BIOACTIVE PRINCIPLES FROM PANAMANIAN MEDICINAL PLANTS

Thesis presented by

Pablo Narclso Solis Gonzalez

for the degree of

Doctor of Philosophy

In the Faculty of Medicine of the University of London

Department of Pharmacognosy The School of Pharmacy University of London

1994 ProQuest Number: 10105140

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 A tribute to my Grand Parents,

Pablo and Conce

and to my Parents,

Nato and Gladys

my models of fortitude. ABSTRACT

Plants are widely used in Panama and Central America to cure different diseases, including malaria and amoebic dysentery. Cephaëlis ipecacuanha of the famiiy Rubiaceae contains emetine, which is used in the treatment of severe amoebic dysentery. Species of Rubiaceae, Simaroubaceae, Meliaceae and Menispermaceae have been examined, they are known to contain isoquinoline alkaloids, quassinoids, limonoids and bisbenzylisoquinoline (bbiq) alkaloids, respectively, with activity against Plasmodium falciparum.

In order to obtain antiprotozoal natural products such as quassinoids, limonoids, bbiq’s and emetine reiated alkaloids it was decided to investigate the following plants: Guarea macropetaia, G. rhopalocarpa, Ruagea glabra (Meliaceae); Abuta dwyerana (Menispermaceae); Cephaëlis camponutans, 0. dichroa, 0. dimorphandrloldes, 0. glomerulata, Laslanthus panamensis (Rubiaceae); PIcramnIa antldesma subsp. fessonia, P. teapensis (Simaroubaceae).

Extracts from different parts of the plants were fractionated and tested against brine shrimp and KB ceils. Four plants were chemically studied and the isoiated compounds were characterized by mass spectra, infrared, ultraviolet, and NMR, also, two dimensional NMR experiments such as COSY-45, HMQC, HMBC and ROESY. Circular dichroism was used to establish the absolute configuration of some of the compounds.

C. camponutans yielded benz[g]isoquinoline-5,10-dione, isolated for the first time from the plant kingdom, and a the new 1-OH- benzoisochromanquinone. Both compounds showed activity against brine shrimp, KB ceils and Plasmodium falciparum, in vitro. C. dichroa yielded six indole monoterpenoid aikaloids, including strictosidine, strictosamide, angustine, vallesiachotamine, iso-vallesiachotamine and the novel vallesiachotamine lactone. In contrast, 0. glomerulata yielded three new quinoline alkaloids. named, glomerulatine A, B and C. The taxonomic position of Cephaëlis is unclear and more detailed information on the constituent alkaloids may prove valuable in unravelling this problem.

The butanolic fraction from de leaves of P. antldesma subsp. fessonia, showed strong activity against KB cells and yielded two known anthraquinones (aloe-emodin, aloe-emodinanthrone) and three new C-glycoside of aloe-emodin, named picramnioside A, B and C. The C-glycosides showed activity against KB cells.

A cytotoxic microwell test using Anemia salina (brine shrimp) was developed in order to overcome some of the disadvantages of the previously described method. The test was predictive of cytotoxicity and compounds with activity on the nervous system were inactive. The activity of the quassinoids against KB cell and P. falciparum parallel the activity against brine shrimp. ACKNOWLEDGEMENTS

I would like to thank my supervisor Professor J. David Phillipson for his precious help and friendship without which this work would not have been possible. I am also indebted to Professor Mahabir P. Gupta, Departamento de Quimica Medicinal y Farmacognosia, Facultad de Farmacia, Universidad de Panama for his encouragement and guidance.

I would like to express my appreciation and thanks to Professors Don Antonio Gonzalez and Don Angel Gutierrez Ravelo and colleagues in the Institute de Productos Maturates Organicos, Universidad de La Laguna, Tenerife, Spain, where part of this work was carried out.

Also, I am indebted to Professor Mireya Correa and Mrs Carmen Galdames for their valuable help in collection, identification and plant preparation. I am grateful to Mr. Rutilio Paredes, Research Coordinator of Program of Ecology and Management of Wild Areas of Kuna Yala (PEMASKY), who very kindly allowed access for plant collection. I thank Mrs. J. Hawkes for running the NMR experiments in the AMX400 and WM250 NMR, University of London Intercollegiate Research Service at King’s College and Dr. K. Welham and his colleagues for running the mass spectra at the Mass Spectrometry Unit at The School of Pharmacy, University of London.

I am grateful to my colleges in the Department of Pharmacognosy, The School of Pharmacy with whom I have collaborated in various aspects of this work, particularly Miss Maria Camacho, Miss Caroline Lang’at and Dr. Colin Wright. I also wish to thank Drs. D.C. Warhurst and G.C. Kirby, Department of Medical Parasitology, The London School of Hygiene and Tropical Medicine for running the antimalarial activity of some of the isolated compounds.

I am grateful to Dr. R.T. Brown, University of Manchester for generous gifts of strictosidine, vincosidine and their acetylated derivatives and for their NMR spectra. Also, I would like to thank Mrs. M. Pickett for technical help and Mrs A. Cavanagh for excellent graphical work in the preparation on slides and posters for use at conferences.

Undoubtedly, I am eternally indebted to my wife and lover, Nilka, and my children, Nilkita, Pablito and Paola, for their daily encouragement, comprehension and patience during these three difficult years of my life.

I am grateful to the Commission of the European Community for the provision of a research scholarship (B/CI1*-913064). CONTENTS

Page

Abstract 3 Acknowledgement 5 List of Abbreviations 12 List of Figures 15 List of Tables 16

SECTION 1 INTRODUCTION 18 1.1 Health care in Panama 19 1.1.1 The country 19

1.1.2 Treatment of diseases 20 1.1.2.1 Health care coverage in Panama 20 1.1.2.2 Major health problems in Panama 21 1.1.3 Ethnomedicine in Panama 25 1.1.4 Natural resources and conservation 28

1.2 Selection of plants for chemical and biological investigation 36

1.2.1 Traditional medicine 36 1.2.2 Random selection of plants for chemical investigation 38

1.2.3 Chemotaxonomy 39

1.2.3.1 Chemotaxonomy background 41

1.3 Biological testing of plant extracts 42 1.3.1 Advantages and disadvantages of in-vivo and in-vitro testing 43

1.3.2 Simple bioassays 45

1.3.2.1 Brine shrimp assay 45

1.3.2.2 KB cells assay 46

1.3.2.3 P. falciparum assay 47

1.4 Aims of this study 49 SECTION 2. PLANTS SELECTED FOR INVESTIGATION 50

2.1 Meliaceous plants 51 2.2 Menispermaceous plants 57

2.3 Rubiaceous plants 58

2.4 Simaroubaceous plants 67

SECTION 3 TESTING FOR BIOLOGICAL ACTIVITY 72

3.1 Materials and methods 73

3.1.1 New microwell brine shrimp assay 73 3.1.2 Brine shrimp assay 74

3.1.3 KB cells assay 74 3.1.4 P. falciparumàssay 76

3.2 Results and discussion 76 3.2.1 Evaluation of brine shrimp assay 76 3.2.2 Activity of the plant crude extracts against brine shrimp and KB cells 80

SECTION 4 PHYTOCHEMISTRY 89

4.1 Material and general methods 90

4.1.1 Plant material 90 4.1.2 Solvents 90

4.1.3 Chromatography techniques 92

4.1.4 Spectrometric methods 92

4.1.4.1 Ultraviolet spectroscopy (U V ) 92

4.1.4.2 Infrared spectroscopy (IR ) 92

4.1.4.3 Mass spectrometry (M S) 93

4.1.4.4 Nuclear magnetic resonance (N M R ) 93

4.1.4.5 Polarimetry 93

4.1.4.6 Circular dichroism (CD) 94

4.1.5 Preparation of acetyl derivatives 94

4.2.1 Preparation of crude plant extracts for

biological test 94

8 4.2.1.1 Results 94

4.3 Isolation and identification of bioactive compounds from Cephaëlis camponutans {Psychotria camponutans) 98

4.3.1 Extraction procedure 98

4.3.2 Identification of the isolated compounds 98

4.3.3.1 1 -Hydroxybenzoisochromanquinone 98

4.3.3.1.1 Spectral data 98 4.3.3.1.2 1 - Acety Ibenzoisochromanquinone 99 4.3.3.1.2.1 Spectral data 99

4.3.3.2 Benz[g]isoquinoline-5J0 dione 99

4.3.3.2.1 Spectral data 99

4.3.4 Discussion 1 0 2 4.3.5 Biological activity of the isolates 108 4.3.5.1 Results and discussion 108

4.4 Isolation of alkaloids fromCephaëlis dichroa 1 1 0

4.4.1 Extraction and separation procedure 1 1 0

4.4.2 Identification of isolated compounds 1 1 0

4.4.2.1 Vallesiachotamine lactone 1 1 0

4.4.2.1.1 Spectral data 1 1 0

4.4.2.2 Vallesiachotamine 1 1 1

4.4.2.2.1 Spectral data 1 1 1

4.4.2.3 Strictosidine 1 1 2

4.4.2.3.1 Spectral data 1 1 2 4.4.2.4 Strictosidine lactam 113

4.4.2.4.1 Spectral data 113

4.4.2.4.2 Acetyl strictosidine lactam 114

4.4.2.4.2.1 Spectral data 114

4.4.2.5 Angustine 115 4.4.2.5.1 Spectral data 115

4.4.3 Discussion 117 4.5 Isolation and identification of alkaloids from

Cephaëlis glomerulata (Psychotria glomerulata) 121

4.5.1 Extraction and separation procedure 121

4.5.2 Identification of the isolated compounds 121

4.5.2.1 Glomerulatine A 121

4.5.2. 1 . 1 Spectral data 121

4.5.2.2 Glomerulatine B 122

4.5.2.2.1 Spectral data 122 4.5.2.5 Glomerulatine C 123 4.5.2.3.1 Spectral data 123

4.5.3 Discussion 124

4.6 Isolation and identification of bioactive compounds

from Picramnia antldesma var. fessonia 131 4.6.1 Extraction and separation procedure 131

4.6.2 Identification of the isolated compounds 132

4.6.2.1 Picramnioside A 132

4.6.2.1 . 1 Spectral data 132

4.6.2.2 Picramnioside B 133

4.6.2.2.1 Spectral data 133

4.6.2.3 Picramnioside C 135

4.6.2.3.1 Spectral data 135

4.6.2.4 Aloe-emodin 136

4.6.2.4.1 Spectral data 136 4.6.2.5 Aloe-emodinanthrone 136

4.6.2.5.1 Spectral data 136

4.6.3 Discussion 137

4.6.4 Biological activity of the isolated compounds 144

SECTION 5 GENERAL DISCUSSION 145

5.1 Introduction 146

10 5.2 Plants selected for this study 147

5.3 Testing for biological activity 148

5.3.1 Brine shrimp assay 148 5.3.2 Biological activity of plant extracts 149 5.4 Phytochemistry 149

5.4.1 Cephaëlis species 149 5.4.2 Picramnia antidesma subsp. fessonia 156 5.5 Are plants predictable in their chemotaxonomy? 157

SECTIO N 6 CONCLUSIONS 159

SECTIO N 7 REFERENCES 162 A PPEN D IX I SPECTRA 185 APPENDIX n LIST OF SCIENTIFIC PAPERS 247

II List of Abbreviations

pCi Microcurie pM Micromolar Dept Distortionless enhancement by polarization transfer (differentiation

between CH, CHj, and CH 3 using the improved sensitivity of

polarisation transfer)

Carbon thirteen

'H Proton 2D Two dimensional BBIQ Bisbenzylisoquinoline alkaloid BuOH n-Butanol br Broad Centigrade QD, Deuterobenzene CC Column chromatography CD Circular dichroism

CD 3 OD Deuteromethanol

CDCI3 Deuterochloroform CIMS Chemical ionization mass spectrometry cm'^ Centimetre to the minus one COSY-45 Two dimensional correlation spectroscopy d Doublet

DCCC Droplet counter current chromatography dd Double doublet ddd Double double doublet

DMSO-Dg Deuterodimethylsulphoxide

EDgo Effective dose fifty EIMS Electron impact mass spectrometry

EtOAc Ethylacetate

EtOH Ethanol eV Electron volt = 94.487 kJ/mol = 23.06 kcal/mol

12 FABMS Fast atom bombardment mass spectrometry FDM S Field desorption mass spectrometry

FAO Food and Agricultural Organization g Gram GC Gas chromatography

HETCOR Heterocorrelation

HMBC Heteronuclear multiple bond connectivity

H M Q C Heteronuclear multiple quantum coherence

HPLC High performance liquid chromatography HRMS High resolution mass spectrometry hrs Hours H z Hertz

IR Infrared J Coupling constant

Kg Kilogram Km^ Square kilometre

1 Litre

LC 50 Liquid concentration fifty m Multiplet M Molar

N Normal NCI National Cancer Institute max Maxima mg Milligram ml Millilitre miz The mass of the ion in dalton divided by its charge

MegCO Acetone

MeOH Methanol

MHz Mega Hertz

MNOBA Mononitro-ortho-benzoic acid MS Mass spectrometry/spectrum nm Nanometre

13 nM Nanomolar N M R Nuclear magnetic resonance nOe Nuclear Overhauser effect NOESY Two dimensional nuclear Overhauser effect spectroscopy ppm Part per million

pTLC Preparative thin-layer chromatography

ROESY Rotating frame nuclear Overhauser effect spectroscopy

s Singlet sd Standard deviation sh Shoulder

t Triplet TEA Trifluoroacetic acid

TLC Thin-layer chromatography

TMS Tetramethylsilane

UV Ultraviolet WHO World Health Organization

14 LIST OF FIGURES Page Figure 1.1 Distribution of the Indigenous population in Panama 27 Figure 1.2 Different vegetation zones in Panama 32 Figure 1.3 Natural parks and protected areas in Panama, 1985. 35 Figure 4.1 Extraction and fractionation procedure 95 Figure 4.2 Compounds isolated from Cephaëlis camponutans and the acetyl derivative of 1-hydroxibenzoisochromanquinone 103 Figure 4.3 Putative mass spectral fragmentation of

1 -hydroxybenzoisochromanquinone 106 Figure 4.4 Putative mass spectral fragmentation of benz[g]isoquinoline-5,10-dione 107 Figure 4.5 Alkaloids isolated from C. dichroa showing their possible biosynthetic relationship 116 Figure 4.6 Putative mass spectral fragmentation of vallesiachotamine lactone 118 Figure 4.7 Possible structures of the alkaloids isolated from Cephaëlis glomerulata 128 Figure 4.8 Anthraquinones isolated from P. antidesma subsp. fessonia 138 Figure 4.9 C-Glycoside isolated from P. antidesma subsp. fessonia showing the most relevant ROESY effects 139 Figure 4.10 Picramniosides B and C isolated from P. antidesma subsp. fessonia showing the most relevant ROESY effects 140 Figure 4.11 Putative mass spectra fragmentation of picramniosides A and C 142 Figure 5.1 Biosynthetic pathway of indole and isoquinoline monoterpenoid alkaloids 151 Figure 5.2 Putative biosynthetic pathways of non-iridoid alkaloids of the quinoline- and polyindolenine-type typical of the tribe Psychotriae 153 Figure 5.3 Biosynthetic relationship of the alkaloids produced in sub-genera Heteropsychotria and Psychotria 155

15 LIST OF TABLES Page Table 1.1 Health care coverage in Panama, in 1991 22 Table 1.2 Health indicators in urban and rural areas 23 in Panama, 1990 Table 1.3 Number of cases of some life threatening diseases in Panama 24 Table 1.4 Major life forms of the vegetation in Panama 29 Table 1.5 General information on the Panamanian flora 30 Table 1.6 Geographic distribution of plant species in Panama 31 Table 1.7 Plant families in Panama with more than 80species 33 Table 1.8 Advantages and disadvantages of in vivo and in viti'o bioassays 44 Table 1.9 Panamanian medicinal plants selected for chemical and biological studies 49 Table 2.1 Chemical compounds isolated from the genus Guarea 52 Table 2.2 Chemical compounds isolated from Abuta species 59 Table 2.3 Chemical compounds from Psychotria species 65 Table 2.4 Compounds isolated from species of Picramnia 68 Table 3.1 Comparison of the values obtained in the brine shrimp micro-technique with previously reported data 78 Table 3.2 Comparison of brine shrimp toxicity against in vitro KB cell cytotoxicity of compounds with different mechanisms of action 79 Table 3.3 Activities of quassinoids against brine shrimps, KB cell and P falciparum in vitro 81 Table 3.4 Activity against brine shrimp of the extracts from Guarea macropetaia 82 Table 3.5 Activity against brine shrimp of the extracts from Guarea rhopalocarpa 82 Table 3.6 Activity against brine shrimp of the extracts from Ruagea glabra 83 Table 3.7 Activity against brine shrimp of the extracts from Abuta dwyerana 83

16 Table 3.8 Activity against brine shrimp of the extracts from Cephaëlis camponutans 84 Table 3.9 Activity against brine shrimp of the extracts from Cephaëlis dichroa 84 Table 3.10 Activity against brine shrimp of the extracts from Cephaëlis dimorphatidrioides 85 Table 3.11 Activity against brine shrimp of the extracts from Cephaëlis glomerulata 85 Table 3.12 Activity against brine shrimp of the extracts from Laslanthus panamensis 86 Table 3.13 Activity against brine shrimp of the extracts from Picramnia antidesma subsp. fessonia 86 Table 3.14 Activity against brine shrimp of the extracts from Picramnia teapensis 87 Table 3.15 Activity against KB cells of extracts selected on the basis of previously activity against brine shrimp 88 Table 4.1 Plants collected in Panama 91 Table 4.2 Crude extracts for biological test 96-97 Table 4.3 N M R Spectral data of compounds isolated from C. camponutans 100 Table 4.4 N M R spectral data of the compounds isolated from C. camponutans 101 Table 4.5 Activity of the compounds isolated from P. camponutans against brine shrimps, KB cells and P. falciparum 108 Table 4.6 Comparison of the Spectral Data of Calycanthine-Type Alkaloids 126 Table 4.7 ^^C N M R spectral data of the compounds isolated from P. antidesma subsp. fessonia 134 Table 4.8 Activity of the compounds isolated from P. antidesma subsp. fessonia against KB cells 144 Table 5.1 Compounds expected and those found in the plants selected for this study 158

17 I. INTRODUCTION. 1.1 HEALTH CARE IN PANAMA. 1.1.1 The Country

The Republic of Panama is in the northern hemisphere between the coordinates 7°12’07” of latitude north, and the coordinates 77°09’24” and 83®03’07” of longitude west, in the intertropical zone next to the equator. Panama limits with the Caribbean sea in the north, eastward with the Republic of Colombia, in the south with the Pacific ocean and the Republic of Costa Rica in the west. It is isthmus that joins South America to the rest of the American continent.

The total land surfaces of the Republic is 75,517 Km^, including 1023 islands in the Caribbean sea and 495 in the Pacific side. The climate is characterized by moderately high and constant temperatures around the year (22-31 °C) with abundant rainfall during the year, averaging 1.7 m/year.

Panama is recognized for its extraordinarily rich diversity of plants and animals (see section 1.1.4) and serves as a biological bridge in the migration of species between north and south America. Moreover, Panama has been an important point in the evolution of the species, for example, there are more species of birds, mammals, reptiles and plants species in Panama than in Canada, United States and north Mexico together (Anonymous, 1993a).

There is a population of 2.3 millions inhabitants, with a density of 30.8 inhabitants/Km^ (Panama city capital 76.0 inhabitants/Km^, rest of the country 18.0 inhabitants/Km^), about 46.3 % of the Panamanian population lives in rural areas. The Panamanian population is a mixture of Indians, Africans and Spanish. In 1991, it was estimated that the 10.7 % of the population were illiterate.

19 The gross national product per capita was US $ 2,240 in 1991, while the inflation reached only 2.8% in 1991 with respect to 1987. However, according to the Ministerio de Planificacion y Politica Economica, the level of poverty of the population is estimated at 33.6 %. The percentage of unemployed in 1991 was 16.1 (19.5 % in Panama city capital, 11.4 rest of the country) (Anonymous, 1992).

1.1.2 Treatment of Diseases.

1.1.2.1 Health Care Coverage In Panama.

The health care coverage in Panama is a government priority with US $370.7 millions assigned in 1993, for health care, corresponding to 7.4 % of the gross national product. More than US $ 64.97 millions were spent on drugs, representing a drug consumption per capita of approximately US $ 26 (Anonymous, 1993b)

In the last 20 years the health care coverage has considerably increased, the country has 56 hospitals, 181 health centres and clinics and 433 health subcentres. In 1973, 68.9 % of births were assisted by physicians, whereas, in 1991 this number increased to 88.6 % (urban 99.6 %, rural area 78.6 %). Higher priority is given to the vaccination programs and, in 1991, 70 % of the children were vaccinated against polio myelitis, DPT (diphtheria, pertussis and tetanus) and BCG (Bacilli Calmette Guerini) (Anonymous, 1992).

The health sector is composed of public and private sectors; within the public sector the Ministry of Health is financed by the government and the social security is financed by employee and employer. Social security covered 48.7 % of the population, in the year 1991, while, the private sector covered only 15 % (Anonymous, 1992). Table 1.1 (p.22) shows the health care coverage in Panama, in 1991.

20 While, in Panama city, between 1979 and 1986, the total population covered by Ministry of Health and social security was 75.2 %, in Darien this number only reached 10.8 %. In Panama city, in 1991, there was an average of 10.7 physicians per 10,000 inhabitants and 5.2 beds in hospitals per 1,000 inhabitants, whilst in Darien these numbers were 4.0 and 2.7, respectively. These statistics illustrate the difference between the more and less developed areas in the country (Anonymous, 1992).

1.1.2.2 Major Health Problems In Panama.

The death rate, in 1990, was 4.1 per 1,000 inhabitants and the child death rate was 18.9 per 1,000 live birth, whereas the child birth rate was 24.8 per 1,000 inhabitants and the male expectation of life, reached 72.2 years. Table 1.2 (p.23) shows these health indicators during 1990 and compares the urban and rural areas (Anonymous, 1992).

The main causes of death in 1990 were: malignant tumours (15.5 %), accidents and other violent deaths (14.1 %), cerebrovascular diseases (10.8 %), acute myocardial failures (8.2 %), other heart diseases (5.1 %), pulmonary circulation and related diseases (5.8 %), diabetes mellitus (3.1 %), pneumonia (3.1 %) and congenital abnormalities (2.9 %). Ten percent of child deaths, between 1 and 4 years old, was caused by malnutrition and anaemia. The major causes of death in children under one year old include intestinal infections, anaemia, pneumonia and congenital abnormalities.

Panama has being threatened by many infectious diseases in recent years. Diseases such as haemorrhagic dengue and yellow fever, which are viral diseases transmitted by the mosquito Aedes aegypti. The haemorrhagic dengue is causing disasters in neighbouring countries such as Colombia and more recently in Costa Rica. In Panama, both diseases, have been effectively controlled, although the infestation of mosquitoes, in Panama city, is above the minimum levels needed to spread these diseases.

21 indicator Number

Percentage of the population covered 48.7 by the social security.

Number of health institutions

Hospitals 56

Health centres and Clinics 181

Health subcentres 433

Number of health professionals/10,000 inhabitants

Physicians 11.5

Dentists 2.2

Nurses 10.3

Beds in hospitals/1000 inhabitants 3.0

Percentage of hospital beds occupied 58.7

Total of vaccinations^ 1,351,879

Number of medical consultations^ 5,424,641

^Include Ministry of Health and social security only, in 1991. ^Source: Anonymous 1992.

Table 1.1 Health care coverage in Panama, in 1991‘

22 Health Indicator Total Urban Rural

Child birth rate/1,000 inhabitants 24.8 21.4 28.6

Death rate/1,000 inhabitants 4.1 4.1 4.0

Child death rate/1,000 live births 18.9 17.9 19.7

Maternal death rate/1,000 lives birth 0.5 0.3 0.8

Professional assistance during the child birth, in percentage 86.3 99.3 75.4

Expectation of life in years (male) 72.4 74.1 69.9

^ Source: Anonymous 1992.

Table 1.2 Health Indicators In Urban and Rural Areas in Panama, 1990\

In January, 1991, epidemic choiera emerged in Peru and was spread to northern countries such as Colombia and imported to Central American countries such as El Salvador and Nicaragua (Glass et al., 1991). The first case of cholera in Panama, was reported in September 1991 and it reached a total of 1180 cases, in the same year, including 29 deaths, this number was increased to 2,418 cases in 1992; whereas in 1993 the total was 42 with 4 deaths and the last case was reported in May of the same year (Anonymous, 1993c). This case may reflect the capacity of the health authorities in Panama with respect to the neighbouring countries, where cholera is still a major problem.

Levels of infestation of the mosquito Anopheles, the malaria transmitter, is

23 low in the urban areas in Panama. Two third of the malaria cases, in the Republic, occur in the Province of Darien and the Indigenous reservation in San

Bias, both bordering with Colombia, where malaria is an endemic disease.

Since 1984, when AIDS (Acquired Immune Deficiency Syndrome) appeared in Panama, a total of 606 cases and 352 deaths has been reported until the end of 1993. Two percent of the cases have been transmitted by blood transfusion, 3.0 % by perinatal transmission, 4.1 % by illegal drug users sharing their syringes and 83.2 % by sexual contact.

Tuberculosis, is one of the diseases growing throughout the world, and in Panama, the rate of cases seems to be constant, with about 30 cases per 100,000 inhabitants, in the last three years. The number of cases of some infectious threatening diseases is presented in Table 1.3 (p.24).

Year Diseases 1989 1990 1991 1992 1993

AIDS 77 64 70 N.A 78'

Cholera N.C. N.C. 1180 2418 42

Malaria 427 381 1115 727 300'

Tuberculosis 676 799 824 714 731'

N.A.= Data not available. N.C.= No reported cases. ^Data until October 1993. ^Source: Anonymous 1992. and Anonymous 1993c.

Table 1.3 Number of Cases of Some Life Threatening Diseases in Panama^

24 1.1.3 Ethnomedicine in Panama.

In Panama, traditional healers are prohibited by law to practice their healing arts. They are, nevertheless, active in both rural and urban areas and the Government has no plans to include folk healers in health services. Traditional medicine is more frequently used by the poor section of the Amerindian population, and the rural populace and some herbs are sold regularly in the public market and in the drug stores. Despite the World Health Organization policy urging countries to utilize their traditional system of medicine in order to achieve the policy of health for all by the year 2000 (Akerele, 1991) there is no general policy or guidance on the use of traditional medicine, the cultivation, sale or use of medicinal plants.

Today in Panama there are five Amerindian groups (Torres de Arauz 1980):

1.- Kuna live in the Archipelago of San Bias, and around Bayano, Chucunaque and Tuira rivers. They are some 54,000 Amerindians (Guionneau-Sinclair, 1989) who practice horticulture, fishing, hunting, and collecting fruits, wild leaves and medicinal plants. 2.- Embera and Waunana are descendants of Colombian Chocoes and they are concentrated around the borders of Darien Rivers, with a total of 17,264 Amerindians. 3.- Ngawbes or Guaymi proper is the most important group settled in the Provinces of Chiriqui, Bocas del Toro and Veraguas. This is the most numerous group with a total of 123,626 and they are the poorest indigenous group. 4.- Bokotas live in the Provinces of Bocas del Toro and Veraguas, with only 3,784 Indians. 5.- Teribes live on the banks of Rio Teribe and San Durvi in the Province of Bocas del Toro and in the Costarican side. In Panama there are 2,194 Indians.

The total population of the five Amerindian groups is approximately 9% of

25 the total population of the country. Although the Government of Panama has made efforts to provide health care through health centres, health posts and hospitals, these ethnic groups continue to use a variety of medicinal plants for curing their ailments. Figure 1.1 (p. 27) shows the distribution of the different indigenous groups in Panama.

Ethnobotanical and ethno-ecological studies have been done with the human groups in eastern Panama, especially the Kuna and Embera Indians, as part of the sea level canal studies (Duke, 1967; Duke, 1968; Duke, 1972; Torres de Arauz, 1972). Folk herbal remedies of the Afro-AntiMeans in Colon have been reported (Angermuller, 1968). Similarly, there have been ethnobotanical and phytochemical evaluations of other human groups in Panama, especially mestizo groups of the central provinces (Gupta et al., 1979; Joly, 1981 ; Gupta et al., 1986).

More recently, two major ethnobotanical inventories have been done with the Guaymi group in western Panama (Joly et al.,1987; Joly et al., 1990) and the Kuna Indians in San Bias, eastern Panama (Gupta et al., 1993a), using the questionnaire recommended by World Health Organization.

The ethnobotanical inventory with the Guaymi (Joly et al.,1987; Joly et al., 1990) was carried out in collaboration with four "curanderos" (witch doctors). The "curanderos" guided the daily collection trips. Interviews were taken in the Guaymi language and translated into Spanish. A total of 104 plants was identified and their uses and preparation were reported.

In the ethnobotanical study of the Kuna Indians (Gupta et.al., 1993a), 90 species were reported. In this study 54.4% of the plants were used topically, 26.7% used internally and 18.9% were used externally and internally. The common illnesses for which these medicinal plants are used, include muscle and joint aches, fever, skin infections, eye infections, asthma, wound healing, snakebite, childbirth, acne, colds and general weakness.

26 KUNA GUAYMI

EMBERA BOKOTAS

TERIBES

Figure 1.1 Distribution of the Indigenous Population in Panama.

27 1.1.4 Natural Resources and Conservation.

The Flora of Panama is one of the richest in the world (Schultes 1972), as plants from North and South America can be found there. About 750 botanists or naturalists have collected plants in Panama for herbarium collections. According to Dwyer (1964; 1985), there are about one half million specimens, excluding duplicate materials, of Panamanian plants in herbaria throughout the world. The Missouri Botanical Garden has the largest collection. There have been three distinct periods of plant collection in Panama: 1700-1914, 1915 to 1957 and 1958-1981. During the first period, Europeans dominated the scene. Berthold Seemann wrote the first flora of Panama and Henry Pittier’s collections are noteworthy (Dwyer, 1985). During the second period, Paul Standley made a significant contribution and wrote Flora of Barro Colorado Island (Standley 1927) and Flora of the Canal Zone (Standley 1928). During the second and third period the Missouri Botanical Garden has been the most active. Although publication of the Flora of Panama has been completed, new species are still being found and many areas remain to be explored. The Flora of Panama recognized 5314 species and a total of 5597 taxa of species rank or below, by 1987 this number was increased to 7345 species and included 195 families (D'Arcy, 1987).

The total number of vascular species in Panama is estimated between 8,000 and 10,000 species, of which 17.3% are endemic. There are also large numbers of non vascular species in Panama, but they have not been investigated fully. Flowering plant-richness and biodiversity of Panama ranks fourth in North and Central America (World Conservation Monitoring Centre, 1992). In Panama there are over 891 species of ferns alone and about 1000 species of orchids, of which 10% are endemic. The epiphytes, the climbers, and lianas are a major component of the Panamanian tropical forest. Mosses abound in moist forests as well as other parts of Panama. Mangroves are characteristic plant formations of tropical seacoasts. Tables 1.4 - 1 .7 (p.29, 30, 31, 33) provide general information on the Panamanian Flora including major

28 life forms, major plant families and geographic distribution. Figure 1 . 2 (p.32) shows the different vegetation zones in Panama.

The Isthmus of Panama constitutes the narrowest territorial portion of the American continent where North and South America join. Panama, situated in biogeographic regions of the Neotropics, plays an important role as a biological bridge between the northern and southern lobes of the Continent. Indeed, three of the four principal routes of bird migration converge in its territories (Cobos-Moran, 1992).

Number Habit Percentage^

895 Climber 12.3

195 Epiphyte 2.7

2 Hemiepiphyte 0.03

3,126 Herb 42.9

1 1 Parasite 0 . 2

1,952 Tree 26.8

53 Treelet 0.7

56 Without Habit (not available from present data)

'Taken from D’Arcy, (1987).

^Do not total to 1 0 0 % because of multiple categories

Table 1.4 Major life forms of the vegetation In Panama'.

29 NUMBER OF Percentage" SPECIES"

Monocotyledons 2,246 27.6

Dicotyledons 5,099 62.6

Total of native species 7,123 87.4

Endemic species 1,230 17.3

Introduced species 2 2 2 2.7

Species of flowering plants 7,345 90.2

Pteridophytes species 800 9.8

Total number of vascular plants 8,145 1 0 0

^Taken from D’Arcy, (1987). Arcy, W. G. (1987), "Since 1981 over 330 species of flowering plants have been newly described from material collected in Panama, and no diminution of this rate of discovery has been seen to date. Thus, the total flora of Panama may include 8,500-9000 flowering plants and 900 pteridophytes. These numbers will decrease as habitats are eliminated from Panama and species are extirpated or extinguished".

t o not total 1 0 0 % because of multiple categories.

