm axcm 'l; 1501 and 878, three CO bands at 1734, 1711 and~~

~~1645, the last band being due to an a,P-unsaturated C =0). The l^c-N M R spectra~~

~~reported in Table IX . 1 confirm the presence o f 26 carbon atoms, eleven o f which show~~

~~resonances in the chemical shift range of S > 77 ppm. The DEPT spectrum (Figure DC. 1)~~

~~exhibited 5 methyls, 3 methylenes and 9 methine signals, while the remaining 9 signals in~~

~~the broad-band spectrum were due to quaternary carbon atoms. The downfield signals~~

~~are three singlets for the quaternary carbonyl carbons C-1, C-16 and C-3. The upfield absorptions o f 5 < 77 are characterized by four singlets (C-14, C-8, C-10 and C-13),~~

~~three doublets (C-5, C-9 and C-15), three triplets (C-6, C-11 and C-12), and five quartets~~

~~for aliphatic C-methyls (C-18, C-19, C-28, C-29 and C-30). The methyl and quaternary~~

~~carbon l^C-NMR signals were unambiguously assigned by employing the conventional~~

~~ID and 2D NMR techniques.~~

~~The high field ^H-NMR o f obacunone (45) showed two sharp singlets, one at~~

~~3.65 ppm due to the proton at the epoxy carbon C-15, and the other at 5.45 ppm, a~~

~~slightly broadened singlet caused by the proton at C-17, adjacent to the furan ring. The~~

~~C-15 proton is clearly influenced by the anisotropic effect of the C-7 keto carbonyl and~~

~~must be deshielded by the keto group in order to account for the observed downfield~~

~~shift, when compared to shift values o f analogues w ithout the C-7 carbonyl group~~

~~(Dreyer, 1965). Two doublets at 6.51 and 5.96 ppm due to the protons on a~~

~~disubstituted double bond (C-1 and C-2) showed an AB-type system. The contour plot~~

~~of the ^H-‘H-COSY-45 spectrum (Figure IX.2) showed that H-1 resonating at 6.51 ppm~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 170~~

~~was scalar-coupled only with H-2 proton resonating at 5.96 ppm and no further~~

~~couplings to other protons was indicated. Cross peaks between H 3-I 8 and H-15a~~

~~occurred in the COSY spectrum. However, the COSY map contained ambiguities o f the~~

~~remaining methyl resonances which could only be eliminated by identification o f the~~

~~spatial proximities as detected in 2D NOESY-45 spectrum (Figure IX.3). The ^H NMR~~

~~spectrum o f 45 showed three methyl proton signals at 1.12, 1.24, 1.45 ppm, and two~~

~~overlapping absorptions at 1.46 ppm, which showed correlation peaks in the HETCOR~~

~~map (Figure IX.4) with five carbon quartets in the ^^C-NMR spectrum at 16.4, 22.1,~~

~~20.5, 26.8 and 32.5 ppm. The methyl resonance at 1.12 ppm was assigned to H 3-I 8~~

~~due to fact that in the COSY spectrum showed cross peaks w ith H-17a; in the NOESY~~

~~spectrum it showed spatial interactions with three protons, H-9o, H-21 and H-22 and in~~

~~the COLOC map (Figure IX.5), a correlation peak with the quaternary carbon C-14 was~~

~~observed. The methyl singlet at 1.24 ppm was ascribed to H 3-3O because it showed a cross peak with C-14 carbon in COLCXZ spectrum and spatial interactions with H-15a,~~

~~H-6p, and one or both o f the methyl groups at 1.46 ppm in the NOESY spectrum. The~~

~~methyl proton resonance at 1.45 ppm showing spatial interactions with H-5a and H- 6a~~

~~and was assigned to H 3-28 . The quaternary carbon resonance at 52.9 ppm was assigned~~

~~C-8 because it showed three-bond coupling to H 3-3O methyl in the COLOC spectrum.~~

~~The quaternary carbon resonance at 83.4 ppm showed correlation peaks w ith H 3-28 and~~

~~H3-29 methyl groups in the COLOC spectrum and was assigned to C-4.~~

~~The comparison between COSY and NOESY spectra (Figures IX.2 and IX.3)~~

~~revealed that several J coupling cross peaks have been effectively suppressed in the~~

