BIOACTIVE PRINCIPLES FROM PANAMANIAN MEDICINAL PLANTS

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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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 A CTIVITY 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. falciparum àssay 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 (UV) 92

4.1.4.2 Infrared spectroscopy (IR) 92

4.1.4.3 Mass spectrometry (MS) 93

4.1.4.4 Nuclear magnetic resonance (NMR) 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


4.3.4 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 from Cephaëlis 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


4.4.2.3 Strictosidine 1 1 2


4.4.2.4 Strictosidine lactam 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


4.4.3 Discussion 117

4.5 Isolation and identification of alkaloids from

Cephaëlis glomerulata (Psychotria glomerulata) 121

4.5.1 Extraction and separation procedure 121


4.5.2.1 Glomerulatine A 121

4.5.2.1 . 1 Spectral data 121

4.5.2.2 Glomerulatine B 122


4.5.2.5 Glomerulatine C 123



4.6 Isolation and identification of bioactive compounds

from Picramnia antldesma var. fessonia 131

4.6.1 Extraction and separation procedure 131


4.6.2.1 Picramnioside A 132

4.6.2.1 . 1 Spectral data 132

4.6.2.2 Picramnioside B 133


4.6.2.3 Picramnioside C 135


4.6.2.4 Aloe-emodin 136


4.6.2.5 Aloe-emodinanthrone 136



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

SECTION 6 CONCLUSIONS 159

SECTION 7 REFERENCES 162

APPENDIX I SPECTRA 185

APPENDIX n LIST OF SCIENTIFIC PAPERS 247

II

List of Abbreviations

pCi

pM

Dept

'H

2D

BBIQ

BuOH

br

QD,

CC

CD

CD3 OD

CDCI3

CIMS

cm'

COSY-45

d

DCCC

dd

ddd

DMSO-Dg

EDgo

EIMS

EtOAc

EtOH

eV

Microcurie

Micromolar

Distortionless enhancement by polarization transfer (differentiation

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

polarisation transfer)

Carbon thirteen

Proton

Two dimensional

Bisbenzylisoquinoline alkaloid

n-Butanol

Broad

Centigrade

Deuterobenzene

Column chromatography

Circular dichroism

Deuteromethanol

Deuterochloroform

Chemical ionization mass spectrometry

Centimetre to the minus one

Two dimensional correlation spectroscopy

Doublet

Droplet counter current chromatography

Double doublet

Double double doublet

Deuterodimethylsulphoxide

Effective dose fifty

Electron impact mass spectrometry

Ethylacetate

Ethanol

Electron volt = 94.487 kJ/mol = 23.06 kcal/mol

12

FABMS Fast atom bombardment mass spectrometry

FDMS Field desorption mass spectrometry

FAO Food and Agricultural Organization

g Gram

GC Gas chromatography

HETCOR Heterocorrelation

HMBC Heteronuclear multiple bond connectivity

HMQC Heteronuclear multiple quantum coherence

HPLC High performance liquid chromatography

HRMS High resolution mass spectrometry

hrs Hours

Hz Hertz

IR Infrared

J Coupling constant

Kg Kilogram

Km Square kilometre

1 Litre

LC 5 0 Liquid concentration fifty

m Multiplet

M Molar

N Normal

NCI National Cancer Institute

max Maxima

mg Milligram

ml M illilitre

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

NMR 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


benz[g]isoquinoline-5,10-dione 107

Figure 4.5 Alkaloids isolated from C. dichroa showing their

possible biosynthetic relationship 116


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

Table 1.1 Health care coverage in Panama, in 1991

Table 1.2 Health indicators in urban and rural areas

in Panama, 1990

Table 1.3 Number of cases of some life threatening diseases

in Panama

Table 1.4 Major life forms of the vegetation in Panama

Table 1.5 General information on the Panamanian flora

Table 1.6 Geographic distribution of plant species in Panama

Table 1.7 Plant families in Panama with more than 80 species

Table 1.8 Advantages and disadvantages of in vivo and in viti'o

bioassays

Table 1.9 Panamanian medicinal plants selected for chemical and

biological studies

Table 2.1 Chemical compounds isolated from the genus Guarea

Table 2.2 Chemical compounds isolated from Abuta species

Table 2.3 Chemical compounds from Psychotria species

Table 2.4 Compounds isolated from species of Picramnia

Table 3.1 Comparison of the values obtained in the brine

shrimp micro-technique with previously reported data

Table 3.2 Comparison of brine shrimp toxicity against in vitro

KB cell cytotoxicity of compounds with different

mechanisms of action

Table 3.3 Activities of quassinoids against brine shrimps,

KB cell and P falciparum in vitro

Table 3.4 Activity against brine shrimp of the extracts from

Guarea macropetaia


Guarea rhopalocarpa


Ruagea glabra

Table 3.7 Activity against brine shrimp of the extracts

from Abuta dwyerana

Page

22

23

24

29

30

31

33

44

49

52

59

65

68

78

79

81

82

82

83

83

16


Cephaëlis camponutans 84


Cephaëlis dichroa 84


Cephaëlis dimorphatidrioides 85


Cephaëlis glomerulata 85


Laslanthus panamensis 86


Picramnia antidesma subsp. fessonia 86


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 NMR Spectral data of compounds isolated from

C. camponutans 100

Table 4.4 NMR 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 NMR 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).

Diseases

Year

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

EMBERA

GUAYMI

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

SPECIES"

Percentage"

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 (Bocas del Toro, Chiriqui)

3,921 (53)

Central Provinces (Canal Area, Codé, Colon, Herrera, Los Santos, Panama, Veraguas)

5,146 (70)

Azuero Peninsula (Herrera, Los Santos)

801 (1 1 )

Eastern Panama (Darien, San Bias)

2,563 (35)

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

P rem o n lan e ram forest

H um id low m o n tan e forest

Very tiumid low m o ntane forest Low m ontane rain forest

Very tium id m o n tan e forest I — I M o n tan e rain forest

e m u D ry tropical forest

I I H um id tropical forest

i m c l Very fium id tropical forest

i I D ry p rem o n tan e torest

S B H um id p re m o n tan e forest [ » e a V ery tium id p rem o n tan e forest

1. 2 ,000,000

Figure 1.2 Different Vegetation Zones In Panama.