Table 1.5 General Information on the Panamanian flora\

30 Region Number of Species Percentage^

Canal Area 2,545 (35) Chiriqui 3,057 (42) Panama 3,246 (44) Western Panama 3,921 (53) (Bocas del Toro, Chiriqui) Central Provinces 5,146 (70) (Canal Area, Codé, Colon, Herrera, Los Santos, Panama, Veraguas)

Azuero Peninsula 801 (1 1 ) (Herrera, Los Santos) Eastern Panama 2,563 (35) (Darien, San Bias)

All Panama 7,345 (1 0 0 )

^Taken from D’Arcy (1987).

^Do not total 1 0 0 % because plant species had been collected in different regions.

Table 1.6 Geographic distribution of plant species in Panama^

31 Premonlane ram forest em u Dry tropical forest Humid low montane forest I I Humid tropical forest Very tiumid low montane forest im cl Very fiumid tropical forest Low montane rain forest i I Dry premontane torest Very tiumid montane forest S B Humid premontane forest I — I Montane rain forest [»ea Very tiumid premontane forest

1. 2 ,000,000

Figure 1.2 Different Vegetation Zones In Panama.

32 FAMILY NATIVE SPECIES ENDEMIC SPECIES^

MONOCOTYLEDONS ORCHIDACEAE 891 113

GRAMINEAE 344 1 1 ARACEAE 234 95

CYPERACEAE 164 1

BROMELIACEAE 136 1 2 PALMAE 95 26 SUBTOTAL 1,864 (85%)' 258 (85%)'

DICOTYLEDONS RUBIACEAE 431 194 LEGUMINOSAE 430 35 COMPOSITAE 272 42 MELASTOMATACEAE 244 43 PIPERACEAE 213 80 GESNERIACEAE 159 63 SOLANACEAE 138 27 MYRSINACEAE 134 95 EUPHORBIACEAE 134 7

ACANTHACEAE 1 1 0 15 MORACEAE 96 4 SAPINDACEAE 85 23 VERBENAGEAE 81 5 ERICACEAE 81 41 BIGNONIACEAE 80 4

SUBTOTAL 2 , 6 8 8 (53%)' 678 (69%)' TOTAL IN THIS GROUP 4,552 (64%)' 936 (72%)'

^Taken from D’Arcy, (1987). ^The number of endemic species is higher than that reported in the Flora of Panama because of the inclusion of 60 varieties. ^The figures in parenthesis represent the percentage with respect to the total number of species in the Flora of Panama.

Table 1.7 Plant families In Panama with more than 80 species^

33 Panama] comprises a terrestrial bridge of extreme biological importance. Presently, throughout the Isthmus, there is an amalgam of biotas of the North and of the South. A consequence of this mixing is a vast plant and animal diversity. This diversity is supported by a unique collection of environmental factors, which influence and allow the coexistence of so many species (Cobos- Moran, 1993). Therefore, its integrity as a biological bridge is necessary for the continued existence and evolution of the species of this important region of the planet.

In Panama, in 1947, 70% of the territory, excluding the Panama Canal Area, was covered by forests. In 1980, primary forests covered a land area of 3,549,700 hectares. More recently, the National Institute of Renewable Natural Resources of Panama (INRENARE) estimated that this number in 1991 was reduced to only 40% and it is estimated that by the year 2000 this percentage will be reduced to 33%. The annual rate of forest loss is estimated at 65,000 hectares (Cobos-Moran, 1992). Therefore, it is vital to study this flora before the species become extinct.

There are two major institutions responsible for conservation in Panama; the National Institute of Renewable Resources (INRENARE) on the Panamanian Government behalf and a private group called Ancon. INRENARE and Ancon are managing fourteen national parks that cover a land surface of 1,389,463 hectares that represent 18.4 % of the national territory; seven forest reserves; six wild life refugees and seven other minor categories of management. The total land surfaces under conservation by law reach 27.5 % (2,077,914 hectares) of the total surface of the country. This number does not include the indigenous reserves that are conservationist by nature; noteworthy are the Kuna Indians recognized by the WWF (World Wild Foundation) as an example of how mankind can live in harmony with the wild life. Figure 1.3 (p.35) shows the protected areas in Panama during the year 1985, whilst Figure 1 . 2 (p.32) shows the distribution of the indigenous population.

34 I National park or wild life refuge by law

! Forest reserve by law

National park or wild life refuge proposed ^ ~^Poflobalo

I ( g o G alun f . } P N PN Ls Amisied PN >barsnla

",P N /-' Main

Palo Seco - R V S kilo* da Campana' Isia Taboga Voicàn Barù lOmar Torrijo^ PN Islade Lae Perlae

la Veguada

Chorogo.. —

A N R - Natural area for recreation B P - Protector forest P.N. - National park P.N.R.- Recreative national park

R.F. - Forestal reserve PN El M oniui R.V.S - Wild life refuge

I R V S 'Ida Iguana

^ T io n o s a

P N > l: 2,000,000 CarroHoyaV

Figure 1.3 Natural Parks and Protected Areas In Panama, 1985.

35 1.2 Selection of Plants for Chemical and Biological Investigation.

Some of the most important drugs of the past 50 years or so, which have revolutionized modern medical practices, have first been isolated from plants, and often from plants that for one purpose or another have been employed in primitive or ancient society (Schultes, 1986). These drugs include, vinca alkaloids used to treat leukaemia, quinine that is effective against malaria and served as prototype in the development of synthetic antimalarial drugs, podophyllotoxin in the development of synthetic anticancer drugs, morphine alkaloids used as analgesic and antitussive drugs and in the study of the biochemistry of pain, digoxin and other cardioactive glycosides, to mention some examples. More recently, taxol, another drug obtained from plants is used in the treatment of colon and breast cancer. Furthermore, in 1973, in United State 25.2 % of the prescriptions contained one or more active constituents obtained from higher plants, including 76 different chemical compounds of known structure derived from higher plants (Farnsworth and Bingel, 1977). Therefore plants should be considered as a primary source of drugs to treat different diseases, of chemical templates to build up more effective drugs and of compounds which may be used as pharmacological tools to give a better understanding of biological processes.

There are three principal approaches used to select plants for chemical and biological investigations, namely, the use of plants in traditional medicine, random selection of plants for massive studies and the chemical relationship of the plant species, chemotaxonomy.

1.2.1 Traditional Medicine.

Plants have been used by man kind since early times and it seems that Neanderthals used plants as medicine (Solecki, 1975). Also, a large number of plants have been used for more than 3,000 years in the Chinese traditional medicine. Ayurvedic medicine and Unami medicine (Farnsworth and Soejarto,

36 1991).

The World Health Organization (Penso, 1983) has attempted to identify all plants used in medicine around the world. More than 20,000 species have been listed providing the latin name of the plants and the country where the plants were used. More recently, a data base, named NAPRALERT, has been established at the University of Illinois, where ca of 9,200 species have been documented (Farnsworth, 1983; Farnsworth and Soejarto, 1991). This data base includes the scientific name of the plants, synonyms, the use, how they are used including the dose in some cases, vernacular names, country where the species are used and the reference where the statement was published.

It is estimated that 74% of the 121 biologically active plant-derived compounds presently in use worldwide, have been discovered by studies that based the plant selection on ethnomedical information (Farnsworth et ai 1985). There is a great deal of information regarding the medicinal use of plants and the reliability of the information should be taken in account to select a plant for chemical investigations. How this information was collected, who is the informant and if a particular species or genus is used for different population groups to treat a particular disease. Also important is the interpretation of the data, as usually a symptom is reported rather than a disease; for example, the term intermittent fever may suggest malaria whereas the term backache could suggest either renal disease or muscular distention.

The way in which the information is collected is crucial in order to have valuable information. Some foreigner researchers go to live with aborigine populations, to gain their confidence and a few months later a paper is submitted to a scientific or medical journal. Most aborigines are reluctant to give information and although, sometimes, they are paid for it, the information lacks value because it is not possible to buy their confidence. In the last two ethnobotanical inventories in Panama (Joly et al., 1987; Joly et al., 1990; Gupta et al., 1993a) a descendant from the community to be studied was chosen

37 among the final year students at the University of Panama, to overcome the confidentiality task and the language limitations. Many differences were found between the work carried out by Duke (1975) and that more recently reported by Gupta (1993a) with the Kuna Indians. Similarly, Joly (1990) found some differences between her work and that from Hazlett (1986) with the Guaymi Indians.

The information on medicinal plants may be obtained from a variety of forms. Among the firsts to write about healing properties of plants were the Chinese and Ayurvedic "doctors", which received long training before they are allowed to prescribe. Then, there are the Shaman in Peru and Sukias and Inadulet in Panama, who received some training before they were allowed to prescribe. Also there is information on those practitioners that receive little or no instruction and the knowledge which they receive has been handed down from the father to the son.

1.2.2 Random Selection of Plant for Chemical Investigation.

Perhaps, the random selection approach is the most expensive and unscientific way to select plants for chemical and biological evaluations. The limitations of random selection of plants have been described (Spjut, 1985). Nevertheless, some promising antitumours agents have been discovered through this approach under the program of the National Cancer Institute. This program, started to screen plant extracts in 1960, twenty years later, in 1980, 114,045 extracts had been screened and 4.3% of the extracts showed activity. This massive program yielded seven compounds, which were in clinical trial by 1980 (Suffness and Douros, 1982).

These compounds included bruceantin and maytansine that showed weak activity and they were not developed further (Suffness and Douros, 1982). Although, indicine N-oxide, in phase II of the clinical trial, showed activity in acute leukaemia; it is ineffective in the treatment of osteosarcoma.

38 neuroblastoma and paedriatic brain tumours, and hepatotoxicity has been shown at therapeutic doses (Miser et al., 1992; Miser et al., 1991). Homoharringtonine, a cephalotaxine alkaloid, is safe and effective for patients with acute myelogenous leukaemia (Feldman et al., 1992). Phyllantoside, isolated in 1977 (Kupchan, et al., 1977) is active against the B16 melanoma. 4p-Hydroxywithanolide E with an a oriented side chain at C-17 was undergoing formulation studies for toxicology (Suffness and Douros, 1982). Finally, taxol isolated from Taxus brevifolia (Wani, et al. 1971 ) has remarkable anti neoplastic qualities against ovarian cancer, melanoma and colon cancer (Edington, 1991 ).

1.2.3 Chemotaxonomy

Secondary metabolites present in plant cells represent not only a chemical entity, but are the manifestation of a whole series of enzymes, which in turn are the genetic expression. Hence, chemical characters could show the relationship between plant individuals and their evolution. Since the presence of secondary metabolites in plants could predict the relationship between plant species, this approach could be used to select plants for chemical and biological investigations. It is mainly useful to find new or more rich sources of a particular compound, or to find more active or selective structural analogues.

The foxglove (Digitalis purpurea) was introduced into modern medicine on the basis of its use as heart stimulant in folk medicine, but the active principle, digitoxin, has short latent periods of action and low cumulative effects. Synthetic variants of digitoxin have proved not to be as effective as natural variants of the drug in related species of Digitalis. Thus, D. lanata, a species native to southeastern Europe, was found to contain three to five fold greater concentration of active principles than the foxglove (Hansel, 1972) and one of the active principles, digoxin, has relatively better pharmacokinetics properties than digitoxin.

The first report of the relationship between chemical components and races

39 of plant was written in 1673 by Nehemiah Grew, who used medicinal plants in his examples. The second, James Petiver, an eminent London apothecary, who wrote, in 1699, "some attempts made to prove that herbs of the same make or class for the generality, have the like virtue and tendency to work the same effects" (Gibbs, 1963).

However, it was not until the beginning of this century when Gresshoff, in 1909, demanded that every accurate description of a genus or of a new species should be accompanied by short chemical description of the plant (Gibbs, 1963). Later, McNair attempted to apply comparative chemistry generally to taxonomy utilizing the fats and oils content of the plants (McNair, 1929). Also, McNair, paid attention to other groups of compounds including the alkaloids of aconite (McNair, 1935). There have been a number of treatises on chemotaxonomy including Manske and Holmes (1950-1958), Hutchinson (1959) and Hegnauer (1962-1994). The chemotaxonomy of the Papaveraceae (Santavy, 1970) and Leguminosae (Harborne, etal., 1971) have been reviewed.

Modern techniques for the isolation of natural products, such as chromatography (TLC, CC, HPLC, GC, DCCC, etc.), and physical methods for structure elucidation, such as mass spectrometry (EIMS, FABMS, CIMS, FDMS, etc.) and nuclear magnetic resonance (^H, 2D experiments such as COSY 45, HMQC, HMBO, NOESY, etc.) now make it possible to fully characterize small amounts of compounds present in plants. In addition, the readily available literature on natural products allows for correlations to be made between the chemical composition and evolution within the plant kingdom.

Plants are widely used in Panama and Central America to cure different diseases, including malaria and amoebic dysentery (Morton, 1981 ; Joly et al., 1987; Joly et al., 1990; Gupta et al., 1986). Species of Simaroubaceae, Meliaceae and Menispermaceae are known to contain quassinoids, limonoids and bisbenzylisoquinoline (bbiq) alkaloids, respectively, with activity against

40 Plasmodium falciparum. In addition, Cephaëlis ipecacuanha is known to contain emetine, which is used in the treatment of severe amoebic dysentery (Tyler et al., 1988). Interest in antiprotozoal natural products and the possibility of obtaining quassinoids, limonoids, bbiq’s and emetine related alkaloids led to the decision to investigate the plants listed in Table 1.9 (p.49).

1.2.3.1 Chemotaxonomy background

Taxonomy is a study aimed at producing a system of classification of organisms, which best reflects the totality of their similarities and differences (Cronquist, 1968) and chemotaxonomy incorporates the principles and procedures involved in the use of chemical evidence for classification purposes. Features used in a classification are termed taxonomic characters. All sources of taxonomic evidence are scanned in the search for taxonomic characters and among the richest have been the fields of morphology, anatomy, cytology, ecology and genetic. The use of chemical data as taxonomic characters marks a recent extension of the range of recognized sources of taxonomic evidence (Smith, 1976).

Taxonomic characters should be easy to assay and the unambiguity of modern chemical analytical procedures enables accurate identification of individual compounds. Also, the taxonomic characters, should be consistently present in a taxon, bearing in mind the nature of natural grouping, which allows exceptions to any generalization. To affirm that a characteristic is consistently present in a taxon implies that it is to be expected in a high proportion of its members. In addition, a taxonomic character, should not be affected by environmental factors, unless the taxonomist is equipped to detect the property of variability itself and use it taxonomically (Heslop-Harrison, 1963). The chemotaxonomic character, in turn, should be easily detected or identifiable, with limited distribution i.e. within a genus of a family, the biosynthetic pathway should be fully understood and its presence should not be seasonal or climatic- condition dependant.

41 1.3 Biological Testing of Plant Extracts.

Unfortunately, there was, and still is, little communication between phytochemists and pharmacologists. New compounds are isolated from plants and their structures elucidated, but they lie dormant on the phytochemist’s shelf. Usually, minute quantities of the compounds are isolated and then is not enough for biological activity testing (McLaughlin, 1991). Pharmacologists, on the other hand, are reluctant to test tarry plant extracts.

Isolation of active compounds from plants by means of bioactivity guided fractionation are time consuming and expensive. Nevertheless, it is the way to obtain meaningful and significant results. Using a bioactivity-guided fractionation in the isolation of a natural product not always ends with a novel compound, but, perhaps, with known compounds with a novel application in medicine.

Many scheme fractionations have been proposed to follow up the activity of plant extracts ( e.g. Ferrigni et al., 1984; Samuelsson et al., 1985; O’Neill et al., 1987). Although, in traditional medicine most of the remedies are prepared by extraction with water, either by boiling the plant parts or by soaking them in cold water, most of the researchers use polar or moderately polar organic solvents. Aqueous extracts tend to be avoided due to their complexity and difficulties in developing suitable work up procedures (Samuelsson et al., 1985). Also, strategies for pharmacological evaluations of crude drugs prescribed in traditional medicine have been reported (e.g. Kyerematen and Ogunlana, 1987). However, the most important is to biologically test all the fractions at each step and all the isolated compounds should be tested in different bioassays. Therefore, the assays designed to guide the fractionation should be simple, rapid, reliable and inexpensive.

The bioassay should be highly sensitive because most natural products are present in the crude extract at dilutions of 1 :1 , 0 0 0 or more even up to

42 1:1,000,000. It is highly likely that in vivo screening alone is going to miss compounds that may be quite active but are not potent enough of not concentrated enough in a crude extract for detection. That is one of the main reason why in vitro methods are preferred to in vivo screens (Suffness and Douros, 1982). However, the bioassay used in screening crude plant extracts must be insensitive to the many compounds or ubiquitous compounds, which give false actives. The National Cancer Institute, for example, dropped the Walker 256 screen from use in the plant programme because it was highly sensitivity to tannins (Suffness and Douros, 1982). Apart from the general requirements of any good assay, such as validity, predictability, correlation, reproducibility and reasonableness of cost, the following considerations should be taken in account in bioassays used to screen plant crude extracts (Suffness and Douros, 1982):

1 .- Selectivity: The assay must be selective enough to limit the number of false positives.

2 .- Sensitivity: The assay must be very sensitive in order to detect active compounds in low concentrations. 3.- Methodology: The assay must be adaptable to materials that are highly coloured, tarry, poorly soluble in water and chemically complex.

1.3.1 Advantages and Disadvantages of in vivo and in vitro Testing.

Table 1 . 8 (p.44) summarize the advantages and disadvantages of the in vitro and in vivo bioassays. Research with experimental animals does not provide the only major route to advances in biological understanding and delivery of medical benefits, but it does provide an approach that is very important, such as pharmacokinetic and bioavailability, and one that is likely to remain so for the foreseeable future. Animal usage might become, progressively, superseded in the preparation of antibody-like molecules.

43 however they have to be made by immunisation of animals (Rees, 1992) and in the search of drugs targeting specific peptide receptors such as analgesic drugs acting on bradykinin receptors, but still isolated from rat uterus (Snell and Snell, 1989).

Advantages Disadvantages

In vivo Activity data Long turn over Expensive Often less sensitive Relatively large sample needed

In vitro Speed In vitro data only Less costly Activity may not Sensitivity correspond to in vivo Small sample size activity.

Table 1.8 Advantages and Disadvantages of in vivo and In vitro Bloassays.

A criticism often repeated by opponents of animal experimentation is that "animal and human diseases are rarely, if ever, identical. Using an animal model that is not identical to human disease is a logically flawed process" (Anonymous, 1991 ). There are metabolic and toxicological differences between mouse and man and the data from mice has been found misleading, specially in cytotoxic anticancer drugs. It is now considered desirable to carry out such studies as soon as possible in human cancer patients following the minimum of animal studies to ensure some degree of relative efficacy/safety of the drugs for man (Parke, 1983).

44 Similar anomalies in rodent studies of the carcinogenicity of chlorinated hydrocarbon pesticides led the Joint Meeting of FAQ and WHO on Pesticides Residues to reject carcinogenicity tests in the mouse as being predictive of potential hazard to man (Anonymous, 1981 ). The lack of relevance of the long­ term animal carcinogenicity to man and the need for more rapid and less expensive assays has led to the development, and acceptance by regulatory authorities, of a number of short-term in vitro tests (Parke, 1983). Among these, the Ames test (Ames et al., 1975) is the most popular and successful and has been adopted by the Committee on Safety of Medicine as an alternative to in vivo carcinogenicity studies in experimental animals (Parke, 1983).

In vitro assays may require the presence of mammalian metabolic activation preparations to reproduce some of the aspects of whole animal metabolism of foreign compounds. The procedures for such activation mixes with mammalian ceils are far from optimal and their presence often leads to cellular toxicity (Scott, 1982).

1.3.2 Simple Bloassays.

1.3.2.1 Brine Shrimp Assay.

Artemia salina Leach (brine shrimp) is a small shrimp commercially used as a food for tropical fish and it has been used at different life stages in experimental researches. Brine shrimp has been used in the analysis of pesticides residues (Tarpley, 1958), mycotoxins (Reiss, 1972; Tanaka et al., 1982; Hoke et al., 1987), metals (McRae and Pandey, 1991), protein biosynthesis inhibitor (Robyn and irvin, 1980), cocarcinogenic phorboi esters (Kinghorn et ai., 1976), anaesthetics (Robinson et ai., 1965) and dinofiagellate toxins (Granade et al., 1976).

45 Also brine shrimp has been used to guide fractionation and isolation of mycotoxins (Eppley and Bailey, 1973), plant neurotoxins (Greig et al., 1980) and antibiotics (Hamill et al., 1969). It has been used in field evaluation of traditional medicine (Trotter et al., 1983; Beioz, 1992) and placed in the second level of evaluation of traditional materia medica (Kyerematen and Ogunlana, 1987). Furthermore, Artemia salina has been subjected extensively to comparative biochemical and anatomical studies ( McRae et al., 1989; Warner et al., 1988).

In 1982 a simple method, using brine shrimp, was developed for screening and fractionation of active materials from higher plants (Meyer et al., 1982) and demonstrations of the use of brine shrimp bioassay in the isolation of bioactive natural products have been reviewed (McLaughlin et al., 1993). The use of potato disc and brine shrimp bioassays to detect activity and isolate antileukaemic natural products have been reported (Ferrigni et al., 1984). In a blind comparison of brine shrimp and human tumour cell cytotoxicities as antitumour prescreens, brine shrimp prove to be superior or equally accurate as the in vitro human solid tumour cell lines (Anderson et al., 1991a). A convenient bioassay to detect antiparasitic avermectin analogues has been reported (Blizzard et al., 1989).

As the brine shrimp test (Meyer et al., 1982) requires relatively large quantities of material (20 mg for crude extracts and 4 mg for pure compounds) and the preparation of dilutions is time-consuming thus limiting the number of samples and dilutions that can be tested in one experiment, it will be attempted to develop a microdilution technique to overcome the above disadvantages.

1.3.2.2 KB Cells Assay.

Since 1960 the in vitro KB assay has been used by the National Cancer Institute (NCI) as a preliminary screen for cytotoxicity and for fractionating plant samples before carrying out assays for in vivo activity. This in vitro system is

46 an excellent bioassay but it is a poor screen because of the sensitivity of the cells to cytotoxic substances that are devoid of in vivo activity (Suffness and Douros, 1982).

The KB cell in vitro test was initially described by Eagle in 1955 and Oyama and Eagle in 1956. The assay has since been standardised by the NCI (Geran and Greenberg, 1972) and later modified by Wall et al. (1987). More recently, a microdilution technique was developed for the assessment of in vitro cytotoxicity against KB cells derived from a human epidermoid carcinoma of the nasopharynx (Anderson et al., 1991b).

1.3.2.3 Plasmodium falciparum Assay.

Malaria in humans is caused by four species of protozoal parasites of the genus Plasmodium (P. falciparum, P. malariae, P. ovale and P. vivax). It is characterized by fever and other symptoms at the time when the merozoites are released from ruptured red cells so that intermittent fever is produced. Anaemia occurs due to haemolysis and other factors. P. falciparum infection is particularly dangerous as cerebral malaria may occur (Cattani, 1993).

The prophylaxis and treatment of malaria have become increasingly complex and difficult because of the widespread resistance of P. falciparum to drugs (Payne, 1987). Multidrug resistance to antimalarials, such as chloroquine and quinine, and adverse effects of drugs, such as pyrimethamine-sulphadoxine combination, have severely limited available therapy (Peters, 1985). New drugs are urgently needed as resistance has occurred to the more recently introduced mefloquine into clinical use. Resistance has been produced in the laboratory against artemisinin and other antimalarials under development, so that new drugs such as halofanthrine (Horton, 1988) may not have a long-term future unless their use is strictly controlled (Warhurst, 1985).

47 The antiplasmodial in vitro test described by O’Neill et al (1985), with modifications described by Ekong et al (1990), is based on the method of Desjardins et al (1979). Cultures of P. falciparum are maintained in vitro in human erythrocytes by a method described by Trager and Jensen (1976) and later modified by Fairlamb et al. (1985). The technique measures the incorporation of ^H-hypoxanthine into drug-treated infected red blood cells compared to untreated infected red blood cells. The in vitro testing of plants extracts has been reviewed (Phillipson et al., 1993, Phillipson et al., 1994). More recently, a colorimetric assay has been described measuring the parasite lactate dehydrogenase activity, which is distinguishable from the host lactate dehydrogenase activity using the 3-acetyl pyridine adenine dinucleotide analogues of nicotinamide adenine dinucleotide (Makler et al., 1993).

48 1.4 Aims of this Study.

The aims of this study are:

1 .- To investigate a small selected number of Panamanian plants. The chosen plants are showed in Table 1.9 (p.49). 2.- To use bioassay guided fractionation procedures. 3.- To develop a brine shrimp microwell assay

4 .- To isolate and identify known compounds as well as to characterise and to determine the chemical structures of novel compounds.

Plant Family Plant species

Meliaceae Guarea macropetala Pennington Guarea rhopalocarpa Radlkofer Ruagea glabra Triana & Planchon

Menispermaceae Abuta dwyerana Kruk. & Barneby

Rubiaceae Cephaëlis camponutans Dwyer & Hayden Cephaëlis dichroa (Standley) Standley Cephaëlis dimorphandrioides Dwyer Cephaëlis glomerulata J. Donnel Smith Lasianthus panamensis Dwyer

Simaroubaceae Picramnia antidesma subsp. fessonia (DC) W.Thomas Picramnia teapensis Tul

Tabie 1.9 Panamanian Medicinai Riants Seiected for Chemical and Biological Studies.

49 Section 2. Plants Selected for Investigation 2.1 Meliaceous Plants.

jpredominantly A tropical and subtropical family/of the Old World, the Meliaceae comprises 50 genera and more than 1000 species of herbs, shrubs and trees. It has been divided into five subfamilies, based on characters of the stamens and seeds (Schultes and Raffauf, 1990).

The family Meliaceae is known to contains limonoids (Connolly, 1983), tetranortriterpenoids (Banerji and Nigam, 1984) and the chemistry of the family as a whole has been reviewed (Taylor, 1983). Limonoids are a group of oxidized triterpenes closely related to the quassinoids which occur in species of Simaroubaceae (Connolly, 1983; Taylor, 1983; Banerji and Nigam, 1984) and several showed moderate antimalarial activity, in vitro (Bray et al., 1990). The most active of these, gedunin, had activity around three times higher than that of chloroquine (Khalid et al., 1986; Bray et al., 1990).

The genus Guarea has 150 species of trees and shrubs in tropical America

and 20 in Africa (Schultes and Raffauf, 1990); 8 of them occur in Panama

(D’Arcy, 1987). Table 2 . 1 (p.52) shows the species of Guarea that have been investigated chemically.

Biological activity for extracts of Guarea species: A water extract of the leaves of G. guidonia showed no activity on guinea pig atrium (Carbajal et al., 1991), whereas, an ethanolic extract of the seed showed antiinflammatory activity in rats (Oga et al., 1981). Different extracts and fractions of the fruits, stem bark and stem wood of G. multiflora showed no activity against Plasmodium falciparum, in vitro (Bray et al., 1990). A fluid extract from the bark of G. rusbyi was active against Mycobacterium tuberculosis, in vitro (Fitzpatrick, 1954). The ether extract of the flower of G. sepium showed activity against various species of helminths including Strongiloides stercoralis, Ancylostoma caninum and A duodenale (Gilbert et al., 1972). The aqueous

extract of the bark of G. thompsonii showed a LC5 0 150 mg/Kg when

51 Plant Species Compounds Isolated References Guarea carinata Diterpenes: h0xadec-2-en-1-ol,3-7-11-15 tetramethyl palmitate, manoyl Pereira et al., 1990 oxide, 13-epi-manoyl oxide, phytol palmitate. Guarea cedrata Triterpenes: 2-OH-rohitukin, 3-4-seco-tirucalla-4(28)-7-4-triene-3-21 - Akinniyi et la., 1980 dioic acid, 3-methyl ester,3-4-seco tirucalla-4{28)-7-24-triene-3-

2 1 -dioic acid.

Guarea glabra Triterpenes: glabretal, glabretal angelate, glabretal did, glabretal Ferguson et al., 1975 methacrylate, glabretal tiglate, glabretal-2-OH-3-methyl butyrate, glabretal-3-one. Steroids: p sitosterol. Guarea kunthiana Triterpenes: euadorin Mootoo et al., 1992 Guarea thompsonii Triterpenes: drageanin Connolly et al., 1976 limonoid B Pettit et al., 1983 Guarea trichilioides Triterpenes: angustinolide Zelnik and Rosito, 1971 25-OH-cycloart-23-en-3-one, cycloart-23-ene-3-25-diol, 3p- Furlan et al., 1993 cycloart-24(31 )-25(26)-diene, 3P-OH-21 -22-23- tetrahydroxycycloart-24-en-24-one, 23-OH-cycloart-24-en-3-one, epi-23-OH-cycloart-24-en-3-one, cycloart-24-ene-3-23-dione, 3p-21-dihydroxy cycloartane, 22(R)OH-cycloartane, 7-oxo- genudin prieurianin, 15-15P epoxy prieurianin Lukacova et al., 1982 Steroids: p-sitosterol Zelnik and Rosito, 1971

Table 2.1 Chemical Compounds Isolated from the Genus Guarea.

52 administered intraperitoneaiiy in rats (Sandberg and Cronlund, 1977). An aqueous extract of the fruits of G. trichilioides Vi/as inactive against Plasmodium gallinaceum, in chicken (Spencer et al., 1947) and the chloroformic extract of the root bark showed an ED 50 0.29 pg/ml against LEUK-P388 cell culture (Lukacova et al., 1982).

Guarea macropetala Pennington: There is no previous chemical or biological study of this plant. It is a tree of wet evergreen lowland and lower montane forest, known only from scattered collections in Panama (Pennington et al., 1981).

Botanical description: "Rami noveili graciies, aureo-tomentosi usque villose, tandem pallide cinereo-aibi, glabri, interdum lenticellati, non suberosi. Folia pinnata, usque ad 50 cm ionga, gemmula terminali incremento intermittente; petioius semiteres; rachis teres vel quadranguiaris, primo aureo-

tomentosa; petioiulus 3-6 mm. Foliola usque ad 8 -juga, late obionga vel oblanceolata, apice obtuse cuspidate, attenuate vel acuminata, basi breviter et anguste attenuate, chartacea, 14.5-22.2 [17.4] cm Ionga, 6-9 [7] cm lata; costa supera et nervi secundarii dense pubescentes, lamina glabra sed crebris punctulis exstantibus conspersa; costa infera nervique dense et grosse pubescentes, lamina sparse pubescens usque glabra, nec glanduloso-punctata nec-striata; venatio eucamptodroma, costa impressa; nervi secundarii 11-14 utroque costae latere, adscendentes, arcuati, paralleli, intersecundarii nulli tertiarii oblique, spissi, paralleli. Inflorescentia cauligena, solitaria vel geminate, 5.5-21 cm Ionga, in racemum vel paniculam gracilem ramulos latérales prope basin

gerentem disposita, dense aureo-puberula; pedicellus iatus, 1 - 2 mm. Calyx late cyathiformis, 6-7 mm longus, in alabastro clausus, tunc irregulariter in 3-4 lobes late ovatos, acutos vel obtusos, 2-6 mm longos

53 dehiscens, extus adpresse puberulus. Petala 4, valvata vel paullum imbricata, 15-18 mm Ionga, 4-5 mm lata, lingulata vel anguste elliptica, apice acuta vel obtusa, extus dense aureo-sericea, intus glabra. Tubus stamineus 10-13 mm longus, 3-5 mm Iatus, margine undulatus, glaber; antherae 10-13,2-2.2 mm longae. Nectarium breviter stipitiforme, 0.25- 1.5 mm longum, apice paullum expansum, glabrum. Ovarium dense strigosum, 7-9-loculare, loculis 2 ovula superposita continentibus; stylus strigosus. Capsula (statu immaturo) pyriformis, apice truncate, sensim and basin attenuate, obscure longitrorsus striata, dense papillose, ca.

4.6 cm Ionga, ca. 3.4 cm lata, 8 -valvata, valvis 2 semina superposita tenentibus; pericarpium 5-7 mm crassum. Semen non visum" (Pennington et al., 1981).