~~NOESY spectrum (e.g. the H-17P, -H 3-I 8 ; H-6P, -H-5a; H-6p, -H - 6a ; and H-9a, -H-~~

~~lla ,p , at {fi;f 2 } = {5.45, 1.12}, {2.90, 2.29}, {2.90, 2.60}, {2.15, 1.8 - 1.95}~~

~~(Figure IX.2), respectively. The mixing time of 800 msec allowed for the spatial~~

~~detection o f several cross peaks, the most revealing o f which connect H 3-3O with H- 15a,~~

~~H-6 P and H 3-I 9, H-6P w ith H 3-I 9 and H 3-29, H-6a with H 3-28 , and H 3-I 8 with H-~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 171~~

~~21, H-9a, H-15a and H-17P. The integration intensity o f the peaks overlapping in the~~

~~region 1.8-1.95 ppm indicated four protons, which, on the basis of COSY and NOESY~~

~~spectra were assigned to H-11 and H-12 protons. A model was created using~~

~~PCMODEL (MMX- force field energy minimization) and all experimental nOEs were in~~

~~agreement with the model.~~

~~The high field NMR measurements o f obacunone (45) indicated marginal~~

~~differences in coupling constants with those reported (Okorie, 1982; Kubo et al., 1976).~~

~~The arrangements o f a relatively large number o f functional groups in a framework o f~~

~~rigid stereochemistry causes the protons of 45 to be shielded to different extents. Thus~~

~~many o f the protons appeared in distinctive regions o f the spectrum and there were few~~

~~overlapping bands. Our implementation of high field 2D NMR techniques reduced~~

~~misassigrunents in both and Oc spectra, which are mainly due to overlap of the~~

~~signals in the upfield regions.~~

~~Complete and unambiguous assignments o f all the protons and carbon signals o f pedonin (46), harrisonin (47) and 12P-acetoxyharrisonin (48) and (+)-ononitol (49)~~

~~were obtained through COSY, NOESY, HETCOR and COLOC experiments (Figures~~

~~IX.6-20). Our NMR data were in close agreement with those reported by Kubo et at.~~

~~(1976) and Hassanali et al. (1987). However, NOESY and COLOC spectra of~~

~~obacunone (45) and pedonin (46) strongly indicated that the previous assignments of~~

~~and f H chemical shifts needs revision.~~

~~Pedonin (46) belongs to a growing group o f limonoids in which one or more o f~~

~~the rings o f the cyclopentenophenanthrene skeleton are cleaved (Connolly, 1983). The~~

~~tetranortriterpenoid nature o f pedonin (46) and its close relationship to obacunone (45)~~

~~and harrisonin (47) was apparent from NMR spectroscopic data. The ^H NM R spectrum~~

~~o f 46 (Figure IX.6) showed resonances for five tertiary methyl groups (0.98, 1.24,~~

~~1.38, 1.58 and 1.64 ppm), a disubstituted double bond (6.35 and 6.17, d, J = 10 Hz, H- 1 and H-2), the characteristics o f a |3-substituted furan (6.94,7.34 and 8.59 ppm) and a~~

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~~secondary epoxidîc proton (3.75, s, H -I5). A major difference between the NMR~~

~~spectrum o f 46 and those o f obacunone (45) and harrisonin (47) (Figure DC. 13) was the~~

~~downfield shift (8.59 ppm) o f one o f the furan protons, suggesting the presence o f a~~

~~deshielding anisotropic effect of a C-17 carbonyl group. The NMR spectrum~~

~~indicated that two o f the four carbonyl groups were ketonic and the other two were either~~

~~lactonic or ester, and confirmed the presence o f an a,P-unsaturated furan and a~~

~~secondary, tertiary ether unit.~~

~~The NMR spectrum of harrisonin (47) {Figure DC. 13} differs from that of obacunone (45) in that the former lacks the H-5/H-6ot/H-6P three proton system in the~~

~~region 3.00-2.00 ppm, and that 4.30 ppm peak (the H -I5a) in 47 is downfield in~~

~~comparison to the 3.65 ppm peak in 45 (Table DC.1). The tetranortriterpenoid nature o f~~

~~12P-acetoxyharrisonin (48), which has an a,P-epoxy-6-lactone group in the ring D and~~