32

FAMILY NATIVE SPECIES ENDEMIC SPECIES^

MONOCOTYLEDONSORCHIDACEAE 891 113GRAMINEAE 344 1 1

ARACEAE 234 95CYPERACEAE 164 1

BROMELIACEAE 136 1 2

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

DICOTYLEDONSRUBIACEAE 431 194LEGUMINOSAE 430 35COMPOSITAE 272 42MELASTOMATACEAE 244 43PIPERACEAE 213 80GESNERIACEAE 159 63SOLANACEAE 138 27MYRSINACEAE 134 95EUPHORBIACEAE 134 7ACANTHACEAE 1 1 0 15MORACEAE 96 4SAPINDACEAE 85 23VERBENAGEAE 81 5ERICACEAE 81 41BIGNONIACEAE 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

N ational park or wild life refuge proposed^ ~ ^P oflobalo

g o G a lun f .( I} PN

P N Ls Amisied

PN>barsnla

",P N /- 'Main

Palo Seco - R V S Isia Tabogakilo* da Campana'

Voicàn BarùlOmar Torrijo^

PNIslade Lae Perlae

la Veguada

Chorogo.. —

A N R - N atu ra l a rea for recreation

B P - P rotector forest

P .N . - N ational park

P .N .R .- R ecrea tive national park

R .F . - Forestal reserve

R .V .S - W ild life refugePN

El Moniui

I R V S 'Id a Iguana

^ T io n o s a

l: 2,000,000P N > 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 a i 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 vitroTesting.

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.

jpredominantlyA 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 ReferencesGuarea carinata Diterpenes: h0xadec-2-en-1-ol,3-7-11-15 tetramethyl palmitate, manoyl

oxide, 13-epi-manoyl oxide, phytol palmitate.Pereira et al., 1990

Guarea cedrata Triterpenes: 2-OH-rohitukin, 3-4-seco-tirucalla-4(28)-7-4-triene-3-21 - dioic acid, 3-methyl ester,3-4-seco tirucalla-4{28)-7-24-triene-3- 2 1 -dioic acid.

Akinniyi et la., 1980

Guarea glabra Triterpenes: glabretal, glabretal angelate, glabretal did, glabretalmethacrylate, glabretal tiglate, glabretal-2-OH-3-methyl butyrate, glabretal-3-one.

Steroids: p sitosterol.

Ferguson et al., 1975

Guarea kunthiana Triterpenes: euadorin Mootoo et al., 1992Guarea thompsonii Triterpenes: drageanin Connolly et al., 1976

limonoid B Pettit et al., 1983Guarea trichilioides Triterpenes: angustinolide Zelnik and Rosito, 1971

25-OH-cycloart-23-en-3-one, cycloart-23-ene-3-25-diol, 3p- 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

Furlan et al., 1993

prieurianin, 15-15P epoxy prieurianin Lukacova et al., 1982Steroids: 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 ED5 0 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).


"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).


"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 ReferenceA. bullata Isoquinoline alkaloids: saulatine, dihydro saulatine. Hocquemiller and

Cave, 1984A. grandifolia Isoquinoline alkaloids: grandirubrine Menachery and Cava,

1980A. grisebachii Isoquinoline alkaloids: grisabine Ahmad and Cava,

1977macolide, macoline, peinamine, 7-0-demethyl-peinamine, 7-0-demethyl-N- methyl-peinamine

Galeffi et al., 1977

imelutine, imenine, imerubine, homo-moschatoline, rufescine, nor-rufescine. Cava et al., 1975aA. pahni Isoquinoline alkaloids: (+) coclaurine, (-)daurisoline, 2’-N-nor-daurisoline, (-)

lindoldhamine, 2'-N-methyl-lindoldhamine, dimethyl-lindoldhamine, stepharine, thalifoline

Dute et al., 1987

A. panurensis Isoquinoline alkaloids: panurensine, nor-panurensine Cava et al., 1975bA. racemosa Triterpenes: dammara-20-25-dien-3p-24a diol Hammond et al., 1990A. rufescens Isoquinoline alkaloids:imeluteine, imenine, imerubine, rufescine, nor-rufescine Cava et al., 1975a

homo-moschatoline, splendidine Skiles et al., 1979A. splendida Isoquinoline alkaloids: aromoline, homo-aromoline, krukovine Saa et al., 1976A. 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).


"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).


"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).


"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).


"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 ReferencesP. adenophylla Triterpenes: a-amyrin, baurenol, baurenol acetate, betuiin, betulinic

acid, friedenn, ursolic acid.Steroids: 0-sitosterol

Dan and Dan, 1986

P. bacteriophila Steroids: 0-sitosterol (from callus tissues) Dohnal et al., 1989P. duoarrei Inorganics: nickel Kersten et al., 1980P. emetica Alkaloids: cephaeline, psychotrine Wehmer, 1931P. forsteriana Polyindolenines alkaloids: chimonanthine, hodgkinsine, psychotridine,

iso-psychotridine, quadrigemine A, quadrigemine BAdjibade et al., 1989

meso-chimonanthine Adiibade et al., 1992iso-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, 1961P. manillensis Monoterpenes: asperuloside Inouye et al., 1988P. oleoides Polyindolenine alkaloids: hodgkinsine, psychotridine, iso-psychotridine

A, iso-psychotridine B, quadrigemine CLibot et al., 1987

psycholeine Gueritte-Voegelein et al., 1992P. rubra Monoterpenes: asperuloside Inouye et al., 1988

Naphtoquinones: psychorubin sesquiterpenes: helenalin

Hayashi et al., 1987

Steroids: 0-sitosterol Hui and Yee, 1967P. serpens Monoterpenoids: asperuloside Inouye et al., 1988

Steroids: 0-sitosterol, stigmasterol Hui and Yee, 1967Triterpenes: 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).