Guarea rhopalocarpa Radlkofer; Synonymous: Guarea tuisiana. There is no previous chemical or biological studies on this plant. It is a tree of lowland tropical rain forest and montane rain forest in Costa Rica and Western and Central Panama (Pennington et al., 1981).

Botanical description: "Young branches minutely appressed puberulous less frequently pubescent, soon glabrous, greyish-white. Leaves pinnate with a terminal bud showing intermittent growth, to 55 cm long; petiole semiterete, rachis terete or quadrangular, minutely puberulous or less frequently pubescent at first, soon glabrous; petiolule 7-12 mm long. Leaflets 4-5 pairs, broadly attenuate, often decurrent into petiolule, chartaceous, 12.5-21 [16.4] cm long, 4.8-8.7[6.2] cm broad, upper surface glabrous, but with numerous minute raised dots, lower surface usually glabrous, less frequently finely puberulous on midrib and veins, usually glandular- punctate and -striate; venation eucamptodromous, midrib flat or slightly

54 sunken; secondaries 9-12 on either side of midrib, ascending, usually arcuate and slightly convergent, rarely straight and parallel; intersecondaries absent, tertiaries oblique. Flowers unisexual, plants dioecious; inflorescences mainly cauliflorous and ramiflorous, with a few from leaf axils, 10-30 cm long, a densely-flowered pendulous spike, minutely puberulous, flowers ± sessile. Calyx usually patelliform, rarely short cyathiform, 2-3 mm long, with 3-4 irregular, ovate, acute or obtuse lobes 0.5-3 mm long, indumentum of minute scattered appressed hairs. Petals 4-6, valvate, 9.5-14 mm long, 1.5-3.5 mm broad, strap-shaped, apex acute of obtuse, minutely appressed puberulous on outer surface, glabrous inside. Staminal tube 8-12.5 mm long, 2-3.5 mm broad, margin orenulate, glabrous; anthers 8-9,1-1.5 mm long; antherodes similar, not dehisced, without pollen. Nectary a short stipe expanded at apex to form a small annulus below ovary, 0.5-1 mm long, stipe glabrous, expanded portion puberulous. Ovary 4-5(-6)-locular, loculi with 2 superposed ovules, appressed puberulous to pubescent; style puberulous of short pubescent; pistillode similar, with a longer and more slender style, containing well-developed, non-functional ovules. Capsule pyriform to ellipsoid, apex acute, rounded or emarginate, base long tapering, densely papillose-puberulous, 4-7.5 cm long, 3-4 cm broad, 4-5 valved, valves broadly and shallowly 3-ribbed, ribs sometimes torulose,

valves with 2 superposed seeds; pericarp 6 - 1 0 mm thick. Seed truncate at base or apex, 1.5-2 cm long, 0.6-1 cm broad, surrounded by a thick fleshy sarcotesta; seed coat thin, cartilaginous; hilum narrow, extending length of seed. Embryo with plano-convex, superposed cotyledons; radicle abaxial, extending to surface"(Pennington et al., 1981).

Ruagea glabra Triana & Ptanchon: Synonymous: Guarea trianae, G, weberbaueri, Ruagea jelskiana, R. augusti, R. weberbaueri, R. trisperma, R. floribunda.

55 There is no previous chemical or biological studies on this genus and it is represented by five species confined largely to montane rain forest and clouds forest from Guatemala to Panama, and Andean South America as far south as Peru (Pennington et al., 1981).

Botanical description: "Young branches usually slender, minutely appressed puberulous when young soon glabrous, pale greyish-brown, usualiy with a few lenticels. Bud scales absent. Leaves imparipinnate or paripinnate, with or without some limited apicai growth, 8-55 cm long; petiole semiterete, rachis often canaiiculate above, sometimes narrowiy winged, puberulous of glabrous; petiolule of lateral leaflets 1-7 mm long, petiolule of terminal

leaflet 1 0 - 1 2 mm long. Leaflets opposite or alternate, (5-)8-15(17), elliptic, oblong of oblanceolate, apex usually short, narrowly attenuate, lees frequently acute to obtuse rarely rounded, base acute, cuneate or attenuate, margin rarely revolute, usually chartaceous, 8-20(-32)[14.6] cm long, 3-9(-16)[5.9] cm broad, upper midrib sometimes puberulous, lower surface glabrous or with sparse to dense soft short pubescence on midrib and veins and scattered hairs on lamina,sometimes mixed with a few red papillae; venation eucamtodromous or rarely brochidodromous; secondaries (8-)10-14(-18) on either side of midrib, broadiy spreading, arcuate at tip, parallel or slightly convergent; intersecondaries often rather iong; tertiaries usuaily obscure. Flowers unisexual, plants dioecious; inflorescence axillary, 5-30(-60) cm long, usually a slender panicle with short basal branches less frequently with widely spreading

branches to 1 0 cm long, flowers in smali ciusters or racemose, minuteiy puberulous or subglabrous; pedicel 0-1 mm long. Calyx patelliform, sepals 0.75-2 mm long, ovate to orbicular, sparsely puberulous or glabrous, dilate. Petals 5-7(-8) mm long, 1.5-2.5 mm broad, elliptic, oblong or spathulate, apex rounded, glabrous. Staminal tube cyathiform

56 or short cylindrical, 2-6 mm long, 1.5-3 mm broad, margin crenulate or

with emarginate lobes, glabrous or sparsely barbate inside; anthers ( 8 - 10(-11 ), 0.75-1 (-1.1) mm long, sometimes with a short basal appendage, glabrous; antherodes narrower, without pollen not dehiscing. Nectary a short stout stipe, sometimes swollen below the ovary, 0.25-1 (-2) mm long, glabrous. Ovary (2-)3(-4)-locular, loculi with (1-)2 superposed ovules, glabrous or sparsely pubescent near the base; style stout, glabrous; style stout, glabrous; style-head discoid; pistilloide similar with well-developed non-functional ovules. Capsule globose or short pyriform, smooth, dark brown (when dry) with large pale lenticels, glabrous, 2-3 cm diam., 3-valved, valves 1-seeded; pericarp 0.5-2 mm thick. Seed ca. 1.5 cm long, 0.7 cm broad, ± ellipsoid, with a thick, fleshy, basal sarcotesta; seed coat thin, membraneous. Embryo with plano-convex, collateral cotyledons; radicle apical, extending to surface" (Pennington et al., 1981).

2.2 Menispermaceous Plants.

The Menispermaceae comprises 65 genera and approximately 400 species native to the tropics and subtropics of both hemispheres. They are mostly lianas, sometimes small trees or shrubs and occasionally perennial herbs and the family is divided into eight tribes based on characters of the fruits and seeds (Schultes and Raffauf, 1990). The major alkaloids in Menispermaceae are the bbiq alkaloids and some have been found to exhibit a variety of pharmacological activities (Schiff, 1983; Schiff, 1987). Bbiq alkaloids showed to be more active against P. falciparum than against Entamoeba histolytica an6 KB cell, in vitro (Marshall et al., 1994).

Phaeanthine, a bbiq alkaloid, isolated from the woody part of Triclisia

57 patens was found to be twice as potent against a chloroquine-resistant P. falcipamm strain (K1) as against a chloroquine-sensitive clone (Ekong et al., 1991). Tetrandrine, the enantiomer of phaenthine, was more active against chloroquine-resistant than against a choloroquine-sensitive strains of P. falcipamm (Ye and Van Dyke, 1989); also, tetrandrine and structurally related alkaloids have shown to be effective in reversing resistance in chloroquine- resistant P. falciparum and when used in combination with artemisinin, tetrandrine is said to provide a long-acting and synergistic activity (Van Dyke, 1991). Similarly, Shiraishi et al. (1987) have shown that some bbiq alkaloids are more active against a multidrug-resistant cancer line than they are against a drug-sensitive line.

The species of Abuta are shrubs, small trees or woody lianas natives to tropical South America and comprises some 35 species (Schultes and Raffauf, 1990). The genus contains alkaloids characteristic of the family and the chemical components isolated from this group of plants are presented in Table 2.2 (p.59).

Abuta dwyerana Kruk. & Barneby: There is no previous chemical or biological studies on this plant and there are three Abuta species occurring in Panama.

2.3 Rubiaceous Plants.

One of the largest of the plant families, the Rubiaceae has 7000 species distributed in 500 genera. Although, it is cosmopolitan most of the species are tropical and is a very natural group, but distinction between some of the genera is frequently difficult and controversial. The family has been divided into three subfamilies and 29 tribes based of a variety of characters (Schultes and Raffauf, 1990).

58 Plant species Isolated compounds Reference A. bullata Isoquinoline alkaloids: saulatine, dihydro saulatine. Hocquemiller and Cave, 1984 A. grandifolia Isoquinoline alkaloids: grandirubrine Menachery and Cava, 1980 A. grisebachii Isoquinoline alkaloids: grisabine Ahmad and Cava, 1977 macolide, macoline, peinamine, 7-0-demethyl-peinamine, 7-0-demethyl-N- Galeffi et al., 1977 methyl-peinamine imelutine, imenine, imerubine, homo-moschatoline, rufescine, nor-rufescine. Cava et al., 1975a A. pahni Isoquinoline alkaloids: (+) coclaurine, (-)daurisoline, 2’-N-nor-daurisoline, (-) Dute et al., 1987 lindoldhamine, 2'-N-methyl-lindoldhamine, dimethyl-lindoldhamine, stepharine, thalifoline A. panurensis Isoquinoline alkaloids: panurensine, nor-panurensine Cava et al., 1975b A. racemosa Triterpenes: dammara-20-25-dien-3p-24a diol Hammond et al., 1990 A. rufescens Isoquinoline alkaloids:imeluteine, imenine, imerubine, rufescine, nor-rufescine Cava et al., 1975a homo-moschatoline, splendidine Skiles et al., 1979 A. splendida Isoquinoline alkaloids: aromoline, homo-aromoline, krukovine Saa et al., 1976 A. velutina Triterpenes: abutasterone Pinheiro et al., 1983

Table 2.2 Chemical Compounds Isolated from Abuta species.

59 This is the largest family of dicotyledons in Panama with about 431 species in 87 genera (D’Arcy, 1987). The vast majority of Rubiaceae in Panama are trees or shrubs with the genus Psychotria representing almost one quarter of the species. Subshrubs or rarely herbs like Borreria, Diodia and Spermacoce are particularly difficult to identify as to species (Dwyer, 1980).

The family is rich in chemical constituents of potential in medicine and industrial uses, including iridoids, several type of alkaloids, triterpenes, sterols, quinones, naphthalene derivatives, polyphenols and tannins. The most important medicinally compounds of the family including quinine from Cinchona species, emetine from Cephaëlis species and the widely distributed quinones. The three major type of alkaloids from the family are all derived from the iridoid precursor, ioganin, and contain either indole, tetrahydroisoquinoline or quinoline moieties, however, non iridoid alkaloids are present. The alkaloids of Rubiaceae have been reviewed (Hemingway and Phiilipson, 1980).

There are some 2 0 0 species of Cephaëlis occurring throughout the tropics, of which 21 occur in Panama (Dwyer, 1980). The taxonomic position of Cephaëlis species is unclear. The study of Cephaëlis Swartz and Psychotria Linnaeus of the Northeastern South America (Steyermark, 1972) and Venezuela (Steyermark, 1974) demonstrated that Cephaëlis as generally circumscribed in South America is polyphyletic and represents no more than a convergent assortment of species and species groups, each of them more closely related to species of Psychotria than to other species of Cephaëlis (Taylor et ai., 1991). in Central America, where fewer intermediate taxa grow than in South America, Cephaëlis has been maintained as a genus (Dwyer, 1980; Standley and Williams, 1975). More recently however, in the preparation of a monograph on the Rubiaceae in Costa Rica (Taylor, 1991), Cephaëlis species have been included in Psychotria, subgenus Heteropsychotria. The genus Psychotria is constituted of two principal subgenera, Psychotria and Heteropsychotria, and they have been combined mostly because both of them

60 have tiny white flowers and the species of both are numerous and difficult to separate (Taylor, 1993).

As not all the new names for the species selected for this study have been published, the Cephaëlis name will be maintained, however, all the synonymous are showed under each plant description. In addition, and to further our knowledge of this group of plants, a review of the chemical compounds present in Psychotria species is presented in this section.

Most treatises of pharmacognosy, ie. Tyler et al. (1988) state that the Panamanian ipecac is Cephaëlis acuminata Karsten, however, it has been shown that this name appears to be a nomen nudum, ie. it has never been validly published from a taxonomic point of view. Then, the correct name for the commercial source of emetine be C. ipecacuanha (Broth.) Rich. (Hatfield et al., 1981) therefore, all the scientific reports about C. acuminata should be considered as C. ipecacuanha.

Some 24 monoterpenoid isoquinoline alkaloids have been isolated from C. ipecacuanha (Wiegrebe, et al., 1984; Itoh, et al., 1989; Itoh, et al., 1991 ; Nagakura, et al. 1993). Emetine is used in the treatment of amoebic dysentery and the extract of the roots as expectorant in cough syrup and in the treatment of poisonings (Tyler et al., 1988). The simple indole alkaloid gramine has been isolated from C. stipulacea (Yulianti and Djamal, 1991). None of the other Cephaëlis species have been investigated for its chemical constituents or biological activities.

Cephaëlis camponutans Dwyer & Hayden. The accepted name of this species is Psychotria camponutans (Dwyer & Hayden) Hammel and occurs in Costa Rica and Panama (Taylor et al, 1991). Botanical description: "Erect, glabrous to pu be ru lent, somewhat succulent, suffruticose herbs

61 0.5-1.25 m tall, unbranched; stems quadrate with rounded corners. Leaf blades oblong to obovoid-oblong, 18-25 cm long, 7-9 cm wide, acute at apex, acute to attenuate at base, coriaceous, dark green above, paler and below; secondary veins 9-11 pairs, not looping to interconnect, indistinct but midrib keeled above, prominulous below, without domatia; petioles 25-55 mm long; stipules deciduous, connate interpetiolarly, triangular, succulent, divergent, 4-5 mm long minutely bilobed. Inflorescences pseudoaxillary, with cymes capitate, green, puberulent, 5-10 mm in diameter, subsessile; involucral bracts green, ovate, 10 mm

long, floral bracts lanceolate, 6 - 8 mm long, acuminate; flowers sessile, 5-merous; calyx limb ca.1 mm long, lobed to base; corolla white, funnelform, glabrous, tube ca. 3 mm long, lobes 1-1.5 mm long. Fruits obovoid to ellipsoid, 7-8 mm long, succulent, red; pyrenes planoconvex, 6-7 mm long, 4-5-angled on back, smooth with a median sulcus ventrally." (Taylor, 1991).

Cephaëlis dichroa (Standley) Standley. This species is known only from Panama and Evea dichroa has been used as synonymous (Dwyer, 1980). Botanical description:

"Glabrous shrubs, to 2 m tall. Leaves elliptic, 8.5-13.0 cm long, to ca. 3.2 cm wide, acuminate at the apex, cuneate at the base, the lateral veins

ca. 1 2 , stiffly papyraceous, concolorous, often drying yellowish, glabrous; petioles to 3 cm long, wiry, often flexuous; stipules connate, ca. 3.5 mm

long, the body compressed cylindrical, each part with 2 widely spaced, erect, obtuse lobes about as long as the sheath. Inflorescences terminal, the peduncle 5-8(-9) cm long, terminated by 3 heads of flowers,

the trio subtended by 2 large obovate bracts, to 2 . 2 cm long, ca. 1 . 8 cm wide. Fruits subrotund to 0.8 cm wide." (Dwyer, 1980).

62 Cephaëlis dimorphandrioides Dwyer. This species is known only from Panama and is distinguished from all other Cephaëlis by the cylindrical spikelike inflorescence which bears a resemblance to the inflorescence of the legume genus Dimorphandra (Dwyer, 1980).

Botanical description: "Shrubs \o 1.4 m tall. Leaves elliptic oblong, often falcate oblong, 17-30 cm long, 6-14 cm wide, deltoid or obtuse toward the apex, acute or abruptly acute toward the base, the costa slender above, prominent beneath, the lateral veins 13-17, arcuate, often with one smaller and

shorter vein between 2 lateral veins, the intervenal areas spreading reticulate, rigidly chartaceous, discolours, glabrate above, minutely puberulent beneath; petioles lignose, to 4.5 cm long, twisted, puberulent; stipules (only one pair seen at tip of twig) oblong rotund, to 0.7 mm long,

deeply 2 -lobed, coriaceous, the lobes rounded, farinose, glabrous within. /n//orescencesterminal, sessile, cylindrical, 5-12 cm long, to 4 cm wide,

obtuse at the apex, black when dry, the flowers more than 1 0 0 . Flowers perhaps sessile, the hypanthium short, swollen, puberulent, the calycine cup short, ca. 5 mm long, the teeth subulate, to 6.5 mm long, unequal, acute, puberulent outside, glabrous inside, carnose, with 3 longitudinal veins, with 2-4 oblong glands, to 0.3 mm long, at the sinuses; corolla red outside, the tube narrowly cylindrical, 10-14 mm long, puberulent outside, glabrous within, subcarnose, the lobes 5, yellow within, lance oblong, to 3 mm long, carnose, puberulent outside; stamens 5 , the anthers narrow oblong, 2,8-3.0 long, minutely apiculate, slender, attached below the middle of the tube; ovarian disc 4 lobed, to 0.8 mm long, the style linear, 9-14 mm long, the stigmas 2, reflexed, linear

oblong, to 0 . 8 mm long. Fruits (immature) oblong, to 5 mm long, to 0.3

wide, truncate, each pyrene smooth, black, 1 0 -ribbed, the hairs weak, white, arachnoid." (Dwyer, 1980)

63 Cephaëlis glomerulata J.Donnell Smith. The proper name of this species is Psychotria glomerulata (J. Donnell Smith) Steyermark and occurs from Guatemala to Panama, in wet forests (Taylor, 1991). Botanical description:

"Erect glabrous shrubs 0.5-1 (- 2 ) m tall, much-branched; stems quadrate. Lea/ blades narrowly elliptic to oblong, (5-)10-13 cm long, (15-)25-55 mm wide, acuminate at apex, attenuate at base, chartaceous, green on both surfaces; secondary veins 12-16(-20) pairs, looping to interconnect near margins, prominulous above and below, without domatia; petioles 5-15 mm long; stipules persistent, membranaceous, appressed, connate interpetiolarly and intrapetiolarly, truncate, 1.5-3 mm long, with two groups of 6-12 caducous awns 0.5-1 mm long. Inflorescences terminal, with cymes capitate, ovoid, pale green to purple, 1.5-2.5 cm long, glabrous; peduncles 0-15 mm long; involucral bracts broadly ovate, 10- 15 mm long, rounded, floral bracts ovate, 8-10 mm long; flowers distylous, 5-merous; calyx limb 0.5-1 mm long, partially lobed; corolla white, funnelform, glabrous, tube 10-15 mm long, lobes 2.5-3 mm, long. Fruits ovoid to cylindrical, 10-15 mm long, ca. 5-6 mm in diameter, succulent, bright blue; pyrenes planoconvex, 3-5 mm long, 5-ribbed dorsally, smooth with a median sulcus ventrally". (Taylor, 1991).

Table 2.3 (p.65) shows the chemical compounds isolated from the genus Psychotria, however, some early reports (Wehmer, 1931; Willaman and Schubert, 1961) of the presence of emetine and related alkaloids in this genus should be treated with prudence.

64 Plant Species Compounds Isolated References P. adenophylla Triterpenes: a-amyrin, baurenol, baurenol acetate, betuiin, betulinic Dan and Dan, 1986 acid, friedenn, ursolic acid. Steroids: 0-sitosterol P. bacteriophila Steroids: 0-sitosterol (from callus tissues) Dohnal et al., 1989 P. duoarrei Inorganics: nickel Kersten et al., 1980 P. emetica Alkaloids: cephaeline, psychotrine Wehmer, 1931 P. forsteriana Polyindolenines alkaloids: chimonanthine, hodgkinsine, psychotridine, Adjibade et al., 1989 iso-psychotridine, quadrigemine A, quadrigemine B meso-chimonanthine Adiibade et al., 1992

iso-psychotridine 0 Roth et al., 1985 Quinoline alkaloids: calycanthine Adjibade et al., 1991 (-) calycanthine, iso-calycanthine Adiibade et al., 1992 P. granadensis Isoquinoline alkaloids: (-) emetine Willaman and Schubert, 1961 P. manillensis Monoterpenes: asperuloside Inouye et al., 1988 P. oleoides Polyindolenine alkaloids: hodgkinsine, psychotridine, iso-psychotridine Libot et al., 1987 A, iso-psychotridine B, quadrigemine C psycholeine Gueritte-Voegelein et al., 1992 P. rubra Monoterpenes: asperuloside Inouye et al., 1988 Naphtoquinones: psychorubin Hayashi et al., 1987 sesquiterpenes: helenalin Steroids: 0-sitosterol Hui and Yee, 1967 P. serpens Monoterpenoids: asperuloside Inouye et al., 1988 Steroids: 0-sitosterol, stigmasterol Hui and Yee, 1967 Triterpenes: ursolic acid Lee et al., 1988 P. tomentosa Isoquinoline alkaloids: (-) emetine Wehmer, 1931

Table 2.3 Chemical Compounds from Psychotria Species.

65 Lasianthus panamensis Dwyer. This plant belongs to the tribe Psychotriae and is the only species of this genus in Panama, formerly named Dressleriopsis panamensis (D’Arcy, 1987).

Botanical description:

"Subshrubs to 1 m tall, often with a pair of slender, rigid, opposite branches. Leaves oblong, to 19 cm long, to 9 cm wide, deltoid to subobtuse, short acuminate, basally cordate, truncate, or auriculate, the lateral veins ca. 15, arcuate, forming a conspicuous submarginal vein, the intervenal areas, spreading reticulate, rigidly chartaceous,

discolorous, pilose petioles to 6 cm long; stipules triangular, free, ca. 7

mm long, ca. 6 mm wide, carnose, glabrous inside, diffusely hirsute outside. Inflorescences axillary, the flowers congested into sessile

globose heads 2 cm in diam.; bracteoles subulate, to 2 mm long.

Flowers subsessile; hypanthium subrotund, to 2 mm long hirsute, the

hairs septate, lobes 4, to 4 mm, ca. 1 . 6 mm wide, basally attenuate glandular inside at the base; corolla white, cylindrical, ca. 3.5 cm long, glabrous within, the lobes 5, often horizontally expanded, oblong, ca. 4 mm long, villose, attenuate; upwards anthers 5, slightly exserted,

subsessile, oblong, ca. 1 mm long, glabrous; disc cushion shaped, ca.

0.5 mm wide, the locules 8 , a single linear oblong axile ovule filling each

locule, the style narrowly, to 5 mm long, the stigmas 8 , radiate. Fruits

(?immature) berrylike, globose, to 1 . 1 cm in diam., not ribbed, compressed at the apex, the internal pulp with abundant raphides, the

pyrenes 8 , pear shaped or somewhat hemispherical, ca. 3 mm long, hard, orange, with an oblong scar on the concave side". (Dwyer, 1980).

Chemical compounds isolated frtm species of Lasianthus: L. curtisii, L. cyanocarpus, L. fordii, L. obliquinervis and L. plagiophyllus contain asperuloside (monoterpenoid) (Inouye et al., 1988). Betuiin (triterpene), damacanthal (quinone), (3-sitosterol and stigmasterol (steroid) have been

66 isolated from L chinensis (Hui et a!., 1967). The flavonoids eriodictyol and eriodictyol-7-O-p-D-glucoside and the coumarin scopeletin have been isolated from L japonica (Ishikura et al., 1979).

2.4 Simaroubaceous Plants.

This family of tropical and subtropical trees has about 30 genera and 2 0 0 species (Schultes and Raffauf, 1990). Antimalarial activity has been reported for quassinoids (Trager and Polonsky, 1981; Guru et al., 1983; O’Neill et al., 1987; Chan et al., 1986; Bray et al., 1987; O’Neill et al., 1988) and for indole alkaloids (Kardono et al., 1991 ; Pavanand et al., 1988) isolated from Simaroubaceous species, but it is the former group of compounds which is the more potent.

The in vitro testing of 40 individual quassinoids against Plasmodium falciparum (K1 ), a chloroquine-resistant strain, has been reported (O’Neill et al., 1986). Also, quassinoids have been shown to have in vitro activity against Entamoeba histolytica (Gillin et al., 1982; Wright et al., 1988) and Leishmania donovani (Robert-Gero et al., 1985).

There are some 60 species of Picramnia in Central and South America and the West Indies (Schultes and Raffauf, 1990), of which six occur in Panama (Porter, 1973). Quassinoids have not been found, so far, in species of

Picramnia. Table 2.4 (p. 6 8 ) shows the chemical compounds isolated from this genus.

Biological activity for extracts of Picrmania species: The chloroformic extract of the branches of P. antidesma was active against Plasmodium gallinaceum in vivo, but the aqueous extract was inactive (Spencer et al., 1947), and the methanolic extract of the leaves were inactive in plant germination inhibition (Dominguez and Alcorn, 1985). The chloroformic and

67 aqueous extracts of P. carpinterae (P. teapensis), P. locupes, P. longissima, P. pentandra, P. sellowii and P. xalapensis were inactive against P. gallinaceum, in vivo (Spencer et a!., 1947). An aqueous extract of the leaves of P. macrostachys was active against Staphylococcus aureus and Streptococcus sp. (Arana and Juica, 1986). The ethanolic extract of the leaves of P. pentandra showed spasmogenic activity in isolated ileum of guinea pig and vasodilator activity in isolated hind quarter of rat (Feng et al., 1962).

Plant species Compounds Isolated References P. antidesma Steroids: p-sitosterol Dominguez and Alcorn, 1985.

P. macrostachys Quinones: chrysophanic acid, Arana and JuIca, physcion 1986 P. parvifolia Quinones: aloe-emodin, Popinigis et al, chrysophanic acid, 1980 emodin, physcion, rhein

P. pentandra Triterpenes: 3-epi-betulinic acid Herz et al., 1972

P. sellowii Quinones: chrysophanic acid, Leon, 1975 emodin, physcion. Triterpene: betulinic acid, 13-epi- betulinic acid

Table 2.4 Compounds Isolated from Species of Picramnia.

Picramnia antidesma subsp. fessonia (DC) W. Thomas:

68 Synonymous; P. fessonia, P. antidesma var. pubescens, P. andicoia, P. bonplandiana, P. tetramera, P. quaternaria, P. seemannaina, P. brachybotryosa, P. pistaciaefolia, P. locuples, P. velutina, P. aiienii.

This species is the most common and variable taxon of Picramnia in Mexico and Central America. This variability, particularly in flower merosity, leaflet number and shape, and features of the indûment, is responsible for the long list of synonymous (Thomas, 1988). The very bitter, liquorice-flavoured bark and leaves have been much used in Central America and the West Indies as remedies for malaria and stomach and intestinal ailments (Standley and Steyermark, 1958). The bark was formerly exported to Europe for use in treating erysipelas and venereal diseases (Morton, 1981).

Botanical description: "Shrubs or small trees, 1-7 m high; branch lets yellowish appressed- tomentulose, becoming glabrate. Leaves alternate, pinnate, 18-24,5 cm long; petiole and the rachis yellowish appressed-tomentulose, becoming

almost glabrate; leaflets 1 2 -2 0 , mainly and even number, alternate to lowermost subopposite, ovate to elliptic, narrowly acuminate apically, rounded and markedly inequilateral basally, the margins entire, revolute, chartaceous, yellowish appressed-tomentulose above and beneath on the main vein and marginally, the blade sparingly pubescent, 22-75 mm long and 8-23 mm wide, the terminal leaflets largest, the lowermost smallest and reflexed, the petiolules yellowish appressed-tomentulose,

2 - 3 mm long. Staminate racemes subterminal, becoming axillary, aggregated to solitary, simple or rarely once-branched, densely-flowered,

yellowish appressed-tomentulose, to 38 cm long. Staminate flowers, 3 -

merous, 1 -several aggregated in clusters, white or greenish, the spreading, yellowish appressed-tomentulose, to 3 mm long, sepals 3,

ovate, apiculate, appressed-pubescent, ca. 15 mm long and 1 mm wide, petals 3, narrowly elliptic, apiculate, glabrous to rarely sparingly

69 pubescent, longer than the sepals, ca. 1.5 mm long; stamens 3, exserted, the filaments filiform, glabrous, ca. 2.5 mm long, ovary absent. Carpellated racemes solitary or in pairs, yellowish appressed- tomentulose, to 28 cm long. Carpellate flowers 3-merous, the pedicels appressed-tomentulose, articulated basally, 1-1.5 cm long in fruit; sepals 3, broadly triangular, appressed-pubescent, persistent and spreading in

fruit, ca. 2 mm long and 1 mm wide; style bilobed, the lobes recurved and persisting in fruit. Berries red or orange, ellipsoid to obovoid, appressed-pubescent to glabrate, to 1.5 cm long; seeds 1". (Porter, 1973).

Picramnia teapensis Tul Synonymous: P. carpinterae, P. antidesma var. carpinterae. This species is found in cool moist forest from Mexico to Venezuela and it is most common in Costa Rica and Panama (Thomas, 1988). In Costa Rica, a decoction of the bitter bark of the trunk and roots is taken as a tonic to stimulate the appetite and digestion, and as a febrifuge (Nunez-Melendez, 1975).

Botanical description: "Sma// trees, 5-12 m high, the crown flat; branch lets yellowish-villous. Leaves alternate, pinnate, to 20 cm long; petiole and the rachis yellowish-villous; leaflets 9-11, alternate to subopposite, obtuse or acute and acuminate apically, more or less inequilateral, the uppermost and middle leaflets elliptic to ovate and cuneate basally, the lowermost ovate and cordate basally, paler beneath, the margins repand, revolute, subcoriaceous, villous marginally and beneath, especially on the veins, to 11 cm long and 5.5 cm wide, the lowermost pair much smaller than

the others, the petiolules villous, 1 - 2 mm long. Staminate racemes

subterminal, becoming axillary, solitary, simple, yellowish-villous, 1 2 - 2 2

70 cm long. Staminate flowers 3-merous, numerous, 1 -several aggregated in clusters, the pedicels glabrous, 1-3 mm long, sepals 3, obovate, sparingly pubescent, 1.5 mm long and 1 mm wide, petals 3, narrowly obovate, ca. 1 mm long; disc 3-4 lobed; stamens 3(-4), exserted, the filaments subulate, glabrous, ca. 2.5 mm long, ovary rudimentary, conical, villous apically, the style and stigma absent. Carpellate racemes solitary, yellowish-villous, 23-27 cm long in flower, to 30 cm long in fruit. Carpellate flowers 3-merous, white, the pedicels villous to glabrate, 2-4 mm long in flower, 4-12 mm long in fruit, articulated basally; sepals 3, broadly triangular, densely villous, persistent and spreading in fruit, ca. 1.5 mm long and 1.5 mm wide; petals 3, narrowly obovate, apiculate, spreading, ca. 2 mm long; staminodes 3, opposite the petals; disc 3- lobed, glabrous; ovary ovoid, densely pubescent, 1 - 2 mm high, 2 - loculed, the ovules 2 per locule, the style bilobed, the lobes recurved and persisting in fruit. Berries red, ellipsoid, sparingly pubescent; seeds 1-2". (Porter, 1973).

71 SECTION 3. TESTING FOR BIOLOGICAL ACTIVITY. 3.1 Materials and Methods 3.1.1 New microwell brine shrimp assay

Brine shrimp (Artemia salina) eggs obtained locally (Interpet Ltd. Dorking, England) were hatched in artificial sea water prepared from sea salt (Sigma

Chemical Co., U.K.) 40 g/l of distilled water supplemented with 6 mg/l dried yeast and oxygenated with an aquarium pump. After 48 hours incubation in a warm room (22-29°C) nauplii were collected with a pasteur pipette after attracting the organisms to one side of the vessel with a light source. Nauplii were separated from the eggs by pipetting them 2-3 times in small beakers containing fresh sea water.

Samples for testing ( 0 . 6 mg) were made up to 1 mg/ml in artificial sea water except for water insoluble compounds which were dissolved in 50 pi DMSO prior to adding sea water.

Serial dilutions were made in 96-well microplates (ICN Flow, U.K.) in triplicate in 100 pi sea water. Control wells with DMSO were included in each experiment. The nauplii were place in a small beaker and air was pumped to

oxygenate and keep a homogenous suspension of larvae, 1 0 0 pi containing 10-15 organisms was added to each well and the covered plate incubated at 22-29°C for 24 hours. Plates were then examined under a binocular microscope (x12.5) and the number of dead (non-motile) nauplii in each well were counted. 1 0 0 pi methanol was then added to each well to kill all the shrimps and after 15 minutes the total numbers of organisms in each well were counted. LCgo values were then calculated by Probit analysis (Finney, 1971).