~~an a,p-unsaturated lactone group in ring A was clarified by ^H- and ^^C-NMR analyses.~~

~~The only difference between harrisonin (47) and 12P-acetoxyharrisonin (48), was the~~

~~presence o f an additional acetoxy group. The appearance o f a CH-OAc signal at 4.76~~

~~ppm (iR NMR, Figure DC. 16) and a methine peak at 72.9 ppm (l^C NMR, Figure~~

~~DC. 19) limited the attachment of the acetoxy group to either C-11 or C-12. The~~

~~assignment o f the acetoxy group at C-12 was based on the chemical shifts o f the adjacent~~

~~carbons. Namely, C-11 and the quaternary carbon (C-13) peaks at 15.3 and 39.7 ppm,~~

~~respectively, in harrisonin (47) were shifted downfield to 26.1 and 42.7 ppm in 48 due to the well-known acétylation or estérification on the P-carbon. Unambiguous~~

~~assignment o f 12p-acetoxy group configuration was based on the analyses o f NMR~~

~~data as well as COSY (Figure DC. 17) and NOESY (Figure DC. 18) spectra. The 14 Hz and the 5 Hz J values o f the H-9a 2.96 ppm signal showed that the 2.35 ppm signal~~

~~(Jg.li = 5 Hz) and 1.76 ppm signal (J g ji = 14 Hz) should be assigned to H-1 la (eq)~~

~~protons, respectively; since the 4.75 ppm H-12 peak is coupled to H-1 Ip and H-1 la by~~

~~1 Hz and the 7 Hz, respectively, its configuration is a , i.e., the 12-acetoxy group is P-~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 173~~

~~oriented. The two hydroxyl groups of the 123-acetoxyharrisonin (48) are both involved~~

~~in hydrogen bonding as evidenced by two sharp NMR singlets at 3.65 and 5.11 ppm~~

~~(inCDCIs). The stereoscopic illustration of (+)-ononitol (49) is shown below. The atomic~~

~~parameters are listed in Table IX.2. Bond lengths, selected bond lengths and torsion~~

~~angles are listed in Tables IX .3.1-3, respectively. It is worth noting that there is no~~

~~intramolecular hydrogen bond in (+)-ononitol (49), but intermolecular hydrogen bonds~~

~~exist between water molecule and hydroxyl group as well as between hydroxyl groups~~

~~(Kailiang et al., 1988). The six membered ring o f 49 takes a chair conformation and the~~

~~conformation o f the 4-0-methyl-myo-inositol molecule is described by the torsion angles~~

~~given in Table DC.3.3. A ll C-0 bonds are equitorial-bonds, except the bond C(6)-0(6)~~

~~which is axial-bond. The C-C and C-0 distances falling in the range o f 1.520-1.530 and~~

~~1.420-1.430 Â respectively, are typical o f single bonds. The absolute configuration of~~

~~(4-)-ononitol (49) is yet to be determined. Preliminary bioassay has shown that 49 has~~

~~some curative effect on blood vessel dilatation o f the heart (Kailiang et aL, 1988).~~

~~IX.3.1. Effect of (+)-ononitol (49) and Limonoids on Germination of S.~~

~~hermonthica Seeds. Preliminary bioassays indicated that obacunone (45), pedonin~~

~~(46), harrisonin (47) and (-t-)-ononitol (49) induce germination of conditioned Striga~~

~~hermonthica seeds collected in Bida during the 1993 harvesting season (Table IX.4).~~

~~This is encouraging because many limonoids are potentially available in very large~~

~~quantities. For example, the timber o f some species may yield 1% o f an isolated~~

~~crystalline limonoid; while a single tree ofEntadrophragma angolense may contain more~~

~~than 100 Kg. of gedunin (50), much of it easily recoverable from timber m ill offcuts~~

~~(Lavie and Troke, 1967).~~

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~~Table IX.1. correlation of obacunone (45) (400 MHz, CDCI 3)~~