"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 fr tm 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,

physcion

Arana and JuIca,

1986

P. parvifolia Quinones: aloe-emodin,

chrysophanic acid,

emodin, physcion, rhein

Popinigis et al,

1980

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

P. sellowii Quinones: chrysophanic acid,

emodin, physcion.

Triterpene: betulinic acid, 13-epi-

betulinic acid

Leon, 1975

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).


"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 TulSynonymous: 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).


"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, LC5 0 values determined by the two methods are similar.

However, when thymol was tested in our laboratory using the test tube method

a LC5 0 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 LC5 0 values determined for a number of

biologically active compounds which have various modes of action together with

their reported ED5 0 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

LCso(pM) ±sd

REPORTED VALUES

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

LCso (pM) ± sd

KB Ceil

EDso (pM)

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

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

CHLOROQUINE

DIPHOSPHATE

>1 0 0 0 . (2 ) 153*

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

LCjo (pM) ± sd

KB CELL

EDso (pM)»

P. falciparum

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 (Code N°)

EXTRACT BRINE SHRIMP LC5 0 pg/ml

MeOH 93.6

CHCI3 89.3

BranchBuOH 395.8

HgO-FRAC. > 1 0 0 0 .(3a) H2 O-EXT. > 1 0 0 0 .

MeOH 154.3

CHCI3 141.5

LeavesBuOH >1 0 0 0 .

H.O-FRAC. > 1 0 0 0 .(3b) H 2 O-EXT. > 1 0 0 0 .

PLANT PART EXTRACT BRINE SHRIMP(Code N°) LC5 0 pg/ml

MeOH 22.2

CHCI3 23.8

BuOH >1000.Branches HgO-FRAC. >1000.

(la) HgO-EXT. >1000.MeOH 5.8CHCI3 8.2

Wood BuOH >1000.H2O-FRAC. >1000.

(1b) H2O-EXT. >1000.MeOH N/TCHCI3 39.6

Leaves BuOH >1000.HgO-FRAC. >1000.

(1c) HgO-EXT. >1000.

Table 3.4 Activity Against Brine Shrimp of the

Extracts from Guarea macropetalaTabie 3.5 Activity Against Brine Shrimp of the

Extracts from Guarea rhopalocarpa

82

PLANT PART (code N*)

EXTRACT BRINE SHRIMP LC5 0 pg/mi

MeOH 324.4

CHCI3 197.1BuOH >1000.

Leaves H2O-FRAC. >1000.

(2 a) HgO-EXT. >1000.

MeOH 228.8CHCI3 44.9BuOH >1000.

Stem-bark H 2 O-FRAC. >1000.

(2 b) H.O-EXT. >1000.


EXTRACT BRINE SHRIMP LC« pg/mi

MeOH 313.9CHOU 411.5

Leaves BuOH 486.6(4a) H9 O-FRAC. > 1 0 0 0 .

H,0-EXT. > 1 0 0 0 .MeOH > 1 0 0 0 .

CHOU 911.Stems BuOH > 1 0 0 0 .(4b) H.O-FRAC. > 1 0 0 0 .

H.O-EXT. > 1 0 0 0 .

MeOH 755.CHOU 263.

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

HgO-EXT. > 1 0 0 0 .


Extracts from Ruagea glabraTabie 3.7 Activity Against Brine Shrimp, of theExtracts from Abuta dwyerana

83



MeOH > 1 0 0 0 .

CHCI3 579.Aerial BuOH > 1 0 0 0 .parts

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

MeOH 275.

CHCig 62.1Roots BuOH > 1 0 0 0 .

(1 0 b) H2O-FRAC. > 1 0 0 0 .

H.O-EXT. > 1 0 0 0 .



MeOH >1 0 0 0 .

CHCI3 >1 0 0 0 .Aerial BuOH >1 0 0 0 .parts

H2O-FRAC. > 1 0 0 0 .(8a) H2O-EXT. > 1 0 0 0 .

MeOH >1 0 0 0 .

CHCI3 >1 0 0 0 .

BuOH > 1 0 0 0 .Roots

H2O-FRAC. > 1 0 0 0 .(8b) H2O-EXT. > 1 0 0 0 .

Table 3.8 Activity Against Brine Shrimp of theExtracts from Cephaelis camponutans

Tabie 3.9 Activity Against Brine Shrimp of theExtracts from Cephaëlis dichroa

84


EXTRACT BRINE SHRIMP LC^ pg/ml

MeOH > 1 0 0 0 .

CHCI3 605.

Leaves BuOH 514.

(9a) H2O-FRAC. > 1 0 0 0 .

HgO-EXT. > 1 0 0 0 .

MeOH > 1 0 0 0 .CHCI3 > 1 0 0 0 .BuOH >1000.

Stems H 2 O-FRAC. >1000.

(9b) H 2 O -E X T. >1000.

MeOH > 1 0 0 0 .CHOU 876.

Roots BuOH > 1 0 0 0 .H,0-FRAC. > 1 0 0 0 .

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

PLANT PART (Code N®)

EXTRACT BRINE SHRIMP LC5 0 pg/ml

MeOH >1 0 0 0 .

CHCI3 > 1 0 0 0 .

BuOH > 1 0 0 0 .Aerial parts

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

MeOH > 1 0 0 0 .

CHCI3 772.8

BuOH >1000.Roots

H 2 O-FRAC. >1000.

(1 1 b) H 2 O -E X T. >1000.

Table 3.10 Activity Against Brine Shrimp of

the Extracts from Cephaëlis dimorphandrioidesTable 3.11 Activity Against Brine Shrimp of

the Extracts from Cephaëlis glomerulata

85

PLANT PART {Code N°)


MeOH >1000.

CHCI3 >1000.

Aerial BuOH >1000.parts

HgO-FRAC. >1000.(7a) H2O-EXT. >1000.

MeOH >1000.

CHCI3 >1000.

BuOH >1000.Roots

H 2 O-FRAC. >1000.(7b) H 2 O -E X T . >1000.