Test compounds: Bruceines A, C and D and brusatol were isolated as previously described (O’Neill et al., 1987). Villastonine was isolated from Alstonia angustifolia as previously reported (Wright et al., 1992). Homoharringtonine, isoharringtonine and cephalotaxine were kindly supplied by Dr. Powell, United States Department of Agriculture, Illinois, and quassin was

73 a gift from Bush, Boake and Allen Ltd. U.K., thymol was obtained from May and Baker Ltd, U.K., actinomycin D, from Aldrich Chemical Co. U.K., and atropine sulphate, chloramphenicol, quinidine sulphate and quinine hydrochloride were supplied by BDH, U.K. All other compounds were obtained from Sigma Chemical Co. U.K.

3.1.2 Brine shrimp assay

Brine shrimp assay was carried out in test tube as previously reported (Meyer et al., 1982). The eggs of Artemia salina were hatched as described in Section 3.1.1 and samples for testing were made up to lOrng/m/ in artificial sea water or in methanol for water insoluble compounds. Appropriate amounts of solution were transferred into test tubes to make a final concentration of 1000, 100 and 10 pg/ml of compound for a total volume of 5 ml. Three replicates were prepared for each concentration. When methanol was used to dissolve the samples it was evaporated under a stream of nitrogen and kept under vacuum overnight. Control vials were prepared using only the solvent.

Ten nauplii were transferred to each sample test tube and artificial sea water was added to make 5 ml. The mixtures were incubated in a warm room (22-29 °C) and after 24 hrs survivors were counted and the percent of deaths at each concentration and control were determined. The LCgg values were calculated by Probit analysis (Finney, 1971).

3.1.3 KB cells assay

The microdilution technique for KB cells assay was carried out as previously described by Anderson et al. (1991b). Human nasopharyngeal KB cells were maintained in Eagles Minimum Essential medium with Earle's salts and sodium bicarbonate (0.85 g/l), supplemented with 1% foetal bovine serum, L-glutamine (2 mM) (ICN Flow, UK.), penicillin (50 units/ml) and streptomycin (50 pg/ml) (ICN Flow, UK.).

74 When the monolayer became fully confluent the cells were subcultured using 0.25 % trypsin solution (ICN Flow, UK.). The detached cells were transferred to sterile centrifuge tubes (10 ml) and spun at 400 rpm for approximately 15 seconds. After twice washing with complete medium, the cells were resuspended in 1 ml of complete medium and mixed thoroughly to break down any cell aggregates. The cells were counted using a haemocytometer and the appropriate number of cells added to fresh culture vessels containing complete medium to give a final concentration of 1 0 ® cells/ml.

A 50 pi aliquot of cells suspension (1x10® cells/ml) was mixed with 50 pi aliquot of serial dilution of compounds, in triplicate each time. In each experiment two controls were used, one without drug as negative control and emetine HCI (Sigma Chem. Co. UK.) as positive control. Incubation for 48 hrs at 37 °C was done in atmosphere of 5 % of COg in air. After incubation, the cells were fixed by adding 6 6 pi of 25 % trichloroacetic acid (Sigma Chem. Co. UK.) at 4 °C to each well and refrigerated at the same temperature for one hour. The plates were then rinsed with water 5 times and left to dry. 1 0 0 pi of 1 % aqueous eosin B stain (Sigma Chem. Co., UK.) was added to each well, after an hour the plate was washed 5 times with 1 % acetic acid.

The plates were dried overnight before 200 pi of 5 mM sodium hydroxide solution was added to each well and kept for 2 0 minutes to release the dye. The optical density of the solution in each well was determined at 490 nm by a microplate reader, model MR-700 (Dynatech Labs Inc. UK.). The percentage inhibition of KB cell growth was calculated by comparison of the absorbance of the control and test samples. The ICgo was calculated by a linear regression of the percentage of inhibition against the logarithms of the concentration of sample being tested, using a computer program (Minitab, statistical software, PC version, release 8,1991. Quickset Inc. Rosemont, PA, USA).

75 3.1.4 Plasmodium falciparum assay.

This was carried out as described previously (Ekong et a!., 1990) by Drs. D.C. Warhurst, G.C. Kirby, Department of Medical Parasitology, the London School of Hygiene and Tropical Medicine, and Miss C. Lang'at. Department of Pharmacognosy, The School of Pharmacy, University of London.

Cultures of P. falciparum (chloroquine-resistant strain K- 1 ) were maintained in human erythrocytes, suspended in RPMI 1640 medium supplemented with D-glucose, NaHCOg, TES and human serum. Two fold dilutions of the sample were prepared in 96-microwell microtitre plates and 50 pi of a suspension of infected human red blood cells (1% parasitaemia) was added to each well. The mixture was incubated in a 3% Og, 4% COg and 93% N atmosphere at 37 °C for 24 hrs before adding 5 pi of [^H]-hypoxanthine (40 pCi/ml) and continuing incubation for a further 18-24 hrs. Chloroquine diphosphate was tested simultaneously to monitor the sensitivity of the parasite strain. Two series of controls were performed: one with infected red blood cells in the absence of drug and the other with uninfected red blood cells.

3.2 Results and Discussion 3.2.1 Evaluation of brine shrimp assay

The method described above (3.1.1) provides a simple and inexpensive screening test for cytotoxic compounds. It has the advantages of requiring only small amounts (0.6 mg) of compounds, in contrast with 50 to 20 mg in the test tube method (3.1.2), and the employment of microplate technology facilitates the testing of large number of samples and dilutions. The use of methanol to kill live shrimps at the end of the experiment enables rapid counting of the total numbers of shrimps.

The results obtained with a number of pharmacologically active compounds, some of which are cytotoxic, are comparable with results reported using a

76 previous method in which test tubes were used (Meyer et a!., 1982); with the exception of thymol, LC 50 values determined by the two methods are similar. However, when thymol was tested in our laboratory using the test tube method a LC50 value of 46.7 ± 12.0 pM was obtained which is in close agreement with that found using the new microplate test.

The test was predictive of cytotoxicity for all the compounds listed in Table

3 . 1 (p.78) except for cyclophosphamide which is known to require metabolic activation in the liver for activity in man. Since >4. salina does not posses the enzymes required for this process, cyclophosphamide would not be expected to be toxic to this organism.

Table 3.2 (p.79) shows the LC 50 values determined for a number of biologically active compounds which have various modes of action together with their reported ED 50 values against KB cells (human nasopharyngeal carcinoma). Of the six compounds active against KB cells, five were also toxic to nauplii. However, there was little correlation observed in the degree of toxicities seen between the two methods. Emetine was more toxic to KB cells than villastonine but against the brine shrimp the converse was found to be the case. Similarly, although podophyllotoxin and actinomycin D were equitoxic to KB cells, the brine shrimp was >50,000 times more sensitive to the latter than to the former. One cytotoxic compound, 6 -mercaptopurine was not toxic to A. salina and this is explicable as its purine metabolism is markedly different from that in mammalian cells (Van Debos and Finamore, 1974) and it is possible that the intracellular activation which occurs in man may not take place in A. salina.

Quassinoids are constituents of the Simaroubaceae and have potent in vitro activity against P. falciparum (O’Neill et al., 1987). These compounds inhibit protein synthesis in both mammalian cells and malaria parasites (Kirby et al., 1989) and, as shown in Table 3.3 (p.81) were found to be toxic to the brine shrimp. Quassin which does not possess cytotoxic or antiplasmodial activity was, as expected, devoid of toxicity. These results suggest that for this group

77 DRUGS MICRO-TECHNIQUE REPORTED VALUES LCso(pM) ± sd pM

ANISOMICIN 35.2 ±14.2 (2 )* -

ATROPINE SULPHATE > 1 0 0 0 . (3) 987.3"

BERBERINE CHLORIDE 25.2 ± 2.9 (3) 60.5"

CAFFEINE >1 0 0 0 . (3) > 1 0 0 0 ."

CEPHALOTAXINE 549.9 ± 83.7 (3) -

CYCLOPHOSPHAMIDE >1 0 0 0 . (3) -

EPHEDRINE >1 0 0 0 . (3) > 1 0 0 0 ."

HOMOHARRINGTONINE 7.4 ± 4.7 (3) 7.5°

- ISOHARRINGTONINE 13.9 ±6.2 (2 )

NICOTINE > 1 0 0 0 . (2 ) >1 0 0 0 .°

PODOPHYLLOTOXIN 15.0 ±2.1 (2 ) 5.8"

QUINIDINE SULPHATE 518.8 ± 126.9 (3) 287.8"

THYMOL 56.3 ± 10.6 (3) >1000."; 46.7 ±

1 2 .0 "

^Indicates number of experiments performed in triplicate, using the method described in section 3 .1 . 1 ‘'Data obtained from Meyer et a!.,(1982) ®Data obtained from Anderson et a!., (1991a) the previously reported data were transformed to pM for comparison purpose only. Value obtained in our laboratory using the test tube method (3.1.2).

Table 3.1 Comparison of the Values Obtained in the Brine Shrimp Micro- Technique with Previously Reported Data.

78 DRUGS Brine shrimps KB Ceil LCso (pM) ± sd EDso (pM)

ACTINOMYCIN D 3.8x10-^ ± 1.4x10-^ ( 2 )“ 0 .0 1 2 "

CHLORAMPHENICOL > 1 0 0 0 . (2 ) >50."

CHLOROQUINE > 1 0 0 0 . (2 ) 153* DIPHOSPHATE

CYCLOHEXIMIDE 104.6 ± 1.0 (3) 3.31"

EMETINE HCI 29.0 ± 6 . 8 (4) 0.673"

6 -MERCAPTOPURINE > 1 0 0 0 . (2 ) 3.42"

PODOPHYLLOTOXIN 15.0 ±2.1 (2 ) 0.007*

QUININE > 1 0 0 0 . (2 ) 333."

VILLASTONINE 10.8 ±4.8 (3) 1 1 .6 "

“Indicates number of experiments performed, in triplicate, using the method described in section 3.1.1 ‘’Data obtained from Anderson, (1992) "Data obtained from Anderson et.al. (1991b) ‘'Data obtained from Wright et.al. (1992)

Table 3.2 Comparison of Brine Shrimp Toxicity Against in vitro KB Ceil Cytotoxicity of Compounds with Different Mechanisms of Action.

79 of compounds the brine shrimp test is suitable for the screening of plant extracts and fractionation of cytotoxic quassinoids, thus obviating the need for the difficult and expensive antiplasmodial test at every stage in the isolation procedure. Similarly, the brine shrimp test has been used as a convenient assay for the detection of antifilarial activity of avermectin analogs (Blizzard et al., 1989).

This approach may be useful for other types of biologically active compounds where the brine shrimp responds similarly to the corresponding mammalian systems. For example the DNA-dependent RNA polymerases of A. salina have been shown to be similar to the mammalian type (Birndorf et al., 1975) and the organism has a ouabaine-sensitive Na^ and dependent ATPase (Ewing et al., 1974), so that compounds or extracts acting on these systems would be expected to be detected in this assay. Although the assay did not detect those compounds which require metabolic activation in man, it may be conveniently used where, as in the case of the quassinoids, the brine shrimp is a reliable detector of biological activity.

3.2.3 Activity of the plant crude extracts against brine shrimp and KB ceils

Tables 3.4 - 3.14 (p.82-87) show the activity of 135 extracts and fractions obtained from the eleven Panamanian medicinal plants selected for this study. The value represents the average of two experiments in triplicate. Section

4.1.4 shows the extraction and fractionation procedures. Table 3.15 (p. 8 8 ) shows the activity against KB cells of extracts selected on the basis of previously activity against brine shrimp.

80 QUASSiNOiD BRINE SHRIMP KB CELL P. falciparum LCjo (pM) ± sd EDso (pM)» iCso MM

BRUCEINE A 0.887 ± 0.24 (2)“ 0.188 0 .0 2 1 "

BRUCEINE C 0.798 ±0.17 (2) 0.037 0.009"

BRUCEINE D 4.47 ±0.19 (2) 2.82 0.037"

BRUSATOL 1.69 ±0.137 (2) 0.196 0.006"

QUASSIN > 1 0 0 0 . >50. 247"

“Indicates number of tests performed in triplicate, using method described in section 3.1.1 ^ Data obtained from Anderson et.al.(1989) "O'Neill et.al.(1987). ''Data obtained from Anderson (1992)

Table 3.3 Activities of quassinoids against brine shrimps, KB ceil and P. falciparum in vitro.

81 PLANT PART EXTRACT BRINE SHRIMP PLANT PART EXTRACT BRINE SHRIMP (Code N°) LC5 0 pg/ml (Code N°) LC5 0 pg/ml MeOH 22.2 MeOH 93.6 CHCI3 23.8

CHCI3 89.3 BuOH >1000. BuOH 395.8 Branches HgO-FRAC. >1000. Branch HgO-FRAC. > 1 0 0 0 . (la) HgO-EXT. >1000. (3a) H2 O-EXT. > 1 0 0 0 . MeOH 5.8 MeOH 154.3 CHCI3 8.2 BuOH >1000. CHCI3 141.5 Wood H2O-FRAC. >1000. BuOH > 1 0 0 0 . (1b) H2O-EXT. >1000. Leaves H.O-FRAC. > 1 0 0 0 . MeOH N/T (3b) CHCI3 39.6 H 2 O-EXT. > 1 0 0 0 . BuOH >1000. Leaves HgO-FRAC. >1000. (1c) HgO-EXT. >1000.

Table 3.4 Activity Against Brine Shrimp of the Tabie 3.5 Activity Against Brine Shrimp of the Extracts from Guarea macropetala Extracts from Guarea rhopalocarpa

82 PLANT PART EXTRACT BRINE SHRIMP PLANT PART EXTRACT BRINE SHRIMP (code N*) LC5 0 pg/mi (Code N°) LC« pg/mi MeOH 324.4 MeOH 313.9 CHCI3 197.1 CHOU 411.5 BuOH >1000. Leaves BuOH 486.6 Leaves H2O-FRAC. >1000. (4a) H9 O-FRAC. > 1 0 0 0 .

HgO-EXT. >1000. 1 0 0 0 (2 a) H,0-EXT. > .

MeOH 228.8 MeOH > 1 0 0 0 . CHCI3 44.9 CHOU 911. Stems BuOH >1000. BuOH > 1 0 0 0 . Stem-bark (4b) H 2 O-FRAC. >1000. H.O-FRAC. > 1 0 0 0 .

H.O-EXT. >1000. H.O-EXT. > 1 0 0 0 . (2 b) MeOH 755. CHOU 263. Roots BuOH > 1 0 0 0 .

(4c) H,0-FRAC. > 1 0 0 0 .

HgO-EXT. > 1 0 0 0 .

Table 3.6 Activity Against Brine Shrimp of the Tabie 3.7 Activity Against Brine Shrimp, of the Extracts from Ruagea glabra Extracts from Abuta dwyerana

83 PLANT PART EXTRACT BRINE SHRIMP PLANT PART EXTRACT BRINE SHRIMP

(Code N°) LC5 0 pg/mi (Code N°) LC5 0 pg/mi

MeOH > 1 0 0 0 . MeOH > 1 0 0 0 .

CHCI3 579. CHCI3 >1 0 0 0 .

Aerial BuOH > 1 0 0 0 . Aerial BuOH > 1 0 0 0 . parts parts H2 O-FRAC. > 1 0 0 0 . H2O-FRAC. > 1 0 0 0 .

(1 0 a) (8a) H2 O-EXT. > 1 0 0 0 . H2O-EXT. > 1 0 0 0 .

MeOH 275. MeOH >1 0 0 0 .

CHCig 62.1 CHCI3 > 1 0 0 0 .

Roots BuOH > 1 0 0 0 . BuOH > 1 0 0 0 . Roots H2O-FRAC. > 1 0 0 0 . 1 0 0 0 (1 0 b) H2O-FRAC. > . (8b) H.O-EXT. > 1 0 0 0 . H2O-EXT. > 1 0 0 0 .

Table 3.8 Activity Against Brine Shrimp of the Tabie 3.9 Activity Against Brine Shrimp of the Extracts from Cephaelis camponutans Extracts from Cephaëlis dichroa

84 PLANT PART EXTRACT BRINE SHRIMP PLANT PART EXTRACT BRINE SHRIMP (Code N°) LC^ pg/ml (Code N®) LC5 0 pg/ml MeOH > 1 0 0 0 . CHCI3 605. MeOH > 1 0 0 0 .

CHCI3 > 1 0 0 0 . Leaves BuOH 514.

H2O-FRAC. > 1 0 0 0 . BuOH > 1 0 0 0 . (9a) Aerial parts HgO-EXT. > 1 0 0 0 . H2 O-FRAC. > 1 0 0 0 .

( 1 1 a) MeOH > 1 0 0 0 . H2 O-EXT. > 1 0 0 0 .

CHCI3 > 1 0 0 0 . MeOH > 1 0 0 0 . BuOH >1000. CHCI3 772.8 Stems H 2 O-FRAC. >1000. BuOH >1000. (9b) H 2 O-EXT. >1000. Roots H 2 O-FRAC. >1000. MeOH > 1 0 0 0 .

CHOU 876. (1 1 b) H 2 O-EXT. >1000. Roots BuOH > 1 0 0 0 . H,0-FRAC. > 1 0 0 0 .

(9c) HpO-EXT. > 1 0 0 0 .

Table 3.10 Activity Against Brine Shrimp of Table 3.11 Activity Against Brine Shrimp of the Extracts from Cephaëlis dimorphandrioides the Extracts from Cephaëlis glomerulata

85 PLANT PART EXTRACT BRINE SHRIMP PLANT PART EXTRACT BRINE SHRIMP

{Code N°) LC5 0 pg/mi (Code N®) LC5 0 pg/mi MeOH >1000. MeOH 511.6

CHCI3 >1000. CHCI3 > 1 0 0 0 . Aerial BuOH >1000. BuOH 485. parts Leaves HgO-FRAC. >1000. HgO-FRAC. > 1 0 0 0 (7a) (5a) H2O-EXT. >1000. H2O-EXT. > 1 0 0 0 MeOH >1000. MeOH 664. CHCI3 >1000. CHCI3 109.7

BuOH >1000. BuOH > 1 0 0 0 . Roots Stems H 2 O-FRAC. >1000. H 2 O-FRAC. > 1 0 0 0 . (7b) (5b) H 2 O-EXT. >1000. H 2 O-EXT. > 1 0 0 0 .

Table 3.12 Activity Against Brine Shrimp of the Tabie 3.13 Activity Against Brine Shrimp of the Extracts from Lasianthus panamensis Extracts from Picramnia antidesma subsp. fessonia

86 PLANT PART EXTRACT BRINE SHRIMP

(Code N°) LC5 0 pg/mi

MeOH > 1 0 0 0 .

CHCI3 > 1 0 0 0 .

BuOH > 1 0 0 0 . Leaves HgO-FRAC. > 1 0 0 0 .

(6 a) HgO-EXT. > 1 0 0 0 .

MeOH > 1 0 0 0 .

CHCI3 736.2

BuOH > 1 0 0 0 . Roots H2 O-FRAC. > 1 0 0 0 .

(6 b) H2 O-EXT. > 1 0 0 0 .

MeOH > 1 0 0 0 .

CHCI3 395.9

BuOH > 1 0 0 0 . Branches

H2 O-FRAC > 1 0 0 0 .

(6 0 ) H2 O-EXT. > 1 0 0 0 .

MeOH > 1 0 0 0 .

CHCI3 416.2

BuOH > 1 0 0 0 . Stems

H2 O-FRAC. > 1 0 0 0 .

(6 d) H2 O-EXT. > 1 0 0 0 .

Table 3.14 Activity Against Brine Shrimp of the Extracts from Picramnia teapensis

87 KB ceil Plant name Plant part Extract LCso(pg/ml)

Branch MeOH 87.4

Branch BuOH 402.3

Branch CHCI3 38.2

Guarea macropetala Leaves MeOH 96.9

Leaves CHCI3 54.9

Branch MeOH 86.08

Branch CHCI3 38.5

Wood MeOH 50.4 Guarea rhopalocarpa Wood CHCI3 29.1

Leaves MeOH 89.6

Leaves CHCI3 32.6

Leaves CHCI3 81.6 Ruagea glabra Stem bark CHCI3 78.7

Leaves MeOH >500

Leaves BuOH 320 Abuta dwyerana Leaves CHCI3 338.8

Roots CHCI3 54.8

Cephaelis camponutans Roots CHCI3 27.54

Picramnia antidesma Leaves BuOH 8.7 subsp. fessonia Stems CHCI3 127.4

Table 3.15 Activity Against KB Ceils of Extracts Selected on the Basis of Previously Activity Against Brine Shrimp.

88 Section 4. Phytochemistry 4.1 Material and General Methods 4.1.1 Plant material

More than 30 plants species were selected for collection either because of their traditional use or because of chemotaxonomic considerations. The botanical descriptions and the voucher specimens of each plant were carefully studied at the Herbarium of the University of Panama before each trip collection. The trips were organized by Mrs Carmen Galdames, a final year student of botany and who is working at the Centre for Pharmacognostic Research (CIFLORPAN) in the Faculty of Pharmacy, University of Panama. The collections were made, mainly, in three different places including Western, Middle and Eastern Panama.

Eleven plants were collected and a voucher specimen was prepared for each plant each time collected. Sample were deposited at the Herbarium of the University of Panama and authenticated by Prof. Mireya Correa. The plants were separated into different part ie. leaves, roots, stembark, branches, etc., air dried, chopped and powdered (1 mm) using a Wiley mill (Thomas Scientific, USA) before despatch to the School of Pharmacy, University of London. Table 4.1 (p.91) shows the date, place of collection, voucher number, plant part collected and the amount of dried powdered plant collected; the botanical description of each plant has been presented in Section 3.

4.1.2 Solvents

Methanol was purchased in bulk and distilled before use. All other solvents were reagent grade and obtained from May & Baker or BDH.

90 Plant Name Place of Date Voucher Plant Dry Collection Number Part Weight (g) Guarea Pemasky 10/Dec/90 572 Branch 1976 macropetala San Bias Leaves 732 Guarea Fortune 2/Feb/91 643 Branch 994 rhopalocarpa Chiriqui Wood 1040 Leaves 763 Ruagea glabra Fortune 5/Feb/91 691 Leaves 1014 Chiriqui Stembark 679 Abuta dwyerana Cerro Azul 19/Nov/90 544 Leaves 316 Panama Stem 915

Root 1 2 0 Cephaëlis Pemasky 11/Dec/90 578 Aerial parts 1080 camponutans San Bias Root 386

Pemasky 9/Oct/92 1 2 2 0 Leaves 1071 San Bias Stem 431 Root 451 Cephaëlis dichroa Fortune 5/Jun/91 755 Aerial part 1613 Chiriqui Root 451 Cephaëlis Fortune 4/Jun/91 754 Leaves 712 dimorphandrioides Chiriqui Stem 2951 Root 836 Cephaëlis Pemasky 11/Jan/91 599 Aerial part 975 glomerulata San Bias Root 206 Lasianthus Pemasky 12/Dec/90 594 Aerial part 1205 panamensis San Bias Root 174 Picramnia El Valle 18/May/90 331 Leaves 827 antidesma subsp. Code fessonia Stem 619 Picramnia Fortune 3/Feb/91 646 Leaves 509 teapensis Chiriqui Root 357 Branch 1097 Stem 1147 ^Locality and Province ^Voucher number should be referred as for example CIFLORPAN 572 (PMA)

Table 4.1 Plants Collected in Panama

91 4.1.3 Chromatography techniques

Thin layer chromatography (TLC) was carried out on pre-coated silica gel GF-254 aluminum backed plates (Merck Ltd). Column chromatography (00) was performed on silica gel 60, 70-230 mesh (Merck Ltd). Preparative TLO (pTLO) plates were prepared with silica gel GF-254 (Merck Ltd) using a Oamag silica gel spreader adjusted to 0.75 mm; plates were activated by heating at

1 1 0 ° 0 overnight, washed by running in acetone and air dried before use.

Visualization, of both TLO and pTLO, was by means of short (254 nm) and long (365 nm) ultra-violet light and spray reagents were also used. Dragendorff’s reagent, iodoplatinate (Stahl, 1969) and ferric chloride in perchloric acid (Jork et al., 1990) were used to detect alkaloids. Kedde’s reagent, to detect a-p unsaturated five membered lactone, and KOH in methanol, to detect anthraquinones were prepared as previously described (Stahl, 1969).

4.1.4 Spectroscopic methods 4.1.4.1 Ultraviolet spectroscopy (UV)

Spectra were recorded in MeOH using a Perkin Elmer model 402 double beam spectrometer. The alkaloids from Cephaëlis glomerulata were recorded on a Perkin Elmer model 550 SE double beam spectrometer.

4.1.4.2 Infrared spectroscopy (IR)

Spectra were recorded in KBr pellets or preparing a thin film on NaCI cells using a Perkin Elmer model 841 infrared spectrometer. The alkaloids isolated from C. glomerulata \vere recorded on a Perkin Elmer model 1600 FT Fourier transformed infrared spectrometer.

92 4.1.4.3 Mass spectrometry (MS)

Electron-impact mass spectra (EIMS) and accurate mass determination were made on a VG-Analytical instrument ZABI F mass spectrometer at 70 eV with an inlet temperature of 180 - 200 °C unless otherwise stated.

Chemical ionization mass spectra (CIMS) were recorded on a VG massiab 12-250 quadrupole instrument using ammonia as ionising gas unless otherwise stated.

Fast atom bombardment mass spectra (FABMS) were recorded on a VG analytical ZABSE spectrometer with xenon as the atom source at 8 eV using the matrix indicated on each spectrum.

4.1.4.4 Nuclear magnetic resonance (NMR)

For the alkaloids isolated from C. dichroa the NMR spectra were recorded at 250 MHz and the ^^0 NMR at 62.9 MHz on a Bruker WM250 in the solvent indicated with IM S (tetramethylsilane) as internal standard. H-C correlation and COSY 45 spectra were also recorded on a Bruker WM250.

All other ^H NMR spectra were recorded at 400 MHz and the NMR at 100.6 MHz on a Bruker AMX-400 in the indicated solvent with IM S as internal standard. Two dimensional experiments were also recorded on an AMX-400 nuclear magnetic resonance spectrometer.

4.1.4.5 Polar! metry

The [a]° were recorded at 2 0 °C in the solvent and concentration indicated on a Perkin Elmer model 241 polarimeter using a 10 cm cell.

93 4.1.4.6 Circular dichroism (CD)

The circular dichroism spectra were recorded on a Jasco J-600 spectropolarimeter in the solvent and concentration indicated.

4.1.5 Preparation of acetyl derivatives

Acétylation was carried out by taking up the compound in pyridine and acetic anhydride and keeping the mixture in the dark overnight. The mixture reaction was dried in a rotary evaporator by azeotroping with toluene to lower the boiling point of the mixture.

4.2.1 Preparation of crude plant extracts for biological test

Twenty grams of each plant part were extracted twice, successively, by maceration with petrol ether 24 hrs each time. The solvent was removed by filtration under vacuum and both extracts were combined and evaporated in rotary evaporator. The plant material was air dried before further extraction with methanol (MeOH) using the procedure as described above and ultimately the plant material was extracted with distilled water (HgO-Ext.) using the same method.

A portion of the methanolic extract was partitioned between chloroform (CHCy and water (HgO-Frac.); the aqueous layer was , then, extracted with butanol (BuOH). The aqueous extract (HgO-Ext.) and the fraction (HgO-Frac.) were lyophilised. Figure 4.1 (p.95) shows the scheme of the extraction procedure.

4.2.1 . 1 Results A total of 162 extracts were obtained and the amount of each extract is presented in Table 4.2 (p.96-97).

94 Dried Plant Material

Double macer at i on • with pet r ol et her t or 24 hr s

Petrol et h er M or c

D ou bl e mocer et i on with MeOH for 24 hrs

MeOH Marc

Double E vapor at i on macer at i on with H 2 O R esi du e for 24 h rs

Extr act Parution between CHCI 3 / H 2 O 1 /5I i ophyl i zed

CH Cl

/BuOH

BuOH I H 2O F r ac

Figure 4.1 Extraction and Fractionation Procedure.

95 Plant Name Plant Dry Petrol MeOH: MeOH: CHCI3 HjO- BuOH HjO Part Weight ether Frac. Ext*

Guarea macropetala Branch 2 0 0.153 0.961 0.843 0.398 0.191 0.068 0.109

Leaves 2 0 0.545 0.840 0.682 0.290 0.187 0.193 0.234

Guarea rhopalocarpa Branch 2 0 0.126 1 . 0 0 0 0.861 0.113 0.140 0.470 0.107

Wood 2 0 0.080 0.762 0.506 0.123 0 . 1 1 0 0.217 0.081

Leaves 2 0 0.349 0.904 0.788 0.247 0.064 0.094 0.242

Ruagea glabra Leaves 2 0 0.302 1.823 1.628 0.627 0.404 0.589 0.243

Stembark 2 0 0.104 1.629 1.390 0.116 0.229 0.830 0.134

Abuta dwyerana Leaves 15 0.259 1.089 0.734 0.205 0 . 1 2 1 0.247 0.113

Stem 15 0.043 0.903 0.577 0.058 0.136 0 . 2 2 2 0.053 Root 15 0.018 0.678 0.586 0.050 0.214 0.245 0.099 Cephaëlis Aerial parts 20 0.164 2.187 1.528 0.343 0.853 0.286 0.140 camponutans Root 2 0 0.042 1.590 1.398 0.090 0.965 0.172 0.253

^All values in gram ^Total extract obtained ^Portion of the methanolic extract used for liquid partition ^Represent 1/5 of the total extract

Table 4.2 Crude Extracts for Biological Test^

96 Plant Name Plant Dry Petrol MeOH^ MeOH* CHCI3 HjO- BuOH H2 O Part Weight ether Frac. Ext/

Cephaëlis dichroa Aerial part 2 0 0 . 0 2 0 1.323 0.895 0.305 0.171 0.277 0.207

Root 2 0 0.014 1.107 0.616 0 . 1 1 2 0.205 0.167 0.146

Cephaëlis Leaves 2 0 0.257 1.143 1.018 0.626 0.141 0.139 0.275 dimorphandrioides Stem 2 0 0.031 1.008 0.608 0.109 0.260 0.174 0.145

Root 2 0 0.024 0.466 0.306 0.063 0.078 0.083 0.103

Cephaëlis glomerulata Aerial part 2 0 0.063 1.142 0.539 0 . 2 0 1 0.118 0.192 0.154

Root 2 0 0.024 0.752 0.596 0.098 0.230 0.140 0 . 1 2 1

Lasianthus Aerial part 2 0 0.070 2.685 1.758 0.370 0.940 0.350 0.213 panamensis Root 2 0 0.019 1.483 0.961 0.127 0.550 0.143 0.259

Picramnia antidesma Leaves 2 0 0.109 3.610 2.335 0.558 0.466 1.238 0.158 subsp. fessonia Stem 2 0 0.009 1.133 0.834 0.083 0.183 0.569 0.172

Picramnia teapensis Leaves 2 0 0.041 1.795 1.177 0.555 0.260 0.261 0.237

Root 2 0 0.014 1.403 1.089 0.062 0.160 0.790 0.084

Branch 2 0 0.027 1.345 0.881 0.058 0.157 0.556 0.095

Stem 2 0 0.007 1.036 0.829 0.077 0.318 0.388 0.152

Table 4.2 Continued.

97 4.3 Isolation and Identification of bloactlve compounds from Cephaëlis camponutans 4.3.1 Extraction procedure

The dried and powdered woody parts (600 g) of Psychotria camponutans were extracted with MeOH by percolation. The extract was filtered and concentrated in vacuo, the residue (60 g) was partitioned between CHCI 3 and

HgO. The CHCI 3 fraction (3.2 g) showed activity against brine shrimps (see Table 3.8, p.84), and was submitted to a bioactivity-guided fractionation by repeated column chromatography (1.5 x 20 cm), using silica gel 60 (70-230 mesh) and CHCl 3 -EtOAc (9:1) as eluent. Two major compounds were obtained, 1-hydroxybenzoisochromanquinone (87 mg) and benz[g]isoquinoline- 5,10-dione(6img^he structure elucidation was achieved by MS, UV, IR, ^H, and HETCOR NMR spectroscopy.