~~S13c MulL® Sauachedjj Assignment~~

~~156.7 CH 6.51 1 123.0 CH 5.96 2~~

~~166.6 Cb - 3~~

~~83.4 Cb - 4 53.3 CH 2.29 5 39.9 CH2 Ha = 2.60; Hp = 2.96 6~~

~~207.4 Cb - 7~~

~~52.9 Cb - 8 49.2 CH 2.15 9~~

~~43.1 Cb - 10 19.4c CH2 11~~

~~26.8C CH2 } 1.8-1.95 12~~

~~37.3 Cb - 13~~

~~68.5 Cb - 14 57.3 CH 3.65 15~~

~~166.9 Cb - 16 84.0 CH 5.45 17 2 2 .1 CH3 1.12 18 26.8 CH3 1.46 19~~

~~120.1 Cb - 2 0 141.0 CH 7.42 21 109.7 CH 6.36 22 143.2 CH 7.40 23 20.5 CH3 1.45 28 16.4 CH3 1.46 29 32.9 CH3 1.24 30~~

~~^ Carbon multiplicities determined through DEPT experiments. ^ Assignments based on COLOC experiment. ^ Values within the same column may be interchanged.~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 175~~

~~18 =20~~

~~CO2CH3~~

~~29 28~~

~~Obacunone (45) Pedonin (46 )~~

~~OR 23 O~~

~~OH OAcOH~~

~~OAc H Harrisonin (47)~~

~~OAc 12p-acetoxyharrisonin (48) Gedunin (50)~~

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~~o' m o a C_) ro § C_) o LD CJ cn CD § (\i •a CJ S CM 3 o CO o U~~

~~CO (_) I CJ 00~~

~~o* ■ I~~

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~~Table IX.2. Coordinates and equivalent isotropic thermal parameters~~

~~atom X y z Beq(Â2)~~

01 1 1 1 2.43(2) 02 1.1317(3) 0.9603(2) 0.6454(2) 2.38(2) 03 0.8820(3) 0.5325(2) 0.3772(2) 2.45(2) 04 0.8119(3) 0.1515(2) 0.5010(2) 2.96(3) 05 0.6622(3) 0.1862(2) 0.8556(2) 2.48(2) 06 0.5403(3) 0.5892(2) 0.9094(2) 2.27(2) C l 1.0127(3) 0.7965(2) 0.8840(2) 1.74(3) C2 0.9390(3) 0.7727(2) 0.6838(2) 1.70(2) C3 0.9584(3) 0.5538(2) 0.5633(2) 1.74(3) C4 0.7658(4) 0.3473(3) 0.6069(2) 1.88(3) C5 0.8364(3) 0.3728(2) 0.8082(2) 1.78(3) C6 0.8231(3) 0.5934(3) 0.9298(2) 1.77(3) C l 0.6024(6) 0.0091(4) 0.3449(3) 3.65(5) OIW 1.3213(3) 0.4309(2) 1.1991(2) 2.68(3) HI 0.859(7) 1.024(5) 0.964(4) 3.2(6) H2 1.039(8) 1.017(6) 0.597(5) 4.0(7) H3 1.023(8) 0.504(6) 0.309(5) 4.3(8) H5 0.500(9) 0.126(6) 0.786(6) 6(1) H6 0.465(4) 0.548(3) 0.978(3) 1.0(4) H la 1.193(5) 0.793(3) 0.907(3) 1.1(3) H2a 0.772(7) 0.782(5) 0.662(4) 3.1(6) H3a 1.137(4) 0.558(3) 0.582(3) 0.5(3) H4a 0.560(8) 0.342(6) 0.561(5) 4.4(8) H5a 1.017(5) 0.372(4) 0.831(3) 1.9(4) H6a 0.872(5) 0.606(4) 1.038(3) 1.6(4) H7a 0.44(1) -0.010(7) 0.377(7) 7(1) H7b 0.63(1) -0.134(8) 0.293(7) 6(1) H7c 0.63(2) 0.10(1) 0.25(1) 13(2) HIW 1.254(7) 0.293(5) 1.129(5) 3.4(6) H2W 1.45(1) 0.442(7) 1.251(7) 5.3(9)

~~Beq — 8ît^/3 2j Uij a% aj a| • aj~~

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~~Table IX.3.1. Selected bond lengths (Â)~~