PLANT PART (Code N®)


MeOH 511.6

CHCI3 > 1 0 0 0 .

BuOH 485.Leaves

HgO-FRAC. > 1 0 0 0

(5a) H2O-EXT. > 1 0 0 0

MeOH 664.

CHCI3 109.7

BuOH > 1 0 0 0 .Stems

H 2 O-FRAC. > 1 0 0 0 .(5b) H 2 O -E X T. > 1 0 0 0 .


Extracts from Lasianthus panamensisTabie 3.13 Activity Against Brine Shrimp of the

Extracts from Picramnia antidesma subsp. fessonia

86



MeOH > 1 0 0 0 .

CHCI3 > 1 0 0 0 .

LeavesBuOH > 1 0 0 0 .

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

MeOH > 1 0 0 0 .

CHCI3 736.2

RootsBuOH > 1 0 0 0 .

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

MeOH > 1 0 0 0 .

CHCI3 395.9

BranchesBuOH > 1 0 0 0 .

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

MeOH > 1 0 0 0 .

CHCI3 416.2

StemsBuOH > 1 0 0 0 .

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

Plant name Plant part ExtractKB ceil

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

Guarea rhopalocarpaWood MeOH 50.4

Wood CHCI3 29.1

Leaves MeOH 89.6

Leaves CHCI3 32.6

Ruagea glabraLeaves CHCI3 81.6

Stem bark CHCI3 78.7

Leaves MeOH >500

Abuta dwyeranaLeaves BuOH 320

Leaves CHCI3 338.8

Roots CHCI3 54.8

Cephaelis camponutans Roots CHCI3 27.54

Picramnia antidesma Leaves BuOH 8.7subsp. 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 Collection

Date VoucherNumber

PlantPart

DryWeight (g)

Guarea Pemasky 10/Dec/90 572 Branch 1976macropetala San Bias Leaves 732Guarea Fortune 2/Feb/91 643 Branch 994rhopalocarpa Chiriqui Wood 1040

Leaves 763Ruagea glabra Fortune 5/Feb/91 691 Leaves 1014

Chiriqui Stembark 679Abuta dwyerana Cerro Azul 19/Nov/90 544 Leaves 316

Panama Stem 915Root 1 2 0

Cephaëliscamponutans

Pemasky San Bias

11/Dec/90 578 Aerial parts 1080 Root 386

Pemasky 9/Oct/92 1 2 2 0 Leaves 1071San Bias Stem 431

Root 451Cephaëlis dichroa Fortune

Chiriqui5/Jun/91 755 Aerial part

Root1613451

Cephaëlis Fortune 4/Jun/91 754 Leaves 712dimorphandrioides Chiriqui Stem 2951

Root 836Cephaëlisglomerulata

Pemasky San Bias

11/Jan/91 599 Aerial part Root

975206

Lasianthus Pemasky 12/Dec/90 594 Aerial part 1205panamensis San Bias Root 174Picramnia antidesma subsp. fessonia

El Valle Code

18/May/90 331 Leaves 827

Stem 619Picramnia Fortune 3/Feb/91 646 Leaves 509teapensis Chiriqui Root 357

Branch 1097Stem 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 methods4.1.4.1 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 IMS (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 IMS 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

D o u b l e macer at i on • w i t h pet r ol et her

t or 24 hr s

M or c

D ou bl e mocer et i on w i t h M eO H fo r 24 hrs

M a r cMeOH

Doubl e macer at i on w i t h H 2 O fo r 24 h rs

E vapor at i on

R esi du e

Ex t r actP a r u t i o n between C H C I 3 / H 2 O 1 /5 I i ophyl i zed

CH Cl

/ B u O H

BuOH IH 2O F r ac

P e t ro l et h er

D r i e d P l a n t M a t e r i a l

Figure 4.1 Extraction and Fractionation Procedure.

95

Plant Name PlantPart

DryWeight

Petrolether

MeOH: MeOH: CHCI3 HjO-Frac.

BuOH HjOExt*

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 0.140camponutans 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 PlantPart

DryWeight

Petrolether

MeOH^ MeOH* CHCI3 HjO-Frac.

BuOH H2 OExt/

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 0.146Cephaëlis Leaves 2 0 0.257 1.143 1.018 0.626 0.141 0.139 0.275dimorphandrioides 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 0.103Cephaëlis 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 0.213panamensis Root 2 0 0.019 1.483 0.961 0.127 0.550 0.143 0.259Picramnia antidesma Leaves 2 0 0.109 3.610 2.335 0.558 0.466 1.238 0.158subsp. fessonia Stem 2 0 0.009 1.133 0.834 0.083 0.183 0.569 0.172Picramnia 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 0.084Branch 2 0 0.027 1.345 0.881 0.058 0.157 0.556 0.095Stem 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 CHCI3 and

HgO. The CHCI3 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 CHCl3 -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 (CHCl3 -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-CHCl3 (1 :1 ), the extracts

were compared on TLC (CHCl3 -EtOAc 9:1).

4.3.2 Identification of the isoiated compounds

4.3.3. 1 1-Hydroxy benzoisochromanquinone4.3.3.1.1 Spectrai data

UV

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

98

IRD (KBr) cm ’ : 3450, 1660, 1595

Elemental analysisFound: 68.37 % (C), 4.43 % (H), 27.22 % (O); calculated values for C1 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)

HRMSm/z calculated [M]+ 230.0579 (C1 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-Acetylbenzolsochromanquinone4.3.3.1.2.1 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

quinone‘

1 -Acetylbenzoisochroman

qulnone^

Benz[g]isoquinollne-

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

CompoundsCarbon

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(COMe)

- 20.90 (CH;) -

Chemical shifts are in 6 -values in ppm.3'‘ Measured in CDCIj-MeOD.

""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)

HRMSm/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 (C1 1 H7 N); found 153.0574.