More recently collected plant material (October 1992) (see Table 4.1, p.91) was carefully cleaned and entirely dried, in order to determine whether the isolates were from a specific part of the plant or from fungal contamination. Sabouraud plates were inoculated with the plant material and incubated for 72 hrs at 25 °C, the resulting fungal mass was extracted with MeOH, filtered and concentrated, the residue was compared on TLC (CHCl 3 -EtOAc 9:1). The root bark was carefully separated from the wood of 25 g of roots, and both root bark and wood were, separately, extracted with MeOH-CHCl 3 (1 :1 ), the extracts were compared on TLC (CHCl 3 -EtOAc 9:1).

4.3.2 Identification of the isoiated compounds

4.3.3. 1 1-Hydroxy benzoisochromanquinone 4.3.3.1.1 Spectrai data

UV X(MeOH) max. nm: 211, 245, 271 sh

98 IR D (KBr) cm ’ : 3450, 1660, 1595 Elemental analysis

Found: 68.37 % (C), 4.43 % (H), 27.22 % (O); calculated values for C 1 3 H1 0 O4 : 67.81 % (C), 4.38 % (H), 27.81% (O) EIMS (see spectrum 1 in Appendix I) m/z (relative intensity %): [M]" 230 (10), 212 (70), 201 (15), 184 (100), 173 (15), 156 (35), 128 (85), 115 (24), 104 (32), 76 (45) HRMS m/z calculated [M]+ 230.0579 (C 1 3 H1 0 O4 ); found 230.0572. m/z calculated [M-

H2OY 212.0473 (C1 3 H8 O3 ); found 212.0464 NMR

and NMR assignment are given in Table 4.3 (p. 100) and 4.4 (p. 1 0 1 ) respectively. HETCOR was used to achieve this assignment. Appendix 1 shows the and NMR of 1-hydroxybenzoisochromanquinone (Spectra 2 and 3, respectively).

4.3.3.1. 2 1-Acetylbenzolsochromanquinone 4.3.3.1.2.1 Spectral data

NMR (see Spectrum 4 in Appendix I) and NMR are shown in Table 4.3 (p. 100) and 4.4, respectively.

4.3.3.2 Benz[g]lsoqulnollne-5,10-dlone 4.2.3.2.1 Spectral data

UV X (MeOH) max. nm: 209, 249 IR x> (KBr) cm"*: 1680, 1580, 1300,700

99 Compounds

PROTON 1-Hydroxybenzoisochroman 1 -Acetylbenzoisochroman Benz[g]isoquinollne- quinone‘^ qulnone^ 5,10-dione‘'

H-1 5.3 (1H)t (3.6) 6.43 dd (1H) (2.02,4.0) 9.57 (1H) s

H-3 4.84-4.63 (2H) ddt (3.0, 18.5) 4.62-4.78 (2H) ddd (1.56,2.3,18.8) 9.12 d (1H) (5.0)

H-4 2.84-2.64 (2H) ddq (3.0,18.9) 2.78 - 2.91 (2H) m 8.08 d (1H) (5.0)

H-6.9 7.76-7.72 (2H) m 7.73-7.77 (2H) m 8.34-8.30 (2H) m

H-7,8 8.10-8.03 (2H) m 8.13-8.06 (2H) m 7.90-7.83 (2H) m

Me 2.09 (3H) s — —

^Chemical shifts are in 5 values (ppm) with coupling constants J in Hz. ‘’Measured in CDCI3 + MeOD. ‘'Measured in CDCI3.

Table 4.3 NMR Spectral Data" of Compounds Isolated from C. camponutans.

100 Compounds Carbon

la° 2 °

C- 1 90.49 (CH)"* 89.19 (CH) 155.36 (CH)

C-3 57.93 (CHg) 57.95 (CHg) 149.65 (CH)

C-4 28.44 (CHg) 26.11 (CH;) 118.93 (CH)

C-4a 139.97 (C) 138.26 (C) 126.11 (C)

C-5 183.93 (C) 182.70 (C) 182.39 (C)

C-5a 131.87 (C) 131.63 (C) 132.92 (C)“

C- 6 134.04 (CH) 133.87 (CH) 135.01 (CH)

C-7 126.25 (CH) 126.18 (CH) 127.27 (CH)

C" 8 126.56 (CH) 126.46 (CH) 127.32 (CH)

C-9 133.99 (CH) 133.92 (CH) 138.33 (CH)

C-9a 132.12 (C) 131.78 (C) 132.92 (C)

C-10 183.44 (C) 183.05 (C) 182.47 (C)

C-lOa 141.74 (C) 140.66 (C) 138.33 (C)

C- 1 (C=0) - 169.32 (C) -

C-1 - 20.90 (CH;) - (COMe)

Chemical shifts are in 6 -values in ppm. ‘^Measured in CDCIj-MeOD.3' ""Measured in CDCI3. "^Multiplicity from Dept ^^0 NMR experiment. ®This signal integrates for two quaternary carbons in an inverse gated decoupled NMR experiment, giving almost quantitative intensities.

Compound 1 , 1-hydroxybenzoisochromanquinone; 1 a, 1 - acetylbenzoisochromanquinone; 2, benz[g]isoquinoline-5,10-dione

Table 4.4 NMR Spectral Data‘ of the Compounds Isolated from C. camponutans

101 EIMS (see Spectrum 5 in Appendix I) m/z (relative intensity %): [M]" 209 (100), 181 (55), 153 (65), 126 (40), 83 (20), 76 (24), 50 (27) HRMS m/z: calculated [M]" 209.0477 (CigH^NOg); found, 209.0474. m/z calculated [M-28r, 181.0528 (C^gHyNO); found, 181.0521. m/z calculated [M-56]",

153.0578 (C 1 1 H7 N); found 153.0574. NMR and NMR are shown in Table 4.3 (p. 100) and 4.4 (p. 101) respectively.

Spectra 6 and 7 in appendix I shows the and NMR of benz[g]isoquinoline-5,10-dione.

4.3.4 Discussion

Two benzoquinones have been isolated from the woody parts of C. camponutans, as a result of bio-activity guided fractionation using brine shrimps. Figure 4.2 (p. 103) shows the chemical structures of the compounds isolated from this plant and the acetyl derivative of 1 - hydroxibenzoisochromanquinone.

The UV spectrum of 1-hydroxibenzoisochromanquinone showed a conjugated ketone band at 245 nm and the IR spectrum showed absorption typical of C=0 (1660 cm'^). The HRMS showed a [M]^ peak at m/z 230.0572 corresponding to the molecular formula C 1 3 H1 0 O4 . The EIMS spectrum showed a weak [M]^ m/z 230 (10 %), loss of water [M-18r m/z 212 (70 %) and consecutive loss of two C=0 moieties, typical for quinones (Budzikiewicz et al., 1967), as shown by peaks at m/z 184 (100 %) and 156 (35 %). A portion of the total amount of compound analyzed lost the two C=0 moieties without losing the water, as demonstrated by peaks at m/z 2 0 1 (15 %) \ [M-29r and [M-56]^ m/z 173 (15%). Figure 4.3 (p.106) shows the mass spectral fragmentation of 1 -hydroxybenzoisochromanquinone.

102 8

1 -OH-Benzoisochromanquinone

8

1 -Aoetylbenzolsochromanquinone

Benz[g]lsoqulnollne-5,10-cllone

Figure 4.2 Compounds Isolated from Cephaëlls camponutans and the Acetyl Derivative of 1-Hydroxlbenzolsochromanquinone.

103 The NMR spectrum (CDCI3, 400 MHz) showed four aromatic protons typical of a 1,2 substituted benzene ring and a fine triplet at 5 5.3 ppm assigned for the proton on C-1. A double doublet at Ô 4.84-4.63 ppm (J=3.01 and 18.5 Hz) showing geminal coupling constant, was assigned to protons on 0-3 and the double doublet at Ô 2.84-2.64 ppm, that showed similar coupling constant to the latter, was assigned to the protons on 0-4. ^^0 NMR showed a quasi symmetric molecule in the aromatic region, including two very similar carbonyl signals. The signal at 6 90.49 ppm typical of a carbon supporting two oxygens, and the ^^0 DEPT experiment showed a OH signal for this carbon. Furthermore, the acétylation of 1 -hydroxibenzoisochromanquinone confirmed the presence of an OH group, showing in ^H NMR of 1 -acetylbenzoisochromanquinone a three protons singlet at S 2.09 ppm signal for the methyl of the acetyl group and the triplet, that in 1 -hydroxibenzoisochromanquinone is at 6 5.3 ppm, being shifted 1.1 ppm down field, indicating the position of the OH group. This is the first report of the occurrence of 1 -hydroxybenzoisochromanquinone from natural or synthetic sources.

The UV of benz[g]isoquinoline-5,10-dione showed absorption at 249 nm for a conjugated ketone and the IR showed carbonyl band at 1680 cm'\ The HRMS spectrum of benz[g]isoquinoline-5,10-dione showed a strong [M]^ peak at 209.0474 corresponding to the formula C 1 3 H7 NO2 . EIMS spectrum showed a strong [M]"^ m/z 209 ( 1 0 0 ), consecutive loss of two C=0 moieties m/z 181 (55) and 153 (65), and loss of HCN m/z 126 (40); the mass fragmentation is showed in Figure 4.4 (p. 107). The ^H NMR showed seven aromatic protons, a singlet at 5 9.57 ppm was assigned to the proton on C- 1 , two doublets coupling to each other at Ô 9.12 ppm (J=4.95 Hz) and 5 8.08 (J=5.0 Hz) were assigned to protons on 0-3 and 0-4 respectively; the other two signals integrating for two protons were characteristic of a 1 ,2 -substituted benzene ring. NMR showed a quasi symmetric molecule, including two identical quaternary carbons at 5 132.92 assigned to 0-5a and 0-9a; there were also two very similar signal for carbonyl groups and two pairs of OH for the benzene ring. The isolated compound was identical on TLO (OHOl 3 -EtOAc 9:1) to a

104 commercial sample of benz[g]isoquinoline-5,10-dione, obtained from Aldrich Chemical Co. U.K.; also, the NMR spectra were superimposable.

Benz[g]isoquinoline-5,10-dione does not react with 2% KOH in MeOH, as the other quinones, was not sensitive to Dragendorff’s reagent (only reacting at high concentrations), and only reacted with iodoplatinate when the plate was previously sprayed with 5% H 2 SO4 .

Benz[g]isoquinoline-5,10-dione has been isolated as an impurity from commercial acridine (Walton et al., 1983), but this is the first report of its occurrence in the plant kingdom. However, the skeleton, but not the benz[g]isoquinoline-5,10-dione itself has been found in Nectria haematococca, a fungus (Parisot et al., 1990). In order to ascertain that benz[g]quinone-5,10- dione was a constituent of C. camponutans, and not from a fungal contaminant, the powdered plant material was incubated to produce fungal biomass. Extraction obtained from the sabouraud plates was compared on TLC and showed that 1-hydroxibenzoisochromanquinone and benz[g]isoquinoline-5,10- dione were not present. When the extraction of separated root bark and wood were compared on TLC the results showed that 1 - hydroxybenzoisochromanquinone was mainly in the root bark, while benz[g]isoquinoline-5,10-dione was only detected in the root wood. It is reported that anhydrofusarubin lactol, a compound with a similar skeleton to 1 - hydroxibenzoisochromanquinone can react with ammonia at room temperature to yield bostrycoidin (Parisot et al., 1989), a compound with similar skeleton of benz[g]isoquinoline-5,10-dione.

In order to establish that compound benz[g]isoquinoline-5,10-dione was not an artifact, 1-hydroxibenzoisochromanquinone was suspended in MeOH and concentrated ammonia (1:1) and allowed to stand for 72 hours at ambient temperature. TLC showed that benz[g]isoquinoline-5,10-dione was absent, even after warming the mixture at 100 °C for 2 hrs. Therefore, the possibility that benz[g]isoquinoline-5,10-dione is an artifact formed during extraction or for

105 -H , G

OH m /z 230.0579 m/c 212.0473

CO -CO -H

m fi 201 m lz 184

-CO -CO

OH

;CC^H n tfz 173 m fz 156

m fi 128

Figure 4.3 Putative Mass Spectral Fragmentation of

1 -Hydroxybenzoisochromanquinone

106 O

m fz 209.OS74 C 1 3 H7 N O2

m fz 181.0528 C 12 N O

-co

a :

m à 153.0574 C „ H7 N,

CNH

_ n :

m fz 125.0446 CiQ Hg

Figure 4.4 Putative Mass Spectral Fragmentation of Benz[g]lsoqulnollne-5,10-dlone.

107 the presence of ammonia in nature may be excluded, as well as, the possibility that it is due to fungal contamination.

4.3.5 Biological activity of the isolates 4.3.5.1 Results and discussion

Table 4.5 (p. 108) shows the activity of 1 -hydroxibenzoisochromanquinone, 1-acetylbenzoisochromanquinone and benz[g]isoquinoline-5,10-dione against brine shrimps, KB cells and P. falciparum. Benz[g]isoquinoline-5,10-dione was

three times as active, against brine shrimps, than 1 - hydroxibenzoisochromanquinone. In KB cells the activity of 1- hydroxibenzoisochromanquinone and benz[g]isoquinoline-5,10-dione was

similar, but 1 -acetylbenzoisochromanquinone was less active.

Brine Shrimps KB ceils P. falciparum Compounds LCso±sd (pM/mi) LCso±sd (pM/mi) iCgotsd (pM/mi)

1 31.83±4.30 (2)“ 8.06±0.30 (2) 11.56±2.96 (2)

la 17.24±0.92 (2) 11.80±3.93 (2) 2 2 .1 2 ± 0 . 6 6 (2 )

2 11.72±1.48 (3) 7.75±1.44 (2) 4.02±1.53 (2)

Control'* 36.35±3.42 (3) 1.50±0.56 (3) 0.61±0.01 (2 )

“Number of test performed, in triplicate '’Emetine was used for Brine shrimps and KB cell tests, Chloroquine diphosphate for P. falciparum

Compound 1 , 1 -hydroxybenzoisochromanquinone, 1a, 1 - acetylbenzoisochromanquinone, 2, benz[g]isoquinoline-5,10-dione.

Table 4.5 Activity of the Compounds isolated from P. camponutans Against Brine Shrimps, KB Ceils and P. falciparum.

108 Benz[g]isoquinoline-5,10-dione was some three times more active than 1- hydroxibenzoisochromanquinone and some 5 times more active than 1- acetylbenzoisochromanquinone against multi-drug resistant P. falciparum (K1 ).

1-Hydroxibenzoisochromanquinone was four times more active against KB cells and P. falciparum than against brine shrimps, while its acetylated derivative appears to have the same value in the three bio-assays. 1 - acetylbenzoisochromanquinone was less active against both, KB cells and P. falciparum, than the starting compound 1 -hydroxibenzoisochromanquinone, an increase in lipophilicity, apparently, decreased its activity, but the converse was observed against brine shrimps. Although, benz[g]isoquinoline-5,10-dione has been reported as mutagenic against insects (Walton et al., 1983), the results obtained against the malaria parasite are promising for further studies of this kind of compounds.

109 4.4 Isolation of Alkaloids fromCephaëlis dichroa. 4.4.1 Extraction and separation procedure

Dried and powdered aerial parts (900g) of C. dichroa (Standley) Standley were extracted with MeOH by percolation. The extract was filtered and concentrated to a gum (70.5g) under vacuum and then partitioned between HgO and CHCI3 ; the aqueous layer was extracted with BuOH, the extract was concentrated under vacuum and the residue (11.2g) was taken up in MeOH and precipitated with MegCO. The soluble fraction was submitted to CC using EtOAc-EtOH-HgO (2:1:2 upper layer) as eluent, strictosidine (2.9 g) was obtained and strictosidine lactam was also detected. The remaining aqueous fraction was basified to pH ~9 with NH 4 OH and extracted with CHCI3 , the CHCI 3 extracts were dried on anhydrous NagSO^ and concentrated under vacuum, this fraction (0.676g) was submitted to CC using CHCl 3 -MeOH (9:1) and EtOAc-

EtOH-HgO ( 2 :1 : 2 upper layer) as eluent, vallesiachotamine (59.3 mg), strictosidine lactam (134.1 mg) were obtained, strictosidine and angustine were also detected.

The CHCI3 fraction was extracted with 2 N HCI, filtered, basified to pH-9 using NH 4 OH and re-extracted with CHCI3 ; extracts were dried on Na 2 S0 4 and concentrated under vacuum. This fraction (0.401 g) was submitted to CC using

CHCI3 , CHCl3 -MeOH (9:1 and 8 :2 ) as eluents. Vallesiachotamine (38.7 mg) and angustine (3.4 mg) were isolated, and vallesiachotamine-lactone (1.5 mg) was further purified by p-TLC using CHCl 3 -EtOAc (1 :1 ) as solvent system.

4.4.2 Identification of Isolated compounds 4.4.2.1 Vallesiachotamine lactone 4.4.2.1.1 Spectral data

Chemical reactions on TLC The isolated alkaloid gave a positive reaction with Dragendorff’s reagent as well a giving a pink-purplish colour with Kedde’s reagent, which is typical of a-p five

110 membered lactone (Stahl, 1969).

UV (MeOH) X,max. 225, 285 (sh), 291 nm. IR (KBr) D max. nm \ 3430(N-H), 1750, 1740 and 1730 (C=0), 1620 (C=C).

EIMS (Spectrum 8 , Appendix I) m/z (Relative intensity %) [M]" 364(55), 349(10), 333(22), 319(5), 305(100), 221 (11) 209(25), 169(20), 143(15), 130(15), 115(15). HRMS m/z calculated value: 364.1423 (CgiHgoNgOJ; found: 364.1435. H NMR (Spectrum 9, Appendix I)

(CDCI3, 250MHz, 5): 1.85-1.92 (m, 1 H, H-14a), 2.70-2.99 (m.3H, H-5,H-14p),

3.68 ( 8 , 3H,COOMe-16),3.48-3.75 (m, 2 H, H-6 ), 3.87 (m, 1 H, H-15), 4.28 (dd,

J=12.6, 1H, H-3), 4.87 (m, 2H, H-18), 7.09-7.22 (m, 3H, H-10, H- 1 1 , H-19),

7.35 (d, J=7.64, 1H, H-12), 7.48 (d, J=7.29, 1H, H-9), 7.74 (s, 1 H, H-17), 8.14

(br 8 , 1 H, N-H). A ^H-^H COSY-45 (Spectrum 10, Appendix I) was used to confirm this assignment.

4.4.2.2 Vallesiachotamine 4.4.2.2.1 Spectral data

UV (MeOH) Xmax. 222, 285 sh, 291 IR (KBr) D max. cm '\ 3250, 2840, 2910, 2950, 1680 (0=0), 1660 (0=0), 1635, 1620, 1425, 1310, 1230, 1190, 750.

EIMS (Spectrum 1 1 , Appendix I) m/z (Relative intensity %) 350 (39), 335 ( 2 0 ), 322 (38), 307 (28), 291 (63), 279

(100), 263 (6 ), 249 (27), 221 (67), 209 (27), 184 (22), 170 (32), 169 (28), 156 (22), 154 (21), 144 (16), 143 (16), 143 (15), 128 (22).

I ll HRMS m/ 2 calculated value; 350.1630 (CjiHjaNjOj); found: 350.1641. 'H-NMR (Spectrum 12, Appendix I)

(CDCI3 . 250 MHz, 5): 1.93 (m, 1H, H-14a), 2.10 (d, J„.„=7.28,3H, H-18), 2.17

(m, 1 H, H-14P), 2.86 (m, 2 H, H-6 ), 3.64 (s, 3H, COOCHj-16), 3.73 (m, 2 H, H-

5), 4.02 (dd, J=4.57,1H, H-15), 4.48 (dd, J=11.7,1H, H-3), 6 . 6 8 ( q, J,s.„=7.30,

1 H, H-19), 7.08-7.20 (m, J=1.26, 7.24, 2 H, H-10, H-11), 7.30 (dd,J=1.48, 7.13,

1H, H-12), 7.49 (dd,J=7.25, 1H, H-9), 7.68 (s, 1H, H-17), 7.95 (br S, 1H, N-H),

9.37 (S , 1H, H-21). "C NMR

(62.9 MHz, CDCy 5 132.5 (C- 2 ), 49.4 (C-3), 50.7 (0-5), 2 2 . 1 (0-6), 108.0 (0- 7), 126.8 (0-8) 118.1 (0-9), 119.8 (0-10), 122.1 (0-11), 111.1 (0-12), 136.3(0- 13), 34.1 (0-14), 30.9 (0-15), 94.2 (0-16),168.4 (000-16), 51.1 (-OMe-16), 147.5 (0-17), 15.0 (0-18), 152.8 (0-19), 146.4 (0-20), 195.9 (0-21). 'H-” 0 heterocorrelatlon was used to achieve this assignment.

4.4.2.3 Strictosidine 4.4.2.3.1 Spectral data

UV (MeOH) Xmax. 227, 283, 289. IR (KBr) T) max. cm ’ : 2900,1660, 1630,1450,1325, 1090. FARMS (Spectrum 13, Appendix I) (MNOBA + Na matrix) m/z (relative intensity %) 531 (4), 329 (16), 236 (20), 176 (100), 154(25), 136(24). ’H-NMR (Spectrum 14, Appendix i)

(OD3OD, 400 MHz, 8): 1.90-2.08 (m, 2 H, H-14), 2.64-2.73 (m, 2H, H-6 , H-20),

2.79-2.82 (m, 1 H, H-6 ), 2.91-2.96 (m, 1 H, H-5), 3.00-3.05 (m, 1 H, H-15), 3.23-

3.45 (m, 5H, H-2',H-3', H-4’, H-5', H-5), 3.62-3.69 (m, 1 H, H-6 ’), 3.75 (s, 3 H, H-

23), 3.95-3.98 (m, 2H, H-6 ’, H-3), 4.80 (d, J=7.8, 1H, H- 1 ’), 5.19-5.32 (ddd,

J=0.9, 11.0, 17.4, 2H, H-18), 5.79-5.88 (m, 2 H, H-2 1 , H-19), 6.94-6.97 (ddd.

112 J=0.9,7.2,7.6,1 H, H-10), 7.01 -7.05 (ddd, J=0.9, 6.9, 8.1,1H, H-11), 7.24-7.38 (dd, J=0.9, 7.6, 1H, H-12), 7.37 (d, 1H, H-9), 7.67 (s, 1H, H-17).

” C NMR

(100.6 MHz, CD3OD) 5 136.54 (C-2 ), 52.01 (C-3), 43.66 (C-5), 22.72 (C-6 ),

108.72 (C-7), 128.73 (C- 8 ), 118.92 (C-9), 120.00 (C- 1 0 ), 122.29 (C-11), 112.14

(C-1 2), 137.93 (C-13), 37.38 (C-14), 33.04 (C-15), 110.96 (C-16), 155.71 (C-

17), 119.55 (C-18), 136.26 (C-19), 46.16 (C-20), 97.83 (C- 2 1 ), 170.47 (C-2 2 ),

52.59 (C-23), 100.55 (C-1'), 74.89 (C- 2 ’), 78.91 (C-3’), 71.97 (C-4’), 78.23 (C-

5’), 63.26 (C-6 ’). COSY-45 (Spectrum 15, Appendix I) and 'H-'^C heterocorrelatlon (Spectrum 16, Appendix I) NMR experiments were used to confirm these assignments.

4.4.2.4 Strictosidine iactam 4.4.2.4.1 Spectrai data

UV (MeOH) Xmax. 225, 283, 291 iR (KBr) ucm ’ : 3400, 2920,1650 (C=0), 1590, 1080, 745 EiMS (Spectrum 17, Appendix I) m/z (relative Intensity %) [MJ* 498 (35), 336 ( 1 2 ), 335 (1 2 ), 319 (13), 265 (92),

235 (100), 221 (1 1 ), 206 (50), 169 (58), 143 (32), 127 (24), 115 (33), 85 (33), 73 (44) CiMS

(NH3 gas) m/z (relative Intensity): [M+ 1 ]" 499 (25), 337 (10), 267 (52), 250 (19), 237 (14), 198 (10), 180 (100), 160 (27), 145 (40), 127 (31), 115 (20), 85 (37), 69 (70). HRMS m/z calculated value 498.2002 (CjeHjoNjOe); found 498.2016 ’H NMR (Spectrum 18, Appendix I)

(CD3 OD, 250MHz, 5): 1.97-2.10 (m, 1 H, H-14a), 2.43-3.24 (m,1 OH, H-14P, H-5,

113 H-6 , H-15, H-2 ', H-3', H-4’, H-5’), 3.60-3.67 (d, J=11.7,1H, H-6 ’), 3.83-3.88 (dd,

J=2.3,11.7,1H, H- 6 ), 4.61 (d, J=7.9,1H, H-1’), 4.92-4.90 (m, 1H, H-20), 5.06 (m, 1H, H-3), 5.30-5.41 (m, 3H, H-18, H-21), 5.57-5.72 (m, J=9.8,17.2,1H, H-

19), 6.97 (m, 2 H, H-10, H-1 1 ), 7.32-7.40 (m, 3 H, H-9, H- 1 2 , H-23).

” C NMR

(62.9 MHz, CDCy 8 137.70 (C-2 ), 54.22 (0-3), 43.75 (C-5), 27.20 (C-6 ),

109.14(0-7), 128.66 (C- 8 ), 118.80 (C-9), 122.32 (C-10), 122.04 (C- 1 1 ), 112.21

(C-1 2 ), 134.57 (C-13), 21.64 (C-14), 24.68 (C-15), 110.33 (C-16), 165.14 (17),

120.36 (C-18), 133.35 (C-19), 44.56 (C- 2 0 ), 97.82 (C- 2 1 ), 148.02 (C-23),

100.51 (C-1 ’), 74.12 (C-2 ’), 77.93 (C-3 ), 71.34 (C-4 ), 77.93 (C-5’), 62.74 (C- 6 ’).

4.4.Z4.2 Acetyl strictosidine lactam 4.4.2.4.2.1 Spectral data

UV (MeOH) W ax. 227, 283, 291 IR (KBr) Dcm'^: 3400. 2940, 1760, 1660, 1498, 1220, 1065, 1040, 965. EIMS (Spectrum 19, Appendix I) m/z (relative intensity %) [M]* 6 6 6 (1 0 0 ), 624 (8 ), 564 (1 2 ), 379 (23), 331 (92), 319 (100). FABMS

(MNOBA + Na matrix) m/z (relative intensity %) [M+Na]^ 689 (100), [M]^ 6 6 6

(22), 319 ( 6 ), 265 (15), 169 (46), 109 (34). HRMS m/z calculated value 666.2425 (C 3 4 H3 8 N2 O12) found: 666.2434. H NMR (Spectrum 20, Appendix I) (CDCI3, 250MHz, Ô): 1.21, 1.88, 1.99, 2.07 (s,3H each,Me-Ac),2.03-2.19 (m,2H,H-14), 2.58-2.71 (m, 3H, H-5, H-15),2.99-3.04 (m,2H,H-6), 3.65-3.72 (m, 1H, H-3'), 4.06-4.29 (dd, 2H, H-6') 4.74-4.82 (m, 2H, H-4', H-21 ) 4.94-5.02 (m, 3H, H-3, H-20, H-5'),5.08-5.16 (m,1 H,H-2'), 5.27-5.36 (m, 3H, H-18, H-1 '), 5.53-

114 5.68 (quint, 1 H. J=9.58, 7.8, 13.5, H-19), 7.06-7.12 (t, 1 H, J=1.0, 7.0, H-10), 7.15-7.21 (t, 1H, J=1.0, 7.0, H-11), 7.36 (dd, 1H, H-12), 7.42 (S, 1H, H-17),

7.43 (dd, 1H, H-9), 8.26 (br s, 1 H, N-H). COSY-45 and 'H-” C heterocorrelatlon (Spectrum 22, Appendix I) NMR experiments were used to confirm these assignments.

"C NMR

(62.9 MHz, CDCy S 132.9 (C-2), 53.4 (C-3), 43.7 (C-5), 21.0 (C- 6 ), 108.5 (C-

7), 127.5 (C-8 ), 118.0 (C-9), 119.8 (C- 1 0 ), 122.2 (C-11), 111.3(C-12,136.1 (C- 13), 26.5 (C-14), 23.9 (C-15), 110.5 (C-16), 164.7 (C-16a), 120.8 (C-18), 132.1

(C-19), 42.8 (C-20), 94.9 (C-21), 146.9 (C-17), 94.8 (C-1), 72.0 (C- 2 '), 72.1 (C-

3 ), 70.0 (C-4’), 68.3 (C-5’), 61.7 (C - 6 ), 169.1, 169.5, 1699.9, 170.6 (COO Acetyl), 19.2, 20.5, 20.6, 20.7 (CH, Acetyl).

4.4.2.S Angustine 4.4.2.5.1 Spectral data

UV (MeOH) Xmax. 214, 376, 396.

EIMS (Spectrum 2 2 , Appendix I) m/z (relative Intensity %) [M]* 313 (99), 298 (28), 282 (9), 255 (13), 169 (5), 157 (12), 141 (11), 128 (18), 115 (17), 94 (35), 44 (43), 28 (100). HRMS m/z calculated value 313.1215 (C^gHi^NgO); found 313.1219 'H NMR

(DMSO-Dg, 250MHz, 5): 3.14 (t, 2H, J= 6 .6 , H-6 ), 4.41 (t, 2H, J=6 .6 , H-5), 5.63

(dd, 1H, J= 1.0,11.8 , H-18), 6.07 (dd, 1H, J=1.0,17.5, H-18), 7.10-7.36 (m, 4H,

H-10, H-1 1 , H-14, H-19), 7.47 (dd, 1 H, J= 2.4, 8 . 1 , H-12), 7.63 (d, 1 H, J= 7.8,

H-9), 8 . 8 8 (S, 1H, H-21), 9.24 (s, 1H, H-17), 11.82 (s, 1H, H-1).

115 2 0 jk ' O Ghi COOMe

Me

Is < h ValWmcholmmine

s

COOMe

O -G lu Vallesiachotamine Strictosidine lactam

COOMe

Vallesiachotamine lactone Angustine

Figure 4.5 Alkaloids Isolated from C. dichroa Showing Their Possible Biosynthetic Relationships.

116 4.4.3 Discussion

The structures of the isolated compounds are showed in Figure 4.5 (p.116), unexpectedly all of them are indole derivatives instead of the anticipated emetine-related alkaloids which are found in C. ipecacuanha. This figure also shows the possible biosynthetic relationship of the alkaloids isolated from C. dichroa.

The UV spectrum of vallesiachotamine lactone was characteristic for alkaloids of the vallesiachotamine type (Djerassi et al., 1966) and the IR spectrum showed absorptions typical of NH, C= 0 of ester and five membered lactone ring. The EIMS showed a molecular ion m/z 364 (55) with fragments for the loss of CHg m/z 349 (10), CH3O m/z 333 (22), COOCH3 m/z 305 (100) and the loss of the lactone ring and the ester function giving arise to m/z 2 2 1

(1 1 ), Figure 4.6 (p.118) shows the mass fragmentation. The HRMS spectrum of vallesiachotamine lactone showed a [M]"^ peak at m/z 364.1435 corresponding to the molecular formula C 2 1 H2 0 N2 O4 .

The NMR spectrum (CDCl3,250 MHz,) was found to be closely related to that of iso-vallesiachotamine (Waterman and Shouming Zhong, 1982) and signals were observed for the proton at C-3 and it has an a- configuration (Ô 4.28). A two-proton broad singlet at 5 4.87 was assigned to the 0-18 and one proton in the aromatic region (5 7.09 - 7.22 m,3H) was assigned to the proton on 0-19. Furthermore, ^H-^H OOSY (Spectrum 1 0 , Appendix I) clearly showed long range coupling both between 6 3.87 (0-15H) and the multiplet at Ô 7.09 - 7.22 (0-19H) and between 5 3.87(0-15H) and S 4.87 (2H,0-18H) supporting a lactone ring for this alkaloid attached to position 15. In addition, Kedde reagent gave a clear pink-purplish colour (Stahl, 1969) indicating the presence of the five membered a-p unsaturated lactone; while no colour was given by a mixture of vallesiachotamine and isovallesiachotamine.

The spectral data of vallesiachotamine agreed closely with that previously

117 c o o

m iz 364.1435 mfz 349

m /l 305 mfz 333

m/z 221

Figure 4.6 Putative Mass Spectral Fragmentation of Vallesiachotamine lactone.