~~01-C l 1.420(2) C1-C2 1.527(2)~~

~~02-C2 1.424(2) C1-C6 1.521(2)~~

~~03-C3 1.428(2) C2-C3 1.528(2)~~

~~04-C4 1.428(2) C3-C4 1.534(2)~~

~~04-C7 1.430(2) C4-C5 1.531(2)~~

~~05-C5 1.419(2) C5-C6 1.527(2)~~

~~06-C6 1.426(2)~~

~~Table EX.3.2. Selected bond angles (deg.)~~

~~C4-04-C7 116.3(2) 04-C4-C3 108.8(1)~~

~~01-C1-C2 111.5(1) 04-C4-C5 108.3(2)~~

~~01-C1-C6 110.9(1) C3-C4-C5 110.4(1)~~

~~C2-C1-C6 111.1(1) 05-C5-C4 112.5(1)~~

~~02-C2-C1 109.0(1) 05-C5-C6 110.8(1)~~

~~02-C2-C3 109.7(1) C4-C5-C6 111.1(1)~~

~~C1-C2-C3 110.0(1) 06-C6-C1 108.6(1)~~

~~03-C3-C2 109.0(1) 06-C6-C5 110.0(1)~~

~~03-C3-C4 109.8(1) C1-C6-C5 111.8(1)~~

~~C2-C3-C4 111.7(1)~~

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~~Table IX.3.3. Selected torsion angles (deg.)~~

~~C7-04-C4-C3 -102.7(2) 01-C1-C2-02 58.9(2)~~

~~C7-04-C4-C5 137.3(2) 01-C1-C2-C3 179.3(1)~~

~~C6-C1-C2-02 -176.8(1) 01-C1-C6-06 59.2(2)~~

~~C6-C1-C2-C3 -56.4(2) 01-C1-C6-C5 -179.3(1)~~

~~C2-C1-C6-06 -65.4(2) 02-C2-C3-03 -61.5(2)~~

~~C2-C1-C6-C5 56.1(2) 02-C2-C3-C4 177.0(1)~~

~~C2-C3-C4-04 -175.1(2) 03-C3-C4-04 63.9(2)~~

~~C2-C3-C4-C5 -56.4(2) 03-C3-C4-C5 -177.4(2)~~

~~C1-C2-C3-03 178.6(1) 04-C4-C5-05 -61.4(2)~~

~~C1-C2-C3-C4 57.0(2) 04-C4-C5-C6 173.7(2)~~

~~C3-C4-C5-05 179.6(2) 05-C5-C6-06 -60.4(2)~~

~~C3-C4-C5-C6 54.7(2) 05-C5-C6-C1 178.9(2)~~

~~C4-C5-C6-06 65.4(2) C4-C5-C6-C1 -55.2(2)~~

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~~Table IX.4. Percentage germination o f conditioned Striga hermonthica seeds treated with aqueous solutions o f obacunone (45), pedonin (46), harrisonin (47) and (+)-ononitol (49)~~

~~Compound Concentration (Moles/L) % Germination~~

Obacunone (45) 2.3 X 10-3 M 85 (66.9)*

~~2.3 X 10-4 M 90 (72.5)~~

~~2.0 X 10-5 M 22 (27.9)~~

~~Pedonin (46) 2.0 X 10-3 M 70 (56.9)~~

~~2.0 X 10-4 M 80 (63.7)~~

~~2.0 X 10-5 M 18 (24.9)~~

~~Harrisonin (47) 1.9 X 10-3 M 28 (31.8)~~

~~1.9 X 10-4 M 30 (33.1)~~

~~1.9 X 10-5 M 12 (19.9)~~

~~Ononitol (49) 4.7 X 10-3 M 86 (68.2)~~

~~4.7 X 10-4 M 98 (82.1)~~

~~4.7 X 10-5 M 26 (29.9)~~

* Figures in parentheses are arc sine transformations.

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~~F ig u re K .2 . 2D 'H NMR COSY o f obacunone (45).~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 183~~

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~~Figure IX.3. 2D NMR NOESY spectrum o f obacunone (45).~~

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~~Figure DC7. 2D IH NMR COSY spectrum of pedonin (46).~~

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~~Figure DC.8. 2D NMR NOESY spectrum of pedonin (46).~~

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~~Figure IX.15. 2D NMR NOESY spectrum o f harrisonin (47).~~

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~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 198~~

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~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER X~~

~~FUTURE RESEARCH~~

~~201~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 202~~

~~The five classical plant hormones, namely ethylene (1), auxins (51), cytokinins~~

~~(52), gibberellins (53) and abscissic acid (54), are responsible for the control of plant~~