NMRand 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 C1 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 C1 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 (OHOl3 -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% H2 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

OH

-H , G

m/z 230.0579

CO-H

m/c 212.0473

-CO

m fi 201 mlz 184

-CO

OH

;CC^Hntfz 173

-CO

mfz 156

m fi 128

Figure 4.3 Putative Mass Spectral Fragmentation of 1 -Hydroxybenzoisochromanquinone

106

O

mfz 209.OS74C 13 H7 N O2

mfz 181.0528 C 12 N O

-co

a :

mà 153.0574 C „ H7 N,

CNH

_ n :

mfz 125.0446CiQ 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.

CompoundsBrine Shrimps

LCso±sd (pM/mi)KB ceils

LCso±sd (pM/mi)P. falciparum

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 from Cephaëlis 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 NH4 OH and extracted with CHCI3 , the CHCI3

extracts were dried on anhydrous NagSO and concentrated under vacuum, this

fraction (0.676g) was submitted to CC using CHCl3 -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 NH4 OH and re-extracted with CHCI3 ; extracts were dried on Na2 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 CHCl3 -EtOAc (1 :1 ) as solvent system.

4.4.2 Identification of Isolated compounds

4.4.2.1 Vallesiachotamine lactone


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


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 l l

HRMSm/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


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 iactam4.4.2.4.1 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 lactam4.4.2.4.2.1 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 (C3 4 H3 8 N2 O1 2 ) found: 666.2434.


(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


"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 Angustine4.4.2.5.1 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

20jk' O GhiCOOMe

Me

Is<h ValWmcholmmine

s

COOMe

O -G lu

Strictosidine lactamVallesiachotamine

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 C2 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

co o

mfz 349miz 364.1435

m /l 305mfz 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 NH4 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 OHOl3 -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 + NH4 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


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


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)

HRMSm/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)


( 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 B4.5.2.2.1 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)


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 04.5.2.3.1 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


(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)


(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 DiscussionThe 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 tetrad0 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-

Calycanthlne*

Glomerulatine

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 aa r r -z .T T Z i~ ï^ J .___ ■ - i ü l

71.82 71.66 165.11 (76.51)'

‘'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 C2 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

HjCk

1 CH)

o r

5

G l o m e r u l a t i n e A

o r

G l o m e r u l a t i n e B

" ^ n j I

o rCH, N

CH,

G l o m e r u l a t i n e 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 CDCI3 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 C2 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 fromPIcramnia 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 CHCI3 , CHCl3 -MeOH (9:1,

6 :2 , 7:3) as eluent. Sixty fractions (~ 125 ml) were obtained, fractions 7 to 1 0

were washed with CHCI3 and crystallized from CHCI3 yielding 0.83 g of aloe-

emodin; fractions 18 to 27 were submitted to GO using CHCI3 , CHCI3 -M0 OH

(95:5, 9:1, 8:2) as eluent, yielding, after crystallization from CHCl3 -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 GHGI3 yielded

0.490 g of picramnioside G; whereas from fractions 36-41 after preparative TLG

using GHGl3 -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 GHGI3 ; the

chloroformic fraction was submitted to pTLG using GHGl3 -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


UVX 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 (12 ), [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.


(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)


(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


UVX 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


(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 (C2 2 H2 2 O1 0 + 1 ) found 447.1299. [M-acetyl] m/z

calculated 387.0716 (C2 0 H1 9 O8 ) found 387.0722.


(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)


(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


133

CarbonPicramnioside

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.


(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.


(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


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)

HRMSm/z calculated 270.0528 (CigHioOg); found 270.0536


(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



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 (LC5 0 8.7 pg/ml), however,

this fraction showed no significant activity against brine shrimp (LC5 0 485 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 4.11

(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-emodinanthroneFigure 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 theI

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

OHlO H

Picramnioside CFigure 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

tnlz 509

Cft

OHOH

mji 447

OHOH

mfi 387

H n :

Cl^OH

mil 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.

CompoundsKB ceils

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 Gla

Strktoüidine

NH

lO Gin

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

L - Tryptophan

rCH,

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

Ç'HpNHj

N -H

H-

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

DTI.Dopamine

Me

Me

OMe

OMe

Isoquinoiine monoterpencid alkaloids as those produced by

C ip eca cuan ha , C, co rre a e...OGIu

MI2

Tryptamine

H3Q

CH3

Colycanthine alkaloids fhwn C. g lom erukU a , P . fo rs te r ia n a

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

C tt

Polyindobnine Alkaloids (non-iridoidaD

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-5,10 -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

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182

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183

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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

10195.9085 J80757065605550454835302520

1510

5

93SE270tl xl Bgd=0 01-MRR-93 14 5*01 8pM=8 1=2.4v Hn=900 TIC=173988000 RVÇP4 El 190 DEGC

00 2R8-SE El*RcntULSOP SysDCI

PT=0® CalDRVCRL

58

ilk

128

76

6357

AM

104

83

68

91i i

98

115

U 1 ill till100 120

MRSS=15877000

164184

156

145

|L ui/iikL140

173

212

201230

160 180 200

22

220L 253 267

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 i n c n c i 1 t

V \ \ H v yR 5 5 S 5 5 S 5

I Y I

Spectrum 3. "C NMR spectrum of 1-hydroxybenzoisochromanquinone

190

Current Oeta1 Parew tersNAME acety]cc2CXPNO 10pnocNO >

F2 - A cqu is ition P ir iK ts r s930525

T lw 10.35PULPflOG 2930SOLVENT CDC13AO 1.9661000 s tcFlOflES 0.254313 HzON 60.0 wsecAS 912NUCLEUS IHHLl 1 0801 1.0000000 stcFIOESFOl 400.1366620 MHZSMK 6333.33 HZ10 32766MS 16OS 2

SI 16364SF 400.1343656 MHzNON EMSSB 0L I 0.30 Hz66 0FC 1.00

ID NMR p lo t p o rs M ttrscxFIR 9.600 ppBFI 3641 29 HzF2P -0 400 ppmF2 -160.05 HZPPMCM 0.25000 ppm/cmHZCH 100.03360 Hz/cm

J i.

Me

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 OCICC3 El 138 DEGC PT= 0® CaL BflVCflL MHSS:

12246080289

209188,95.90.85.