118 published (Waterman and Shouming Zhong, 1982) except that the Heterocorrelation experiment indicated that the multiplet at 5 2.86 should be assigned to the C - 6 protons and the multiplet at 5 3.73 to protons on 0-5. The spectra clearly resolve the 0-14 proton into multiplets at Ô1.93 and 5 2.17 corresponding to the a and p protons respectively. In addition, it was observed that the isolated sample of vallesiachotamine after two months in storage was converted to a 1 : 1 mixture of vallesiachotamine and iso­ vallesiachotamine.

Vallesiachotamine was originally isolated from Vallesia dichotoma (Djerassi et al., 1966), and subsequently from Rhazya orientalis and R. stricta all of which are members of the family Apocynaceae (Evans et al., 1968); more recently it has been reported from Strychnos trycalysiodes (Loganiaceae)(Waterman and Shouming Zhong, 1982). Vallesiachotamine has not been reported in any taxa of the Rubiaceae.

The spectral data of strictosidine agreed closely with that previously published (Battersby et al., 1969; Smith, 1968), which is the putative precursor of the vallesiachotaman type alkaloids. Strictosidine was lactamized by adding NagOOj (Battersby et al., 1969), the NMR and mass spectra of the product were similar to that obtained for strictosidine lactam, confirming the identity of the isolated compound as strictosidine. The and NMR were fully assigned by COSY-45 and ^H-^% heterocorrelation.

The UV, IR, and NMR of strictosidine lactam agreed with those recently published (Atta-Ur-Rahman et al., 1991); it has previously been isolated from Nauclea latifolia (Rubiaceae) (Brown et al., 1977; Hotellier et al., 1975) and from Rhazya stricta (Apocynaceae) (Warren, 1970). In the present study, the strictosidine lactam was acetylated and its and ^^0 NMR fully assigned by Heterocorrelation, which showed that the C-3 (S 53.4) previously reported as Ô 53.6 (Cordell, 1981) clearly correlated with the multiplet at 5 4.94-5.02 (3H) rather than with the signal at 5 2.58-2.71 previously reported

119 (Atta-Ur-Rahman et al., 1991). The isolated alkaloid and its acetylated derivative were found to be identical on TLC with an authentic sample of strictosidine lactam, kindly supplied by Dr. R.T. Brown, University of Manchester, and their ^H-NMR were superimposable.

The spectral (UV, EIMS, NMR) data of a yellow fluorescent compound agreed closely with that observed previously for angustine, which occurs widely throughout the Rubiaceae and Loganiaceae (Phillipson et al., 1974; Au et al., 1973). The isolated compound was found to be identical on TLC with an authentic sample of angustine available in the laboratory.

120 4.5 Isolation and Identification of Alkaloids from Cephaëlis glomerulata, 4.5.1 Extraction and separation procedures

Dried and powdered aerial parts (792 g) of Cephaëlis glomerulata J.Don. Smith were extracted with MeOH by percolation. The extract was filtered and concentrated to a gum (49.4 g) under vacuum and partitioned between 2 M HCI and CHCI3. The acidic fraction was filtered, basified to pH~9 using NH 4 OH and re-extracted with CHCI3. The CHCI3 fraction was dried on NagSO^ and concentrated under vacuum and the residue (1.08 g) was submitted to 0 0 using OHOl 3 -MeOH + NH4 OH (98:2 + 0.1 %) as solvent system. A total of 50 (10 ml) fractions were obtained, from fractions 9-11 47 mg glomerulatine A were obtained; fractions 13-27 containing glomerulatine A and 0 were chromatographed on pTLO using EtOAc-MOgOO + NH 4 OH (80:20 + 0.1 %) as solvent system, 5 mg of glomerulatine 0 were, thus, obtained. Fractions 32-38 were chromatographed on pTLO and developed in the same conditions, yielding 4 mg of glomerulatine B.

4.5.2 Identification of the Isolated compounds 4.5.2.1 Glomerulatine A 4.5.2.1.1 Spectral data

UV X max. (EtOH)nm: 274, 304 shoulder IR NaCI \) cm ': 2919, 2860, 1625 (C=N), 1590 (C=C), 1466, 1407, 1272, 1219, 755 [a]“20°C (0=0.36, GHCy -466.1 CD (Spectrum 23, Appendix I) (CH3CN, 0= 105 nM) X nm (A e): 310.3 (-13.49), 296.0 (-13.11), 280.6 (+0.04), 2.68.8 (+12.66), 246.6 (+2.99), 214.7 (+12.17).

121 EIMS (Spectrum 24, Appendix I) m/z (relative intensity %) 342 (100), 327 (3), 313 (10), 298 (11), 284 (20), 270 (14), 256 (16), 255(16), 242 (12), 230 (7), 209 (14), 128 (10). 115 (12) HRMS m/z calculated: 342.18445 (CggHggNJ found:342.18565 NMR (Spectrum 25, Appendix I)

(CeDg , 400 MHz) Ô 1.35-1.39 (dd, J=6.2, 1 H, H-3), 1.90-1.98 (m, 1H, H-3), 2.61 {t, J=8.98, 1H, H-2), 2.92 (s, 3H, N-1 Me), 2.96-3.09 (m. 1H, H-2), 6.62-

6.67 (m, 1H, H-5), 6.76-6.78 (dd, J=7.7, 1.5, 1 H, H-4), 6.93-6.98 (m, 1H, H- 6 ), 7.46-7.48 (dd, 1H, H-7) NMR (Spectrum 26, Appendix I)

( CgDe, 100.6 MHz): 5 30.26 (C-3), 30.87 (N - 1 Me), 48.15 (0-2), 49.09 (C-3a),

122.28 (C-5), 123.68 (C-4), 125.04 (C-7), 126.41 (C-4a), 128.90 (C- 6 ), 147.32

(C-7a), 165.11 (C-8 a). COSY 45, ^H-^^C heterocorrelation (Spectrum 27, Appendix I), ^H-^^C long range heterocorrelation (Spectrum 28, Appendix I) and ROESY (Spectrum 29, Appendix I) NMR experiments were used to confirm these assignments.

4.5 2.2 Glomerulatine B 4.5.2.2.1 Spectral data

UV

X max.(CH3 CN) nm: 279 CD (CHgCN, C= 116 nM) X nm (A e): 305.5 (-12.97), 219.9 (-13.11), 274.3 (+0.098), 262.9 (+12.19), 242.2 (+8.09), 213.9 (+12.27) EIMS (Spectrum 30, Appendix I) m/z (relative intensity %): 328 (100), 313 ( 6 ), 299 (12), 284 (9), 270 ( 8 ), 243 (5), 229 (4), 132 (17), 113 (11), 50 (27)

HRMS m/z calculated: 328.16880 (CgiHgg NJ found 328.16957

122 'H NMR (Spectrum 31, Appendix I)

(CgDg, 400 MHz): 5 1.30-1.34 (m, IN, H-3), 1.46-1.49 (m. 1 H, H-3), 1.92-2.03 (m, 2H, H-3 and 3 ), 2.58 (t, J=8.98,1H, H-2), 2.90 (s, 3H, N-1 Me), 2.93-2.95

(m, 1H, H-2), 3.17-3.19 (m, 1H, H-2 ), 3.29-3.33 (m, 1 H, H-2 ’), 6.58-6.62 (m,

1 H, H-5’), 6.64-6.68 (dd, 1 H, H-5), 6.77 (d. J=7.51, 1H, H-4), 6.88-6.92 (dd,

J=7.63, 7.39,1H, H - 6 ), 6.94-6.98 (dd, J=7.51,1H, H-6 ), 7.10-7.12 (m, J=7.63, 6.15, 2H, H-7’ and 4 ), 7.47 (d, J=7.63, 1H, H-7) "C NMR (Spectrum 32, Appendix i)

(CgDg, 1 0 0 . 6 MHz): 8 30.28 (C-3 and 3’), 30.79 (N - 1 Me), 48.09 (C - 2 and 2 ’),

48.62 (C-3a and 3’a), 120.85 (C-4), 1 2 2 .2 1 “ (C-5), 122.59“ (C-5 ), 124.03" (C- 4 ), 124.37" (C-7’), 125.15" (C-7), 125.42" (C-4’a), 126.14" (C-4a), 128.78" (C-

6 ’), 128.97" (C- 6 ), 147.13 (C-7a and 7’a), 164.68 (C- 8 a and 8 ’a), the superscript indicates interchangeable assignment within the same letter. COSY 45, 'H-"C heterocorrelatlon, 'H-"C long range heterocorrelatlon and "C DEPT NMR experiments were used to confirmed these assignments.

4.5.23 Glomerulatine 0 4.5.2.3.1 Spectral data

UV X max. (EtOH) nm: 274, 304 shoulder IR NaCI u cm ’ : 2952, 2857, 2352, 1629 (C=N), 1588 (C=C), 1470, 1407. CD (CHjCN, C= 65 nM) X nm (A e): 305.1 (-12.08), 287.2 (-12.32), 258.6 (+0.1), 247.9 (+12.47), 228.4 (+9.40), 216.4 (+13.35) EIMS (Spectrum 33, Appendix i) m/z (relative intensity %) 344 (23), 343 (6 8 ), 273 (23), 256 (57), 219 (20), 197 (23), 169 (25), 153 (22), 135 (19),130 (41), 127 (22), 115 (30)

HRMS m/z [M-1]* calculated 343.19227 (Cg^Hg^NJ found 343.19248

123 NMR (Spectrum 34, Appendix I) (CgDe, 400 MHz) 5: 1.52-1.58 (m, IN, H-3'), 1.83-1.87 (m, 1H, H-3), 1.91-1.98

(m, 1H,H-3’), 2.14 (s. 3H, N-1’ Me), 2.17-2.20 (m, 1 H, H-2 ’), 2.30-2.34 (m, 1 H, H-3), 2.63-2.70 (m, 2H, H-2 and 2’), 2.97 (s, 3H, N-1 Me), 2.95-3.02 (m, 1H,

H-2), 3.78 (d, J=4.52,1H, H-8 ’a), 3.86 (s broad, 1 H,H-8 ’) 6.19-6.21 {dd, J=0.88,

7.48, 1H, H-7’), 6.45-6.49 (ddd, J= 1.12, 7.36, 7.52, 1 H, H-5’), 6.74-6.86 (m,

3 H, H-5, 4’ and 6 ), 6.96-7.00 (dd, J=7.68, 7.44, 1 H, H-6 ) 7.01-7.04 (dd, J=1.2, 7.72, 1H, H-4), 7.49-7.51 (dd, J=1.24, 7.76, 1H, H-7) NMR (Spectrum 35, Appendix I)

(CgDe, 100.6 MHz) 6 : 30.73 (N- 1 Me), 31.71 (C-3), 34.02 (0-3’), 39.57 (N- 1 ’ Me), 45.30 (3’a), 48.51 (0-2), 49.15 (0-3a), 50.45 (2’), 76.51 (0-8’a), 114.37 (0-7’), 117.53 (0-4’), 122.11 (0-5), 122.26 (0-4’a), 123.00 (0-4), 124.94 (0-5’), 125.2 (0-7), 127.12(0-6 ), 128.80 (0-6), 129.52 (0-4a), 142.10 (0-7’a), 148.62 (0-7a), 166.49 (0-8a). OOSY-45 (Spectrum 36, Appendix I), DEPT, ^H-'^O long range Heterocorrelation (spectrum 37, Appendix I) and ROESY (Spectrum 38, Appendix I) NMR experiments were used to confirm these assignments.

4.5.3 Discussion The UV spectrum of glomerulatine A showed a band at 274 nm of a conjugated quinoline moiety and the IR spectrum showed adsorption for 0=N (1625 cm'^) and 0=0 (1590 cm'^). The HR mass spectrum of glomerulatine A showed a [M]^ peak at m/z 342.18565 corresponding to the molecular formula

O2 2 H2 2 N4 . While the EIMS showed a strong [M]*" with 1 00% relative abundance, indicative of calycanthine-type alkaloids instead of chimonanthine-type (Adjibade et al., 1992). However, the peak at m/z 231 has no significant abundance suggesting that the iso-calycanthine-form be more likely for this compound (Adjibade et al., 1992).

However, 1 the spectroscopic techniques used, in the present work, in the structure elucidation which include EIMS, ^H, NMR, 2D NMR experiments, [a]° and 0 1 ^ failed to distinguish between the calycanthine- or iso- calycanthine-type. Hence, glomerulatine A is either the 8 -8 a,8 ’-8 ’a

124 tetradehydro(-)calycanthine or the 8-8a,8'-8'a tetrad 0 hydro(-)iso-calycanthine (Figure 4.7, p. 128).

Table 4.6 (p. 126) shows a comparison of the spectroscopic data of caiycanthine and iso-calycanthine previously published and glomerulatine A. It is important to note that the chemical structure of caiycanthine has been established by X-ray diffraction (Hamor et al., 1960). In a more recently published work, Adjibade et al. (1992) characterized the first five membered ring quinoline alkaloid as iso-calycanthine. Their argument were based mainly on slight differences in and NMR, differences in relative abundance in EIMS and the major difference being the values, when comparing iso- calycanthine with caiycanthine.

Furthermore, Adjibade et al (1992) did not find difference in the displacements of the signals in NMR of the C - 2 and C-3, although, the signals in NMR for a five membered ring are shifted down field when compared with a six membered ring. For example, the C-2 and 0-3 in chimonanthine forming a five member ring has shown signals a 5 52.6 and 5 35.7 respectively, whereas, in caiycanthine, as part of a six member ring, they show signals at 5 46.6 and 5 31.7, respectively (Libot et al., 1988). The structure of iso-calycanthine has not been confirmed by X-ray diffraction analysis.

The NMR (CgDg, 400 MHz) spectrum of glomerulatine A showed only 1 1 protons and the ^^0 NMR showed 11 carbons, indicating that the dimeric nature of the alkaloid. There were signals for four aromatic protons, from 7.48 to 6.62 ppm, characteristic of a 1 ,2 -disubstituted aromatic ring; four protons signals from 3.09 to 1.35 ppm, clearly separated from each other, were assigned to protons on 0-2 and 0-3 using OOSY 45 and ^H-^^0 heterocorrelation NMR experiments.

125 Caiycanthine* iso- Glomerulatine Calycanthlne* A

[aŸ -489 -150 -466

uv 250,310 247, 305 274, 304

IR 1600, 1570 1500 1625, 1590

EIMS 346(100) 346 (100) 342 (100)

302 (30) - 298 (11) 231 (77) 231 (20) 230 (7)

'H NMR N-Me 2.41 2.32 3.2Sf

8 a 4.31 4.30 4.2' aromatic 6.25 to 6.57, 6.73, 6.63-6.74 protons 7.03 7.02, 7.05 7.02-7.05"

” C NMR N-Me 43.39 42.95 30.87 (39.57)' 7a 146.20 145.35 147.32

8 a 71.82 71.66 165.11 (76.51)' arr-z.TTZi~ï^J. ___ ■ - i ü l

‘'Measured in CDCI3 ®Value for glomerulatine C measured in CDCI3 ‘Value for glomerulatine C in C^Dg

Table 4.6 Comparison of the Spectral Data of Calycanthine-Type Alkaloids

126 Also, the NMR showed a singlet integrating for three protons at 2.92 ppm for a N-methyl, and long range heterocorrelation NMR experiment showed a correlation between the signal of the N-methyl and the carbon signals at 165.11 (C- 8 a) and 48.15 ppm (C- 2 ), establishing its position on N- 1 . The G-3a shows a signal at 5 49.09 shifted to lower field because it is adjacent to an additional conjugated carbon (C- 8 a), rather than to a methine group as in caiycanthine, which displays a signal at 5 30-40 characteristic of a piperidinoquinoline ring (Libot et al.,1988). Whilst, a ROESY effect was shown between the N-methyl and the proton on 0-4’, and the proton a on C - 2 and C- 4’, which are more feasible in the /so-calycanthine form than in caiycanthine, where the methyl and the protons a on C - 2 are farther from the protons on C- 4’, however, it is possible as well.

The showed that glomerulatine A is levogyre with a value of (-)466, close to that reported for (-) caiycanthine (- 489) (Adjibade et al., 1992), whereas, for /so(-)calycanthine a value of (-)150 has been reported (Adjibade et al., 1992). The CD spectrum indicated a S-configuration for glomerulatine A. Hence, the isolated compound may have a configuration similar to caiycanthine, however, none of the spectroscopic techniques used to elucidate this structure showed further evidence to distinguish between the caiycanthine or iso-calycanthine isomers. Only X-ray crystallography could unravel this problem and, although, the isolated alkaloid was crystallized the crystals were not good enough to complete the experiment. This plant should be collected again in order to isolate more of these compounds to establish the configuration. Figure 4 . 7 (p. 128) shows both possible isomers of the isolated alkaloids from C. glomerulata.

Glomerulatine B showed similar UV and IR spectra to glomerulatine A. The HR mass spectrum showed a [M]^ peak at m/z 328.16957 corresponding to the molecular formula C 2 1 H2 0 N4 . EIMS showed a strong [M]^ with 100% relative abundance, fourteen mass units less than glomerulatine A suggesting that this alkaloid is the structural N-demethyl isomer.

127 H jC k

5 o r

1 CH)

Glomerulatine A

o r

Glomerulatine B

" ^ n j I

o r N CH, CH,

Glomerulatine C

Figure 4.7 Possible Structures of the Alkaloids Isolated from Cephaëlis glomerulata.

128 NMR showed an unsymmetrical molecule and only one signal integrating for three protons at 5 2.90 for a N-methyl group similar to glomerulatine A. The signal for the protons on C-2 and 0-3 were spread from 5 1.30 to 3.33, whereas, the aromatic part of the spectrum showed eight protons indicating two orï/70-disubstituted aromatic rings. Furthermore, NMR in CDCI 3 showed a very broad singlet at ô 2.55 that disappeared after the addition of DgO, indicating the NH at position 1 '. The assignment of this spectrum was achieved by comparison with glomerulatine A, since one monomeric moiety is identical, also for 2D NMR experiments, such as COSY 45, ^H-^^C heterocorrelation and ^H-^^C long range heterocorrelation confirmed assignments made for the demethyl analogue.

^^C NMR showed sixteen signals indicating that some of them were overlapped. In the upper field, the signals for C- 8 a and C-7a were quite similar to those for glomerulatine A. While, the aromatic CH and the C-4a were split showing ten separated signals, but, resembling the values showed for glomerulatine A.

Glomerulatine C showed an UV and IR spectra similar to glomerulatine A and the HR mass spectrum showed a [M-1T at m/z 343.19227 indicating to the molecular formula C 2 2 H2 3 N4 . EIMS showed a relatively weak [M]^ peak with only 23% of relative abundance, but a stronger [M-l]'" with 6 8 % of relative abundance, two mass units less than glomerulatine A, suggesting that this alkaloid has loss one double bond. NMR, as in glomerulatine B, showed an unsymmetrical molecule including two singlets integrating for three protons each, one of them at 5 2.97 as in glomerulatine A and B and the other at Ô 2.14 (52.40 in CDCy as reported for caiycanthine (Libot et al., 1988). Also, a doublet at 5 3.78 and a broad singlet at 5 3.86 for a proton on the carbon between the two nitrogens and for N-H respectively. This data suggested that one of the monomers is similar to glomerulatine A and the other similar to caiycanthine.

129 Furthermore, NMR showed twenty-two carbons, a CH signal at Ô 76.51 for the methine (0-8) and a quaternary carbon at 5166.49, which are in favour of the first hypothesis. long range heterocorrelation NMR experiment showed a correlation between the singlet at 5 2.4 (in ODOy and the carbon at 5 76.5 and 50.45, establishing that this methyl group is on the N-1 '. In addition, the methyl, displaying a signal at 5 2.97, showed the same correlation observed in glomerulatine A. ROESY NMR experiment showed, apart from the signals characteristic of the monomer similar to glomerulatine A a correlation between the doublet at 5 3.78, for the proton on C- 8 'a, and the multiplet at 5 7.01 -7.04, for the proton on 0-4 indicating that this proton is in position a to the six members ring. Therefore it can be concluded that the isolated compound is the 8 '-8 'a dihydro glomerulatine A, named as glomerulatine 0.

It was decided that ^H-^% long range heterocorrelation of glomerulatine 0 may help to distinguish between the two possible isomers, caiycanthine or iso- calycanthine. Since, the proton on 0-8’a (5 3.78) may show a correlation to three bonds to the 0-3 (5 31.7) if the alkaloid is of the calycanthine-type or to the C-3’(5 34.02) if it is of the iso-calycanthine-type. The long range heterocorrelation NMR experiment failed to show this correlation, but it is highly probable that this was due to the fact that only 5 mg of alkaloid was available for the experiment.

130 4.6 Isolation and Identification of Bioactive Compounds from PIcramnia antidesma subsp. fessonia. 4.6.1 Extraction and separation procedure

Dried and powdered leaves (600 g) of P. antidesma subsp. fessonia were extracted with MeOH by percolation. The extract was filtered and concentrated to a gum (127 g) under vacuum, and partitioned between HgO and CHCI3, the aqueous layer was subsequently extracted with BuOH.

The butanolic fraction (59 g) was submitted to CO (silica gel 60, 0.063-0.2 pm, Merck) using a glass column (4.5 x 75 cm) and CHCI 3 , CHCl3 -MeOH (9:1,

6 :2 , 7:3) as eluent. Sixty fractions (~ 125 ml) were obtained, fractions 7 to 1 0 were washed with CHCI 3 and crystallized from CHCI 3 yielding 0.83 g of aloe- emodin; fractions 18 to 27 were submitted to GO using CHCI 3 , CHCI3 -M0 OH

(95:5, 9:1, 8:2) as eluent, yielding, after crystallization from CHCl 3 -MeOH (9:1) 63 mg of picramnioside A; fractions 28 to 30 were submitted to GO using the same eluent as for picramnioside A, yielding, after precipitation from MeOH-

GHGI3 (9:1), forming 0.43 g of amorphous powder of picramnioside B; fractions 31 to 35 were washed with MeOH and a residue soluble only in DMSO was obtained, after repeated precipitations from DMSO by adding GHGI 3 yielded 0.490 g of picramnioside G; whereas from fractions 36-41 after preparative TLG using GHGl 3 -EtOAc ( 1 :1 ) 2 mg of aloe-emodin anthrone were obtained.

Oxidative hydrolysis: 10 mg of picramnioside B and picramnioside G were separately dissolved in 25 ml of 4N HGI containing 1 mg of FeG^, and heated at 100 °G for 4 hrs. The cooled solution was extracted with GHGI 3 ; the chloroformic fraction was submitted to pTLG using GHGl 3 -MeOH (9:1) as solvent system. The isolated compound was identical to aloe-emodin on TLG

(GHGl3 -MeOH 9:1) and their NMR spectra were superimposable.

131 4.6.2 Identification of the isoiated compounds 4.6.2.1 Picramnioside A 4.6.2.1.1 Spectral data

UV X max (MeOH)nm; 221, 273, 302, 382 IR t max (KBr) cm'^: 3440 (OH), 2950, 1725, 1640, 1620, 1600, 1300 FDMS m/z (relative intensity %): [M+ Na]* 531 (1 00), [M]'’ 508 (1 2 ), [M-benzoate+ Na]'’ 409 (18), benzoate 122 (7) FABMS (Spectrum 39, Appendix I) (glycerol/thioglycerol/TFA matrix) m/z (relative intensity %) [M+1]* 509 (17), 387 (54), 256 (46), 239 (26), 207 (40), 181 (53), 165 (37), 149 (34), 131 (34) HRMS (FABMS) [M-benzoate] m/z calculated 387.0716 (CgoHigOg) found 387.0724. The molecular ion was too small to be measured. CD (Spectrum 40, Appendix I) (MeOH, C= 86.5 pM) X nm (A e): 342.8 (+1.68), 314.2 (-0.027), 295.0 (-6.36), 239.0 (+1.70), 230.6 (-3.89), 221.2 (+14.4) H NMR (Spectrum 41, Appendix I)

(DMSG-Dg, 400 MHz): 5 3.45-3.53 (m, 1 H, H-2 '), 3.67 (m, 2 H, H-3' and 4"),

3.85 (dd, Ji..io= 2.1, J i 2 .= 9.9 1H, H-1"), 4.03 (of, 2H, H-11), 4.65 (d, 1H, H-10),

4.91 (d, 1H, H-3'OH), 5.01 (d,1H, 4'OH), 5.20 (t, 1H, H- 1 1 OH), 5.42 (d, 1H, H- 20H), 5.62 (s, 1H, H-5'), 6.62 (s, 1H, H-2), 6.83 (s, 1H, H-4), 6.93 (d, 1H, H-7),

7.15 (d, 1H, H-5), 7.50-7.53 (dd, 2H, H-3"), 7.57-7.61 (dd, 1 H, H-6 ), 7.65-7.69

(dd, 1H, H-4"), 7.71-7.73 (dd, 2H, H-2"), 11.83 (s, 1H, H- 1 ), 11.96 (s, 1 H, H-8 ). long range heterocorrelation NMR experiment (Spectrum 43, Appendix I) proton (carbons) 2 (1a,11), 3 (2, 3), 4 (la, 2, 4a, 10, 11), 5 ( 6 , 7, 8 a, 10), 6

(5a), 7 (5, 6 ), 10 (la, 4, 4a, 5a, 8 a, 1'), 1' (4a), 5’ (V, 3', 5'C=0), 2" (3", 4", 5'C=0), 3" (1")

132 4.6.2.2 Picramnioside B 4.6.2.2.1 Spectral data

UV X max (MeOH) nm: 225, 260, 270, 300, 365 X max (MeOH + NaOH) 230, 265, 375, 390, 455 (sh) IR

0 KBr (cm'^): 3350 (OH), 2730, 1760, 1640, 1620, 1600, 1590, 1295, 1230 FABMS (Spectrum 45, Appendix I) (glycerol/thioglycerol/TFA matrix) m/z (relative intensity %) [M +l^ 447 (48), 387 (16), 351 (46), 256 (100), 239 (39) HRMS (FABMS) lh^+^Y m/z calculated 447.1291 (C 2 2 H2 2 O10 + 1 ) found 447.1299. [M-acetyl] m/z calculated 387.0716 (C 2 0 H19 O8 ) found 387.0722. CD (Spectrum 46, Appendix I) (MeOH, 0= 180 |iM) X nm (A e): 355.0 (+0.27), 336.2 (-0.02), 321.6 (-0.58), 308.6 (-0.004), 294.6 (+1.25), 272.0 (-0.003), 266.0 (-0.94), 240.4 (+0.11), 226.6 (-2.27), 210.8 (+2.43) NMR (Spectrum 47, Appendix I)

(DMSO-6 , 400 m/z MHz) 61.17 (s, 3H, H-S'CH^), 3.34- 3.41 (m, 1 H, H-2 '), 3.46-

3.48 (m, 2H, H-3’ and 4"), 3.64-3.67 (dd, Jvio=2.17, Ji, 2 =9 .7 , 1 H, H-1’), 4.58-

4.62 (m, 3H, H-11 and H-10), 4.85 (^, 1 H, H-3’0H), 4.94 (^, 1 H, H-4'OH), 5.32

(d, 1H, H-2’0H), 5.42 (s, 1H, H-5 ), 5.46 (t, 1 H, H-11 OH), 6 . 8 6 (d, 1H, H-7),

6.89 (s, 1H. H-2), 6.99 (d, 1 H, H-5), 7.07 (s, 1 H, H-4), 7.53-7.57 (dd, 1H, H-6 ),

11.85 (s, 1H, H - 1 OH), 11.89 (s, 1H. H - 8 OH). COSY-45 (Spectrum 48, Appendix I), ^H-^^C heterocorrelation (Spectrum 49, Appendix I), ^H-^^C long range heterocorrelation and ROESY (Spectrum 50, Appendix I) were used to confirm these assignments.

133 Picramnioside Carbon A B c

C- 1 161.38 (C)‘* 161.23 (O f 161.17 [C f C-la 115.58 (C) 115.74 (C) 115.44(C)

C- 2 112.17 (CH) 113.03 (CH) 112.09 (CH) C-3 152.64 (C) 151.84 (C) 152.29 (C) C-4 115.69 (CH) 118.41 (CH) 115.54 (CH) C-4a 141.26 (C) 141.24 (C) 141.07 (C) C-5 120.65 (CH) 118.21 (CH) 120.40 (CH) C-5a 145.97 (C) 146.07 (C) 145.81 (C)

C- 6 135.8 (CH) 136.27 (CH) 135.43 (CH) 0-7 116.12 (CH) 115.74 (CH) 115.79 (CH)

C- 8 161.51 (C) 161.64 (C) 161.19 (C)

C-8 a 117.17 (C) 117.15 (C) 116.90 (C) C-9 193.46 (C) 193.50 (C) 193.21 (C)

C- 1 0 42.44 (CH) 42.74 (CH) 42.30 (CH)

C- 1 1 62.12 (CHg) 62.59 (CHg) 62.25 (CHj)

C-1 ' 81.56 (CH) 80.87 (CH) 80.65 (CH)

c- 2 ’ 66.83 (CH) 66.72 (CH) 66.48 (CH) C-3’ 71.91 (CH) 71.73 (CH) 71.42 (CH) C-4’ 69.39 (CH) 69.30 (CH) 69.01 (CH) C-5’ 94.74 (CH) 93.74 (CH) 93.35 (CH)

C-5’ (C=0 ) 163.36 (C) 167.96 (C) 167.71 (C)

C-5’(CH3) - 20.46 (CHg) 2 0 . 1 0

C-1 ” 129.17 (C) - -

C-2 ” 129.22 (CH)® --

C-3” 128.89 (C)® - -

C-4” 133.71 (CH) - -

“Chemical shifts are in 6 -values in ppm, measured in DMSO-Dg. ‘^Multiplicity from Dept ^^0 NMR experiment. H"he intensity for this signal was twice that of the other CH signals.

Table 4.7 NMR Spectral Data° of the Compounds Isolated from P. antidesma subsp. fessonia.