~~development and the integration o f the activities o f the different plant organs. From our~~

~~rather simple approach mainly based on exogenous treatment o f conditioned Striga spp.~~

~~seeds with germination stimulants, we have shown that germination occurs over a broad~~

~~concentration range suggesting that the stimulants are a new class of hormones.~~

~~However, the central question which remains largely unanswered is: how far does the~~

~~regulation of germination arise from modulation by these “exogenous” hormones?.~~

~~Accurate measurements (Weiler, 1984) o f the concentrations o f hormones in Striga spp.~~

~~seeds w ill provide useful information. Because Striga spp. seeds contain some o f the~~

~~compounds also occurring in host plants, we cannot rule out the possibility o f a specific~~

~~gene-for-gene relationships which may well occur as the rule rather than the exception in~~

~~hostrparasite systems (Person, C., 1959).~~

~~The studies in the isolation and structure elucidation of germination stimulants~~

~~from host and nonhost plants yielded an array of compounds. There appears to be reason~~

~~for confidence that our screening o f plants w ill yield sorely-needed templates for the~~

~~synthesis o f potentially superior germination stimulants. Data on active concentration of~~

~~germination stimulants in the soil needs to be determined by studying the rate o f input to~~

~~the environment (i.e. leachate, root exudates, or decomposition and leaching o f plant~~

~~residues), absorption and adsorption by Striga seeds, fixation by soil components, and~~

~~leaching and microbiol degradation (Huang et al., 1977). Moreover, studies on the~~

~~stability of germination stimulants enhanced by entrapping or encapsulating in a~~

~~biodegradable polymeric matrix (Shasha er a/., 1981) or cyclodextrins (Wang and~~

~~Warner, 1993) which slowly releases the germination stimulant during biodégradation is~~

~~needed.~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 203~~

~~CH2OH~~

~~COOH HN~~

~~Auxins (51) Cytokinins (52)~~

~~,0H~~

~~OH HO COOH COOH~~

~~Gibberellins (53) Abscissic acid (54)~~

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Adams, D. O.; Yang, S. F. Ethylene biosynthesis: Identification of 1- aminocycloprcpane-l-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA, 1979, 76, 170-174.

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Aggarwal, V. D. Latest in cowpea Striga research at HTA. In: Weber, H. C. and W. Forstreuter, eds. Parasitic Flowering Plants. Proceedings o f the Fourth International Symposium on Parasitic Flowering Plants. Marburg: Philips University, 1987, pp. 27-28.

~~Akisanya, A.; Bevan, C. W. L.; Hirst, J.; Halsall, T. G.; Taylor, D. A. H. West Afican Timbers. Part HI. Petroleum extractives from the genus Entandrophragma. J. Chem. Soc., 1960, 302.~~

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Arigoni, D.; Barton, D. H. R.; Corey, E. J.; Jeger, O.; Cagioti, L.; Sukh Dev; Ferrini, P. G.; Glazier, E. R.; Malera, A.; Pradhan, S. K .; Schaffner, K.; Sternhell, S.;Templeton, J. P.; Tobinaga, S. The Constitution of Limonin. Experientia, 1960, 16, 41.

~~Ashton, I. A.; Abulnaja, K. O.; Pallett, K. E.; Cole, D. J.; Harwood, J. L. The Mechanism o f inhibition o f fatty acid synthase by the herbicide diflufenican. Phytochemistry, 1994, 35, 587-590.~~

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~~Babiker, A. G. T.; Hamdoun, A. M.; Rudwan, A.; Mansi, N. G.; Faki, H. H. Influence o f soil moisture on activity and persistence o f the strigol analogue GR 24. Weed Res., 1987, 27, 173-178.~~

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Berenbaum, M. Brementown revisited: interactions among allelochemicals in plants. In: G. A. Cooper-Driver, T. Swain, E. A. Conn, eds. Chemical mediated interactions between plants and other organisms. Rec. Adv. Phytochem., 1985, 19, 139-169.