75.70.65.

25.

??' 239111 119.200 268

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

192

J

J

8

u

Benz[g]isoquinoline-5,10-dione

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

193

s s “i5 55r T «

CCI repeat of 13C using Inverse gated ilecoupiing to give almost q u an t i ta t ive spectrum

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

194

3 ? 2 f0 ? 2 l l x l Bqd=8 2 0 - jn N -9 2 15 48* 0pM=O U 7 .Û V Hm=890 T1C=5B0313024 flV1 X Ï CD3-C [ [ VARIOUS lEHPERAIURES

1 0 8 ZAB2F E l*flcn t ULSOP Sys ERE in s

P 1 = 0 “ CalDRVCRLHMR: 50845000

MASS: 305305100,

7 5 .

60.

4 5 .

3 0 .

3332092 0 .

27731?

. w ll ik llL .illilW lliilklijljlilimylilii hu jjljl L i\lil Lyall! 100 150

319

400300200

Spectrum 8. Mass spectrum of vallesiachotamine lactone

195

CD3-C IN C0CL3 *TMS. MM250 IH

COOMe

PPM/CM

17OMe

15;

14

Spectrum 9. NMR spectrum of vallesiachotamine lactone

196

CD3-C IN C0CL3; HM250 IH COSY SPECTRUM

u

csa

. 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

7 .5

J 8 . 0

PPM

Spectrum 10. COSY-45 NMR spectrum of vallesiachotamine lactone

1 9 7

9 2 2 F 0 4 5 I1 x l Bgd=0 H -J f lN -9 2 10 4 6 *0 Bpn=0 I= 7 .9 m Hn=090 TIC=38297?B944 AV[ 0 2 -H E l 190 DEGC

ZRB2F E l*Rcnt ULSOP Sys IRE 1RS

PT= 8“ Cal ORVCRLHMR: 51504008

MASS 279279100,

9 0 .

7 5 .

6 5 .

6 0 .

5 5 .249

5 0 .

4 5 . 350

307322

3 0 .

2 5 . 209

236

U lM i L> L4 II11

388200 250108 150

Spectrum 11. Mass spectrum of vallesiachotamine

198

C 02-C IN CDCL3 +IM S. NM250 IH

H Z/P I

“COOMe

OMe Me

19

ULi

Spectrum 12. 'H NMR spectrum of vallesiachotamine

199

92SCI72eil xl Bgd=0 18-N0V-92 I1=B>G BpH=8 1=8.5v Hm 28B8 TIC=352BB48B8 HV CDl HNQBfl > Nfl MATRIX

88 ZRB-SE FB*Rcnt-ULSOP SysLMLRFRB

PT= 8* CalFRBCRLHMR:

MASS:55455888

176108,

85.

75.

5 5 .

5 0 .

136 236329

187214128 258 289 307 352 373 309 413 553 604514 648

208 300 408 508

Spectrum 13. Mass spectrum of strictosidine

200

aO Glu

Me(X)C

F2 - A c q u is it io n ParametersDate 930324Tine B.59PULPROG zg30SOLVENT HeOHAQ 1.9660922 secFIORES 0.254314 HzON 60.0 useeRG 512NUCLEUS IHHLl 1 dB01 1.0000000 secPI 9 .5 useeDE 75.0 useeSFOl 400.13B4705 MHZSMH B333.37 HzTO 3276BNS BOS 2

F2 - Processing parametersSI 163B4SF 400.1359667 MHzWOWS5B 0LB 0.00 HZGB 0PC 1.00

10 NMR p lo t parametersCX 40.00 cmF IP 9.600 ppmFI 3B41.31 HZF2P -0.400 ppmF2 -160.05 HzPPMCH 0.25000 ppm/cmHZCH 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 s h i f t c o rre la tio n : 13C garp decoupled

L

T—' ppm

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

203

9 2 2 fB 5 5 ll x l Bgd=0 16 -JR N -92 18 4 9 *0 l = 4 .9 v IU = a s a T IM 1 1 2 G 3 1 0 4 0 AV

C03-H E l VARIOUS TEMPERATURES 108, 235

88 ZAB2F E l*Aa.L ULSOP Sys LREIfIS

PT=8® CalBAVCAL;iUR: 32jJ[038

HASS: 235

498

4 6 7 4 8 0.A -*-288 250 300 358 488 458

Spectrum 17. Mass spectrum of strictosidine iactam

588 5!S

204

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

SF 2 50 .13 4

5 37 5 .0 00 16384 16384 3 75 9 .3 98

r .459SHH Z /P T

PHAOAQAGNSTE

2 .1 7 9200

FW 4700 02 0.1 OP 63L PO

LB

CXCY

4 0 .0 02 3 .0 0

9 .601P - 398P

6 2 .5 27.250

383 8.5 7

F2HZ/CMPPM/CM

18

I I ---------------1................... 1......... . . . I .......... i . . . . , . . . . i ............ . . . I 11 ■■111■«. 111■111. 1. 11. .1 .1. . . 11... . I. . . . 118 .0 0 7 .5 0 7 .0 0 6 .5 0 6 .0 0 5 .5 0 5 .0 0 4 .5 0 4 .0 0 3 .5 0 3 .0 0 - 2 .5 0 2 .0 0 1 .50 1 .0 0 .50 0 .0

PPM

Spectrum 18. 'H NMR spectrum of strictosidine lactam

205

922F1118#1 x l Bgd= BpH=0 I= lB v Hn =890 RCET CD3 ( 1 ) E l 190 DEGC

26-S E P -87 13 55« T IC =9023421440 RV

101

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 .

1 5 .

1 8 .

5 .

379

1=88 ZR82F E hR en t: Sys:LREinS

P T = 0 ® CalDRVCRL

564

446 I X i l l

582

MRSS:58785800

666666

624

i698X

358 458 588 550 688 650 700

Spectrum 19. Mass spectrum of tetraacetyl-strictosidine lactam750

206

A c e trJ -C D 3 IN C0CL3 t lH S . NN2S0 IH

H 3 9 t50 .00 t

HZ/PT

PPM/CM

I j

Spectrum 20. NMR spectrum of tetraacetyl-strictosidine lactam

207

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

A . J1 1

!