134 4.6.2.S Picramnioside C 4.6.2.3.1 Spectrai data UV X max (MeOH) nm: 225, 261 (sh), 270, 300, 365 iR ù (KBr) cm': 3500, 3390, 2990, 2920, 1725, 1640, 1620, 1600, 1300, 1250. FABMS (Spectrum 51, Appendix I) (glycerol/thioglycerol/TFA matrix) m/z (relative intensity %) [M+1]* 447 (63), 387 (48), 351 (26), 256 (100), 239 (40) HRMS [M+1]* m/z calcuiated 447.1291 (CjjHjjO,» +1) found 447.1302. [M-acetyl] m/z calculated 387.0716 (CgoH^O,) found 387.0712. CD (Spectrum 52, Appendix I) (MeOH, 0= 85 pM) X nm (A e): 351.4 (+0.78), 317.0 (-0.019), 294.0 (-5.37), 268.8 (+0.009), 253.0 (+1.1), 230.0 (-3.13), 210.6 (+6.47) 'H NMR (Spectrum 53, Appendix I)

(DMSO-Dg, 400 MHz) 5 1.72 (s, 3H, H-5'CH,), 3.38-3.44 (m, 1 H, H-2'), 3.54-

3.57 (m, 2H, H-3’ and 4'), 3.67-3.70 {dd, J,.,o=2.0, J,..2=9.85, 1H, H- 1 '), 4.51-

4.63 (m, 3H, H-10 and H-11), 4.85 (d, 1H, H-3'OH), 4.93 (d, 1 H, H-4'OH), 5.38

(d 1H, H-2'OH), 5.40 (s, 1 H, H-5 ), 5.48 (f, 1 H, H- 1 1 OH), 6.80 (s, 1H, H-2),

6.91 (d 1H, H-7), 6.98 (s, 1 H, H-4), 7.13 (d 1 H, H-5), 7.55-7.59 (dd, 1H, H- 6 ),

11.84 (s, 1H, H - 1 OH), 11.93 (s, 1 H, H- 8 OH) 'H-'^C long range heterocorrelation NMR experiment (Spectrum 55, Appendix I) proton (carbons) 1 OH ( 1 , 2 ), 2 (1, la, 4, 1 1 ), 4 (2, la , 1 0 , 1 1 ), 5 (1 0 ), 6 (8 ,

5a), 7 (5, 6 , 8 , 8 a) , 8 OH (8 , 8 a) 10 (1, la, 4a, 5a, 8 a, 1'), 11 (2, 3), T (2 '), 2' (3 ), 5' (V, 3'), 5'CH, (5'C=0)

135 4.6.2.4 Aloe-emodin 4.6.2.4.1 Spectral data

UV X max (MeOH) nm: 224, 256, 277, 287 X max (MeOH+NaOH) nm: 232, 247, 292 (sh), 330 EIMS (Spectrum 57, Appendix I) m/z (relative intensity %) [M]" 270 (1 0 0 ), 214 (85), 224 (9), 213 ( 1 1 ), 196 ( 6 ), 168 (10), 139 (22), 128 (10), 121 (15) HRMS m/z calculated 270.0528 (CigHioOg); found 270.0536 NMR (Spectrum 58, Appendix I)

(DMSO-6 , 400 MHz) 5 4.59 (s, 2H, H- 1 1 ), 5.59 (br s, 1 H, OH-1 1 ), 7.19 (d,

J=0.9,1H, H-2), 7.28-7.30 (dd, J= 8 .2 , 0.86, 1 H, H-7), 7.56 (d, J=0.9, 1H, H-4),

7.56-7.60 (dd, J=7.6, 0.86, 1 H, H-5), 7.73-7.74 (dd,J= 8 .2 , 7.6, 1 H, H-6 ), 11.83

(br s, 2H, H-1, H-8 ) '"C NMR

(DMSO-6 , 400 MHz) Ô 161.31 (C-1 ), 114.13 (C- 1 a), 117.03 (C-2 ), 153.69 (C-3),

119.26 (C-4), 132.87 (C-4a), 120.57 (C-5), 133.07 (C-5a), 137.27 (C- 6 ), 124.30

(C-7), 161.61 (C-8 ), 115.59 (C- 8 a), 191.45 (C-9), 181.09 (C- 1 0 ), 62.04 (C-1 1 )

4.62.5 Aloe-emodinanthrone 4.6.2.5.1 Spectral data

EIMS (Spectrum 59, Appendix I) m/z (relative intensity %) [M]+ 256 (100), 238 (32), 226 (31), 210 (32), 197 (24), 181 (12), 165 (7), 152 (16), 139 (15), 127 (16), 115 (10) 'H NMR (CD3OD, 400 MHz) Ô 4.44-4.55 (q, J=14.7,11.2,1H, H-11), 4.64 (s, 2H, H-10),

6.06 (s, 1H, H-2), 6.77 (d, J=7.4,1H, H-7), 6.84 (s, 1 H, H-4), 6.89-6.91 (dd, J=

8.3, 0.7, 1 H, H-5), 7.48-7.52 (dd, J= 8.3, 7.4, 1 H, H-6 )

136 NMR (CD3OD, 400 MHz) 5 163.15 (C-1), 116.66 (C-1 a), 114.90 (C-2), 152.35 (C-3),

117.93 (C-4), 142.12 (C-4a), 119.44 (C-5), 143.72 (C-5a), 137.35 (C- 6 ), 121.42

(C-7), 163.70 (C-8 ), 118.55 (C- 8 a), 193.47 (C-9), 57.52 (C-10), 64.38 (C- 1 1 )

4.6.3 Discussion

A bioactivity guided fractionation of the methanolic extract from P. antidesma yielded two known anthraquinones, aloe-emodin and aloe-emodin anthrone. Figure 4.8 (p. 138), and three new aloe-emodin C-glycosides, named picramnioside A, Figure 4.9 (p. 139), picramnioside B and picramnioside C, Figure 4.10 (p. 140). This plant was selected because of its activity against P. gallinaceum (Spencer et al., 1947) and from the chemotaxonomic stand point the presence of quassinoids was predictable. Only the butanolic fraction (see

Table 3.15, p.8 8 ) proved to be active against KB cell (LC 50 8.7 pg/ml), however, this fraction showed no significant activity against brine shrimp (LC 50 485 ng/ml)

(see Table 3.13, p. 8 6 ).

The UV spectrum of picramnioside A showed four bands (221, 273, 302, and 382 nm) characteristic of highly conjugated system, such as anthraquinones and the IR spectrum showed a band at 1725 cm '' for a ketone group. The FDMS showed a strong peak at m/z 531 (100) [M+Na]^ and m/z 508 (12) for [M]^, and FABMS (glycerol/thioglycerol/TFA matrix) showed a weak

[M+1 ]^ peak at 509 (17). The peaks at m/z 387 (54) indicated the loss of a benzoate moiety [M- 1 22]'’ and at m/z 256 indicated the aglycone. Figure 4.1 1 (p. 142) shows the mass spectra fragmentation of picramniosides A and C. HRMS (FABMS) showed a [M-benzoate]"^ m/z 387.0724 corresponding to the molecular formula CgoHigOg, the molecular ion at 509 was too small to be measured using this technique.

^H NMR of picramnioside A showed two singlets at S 11.96 and 11.83

137 OH

Aloe-emodin

Aloe-emodinanthrone

Figure 4.8 Anthraquinones Isolated from P. antidesma subsp. fessonia.

assigned to the OH groups on C-1 and C- 8 ; there were signals for ten aromatic protons from 5 6.63 to 7.72, and the COSY 45 spectrum showed three separated aromatic rings; a singlet at 5 5.62 was assigned to the proton on C- 5’; four DgO exchangeable protons (5 5.41, 5.20, 5.07 and 4.91) were assigned to the hydroxyl groups in the sugar moiety and to the hydroxyl on C - 1 1 in the aglycone; a double doublets at 5 4.65 was assigned to the proton on C - 1 ’.

The doublet integrating for two protons at 5 4.02 was assigned to the

protons on C - 1 1 and a double doublet at 5 3.85 accounted for the proton on C-

T of the sugar. The coupling constants, Ji._io=2.1 and Jy. 2 =9 .9 , suggested a

138 Picramnioside A Figure 4.9 C-Glycoside Isolated from P. antidesma subsp. fessonia Showing the Most Relevant ROESY Effects. p configuration for the carbon of the sugar bound to the aglycone, close to those reported for aloins (Manitto et al., 1990): the two proton singlet-shaped multiplet at Ô 2.90 was assigned to the protons on C-3’ and 0-4’ and a multiplet at Ô 3.53-3.44 accounted for the protons on C- 2 ’.

The ^^C nmr assignment is presented in Table 4.7 (p. 134), and there are 25 signals. Two CH signals at 5 129.22 and 133.7 showing a double intensity characteristic of a monosubstituted benzene ring, an interesting feature of this C-glycoside in 1 NMR is the low field position of the signal for C-5’ (Ô 94.74), which may indicate the presence of an 0-glycoside. In fact, this sugar residue has the same configuration of xylose indicated by ROESY NMR experiment, but, it has the aloe-emodinanthrone moiety attached to the C - 1 and the I benzoate is attached to C-5 of the xylose moiety.

2D NMR experiments such as COSY 45, ^H-^^C heterocorrelation, ^H-^^C long range heterocorrelation and ROESY were used to confirm these assignments.

139 OH

OH

8 OH

Picramnioside B

OH

OH

lO H OH

Picramnioside C

Figure 4.10 Picramniosides B and C isoiated from P. antidesma subsp. fessonia Showing the Most Reievant ROESY Effects.

140 The long range correlation NMR experiment showed that the singlet at Ô 5.62 corresponding to the proton on C-5' and the double doublet at 5 7.73- 7.65 assigned to the proton on C-2” , were correlating to the carbon at 5 163.36 assigned to the carbonyl of the ester, thus establishing the position of the benzoate on C-5’. Also, the signal at 5 4.65 for the proton on C - 1 0 showed a correlation to the carbon at Ô 81.56 for the C- 1 ’ corroborating the position of attachment between the aglycone and the sugar moiety.

In addition, the ROESY NMR experiment showed a correlation between the signal at 5 5.62 for the proton on C-5’ and the signals at 5 3.85, 3.67 and 5.01 for the protons on C-1’, C-3’ and the OH on C-4’, respectively; indicating that all the substituents on the sugar moiety are in the equatorial position.

Moreover, the proton on C -1 ’ ( 6 3.85) and the signal for the proton on C-4 (5 6.83) showed correlation, and the multiplet at Ô 3.45-3.53 (H-2’) and the doublet at 5 7.15 (H-5). Therefore the configuration of the C - 1 0 is R, as previously reported for aloins (Manitto et al., 1990) and cascarosides (Manitto et al., 1993). Figure 4.9 (p. 139) shows the ROESY effects displayed for picramnioside A, B and C and their absolute configuration. Also, the Circular dichroism agreed with that previously reported for (10R) aloin (Manitto et al., 1990).

The UV spectra of picramnioside B and picramnioside C were similar and the IR spectra showed little differences the carbonyl band which appear at 1725 cm*^ in the spectrum of picramnioside B and at 1760 cm'^ for picramnioside C. Oxidative hydrolysis (Fairbairn and Simic, 1963) of both compounds, yielded aloe-emodin, which was identified by comparison on TLC and ^H NMR with a reference sample. FABMS (glycerol/thyoglycerol/TFA matrix) showed, for both picramnioside A and picramnioside B, a [M+1]^ peak at m/z 447, a peak at m/z 387 indicating the loss of the acetate of the sugar and at m/z 256 for the aglycone. HRMS showed a [M+1]* at m/z 447.1299 for picramnioside B, and at m/z 447.1302 for picramnioside C, both corresponding to the formula

C2 2 H2 2 O1 0 .

141 H |H +

C&OH

Cft

OH OH

tnlz 509 m ji 447

OH OH

m fi 387

H n :

Cl^OH

m il 256

Figure 4.11 Putative Mass Spectra Fragmentation of Picramniosides A and C.

142 NMR showed, in both cases, only two aromatic systems indicating a difference with respect to picramnioside A; instead, both picramnioside B and

picramnioside C showed a methyl group signal at 6 1.7 for an acetyl group, suggesting that picramnioside B and picramnioside C are isomers and have an acetyl substituent at the 5’ instead of the benzoate substituent as in picramnioside A. The NMR of picramnioside B and picramnioside C were only differentiated in the aromatic pattern and the displacement of the signals; picramnioside B showed, from 5 6.86-7.07, a doublet-singlet-doublet-singlet pattern, while, picramnioside C showed, from 5 6.80-7.13, a singlet-doublet- singlet-doublet pattern.

NMR showed little differences between these two compounds, suggesting that they are the C - 1 0 isomers; and this was apparent when, both compounds, after a few hours in solution (DMSO-g) were converted into each other. ^H-^% long range heterocorrelation NMR experiments, for both compounds, showed, as important features, a correlation of the signal at 6 1.7

(methyl) with the carbon at 5 167 for the carbonyl group; the signal at 6 4.6 for the proton on 0-10 correlated to the signal for the protons on C- 1 ’, C-la, C-4,

C-4a, C-5, C-5a and C-8 a. Furthermore, ROESY NMR experiment, as in picramnioside A, indicated the same sugar configuration and, as previously reported for aloins and cascarosides (Manitto et al., 1990; Manitto et al., 1993).

The nOe effect showed by the proton on C- 1 ’ and C-2 ’ helps to differentiate between the R and 8 isomers; picramnioside B showed an interaction between the proton on C - 1 ’ (5 3.64-3.67) and the doublet at 6 6.99 for the proton on C- 5 , whereas the proton on C- 2 ’ (Ô 3.34-3.41) interacted with the singlet at 5 7.07 for the proton on C-4, therefore the absolute configuration of the C-10 in picramnioside B is S. On the other hand, picramnioside C showed, in the

ROESY NMR experiment, a correlation between the signal at 5 3.67-3.70 (C - 1 ’) and the singlet at Ô 6.98 for the proton on C-4, whereas, the signal proton at

Ô 3.38-3.44 (C- 2 ’) correlated to the doublet at 5 7.13 for the proton on C- 5 , showing an R configuration at C - 1 0 in picramnioside C.

143 The circular dichroism spectra, of picramnioside B and picramnioside C showed that they were similar to that reported for ( 1 0 S) aloin and (10R) aloin respectively, establishing, thus, the absolute configuration of picramnioside B as (10S) and of picramnioside C as (10R).

Aloe-emodin was identified for its spectroscopic properties (UV, HREIMS, and NMR) and by comparison of the NMR with a commercial sample (Apin Chemical, UK). Aloe-emodinanthrone was characterized using EIMS, and NMR and by comparison of previously reported values (Rychener and Steiger, 1989).

4.6.4 Biological Activity of the isolated Compounds

Picramniosides A, B, and 0 were tested against KB cells and the result is shown in Table 4.8 (p. 144). Picramnioside A was some four times less active than the acetyl isomers, picramniosides B and 0 and, although, picramniosides B and 0 are chiral isomers appear to have the same value, may be, because they are converted into each other in solution.

KB ceils Compounds LCso±sd (pM/ml)

Picramnioside A 35.7±0.10 (2)®

Picramnioside B 8.74±2.40 (3)

Picramnioside 0 8.32±2.47 (3)

Emetine 1.50±0.56 (3)

^Number of tests performed, in triplicate. Table 4.8 Activity of the Compounds Isoiated from P. antidesma subsp. fessonia Against KB Cells.

144 SECTION 5 GENERAL DISCUSSION 5.1 Introduction

Panama is a small Central American country, with a level of poverty estimated at 33.6 % and with a disparate health care coverage between the rural and urban area. The Panamanian government spent US $ 64.97 millions In drugs, In 1993, and all of these drugs were imported from developed countries. Panama has no plans to Include folk medicine in health services, as the World Health Organisation has urged developing countries to do. Nonetheless, herbs are sold In public markets and In drug stores without any general policy, guidance on the use or quality control of herbal medicines.

There are more than 200,000 (about 9 % of the total population) Amerindians, today In Panama, distributed In five groups, which live in the forest using plants as the major. If not the unique, source of medicine for curing their ailments. Although, there have been two major attempts to compile the ethnobotanlcal Information amongst these Amerindian groups, more surveys are urgently needed to rescue all their knowledge before it become extinct, since the youngsters are moving to the cities and leaving their culture behind.

More than 200 plants species have been reported to be used as medicines (Joly et al., 1987; Joly et al., 1990; Gupta et al., 1993a) amongst the Guaymi and Kuna Indians. Nevertheless, little or nothing has been done to scientifically evaluate these plant species, which may yield effective drugs that can be Introduced Into the Panamanian health care system.

The flora of Panama Is one of the richest In the world (Schultes, 1972) with more than 8 , 0 0 0 plant species and more than one half million voucher specimens being available worldwide (Dwyer, 1964; Dwyer, 1985). However, little Is known about the chemistry and biological activity of these plant species.

Although 27.5 % (2,077,914 hectares) of the Panamanian land surface Is under conservation by law, the annual rate of deforestation is estimated at

146 65,000 hectares. Therefore, it is vital to study this flora before the species become extinct.

5.2 Plants selected for this study

The plants investigated in this study were selected using chemotaxonomic criteria at different taxonomic ranks to limit the range of plants. For the Meliaceous, Menispermaceous and Simaroubaceous plants the family rank was used. Thus, Guarea and Ruagea belongs to the family Meliaceae which is known to contain limonoids (Connolly, 1983) and some of them are active against P. falciparum (Khalid et al., 1986; Bray et al., 1990).

Abuîa belongs to the family Menispermaceae which produces bbiq alkaloids with a variety of pharmacological activities (Schiff, 1983; Schiff, 1987). Some of these alkaloids show more activity against drug resistant strains of P. falciparum (Ye and Van Dyke, 1989) and multidrug resistant cancer cell lines (Shiraishi et al., 1987) than against the drug sensitive strains. The genus Picramnia belongs to the family Simaroubaceae, which is known to contain a bitter group of compounds, named quassinoids, some having activity against the malaria parasite (e.g. O’Neill et al., 1986). Both, P. antidesma subsp. fessonia and P. teapensis have bitter tastes, suggesting the presence of quassinoids in these species.

On the other hand, for the species of Cepfiaëlis, investigated in this study, the rank of genus was used. Cephaëlis ipecacuanha is the commercial source of emetine, which is effective in the treatment of severe amoebic dysentery. Until recently, knowledge of the chemistry of Cephaëlis species was limited to C. ipecacuanhavfh\ch contains monoterpenoid isoquinoline alkaloids (Wiegrebe et al., 1984; Itoh et al., 1989; Itoh, et al., 1991 ; Nagakura, et al., 1993) and to C. stipuiacea which contains gramine (Yulianti and Djamal, 1991), an indole alkaloid. Since this study started two Cephaëlis species from Panama have been studied in coordinated work by the Centre for Pharmacognostic Research

147 (CIFLORPAN) at the University of Panama. C. correae yielded the known iso- dolichantoside, three new indole monoterpenoid alkaloids, and one isoquinoline monoterpenoid glucoside alkaloid (Achenbach et al .,1993) and C. axillaris yielded two known indole monoterpenoid alkaloids, vallesiachotamine and 6 ’- trans-feruloyl-lyaloside (Gupta et al., 1993b).

5.3 Testing for biological activity 5.3.1 Brine shrimp assay

A new microwell method for brine shrimp testing has been developed in the present work and this overcomes some of the disadvantages of the test tube method, previously reported (Meyer et al., 1982). Requiring smaller amount of sample and the employment of microplate technology facilitates the testing of larger number of samples and dilutions.

Although, the use of brine shrimp has been proposed as a general bioassay, detecting a wide variety of pharmacological activities (McLaughlin, 1991), compounds with only cytotoxic activity were detected in this work. In general, drugs acting on the nervous system, such as atropine, caffeine, ephedrine and nicotine were not active. It has been suggested that a brine shrimp test could be used in the evaluation of traditional medicines (Kyerematen and Ogunlana, 1967), nonetheless, the present study clearly indicates that this assay is predictive of cytotoxicity, therefore, medicinal plants containing only cytotoxic compounds would be detected. Plants containing compounds with different pharmacological activities would not be evaluated properly by a brine shrimp assay.

Brine shrimp may be useful to guide the fractionation of plant extracts containing a group of compounds that is known to be active in this bioassay. This was demonstrated using quassinoids, which were active against KB cell as well as against P. falciparum. Thus, plant extracts from species of Simaroubaceae showing activity against brine shrimp are likely to contain

148 cytotoxic quassinoids with activity against P. falciparum.

5.3.2 Biological activity of plant crude extracts

Extract from Guarea macropetala, G. rhopalocarpa and Ruagea glabra (Meliaceae) showed strong activity against both brine shrimps and KB cells, while extracts from Abuta dwyerana (Menispermaceae) showed only weak activity in these two bioassays. Amongst the Cephaëlis species, the chloroformic extract from the roots 0. camponutans showed strong activity against brine shrimps and KB cells. Based on this result it was selected for bioassay guided fractionation in order to isolate the active principles.

Although some extracts from P. antidesma subsp. fessonia showed weak activity against brine shrimp, the butanolic fraction from the leaves was the most active of all the extracts tested against KB cell and was submitted to a bioassay-guided fractionation using KB cell assay.

5.4 Phytochemistry 5.4.1 Cephaëlis species

A bioassay-guided fractionation of the woody part of 0. camponutans, (accepted name Psychotria camponutans) yielded a novel benzoisochromanquinone and a 2 -aza-anthraquinone isolated for the first time from the plant kingdom. C. dichroa extracts showed strong reaction with Dragendorff’s reagents and five indole monoterpenoid alkaloids were isolated, including a novel vallesiachotamine lactone, whereas, C. glomerulata, (accepted name Psychotria glomerulata) yielded three new non iridoid quinoline alkaloids, named, glomeruiatine A, B and C. Interestingly the expected emetine-type alkaloids were not found in these three species.

The major alkaloids of the Rubiaceae are the indole monoterpenoid

149 alkaloids, derived via condensation of tryptamine and secologanin to yield strictosidine, which is the precursor of the whole range of indole monoterpenoid alkaloids. One exception to this biosynthetic pathway occurs in C. ipecacuanha where dopamine condenses with secologanin to yield isoquinoline alkaloids of the emetine type. Figure 5.1 (p.151) summarises the biosynthetic pathway of indole and isoquinoline monoterpenoid alkaloids. The findings, in the present work, clearly indicate that these biosynthetic pathways are not necessarily a characteristic of the genus as a whole, since the three Cephaëlis species studied do not contain isoquinoline-iridoid alkaloids, and only one of the three species contained tryptamine-iridoid alkaloids.

The genus Cephaëlis belongs to the tribe Psychotriae and the major alkaloids found throughout this tribe are of the polyindolenine-type, which are derived via polymerisation of tryptamine. Also non-iridoid quinoline alkaloids of the calycanthine-type have been found In Psychotha species (Adjibade et al., 1992), being derived from two molecules of tryptamine. Figure 5.2 (p. 153) shows the biosynthetic pathway of polyindolenine and calycanthine-type alkaloids.

The taxonomic position of Cephaëlis species is unclear. Steyermark, in 1972 and 1974, grouped the Cephaëlis species within Psychotha. Nevertheless, Dwyer in 1980 and Standley in 1985 retained Cephaëlis as a genus. More recently, however, Cephaëlis species have been included in Psychotha, subgenus Heteropsychotria (Taylor et al., 1994) and they have been combined mostly because both of them have tiny white flowers and the species of both are numerous and difficult to separate (Taylor, 1993).

Cephaëlis, which comprises some 2 0 0 species (Dwyer, 1980), was originally distinguished by its capitate inflorescences and relatively large floral and inflorescence bracts (Taylor, 1991 ). These plants are not clearly separable from Psychotha, however, and in addition apparently represent a widely polymorphic assortment of species with similar inflorescence structures

150 NH, NH O G la

Strktoüidine

NH

lO G in

Dopttmin» Dcacctyi iso iptcoside

Figure 5.1 Biosynthetic Pathway of indole and isoquinoiine Monoterpenoid Alkaloids.

(Steyermark, 1972). According to Taylor (1993) all the Cephaëlis species should be more correctly named Psychotria.

Psychotha is a large genus which comprises some 1650 species (Hamilton, 1989) and poses taxonomic problems. The genus has been divided into two principal sections, Heteropsychotria and Psychotria; species classified as

151 Cephaëlis have been assigned to the subgenus Heteropsychotria (Taylor, 1993). The subgenus Psychotria is pantropical and includes most of the African species and many Asian and Pacific species. These species are separated by its brown or grey drying colour, usually red fruits and deciduous stipules leaving a line of well-developed colleters (Taylor, 1994). Whilst, the Heteropsychotria species are neotropical and are characterized by green or grey-green drying colour, stipules usually persistent at least on the distal 3-6 nodes and bilobed or bidentate and not leaving a ring of colleters, and fruit blue or black (sometime passing through a red or orange intermediate stage) (Taylor, 1994). "In this group fall most (though not all) of the species formerly placed in Cephaëlis" (Taylor, 1994).

If the alkaloid content of the both subgenera Heteropsychotria (former genus Cephaëlis) and Psychotria (former genus Psychotria) is taken into account as a chemotaxonomic character, two major groups may be clearly separated (Figure 5.3, p. 155). The first group producing isoquinoline or indole monoterpenoid alkaloids, including C. ipecacuanha, C. dichroa, C. correae and C. axiiiaris. A second group producing non-iridoid alkaloids, such as polyindolenine and quinoline alkaloids, including C. giomeruiata, P. forsteriana and P. oieoides. Within the first group two sub-groups may be differentiated, those producing isoquinoline alkaloids as C. ipecacuanha and C. correae, and those producing indole alkaloids as C. dichroa, C. axiiiaris and C. correae. As an interesting feature, C. correae produces both isoquinoline and indole monoterpenoids alkaloids, therefore other taxonomic characters should be used to establish the relationship with one of the sub-groups. Fig 5.3 shows the biosynthetic relationships of the alkaloids produced in sub-genera Heteropsychotria and Psychotria.

Although C.camponutans contains a nitrogen-containing anthraquinone, it is not possible to situate it within this scheme, since the biosynthetic pathway to build up this 2 -aza-anthraquinone is unknown, but this species produces anthraquinones which are characteristic of the Rubiaceae as a whole.

152 Tycoon r CH,

L - Tryptophan N'-M«thyl Tryptamine Indolenh» r«dlcal

N-H

Ç'H p H- NHj

CEf^ H

Iso- Calyuvithlnt CaiycamtMn# CMmomanthina

Figure 5.2 Putative Biosynthetic Pathways of Non-iridoid Alkaloids of the Quinoline- and Poiyindoienine-Type Typical of the Tribe Psychotriae.

153 On the other hand, it does not mean, necessarily, that C. camponutans is not closely related to other species of the sub-genus Heteropsychotria or Psychotria. Since the production of alkaloids needs a whole block of active genes and if only one is affected the plant is not able to produce them. Although, perhaps the other genes are identical. That is why the presence of a taxonomic character is likely to be more important for taxonomic purpose than its absence (Cronquist, 1968). Interestingly enough, is the fact that, M. Nepokroeff (Department of Botany, University of Wisconsin), in a unpublished work has shown that C. camponutans should be included in the subgenus Notopleura within the genus Psychotria or maybe in a separate genus Notopleura not closely related to Psychotria at all (Taylor, 1994). This group of plant is recognized by a low, herbaceous, succulent, usually unbranched habit, pseudoaxillaris inflorescences, and strange stipules that are succulent and often deciduous (Taylor, 1994).

The presence of non-iridoid alkaloids in C. giomeruiata may indicate that this species should be better placed in the subgenus Psychotria, unless other taxonomic characters do not indicate so. Cephaëlis species produce different types of alkaloids, however, little is known about the chemistry of this large group of plants, now within Psychotha, and more work is needed to know if, a posteriori, alkaloids are of value as taxonomic characters.

Since Cephaëlis species produce indole and isoquinoline alkaloids and not the polyindolenine alkaloids characteristic of the tribe Psychotriae, it indicates that Cephaëlis species are different from other genera of Psychotriae. Nonetheless, genera should be defined in such way that they can be recognised without recourse to technical characters not readily visible to the naked eye (Cronquist, 1968). Consequently, since the alkaloidal content can not be easily examined, Cephaëlis species may be kept as member of Psychotria, in the sub-genus Heteropsychotria; unless a visible character is correlated to this group of plants.

154 Me DTI. Me OMe Dopamine OMe

Isoquinoiine monoterpencid alkaloids as those produced by C ipecacuanha, C, correae ...OGIu

> 3500 Indole-monoterpenoid alkaloids characteristic of Apocynaceae, Loganiaceae MI2 and Rnbiaceoe. C .dtchroa, C. axUaris, C. correae

Tryptamine

H3Q

C tt CH3

Colycanthine alkaloids fhwn Polyindobnine Alkaloids (non-iridoidaD C. glomerukUa, P. forsteriana from Psychotria species

Figure 5.3 Biosynthetic Reiationship of the Aikaioids Produced in Sub-Genera Heteropsychotria and Psychotria.

155 5.4.2 Picramnia antidesma subsp. fessonia

P. antidesma subsp. fessonia was selected for this study expecting to isolate quassinoids with activity against P. falciparum. As the activity of the quassinoids against P. falciparum and KB cell parallels the activity against brine shrimp (see Table 3.3, p. 8 8 ), it was decided to guide the fractionation using KB cell and brine shrimp. Nevertheless, only the butanolic fraction proved to be active against KB cell, but this fraction showed no significant activity against brine shrimp.

The butanolic fraction was submitted to CC yielding two known anthraquinones, aloe-emodin and aloe-emodinanthrone, and three new aloe- emodin C-glycosides, named Picramnioside A, B and C. Previous work with Picramnia (Leon, 1975; Popinigis et al., 1980; Arana and Juica, 1986) based their search for quinones on oxidative hydrolysis, therefore missing these C- glycosides.

An interesting feature of picramniosides is the that sugar, which has the configuration of xylose, contains an acetate or benzoate attached to position 5’. It is difficult to explain from the biosynthetic point of view, since a reactive group may be expected on position 5' of the sugar to react with acetate or benzoate, but, as far it is known, there is no known xylose substituted with reactive groups, such as hydroxyl, on this position.

In addition, the picramniosides showed strong activity against KB cell, whereas, the aglycone, aloe-emodin per se, is inactive against this cell line. Hence, it is important to continue working on this group of plants to follow up this rare group of C-glycosides and to evaluate them in different biological assays.

156 5.5 Are Plants Predictable In their Chemotaxonomy ?

Chemotaxonomy is the use of chemical evidence for classification of plant species and this is because a secondary metabolites present in plant cells represent not only a chemical entity, but are the manifestation of a whole series of enzymes which in turn are the genetic expression. The systematic position of the species is of excellent predictive value concerning the existence of useful chemicals (Gottlieb, 1982). Then related plant species may produce similar or related secondary metabolites and this criteria has been used to select plants for chemical studies (see Section 1 .2.3). Thus, species from Papaveraceae are expected to contain isoquinoiine alkaloids, species from Rubiaceae are expected to contain indole-monoterpenoid alkaloids and quinones, and species from Menispermaceae are expected to contain bbiq alkaloids.

Cephaëlis species were selected for this study expecting emetine analogues to be present and P. antidesma subsp. fessonia anticipating quassinoids. Although, compounds with activity against brine shrimp, KB cell and P. falcipamm were present none of the anticipated type of compounds were found. Instead, each Cephaëlis species yielded different compounds, namely, indole monoterpenoids alkaloids, aza-anthraquinone, benzoisochromanquinone and quinoline alkaloids; and P. antidesma subsp. fessonia yielded three new C- glycosides. Table 5.1 (p. 158) shows the compounds expected and those actually isolated from the plants selected for this study.

Some researchers do not investigate particular plant species because they are prejudged to contain compounds which are either too toxic or not very active on the basis of their chemotaxonomic relationship to a species which has previously been investigated. However, this result indicates that taxonomically related plants may yield and offer new leads in the discovery of useful drugs.

It is estimated that only about 1 0 % of plants have been investigated for pharmacologically active principles (Farnsworth and Morris, 1976), this figure

157 reflects that little is still known to predict with certainty what kind of compounds or pharmacological activities may have some group of plants.

Plant species Anticipated Found

Cephaëlis Emetine No emetine (Rubiaceae) analogues Indole alkaloids Aza-anthraquinone Quinoline alkaloids

Picramnia Quassinoids Anthraquinone (Simaroubaceae) C-glycosides

Table 5.1 Compounds Expected and those Found In the Plants Selected for this Study.

158 SECTION 6 CONCLUSION 1.- A new microwell method for brine shrimp has been developed that overcomes some of he disadvantages of the previous method.

2 .- Only cytotoxic compounds appear to be active against brine shrimp, while compounds acting on the nervous system are inactive.

3.- The activity of the quassinoids against Plasmodium falciparum and KB cell parallel the activity against brine shrimp.

4.- There are some 200 species of Cephaëlis occurring throughout the

tropics of which 2 1 occur in Panama. Until recently, knowledge of the chemistry of Cephaëlis was limited to C. ipecacuanha which contains monoterpenoids isoquinoiine alkaloids and to C. stipulacea which contains gramine.

5.- Cephaëlis camponutans yielded benz[g]isoquinoline-5,1 0 -dione for the first time isolated from the plant kingdom and a new 1-hydroxibenzoisochromanquinone. Both compounds showed activity against brine shrimp, KB cell and P. falciparum.

6 .- Cephaëlis dichroa yielded a new indole alkaloid, vallesiachotamine lactone, together with four known indole alkaloids, vallesiachotamine, angustine, strictosidine lactam and strictosidine.

7.- Cephaëlis giomeruiata yielded three new quinoline alkaloids, named glomerulatine A, B and C, but none of the spectroscopic techniques used in the structure elucidation distinguished between calycanthine or iso-calycanthine-type. Only X-ray crystallography would unravel this problem.

8 .- Since this study started two Cephaëlis species from Panama have been studied in coordinated work by the Centre for Pharmacognostic

160 Research (CIFLORPAN) at the University of Panama. C. correae yielded the known iso-dolichantoside, three new indole monoterpenoid alkaloids, and one isoquinoiine monoterpenoid glucoside alkaloid and C. axillaris yielded two known indole monoterpenoid alkaloids,

vallesiachotamine and 6 ’-trans-feruloyl-lyaloside.

9.- Picramnia antidesma subsp. fessonia yielded three new C-glycoside anthraquinones, named picramniosides A, B and C, and two known anthraquinone aloe-emodin and aloe-emodinanthrone.

10.- Previous work with Picramnia based their search for quinones on oxidative hydrolysis, therefore missing these type of C-glycosides.

11.- The alkaloids prove to be a good chemotaxonomic character to unravel the taxonomic position of the Cephaëlis species.

1 2 .- The plants studied were selected using the chemotaxonomic approach and even though some compounds has shown activity against brine shrimp, KB cell and P. falciparum, none of the expected compounds were found.