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~~J., Worsham A. D. & Eplee R. E., eds. Proc. Second Intl. Symp. on Parasitic Weeds, North Carolina; Raleigh, USA, 1979, pp. 1-78.~~

~~Cechin, I; Press, M. C. The influence of nitrogen on growth and photosynthesis of sorghum infected with Striga hermonthica from different provenances. Weed Res., 1993, 33, 289-298.~~

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~~Champagne, D. E.; Koul, 0.; Isman, M. B.; Scudder, G. E.; Towers, G. H. N. The biological activity of limonoids from Rutales. Review article number 65. Phytochemistry, 1992, 31, 377-394.~~

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~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A P P E N D IX 1. Letter o f Permission to use Published Materials~~

~~SPECTROSCOPY LETTERS An International Journal for Rapid Communication Editor J.W. ROBINSON Departmentof Chemistry.Louisiana Slate University Baton Rouge, Louisiana 70803~~

~~March 8, 1996~~

~~Mr. Joseph K. Rugutr Department of Chemistry Louisiana State University Box C-12, Choppin Hail Baton Rouge, LA 70803~~

~~Dear Mr. Rugutt:~~

Permission is granted to use any material published in your article "Carbon-13 Assignments and Revision of the Stereostructures of theCa.din.3iLQS 2-Hydroxy-8a- angeloyoixycalamene and 2-Hydroxy-8ot-hydroxycalamene and 1-3C-NMR assignments for tertiary methyls and quaternary carbons of the limonoid obacunone" by J.K. Rugutt in your Ph.D. Thesis.

~~Dr. J.W. Robinson Professor of Chemistry~~

~~JWR/jh~~

~~225~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VITA~~

~~Joseph Kipronoh Rugutt was bom in Kericho District, Kenya on December 29,~~

~~1964 to the Christian parents, M r. Thomas Kiprugutt arap Kamoing and Mrs. Rodah~~

~~Chepmabwai Kamoing. He grew up in Keongo Village and attended Keongo Primary~~

~~School. After graduating in 1979, he was admitted to St. Patrick’s High School Iten in~~

~~Elgeiyo-Marakwet District where he had a uniquely successful career. At Iten, Mr.~~

~~Rugutt was involved in sports, especially volleyball and football. He was an excellent~~

~~leader and was selected the Deputy School Captain and School Captain in 1982 and 1984,~~

~~respectively. He won several awards for outstanding performances in academic and~~

~~leadership. After graduating from St. Patrick’s Iten, Mr. Rugutt joined National Youth~~

~~Service (NYS) Pre-university Training in G ilgil, Naivasha. A t NYS, he was selected the~~

~~Commander in charge of Twiga Barracks which earned him President Daniel Arap M oi’s~~

~~Best Recmit Award. In 1986, Mr. Rugutt joined Moi University, Eldoret for a Bachelor~~

~~o f Science degree in Chemistry and was employed as a teaching assistant after~~

~~graduating. At Moi University Mr. Rugutt met and married his beautiful wife, Janny~~

~~Chepkemoi Koske who is currently studying Computer Science at Louisiana State~~

~~University. On August 18, 1993 the beautiful Ms. Elizabeth Chepchumba was bom to~~

~~the Rugutts family. In January 1991 Mr. Rugutt enrolled at Louisiana State University~~

~~where he taught CHEM 1212 and CHEM 2364. He was considered one o f the best~~

~~teaching assistants in the Department o f Chemistry. In 1994, M r. Rugutt was the~~

~~recipient of the Rockefeller African Dissertation Internship vWard. Currently Mr. Rugutt~~

~~is a candidate for the Doctor of Philosophy degree in Organic Chemistry (Natural~~

~~Products). The Rockefeller Foundation have nominated Mr. Rugutt to receive the 1997-~~

~~1998 African Science-Based Career Development Award (ASBD) to enable him continue~~

~~w ith Striga research under the direction o f Prof. Nikolaus Fischer and in collaboration~~

~~with Dr. Dana K. Bemer of the International Institute of Tropical Agriculture, Ibadan~~

~~Nigeria.~~

~~226~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DOCTORAL EXAMINATION AND DISSERTATION REPORT~~

~~Candidate: Joseph Kipronoh Rugutt~~

~~Major Field: Chemistry~~

~~Control of African Striga Species by Natural Products from Title of Oissertation: Native Plants~~

~~Approved:~~

~~Major Professor and Chairman~~

~~Dean of the Graduate School~~

~~EXAMINING COMMITTEE :~~

~~Date of Examinât ion:~~

~~April 8. 1996~~

~~Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.~~