1I

!i g

11

!1

1•; 1

■ i 11

1 ,

! 1 1

' 1 ' 1

1 I I I ' ,

1 ’ 1' 1 1 '

" i l

’

V , ,

1

B j

C39155 SHXF I PflOJ

C03H 001F2 PflOJ

C03C 001AU PflOG

XHCORflO AUDATE 4-11 -92

SI2 2049S Il 256SK2 6771 930? 0 l 983 09:NOG -

1 .5 M0N2 6NOXl SLB2 8.0006B2 0 .0

2 .0 S301 0MC2 pPLIM flowF I 159 t.ÿ^P

2 .5 F2 16 763PAND COLUMNF lF :

3 .0 DI31 iHPI 17 *01 00101.:'"

3 .5 03p :P4 Ï9 I

9 r04 vOiev J3: *.4HflO 0 0PM 0

4.5 DE 71 2Mb 3205ME 123

5 0 IN 0)1:54?

5 .5

6 0

6 .5

7 .0

7 .5

0 .0

Spectrum 21. NMR spectrum of tetraacetyl-strictosidine lactam

208

92Û 0E 24 I1 x l Bgd=0 0 5 -n flR -9 2 14=3*0=00=00 1 2 -250 E l *BpM=0 1 = 4 .2 v Hn=650 110=398292000 HV Rcnt-ULSOP Sys^STEnOEF C D l-8 IH E SCHOOL OF PHHRMHCY PT= fl® C a lU C H L

100. 20

HMR: 27694000MRSS: 28

55 G3 V

313

298

255

229282

150 200 250

Spectrum 22. Mass spectrum of angustine

L300 358

209

3.000E+01

4.000E+01

5:ZOO . O WL [nm]— CGI (CH3CN, C - 1.05 E-4 M)

N o . Wavalenath Valua1 310.30 nm -3.492E+012 296.00 nm - 3 . 109E+013 280.60 nm 4.011E-024 268.60 nm 2.662E+015 246.60 nm 2.990E+006 . .. 2 1 4 .7 0 .Jim .- 2 .1 7 a E t0 1

400.0

Spectrum 23. Circular dichroism of glomerulatlne A

210

scan 37 ( I . 2 45 mi n i of DPITM: B8 5 3 4 . D

-.1)aciTjTSC

Œ

C R L E D

5 0 1

2 0 91 6 7

3M a s s / C h a r g e

Spectrum 24. Mass spectrum of glomerulatlne A

211

roN)

!23K

zSDUi

%sc3a(Q01cg3<D>

0.42

0.430.480.50

so Z

Î

2.21

0.59

0.56

6.97386.95906.95536.93966.93606.78316.77946.76446.76076.65986.65676.64136.63836.62296.6198

— 6.2431

3.09493.021 r3.00512.99712.98112.97312 .957 L2.92512.63512.61302.5899_2.16 2 L1.97941.95671.94871.93271.92591.90191.39731.38161.36651.35081.3059

000) — 165.1055

I3toG)

B)

t i a>J

147.3194

J12S.9007 125.2713 125.0362 126.^059

' - ^ 1 2 5 . 0 4 1 0 1 ^ 1 2 3 .6 7 6 1 — 122.2509

roCO

0zs330)

1“tc32.(Q0

1I3<D

W

00

CJ

w&)to

49.091248.1509

ZÈ(D

30.869630.2630

L11 il LL

(ppm)

- -

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

4

■LlIJJ. *___ I

* 0 ^

M • .7 ? °

o

c

0o

oâ1 r

, <91»

(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 20PC Date: 24-1 I -1993 Time: 13:59: 22

Scans 14 Pr 0:23 Pra<cs«l75 8ase: Mat$-328.16043, rm=l^28fl85

111, ■ J i . . l i l i i l .1' j. I'll piii'l350

tiass

Spectrum 30. Mass spectrum of glomerulatlne B

217

roœ

faC3w

%zs3(/)■OS

3a(O0

1I3(Dm

o o z

09Z

1

1.32

2,59

1.00

7.02746.91336.89456.87456.77986.77036.76106.75066.74266.73986.71806.71436.6992

3.67743.66023.63683.58763.57133.56303.54703.49633.47503.3007

2.52482.51682.50112.4934

1.80081.78361.77931.77041.75591.73931.7242

— 166.7668 8o

— 146.1073

IC3N

J 128.9094 128.8374 124.5720 124.1019

^ 123.6924 J r 122.9417 - ^ 1 2 2 .6 2 7 0 ^ 1 2 1 . 0 7 2 5

roCD

wo

(/)

1c3aCQ01■ncI3<DCD

— 48.9141

— 32.6717 31.5987

^ 30.4423

S c a n 1 0 4 ( 2 . 6 7 5 m i n ) o f D R T R : B 8 5 8 3 . D100:

90-

8 0 '

7 0 '

60 :

c(tj 50 :

3 40':Œ.

30 :

10:

57

1 3 0

100

SCALED

1691 9 7/

2 1 9y

257

d m M a s s - ' ' C h a r g

d m

Spectrum 33. Mass spectrum of glomerulatlne C

220

3 4 3

7.03417.01537.00086.98306.83976.80996.7911“6.76746.7474-6.48516.46826.21266.1930

IC3%

%z

0)

I3a(Q0

1

I.sO

— 3.8695 3.7890

2.9743-2.70492.68312.65972.30332.1397-1.95981.94191.87361.85891.84071.5486

Z-Œ O O

— 166.9790 S

I-nC3N

MO

148.1450

— 142.5783

r 129.9993 / r 125.6666

y j - 125.4031 J - 123.4761 T h 122.7226 = ^ 1 2 2 .5 8 7 0

— 118.0042 114.2469

33 10)