161 SECTION 7 REFERENCES Achenbach, H., Lottes, M., Waibel, R., Karikas, G.A., and Gupta, M.P.(1993) New alkaloids from Cephaëlis correae. Abstracts of the 41 Congress of the Gesellschaft fur Arzneipflanzenforschung, Society for Medicinal Plants Research. Dusseldorff, Germany. Poster # 50, p. 44.

Adjibade, Y, Weniger, B., Quirion, J.C., Kuballa, B., Cabalion, P. and Anton, R. (1992) Dimeric alkaloids from Psychotria forsteriana. Phytochemistry. 31, 317-319.

Adjibade, Y., Hue, B., Pelhate, M. and Anton, R. (1991) Action of calycanthine on nervous transmission in cockroach central nervous system. Planta Medlca. 57, 99-101.

Adjibade, Y., Kuballa, B., Cabalion, P., Jung, M.L., Beck, J.P. and Anton, R. (1989) Cytotoxicity on human leukaemic and rat hepatoma cell lines of alkaloid extracts of Psychotria forsteriana. Planta Medlca. 55, 567-568.

Ahmad, R. and Cava, M.P. (1977) Grisabine and grisabutine, new bisbenzylisoquinoline alkaloids from Abuta grisebachii. Journal of Organic Chemistry. 42,2271-2273.

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184 APPENDIX I. SPECTRA Spectrum # Page # 1 Mass spectrum of 1-hydroxybenzoisochromanquinone 188 2 NMR spectrum of 1-hydroxybenzoisochromanquinone 189 3 NMR spectrum of 1-hydroxybenzoisochromanquinone 190 4 NMR spectrum of 1-acetatebenzoisochromanquinone 191 5 Mass spectrum of benz[g]isoquinoline-5,10-dione 192 6 NMR spectrum of benz[g]isoquinoline-5,10-dione 193 7 NMR spectrum of benz[g]isoquinoline-5,10-dione 194 8 Mass spectrum of vallesiachotamine lactone 195 9 NMR spectrum of vallesiachotamine lactone 196 10 COSY-45 NMR spectrum of vallesiachotamine lactone 197 11 Mass spectrum of vallesiachotamine 198 12 NMR spectrum of vallesiachotamine 199 13 Mass spectrum of strictosidine 200 14 NMR spectrum of strictosidine 201 15 COSY-45 NMR spectrum of strictosidine 202 16 ^H-^®C NMR spectrum of strictosidine 203 17 Mass spectrum of strictosidine lactam 204 18 NMR spectrum of strictosidine lactam 205 19 Mass spectrum of tetraacetyl-strictosidine lactam 206 20 NMR spectrum of tetraacetyl-strictosidine lactam 207 21 NMR spectrum of tetraacetyl-strictosidine lactam 208 22 Mass spectrum of angustine 209 23 Circular dichroism of glomerulatine A 210 24 Mass spectrum of glomerulatine A 211 25 NMR spectrum of glomerulatine A 212 26 ^^C NMR spectrum of glomerulatine A 213 27 ^H-^% NMR spectrum of glomerulatine A 214 28 ^H-^^C long range correlation NMR spectrum of glomerulatine A 215 29 ROESY NMR spectrum of glomerulatine A 216 30 Mass spectrum of glomerulatine B 217

186 31 NMR spectrum of glomerulatine B 218 32 NMR spectrum of glomerulatine B 219 33 Mass spectrum of glomerulatine C 220 34 NMR spectrum of glomerulatine C 221 35 NMR spectrum of glomerulatine C 222 36 COSY-45 NMR spectrum of glomerulatine 0 223 37 long range heterocorrelation NMR spectrum of glomerulatine 0 224 38 ROESY NMR spectrum of glomerulatine 0 225 39 Mass spectrum of picramnioside A 226 40 Circular dichroism spectrum of picramnioside A 227 41 NMR spectrum of picramnioside A 228 42 NMR spectrum of picramnioside A 229 43 long range heterocorrelation spectrum of picramnioside A 230 44 ROESY NMR spectrum of picramnioside A 231 45 Mass spectrum of picramnioside B 232 46 Circular dichroism spectrum of picramnioside B 233 47 NMR spectrum of picramnioside B 234 48 COSY-45 NMR spectrum of picramnioside B 235 49 heterocorrelation spectrum of picramnioside B 236 50 ROESY NMR spectrum of picramnioside B 237 51 Mass spectrum of picramnioside C 238 52 Circular dichroism of picramnioside C 239 53 NMR spectrum of picramnioside C 240 54 ^®C NMR spectrum of picramnioside C 241 55 ^H-^% long range heterocorrelation NMR spectrum of picramnioside C 242 56 ROESY NMR spectrum of picramnioside C 243 57 Mass spectrum of aloe-emodin 244 58 NMR spectrum of aloe-emodin 245 59 Mass spectrum of aloe-emodianthrone 246

187 93SE270tl xl Bgd=0 01-MRR-93 14 5*01 00 2R8-SE El* 8pM=8 1=2.4v Hn=900 TIC=173988000 RV RcntULSOP SysDCI 15877000 ÇP4 El 190 DEGC PT=0® CalDRVCRL MRSS= 164 101 184 95. 90 128 85 J 80 75 70 212 65 60 55 50 45 76 48 156 35 104 30

25 58 115 20 15 173 201 83 230 10 63 91 145 5 57 98 22 L 253 267 ilk AM ii U 1 ill till |L kL ui/ii 68 100 120 140 160 180 200 220 240 260 Spectrum 1. Mass spectrum of 1-hydroxybenzoisochromanquinone

188 1 OH

1 -GH-Benzoisochromanquinone

_r

4 b 3 3 1 2.5 2 1 1.5 I t t 9 0 0 Spectrum 2. ’H NMR spectrum of 1-hydroxybenzoisochromanquinone

189 18 ÎR '

CC2 in cnci 1 t

R55S55S5 V \ \ H v y I Y I

Spectrum 3. "C NMR spectrum of 1-hydroxybenzoisochromanquinone

190 Current Oeta1 Parew ters NAME acety]cc2 CXPNO 10 pnocNO >

F2 - Acquisition P iriK tsrs 930525 T lw 10.35 PULPflOG 2930 SOLVENT CDC13 AO 1.9661000 stc FlOflES 0.254313 Hz ON 60.0 wsec AS 912 NUCLEUS IH HLl 1 08 01 1.0000000 stc FI OE SFOl 400.1366620 MHZ SMK 6333.33 HZ 10 32766 MS 16 OS 2

SI 16364 SF 400.1343656 MHz NON EM SSB 0 LI 0.30 Hz 66 0 FC 1.00

ID NMR plot porsM ttrs cx FIR 9.600 ppB FI 3641 29 Hz F2P -0 400 ppm F2 -160.05 HZ PPMCM 0.25000 ppm/cm HZCH 100.03360 Hz/cm Me

J i. r

Spectrum 4. *H NMR spectrum of 1-acetatebenzoisochromanquinone

191 93SC2B9I1 xl Bgd=8 01'nnR-93 14 4*0 88 88 ZflB-SE El* Bpn=0 1=1.9v llfl=980 T1C=82444000 HV Acnt UlSOP Sys OCI 12246080 CC3 El 138 DEGC PT= 0® CaL BflVCflL MHSS: 289 188, 209 95. 90. 85.

75. 70. 65.

25.

111 119. ??' 239 200 268

Spectrum 5. Mass spectrum of benz[g]isoquinoline-5,10-dione

192 J

8

Benz[g]isoquinoline-5,10-dione

J u

Spectrum 6. NMR spectrum of benz[g]isoquinorme-5,10-dione

193 sr T s “i5 55 «

CCI repeat of 13C using Inverse gated ilecoupiing to give almost quantitative spectrum

Spectrum 7. "C NMR spectrum of benz[g]isoquinoline-5,10-dione

194 3?2f0?2ll xl Bqd=8 20-jnN-92 15 48* 1 0 8 ZAB2F E l* 0pM=O U 7 .Û V Hm=890 T1C=5B0313024 flV flc n t ULSOP Sys ERE in s HMR: 50845000 1XÏ CD3-C [[ VARIOUS lEHPERAIURES P1=0“ CalDRVCRL MASS: 305 100, 305

7 5 .

60.

4 5 .

3 0 .

333 2 0 . 209

277 31?

319 . w ll ikllL.illilWlliilklijljlilimylilii hujjljl Li\lil Lyall! 100 150 200 300 400

Spectrum 8. Mass spectrum of vallesiachotamine lactone

195 CD3-C IN C0CL3 *TMS. MM250 IH

COOMe

PPM/CM

17 OMe

15; 14

Spectrum 9. NMR spectrum of vallesiachotamine lactone

196 CD3-C IN C0CL3; HM250 IH COSY SPECTRUM

u

csa

7 .5

J 8.0

PPM . I .... I ... I .... I I .... I...... I.... I .... I .... I .... I .■■■ 1 . . I .... I ... I . 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 .5 PPM

Spectrum 10. COSY-45 NMR spectrum of vallesiachotamine lactone

197 922F045I1 xl Bgd=0 H-JflN-92 10 46*0 ZRB2F E l* Bpn=0 I=7.9 m Hn=090 TIC=38297?B944 AV Rcnt ULSOP Sys IRE 1RS HMR: 51504008 [02-H El 190 DEGC PT= 8“ Cal ORVCRL MASS 279 100, 279

9 0 .

7 5 .

6 5 . 6 0 . 5 5 . 249 5 0 . 4 5 . 350

307 322 3 0 . 2 5 . 209

236

U lM i L> L4 II11 108 150 200 250 388 Spectrum 11. Mass spectrum of vallesiachotamine

198 C02-C IN CDCL3 +IMS. NM250 IH

HZ/PI

“COOMe

O M e Me

19

ULi

Spectrum 12. 'H NMR spectrum of vallesiachotamine

199 92SCI72eil xl Bgd=0 18-N0V-92 I1=B>G 88 ZRB-SE FB* BpH=8 1=8.5v Hm 28B8 TIC=352BB48B8 HV Rcnt-ULSOP SysLMLRFRB HMR: 554 5 5 8 8 8 CDl HNQBfl > Nfl MATRIX PT= 8* CalFRBCRL MASS: 176 108,

85.

75.

5 5 . 5 0 .

136 236 329

187 128 214 258 289 307 352 373 309 413 553 514 604 648 208 300 408 508 Spectrum 13. Mass spectrum of strictosidine

200 F2 - Acquisition Parameters Date 930324 Tine B.59 PULPROG zg30 SOLVENT HeOH AQ 1.9660922 sec FIORES 0.254314 Hz ON 60.0 usee RG 512 NUCLEUS IH HLl 1 dB 01 1.0000000 sec PI 9.5 usee DE 75.0 usee SFOl 400.13B4705 MHZ SMH B333.37 Hz TO 3276B NS B OS 2 a F2 - Processing parameters SI 163B4 O Glu SF 400.1359667 MHz WOW S5B 0 LB 0.00 HZ Me(X)C GB 0 PC 1.00

10 NMR plot parameters CX 40.00 cm F IP 9.600 ppm FI 3B41.31 HZ F2P -0.400 ppm F2 -160.05 Hz PPMCH 0.25000 ppm/cm HZCH 100.03399 Hz/cm

Spectrum 14. NMR spectrum of strictosidine

201 CD-I COSY 45 spectrum

-8

ppm

Spectrum 15 COSY-45 NMR spectrum of strictosidine

202 CO-1 in C0300 : inverse mode 1H-13C sh ift correlation: 13C garp decoupled

L

T—' ppm

Spectrum 16. 'H-"C NMR spectrum of strictosidine

203 922fB55ll xl Bgd=0 16-JRN-92 18 49*0 88 ZAB2F E l* l=4.9v IU=asa TIM112G31040 AV Aa.L ULSOP Sys LREIfIS ;iUR: 32jJ[038 C 03-H E l VARIOUS TEMPERATURES PT=8® CalBAVCAL HASS: 235 108, 235

498

4 6 7 4 8 0 .A-*- 288 250 300 358 488 458 588 5!S

Spectrum 17. Mass spectrum of strictosidine iactam

204 H367B8.001 DATE 1 0 -2 -9 2 TIME 12: 09

SF 2 5 0 .1 3 4

5 3 7 5 .0 0 0 16384 16384 SH 3 7 5 9 .3 9 8 HZ/PTr .459 PH AO AQ 2 .1 7 9 AG 200 NS TE FW 4700 02 0.1 OP 63L PO LB

CX 4 0 .0 0 CY 2 3 .0 0 9 .6 0 1P F2 - 398P HZ/CM 6 2 .5 2 7 PPM/CM .250 3 83 8.5 7

18

I I------1...... 1...... I...... i....,....i...... I 11 ■■111■«.111■111.1.11..1.1... 11.... I....1 1 8.00 7.50 7.00 6.50 6.00 5.50 5.00 4.50 4.00 3.50 3.00 - 2.50 2.00 1.50 1.00 .50 0.0 PPM Spectrum 18. 'H NMR spectrum of strictosidine lactam

205 922F1118#1 x l Bgd= 26-SEP-87 13 55« 1=88 ZR82F E h BpH=0 I=lBv Hn=890 TIC=9023421440 RV Rent: Sys:LREinS 5 8 7858 00 RCET CD3 ( 1 ) E l 190 DEGC PT=0® CalDRVCRL MRSS: 666 101 666 9 5 . 9 0 . 8 5 . 8 8 . 7 5 . 7 0 . 8 5 . 60 55 J 58 4 5 . 4 0 . 3 5 . 3 0 . 2 5 .

2 0 . 379 1 5 . 564 624 1 8 . 5 . 698 446 582 I X i l l i X 358 458 588 550 688 650 700 750 Spectrum 19. Mass spectrum of tetraacetyl-strictosidine lactam

206 AcetrJ-CD3 IN C0CL3 tlHS. NN2S0 IH

H 3 9 t5 0 .0 0 t

HZ/PT

PPM/CM

I j

Spectrum 20. NMR spectrum of tetraacetyl-strictosidine lactam

207 ACETY-C03 IN C0CL3. 1H-13C CORRELATION SPECTRUM B j

C39155 SHX F I PflOJ C03H 001 F2 PflOJ C03C 001 AU PflOG XHCORflO AU A. DATE 4 -1 1 -9 2 J SI2 2049 ! S Il 256 SK2 6771 930 1 1 ? 0 l 983 09: NOG - I 1 1 .5 M0N2 6 ! NOXl S LB2 8.000 6B2 0 .0 i g 2 .0 S301 0 MC2 p 1 ! 1 PLIM flow 1 •; 1 FI 159 t.ÿ^P 1 2 .5 F2 16 763P 1 AND COLUMN 1 , F l ■ i 1 F: 3 .0 DI 31 iH PI 17 * ! 1 1 01 00101.:'" 3 .5 03 p : P4 Ï9 I ' 1 ' 1 9 r 04 vOiev J 3: *.4H flO 0 0 PM 0 4.5 DE 71 2 Mb 32 05 ME 123 IN 0)1:54? 1 III' , 5 0

1 ’ 1' 1 1 ' 5 .5 "il 6 0

’ 6 .5

7 .0

V, , 7 .5

0 .0 1

Spectrum 21. NMR spectrum of tetraacetyl-strictosidine lactam

208 92Û0E24I1 xl Bgd=0 05-nflR-92 14=3*0=00=00 12-250 El* BpM=0 1=4.2v Hn=650 110=398292000 HV Rcnt-ULSOP Sys^STEnOEF HMR: 27694000 C D l- 8 IH E SCHOOL OF PHHRMHCY PT= fl® C a lU C H L MRSS: 28 100. 20 313

298

55 G3 V 255 282 2 29 L 150 200 250 300 358 Spectrum 22. Mass spectrum of angustine

209 3.000E+01 No. Wavalenath Valua 1 310.30 nm -3.492E+01 2 296.00 nm - 3 . 109E+01 3 280.60 nm 4 . 0 11 E- 02 4 268.60 nm 2.662E+01 5 246.60 nm 2.990E+00 6 . .. 2 1 4 .7 0 . J im .- 2 .1 7 a E t0 1

4.000E+01 ZO O . O WL [nm] 400.0 5: — CGI (CH3CN, C - 1.05 E-4 M)

Spectrum 23. Circular dichroism of glomerulatlne A

210 scan 37 (I. 2 45 mini of DPITM: B8 5 3 4 . D

CRLED

-.1) a c iTj 5 0 1 TS C

Œ

2 0 9 1 6 7

3 Mas s/Ch arge

Spectrum 24. Mass spectrum of glomerulatlne A

211 6.9738 6.9590 6.9553 6.9396 6.9360 6.7831 0.42 6.7794 6.7644 6.7607 6.6598 6.6567 0.43 6.6413 6.6383 0.48 6.6229 0.50 6.6198

— 6.2431

!2 3 K

z S so Z D Î ro Ui N) % s c 3 3.0949 2.21 3.021 r 3.0051 a 2.9971 (Q 0.59 2.9811 2.9731 0 2 .9 5 7 L 2.9251 1 2.6351 2.6130 c 0.56 2.5899_ 2.16 2 L g 1.9794 3 1.9567 1.9259 1.9019 1.3973 1.3816 1.3665 1.3508 1.3059 00 0) — 165.1055

B) 147.3194

12S.9007 125.2713 I ti a> 125.0362 3 J J126.^059 '-^125.0410 to 1 ^ 1 2 3 .6 7 6 1 G) — 122.2509

0 z s 33 W ro 0) CO 1“t 00 c 3 2. (Q CJ 0 1

w 3I &)

Z È (D 30.8696 30.2630 L11 il (ppm) - - LL ^

0 1 «0

0 0

- 0 oO •0

(ppm)

Spectrum 27. 'H-"C NMR spectrum of glomerulatlne A

214 J ü J J -

$ «

• o * a

(ppm)

Spectrum 28. long range correlation NMR spectrum of glomerulatlne A

215 ■Ll IJJ. *___ I

4 * 0 ^

M •. 7 ? °

o

c

0 o oâ 1 r , <9 »

(ppm)

Spectrum 29. ROESY NMR spectrum of glomerulatlne A

216 f>e«criptior?: P a h \o i CÛ2 ) E /l. 7 0 ev Te*tp. F 2 0 PC Date: 24-1 I -1993 Time: 13:59: 22

Scans 14 P r 0:23 Pra

111, ■ Ji.. liliil .1' j. I'll piii'l 350

tiass

Spectrum 30. Mass spectrum of glomerulatlne B

217 7.0274 6.9133 6.8945 6.8745 6.7798 o o z 6.7703 6.7610 6.7506 6.7426 6.7398 6.7180 6.7143 6.6992

f a C 3 w

% z s 3 09Z (/) ro ■O 1 œ S 3.6774 3.6602 3.6368 3.5876 3 3.5713 3.5630 a 1.32 3.5470 (O 3.4963 3.4750 0 3.3007

1 2,59 2.5248 2.5168 3I 2.5011 (D 2.4934 m 1.00 1.8008 1.7836 1.7793 1.7704 1.7559 1.7393 1.7242 — 166.7668 8o

— 146.1073

128.9094 128.8374 124.5720 J124.1019 IC ^ 123.6924 3 J r 122.9417 - ^ 1 2 2 .6 2 7 0 N ^ 1 2 1 . 0 7 2 5

w o ro (/) CD 1 c 3 a CQ 0

■n1 c

3I

— 32.6717 31.5987 ^ 30.4423 Sc an 104 ( 2 . 6 7 5 m i n ) o f DRTR:B8583.D 100:

57 SCALED 90-

80' 3 4 3

70' 257

60:

c 1 3 0 (tj 50:

3 40': Œ. 169 30: 1 9 7 2 1 9 / y

10:

100 d m d m M a s s - ''C h a r g Spectrum 33. Mass spectrum of glomerulatlne C

220 7.0341 7.0153 7.0008 6.9830 6.8397 6.8099 6.7911“ 6.7674 6.747 4- 6.4851 6.4682 6.2126 6.1930 I C 3 % — 3.8695 3.7890

% z 2.9743- 2.7049 2.6831 0) 2.6597 2.3033 2.1397- 1.9598 1.9419 1.8736 I 1.8589 3 1.8407 a 1.5486 (Q 0 1 I. s O

Z-ŒOO — 166.9790 S

148.1450

— 142.5783

r 129.9993 I-n / r 125.6666 C y j - 125.4031 3 J - 123.4761 T h 122.7226 = ^ 1 2 2 .5 8 7 0 N — 118.0042 114.2469 M O

33 1 0)

1■t c 3 a — 76.9857 (Q 0 1 C I 3 — 50.9244 (D 49.6277 48.9860 O ^ 45.7690 — 40.0392

34.4866 — 32.1852 ^ 31.2052 j j A U i f

0 O (

(> § / ^ « t ; jP caO 3 o 3 y

m

•

o o as o

f o ° °

0 1)

Spectrum 36. COSY-45 NMR spectrum of glomerulatlne C

223 (ppm) -A- J_I i k, jAJ

■ ♦ —

(ppm)

Spectrum 37. long range heterocorrelation NMR spectrum of glomerulatlne C

224 (ppm)

Spectrum 38 ROESY NMR spectrum of glomerulatine C

225 94SE232I1 xl Bgd=l 14-FEB-94 14M7>8=BBiflB ZflB-SE FB* BpM=75 I=4.5v H«=1981 TIC=4G?712932 RV SU Rcnt ULSOP Sys LMFHB 18358888 PRF-3 (flkeV Xe ♦FRB/HS) GLgc/ThlogLyc/TFR natrLx. PT= 8® Cal FRCHL MHSS= 185

I ' 9

589

531 4JJ433 459 475 4M I Î 547 GB2 I iiHmMiHiiinii^i I wiiii 588 G88 788 Spectrum 39. Mass spectrum of picramnioside A

226 l.OOOE+Oi No. WavBlenath Valua 1 342.80 nm 1.682E+00 2 314.20 nm - 2 . 769E-02 3 290.00 nm -6.363E+00 4 239.00 nm 1.698E+00 0 230.80 nm -3.898E+00 . s ___2 2 1 .2 0 nm____l_ ^ 4 4 E t0 1 .

Am

8.000E+00 200.0 WL [nm] 000.0 0: PAF3 (Matanol. c - 8.60 E-O M)

Spectrum 40. Circular dichroism spectrum of picramnioside A

227 OH PAF3

F2 - AcquJilt:»n Parauters OH

zgao

I OH

HOH

10 rMR p lo t

J Lv V

Spectrum 41. NMR spectrum of picramnioside A

228 RI RI g 3 8 ai R Z 3 3 iïv IV V

HOHiC

PAF-3 ; normal 13C 53 Si ;RR 8» 8 !î 9 8PR 5 8 3 2 993999993 \r II 111 V \ w I IV III I

3"

5’ V 10

PP# 100 160 140 120 100 60 40 Spectrum 42. "C NMR spectrum of picramnioside A

229 PAF 3 ; long rango Invtrtt 1H-13C corrilitlon: 13C coupltd u

I*,**:*

Spectrum 43. ’H-” C long range heterocorrelatlon spectrum of picramnioside A

230 PAF3 ROESY spectrum; pha se sensitive; positive peaks red;negative black J

5.0 4.5 4.0 3.5

Spectrum 44. ROESY NMR spectrum of picramnioside A

231 94SE218I1 xl Bgd=l 13-FEB-94 IB 55*8=6 =88 ZflB-SE FB* BpM=lB5 I=4.5v Hm=1341 T10=124392888 RV SU flcntULSOP Sys=LMFflB HMR= PRF-4 CBkeV Xe ♦FfiB/tlS) GLy/ThLogly/TFfl m atrix. PT=8® CalFflCflL MflSS= 258 256

447

387

, I ■ I 300 350 408 450 500 558 658

Spectrum 45. Mass spectrum of picramnioside B

232 3 . O O O E + 0 0 No. WavBlenath Value 10 1 399.00 n m 2.666E-01 2 336.20 n m -2.314E -02 3 321.60 n m -9.670E -01 4 306.60 n m -4.969E -03 5 294.60n m 1 .246E+00 6 272.00 n m -2.644E -03 7 266.00 n m -9.499E -01 a 240.40 n m 1.066E-01 9 226.60n m -2.271E+00 10 210.60 n m __2.4aiE±Q 0 Am

*3, OOOE^OO -I— 1. 200.0 W L [n m ] 6 0 0 . 0 6: PAF4 (Matanol. c - 1. 8E-4 M)

Spectrum 46. Circular dichroism spectrum of picramnioside B

233 ..re n t D#t# Parvaeters name «U01693* EXPNO 1

F2 - Acquisition Psraoetiri OH

OH

F2 - Proctsilng piraaetfn

pp«/4 Me Mz/Cl

10

Spectrum 47. NMR spectrum of picramnioside B

234 PAF4 ; cosy 45

_jT V jl-1 A full J 00 ^ i B dg

i

m » s>- '>

,S OGS

3 /• '/C o >

a 1)S0

7.0 6.5 6.0 5 5 5.0 4.5 4.0 3.5

Spectrum 48. COSY-45 NMR spectrum of picramnioside B

235 . ppa 180 160 140 180 100 80 GO 40 20

Spectrum 49. 'H-"C heterocorrelation spectrum of picramnioside B

236 Ui _ y J L v_

0 «

» ► 0|

100

______jJ

i.f

°0°

5.0 4.5 4.0 3.5

Spectrum 50. ROESY NMR spectrum of picramnioside B

237 94SE233I1 xl Bgd=l M-FEB 94 17=18^8 88 80 ZHB-SE FB* BpM=75 I=582m v H #=1343 T IC = 73 958888 RV SU R cnt ULSOP Sys LHFRB 228 1 8 0 8 PRF-5 (DMSO) (BkeV Xe ♦FRB/HS) Glyc/Thloglyc/ÎFR matrix. PT= 8* Cal FRCRL MRSS: 256 188^ 256 9 5 . 9 8 .

447 6 8 .

5 8 . 4 5 . 239 3 5 . 3 8 . 2 5 . 2 8 .

287 298 548 378 438 2 83 632 314 469 288 388 588 688 Spectrum 51. Mass spectrum of picramnioside C

238 7 . O O O E + O O No . Mavelanatn Vmlua 1 391.40 nm 7.760E-01 2 317.00 nm -1.8G0E-02 3 284.00 nm -5.373E+00 4 298.60nm 8.001E -03 9 253.40 nm i.116E+00 6 230.00 nm -3.132E+00 .7. . -210 .SO.jn n -___5..4ZOEfOO_

G .O O O E + O O 200.0 WL Inn] 7: PAFO (Matsnol. c - 8.5E-S M)

Spectrum 52. Circular dichroism of picramnioside 0

239 C u rrin t Data Paranettrs N»H£ )n 2 « 3 .3 EXPNO n PBOCNO J PAF5

F2 - Acqultitlon Paraaetcrs OH

OH

iPH OH'

F2 - n-ocaastng paraaattri HOH 2'

10 p lo t parameters

Me 140.04770 Hx/i i

10

I .. < ■ ■ I.: 3

Spectrum 53. 'H NMR spectrum of picramnioside C

240 V S w ' V Y N il Y V Y

Ui

? A = R8 : sâ §3 %R s%s ofs ? Rx: «; Asasi&iiP § & zzzg Biz 9 ?s as ss:;£ssssss ss srss % 3 g ai ai gaiaigRagg** %% î W M \ V V Y ST 111 Y \ V I

J##WWMm ill J L mW#wW# ppa 180

Spectrum 54. "C NMR spectrum of picramnioside C

241 PAF5, Ion* ronge 13C-1H c o rre la tio n

" ' • -

' ♦ » t. •

f *

: 5!i>

•

ppm 180 160 140 120 100 80 60 40 20

Spectrum 55. long range heterocorrelatlon NMR spectrum of picramnioside C

242 PAF5; ROESr tppi

ÜJ. I. H iAj.

- " i 1

0 1 • r •M . / -, Vt'w : 4 .

r # \ • •

J

Spectrum 56. ROESY NMR spectrum of picramnioside 0

243 93028111 xl Bgd=0 16-NQV-87 8^8«8 88 80 12-258 Eh BpH=8 I=4.2v Hm=980 TIC=150942800 RV Rcnt= SysSTEHDEF HMR: 2 7 73 2000 PRE 1 El 1B0OEGC PT= 8° CallCRL HHSS: 278

9 5 . 9 8 . 8 5 .

7 8 . 6 5 .

6 8 .

5 8 . 4 5 .

3 5 . 3 8 . 2 5 .

2 0 .

213 224 252

150 208 258 Spectrum 57. Mass spectrum of aloe-emodin

244 Currint Dtti P iriM tiri NAME p i l l EXPNO 10 PROCNO 1

plot pii M itiri

12.600 ppa

0.32500 ppa/ca 130.04430 Hz/ca

Spectrum 58. 'H NMR spectrum of aloe-emodin

245 93SE1253#! xl 23-AUG-93 89 39^ 188 2R8-SE E l* BpH=0 1=4,5v H#: TIC=27965ieB8 RV RcntULSOP SysDCI HHR: 16836088 PflFR E l PT=8® CalBCICRL HRSS: 256 1 8 1 256 9 5 . 9 8 . 8 5 . 88 75 78 6 5 . 68 . 5 5 . 5 8 . 4 5 . 4 8 .

3 5 . 89 3 8 . 8 226 238 197 2 5 . 2 8 . 94

1 5 . 181 1 8 . 165 5 . 83 58 8 llii ii# 4uiIL 128 148 168 188 288 228 248 268 288 308 Spectrum 59. Mass spectrum of aloe-emodianthrone

246 APPENDIX II LIST OF SCIENTIFIC PAPERS 1.- Solis, P.N., Wright, C.W. and Anderson, M.M.: "Brine Shrimp: A Simple Microwell Bench Top Bioassay". The Square Research Journal. January, 1993.

2.- Solis, P.N., Wright, C.W., Gupta, M.P. and Phillipson, J.D.:"Alkaloids from Cephaelis dichroa. Phytochemistry. 33(5) 1117-1119, 1993.

3.- Solis, P.M., Wright, C.W., Anderson, M.M., Gupta, M.P. and Phillipson, J.D.:" A Microwell Cytotoxicity Assay using Artemia salina (Brine Shrimp). Planta Medica. 59(3), 250 - 252, 1993.

4.- Lang'at, C.G., Allen, D., Solis, P.N., Phillipson, J.D., Watt, R.A., Toth, I., Kirby, G.C. and Warhurst, D.C.i'The Activity of Quassinoid Analogues Against Chloroquine Resistant Plasmodium falciparum in vitro. Proceedings of The 2P^ Annual Conference of The U.K. Association of Pharmaceutical Scientists. April, 1993.

5.- Solis, P.N., Wright, C.W., Gupta, M.P. and Phillipson, J.D.: "Cephàelis Species as Sources of Alkaloids". Proceedings of The Annual Conference of The U.K. Association of Pharmaceutical Scientists. April, 1993.

6.- Solis, P.N., Gupta, M.P. and Phillipson, J.D.: "Cephaelis Species, A Chemotaxonomic Dilemma". International Symposium of the Phytochemical Society of Europe. Lausanne, Switzerland. 29-Sept-1-Oct., 1993.

7.-Lang'at, C.C., Solis, P.N., Watt, R.A., Phillipson, J.D., Kirby, G.C. and Warhurst, D.C.:"Quassinoid Analogues: Their in v/Yro Antimalarial and Cytotoxic Activity", international Symposium of the Phytochemicai Society of Europe. Lausanne, Switzerland. 29-Sept-1-Oct., 1993.

8.- Solis, P.N., Lang'at, C., Gupta, M.P., Kirby, G.C., Warhurst, D.C., and Phillipson, J.D."Bio-active Compounds From Psychotria camponutans".

248 Submitted to Planta Medica. 1994.

9.- Solis, P.N., Gutierrez-Ravelo, A., Gonzalez, A.G., Gupta, M.P. and Phillipson, J.D."Bioactive Compounds from Picramnia antidesma subsp. fessonia". Proceedings of The 3"*^ Annual Conference of The U.K. Association of Pharmaceutical Scientists. March, 1994.

10.- Solis, P.M., Gutierrez-Ravelo, A., Gonzalez, A.G., Gupta, M.P. and Phillipson, J.D."Bioactive Anthraquinone Glycosides from Picramnia antidesma subsp. fessonia". Submitted to Phytochemistry. 1994.

11.- Solis, P.N., Lang’at, 0., Kirby, G.C., Warhurst, D.C. and Phillipson, J.D. "Cephaelis Species, Diversity of Bioactive and Novel Compounds". Submitted to the British Pharmaceutical Conference. 1994.

12.- Solis, P.M., Gutierrez-Ravelo, A., Gonzalez, A.G., Gupta, M.P. and Phillipson, J.D "Quinoline Alkaloids from Psychotria glomerulata". In preparation.

249