1■tc3a

(Q01C

I3(DO

— 76.9857

— 50.9244 49.6277 48.9860

^ 45.7690— 40.0392

34.4866— 32.1852 ^ 31.2052

j j A U i

3

f

(0 O

(

t

> § / ; jP caO

^ «

3 o

y

m

•

o oas o

fo ° °

0 1)

Spectrum 36. COSY-45 NMR spectrum of glomerulatlne C

223

- A - J_I i k, jA J (ppm)

■ ♦ —

(ppm)

Spectrum 37. long range heterocorrelation NMR spectrum of

glomerulatlne C

224

(ppm)

Spectrum 38 ROESY NMR spectrum of glomerulatine C

225

94S E 232I1 x l Bgd=l 14-FE B -94 14M 7>8=B B iflB ZflB-SE FB*BpM=75 I= 4 .5 v H«=1981 T IC=4G ?712932 RV SU R cnt ULSOP Sys LMFHB PRF-3 (flk e V Xe ♦FRB/HS) G L g c /Th logLyc /TFR n a t r L x . PT= 8® C a l FRCHL

I ' 9

I

589

433 459 475 4 M 531

MHSS=18358888

185

4JJ I Î 547iiHmMiHiiinii i I wiiii GB2

588 G88 788

Spectrum 39. Mass spectrum of picramnioside A

226

l.OOOE+Oi

Am

8.000E+00200.0 WL [nm] 000.0

No. WavBlenath Valua1 342.80 nm 1.682E+002 314.20 nm - 2 . 769E-023 290.00 nm -6.363E+004 239.00 nm 1.698E+000 230.80 nm -3.898E+00

. s ___2 2 1 .2 0 nm____l_ ^ 4 4 E t0 1 .

0: PAF3 (Matanol. c - 8 .60 E-O M)

Spectrum 40. Circular dichroism spectrum of picramnioside A

227

OHPAF3

F2 - AcquJilt:»n P a rau te rs OHzgao

I OH

HOH

10 rMR p lo t

J Lv V

Spectrum 41. NMR spectrum of picramnioside A

228

i ï v IVRI RI g 3 8ai RZ3 3

V

HOHiC

PAF-3 ; normal 13C

53 Si8» ;RR8 !î 9 8 P R 5 8 3 2 9 9 3 9 9 9 9 9 3

\ r I I 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; phase sensitive; positive peaks red;negative black

J

5.0 4.5 4.0 3.5

Spectrum 44. ROESY NMR spectrum of picramnioside A

231

94S E 218I1 x l Bgd=l 13 -FE B -94 IB 55*8=6 BpM=lB5 I= 4 .5 v Hm=1341 T 10=124392888 RV SU PRF-4 CBkeV Xe ♦F fiB /tlS ) G L y /T h L o g ly /T F fl m a t r ix .

256

=88 ZflB-SE FB*flcntULSOP Sys=LMFflB

PT=8® CalFflCflL

447

387

, I ■ I300 350 408 450 500 558

Spectrum 45. Mass spectrum of picramnioside B

HMR=MflSS= 258

658

232

3 . O O O E + 0 0

10

Am

*3, OOOEOO -I— 1.

200.0 W L [n m ] 6 0 0 . 0

No. WavBlenath Value1 399.00 n m 2.666E-012 336.20 n m -2 .314E -023 321.60 nm -9 .670E -014 306.60 n m -4 .969E -035 294.60 n m 1 .246E+006 272.00 n m -2 .644E -037 266.00 n m -9 .499E -01a 240.40 nm 1.066E-019 226.60 n m -2.271E+00

10 210.60 n m __2.4aiE±Q 0

6: P A F 4 ( M a t a n o l . c - 1 . 8 E - 4 M)

Spectrum 46. Circular dichroism spectrum of picramnioside B

233

..ren t D#t# Parvaeters name «U01693*EXPNO 1

F2 - A cqu is ition P s ra o e tir i

OH

OH

F2 - P roc ts ilng p ira a e t fn

Mepp«/4Mz/Cl

10

Spectrum 47. NMR spectrum of picramnioside B

234

PAF4 ; cosy 45

i

_jT V jl-1J

A ful l

00 ^ iB dg

m »'s>->

OGS ,S

3 /• '/Co >

a 1)S07.0 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

j______J

» ► 0|

0 «

_yJL

100

i .f

°0°

v_

5.0 4.5 4.0 3.5

Spectrum 50. ROESY NMR spectrum of picramnioside B

237

94S E 233I1 x l Bgd=l M -F E B 94 17=18^8 88 80 ZHB-SE FB*BpM=75 I=582m v H#=1343 T IC =73958888 RV SU R cnt ULSOP Sys LHFRB PRF-5 (DMSO) (B keV Xe ♦FRB/HS) G ly c /T h lo g ly c / ÎF R m a t r ix . PT= 8 * C a l FRCRL MRSS:

2281808256

2561889 5 .

9 8 .

4476 8 .

5 8 .

4 5 .

239

3 5 .

3 8 .

2 5 .

2 8 .

287 298 548438378283 632

469314

388

Spectrum 51. Mass spectrum of picramnioside C688588288

238

7 . O O O E + O O

G .O O O E + O OWL Inn]200.0

No . M avelanatn Vmlua1 391.40 nm 7.760E-012 317.00 nm -1.8G 0E-023 284.00 nm -5.373E+004 298.60 nm 8.001E -039 253.40 nm i.116E+006 230.00 nm - 3 . 132E+00.7 . . -210 .SO.jn n -___5..4ZOEfOO_

7: PAFO (M atsnol. c - 8 .5E -S M)

Spectrum 52. Circular dichroism of picramnioside 0

239

C urrin t Data Paranettrs N»H£ )n 2 « 3 .3EXPNO nPBOCNO J PAF5

F2 - A c q u ltit lo n Paraaetcrs

OH

OH

iPH OH'F2 - n-ocaastng p a ra a a ttr i

HOH 2'

10 p lo t parameters

Me140.04770 Hx/i i

10

I ..< ■ ■ I .: 3

Spectrum 53. 'H NMR spectrum of picramnioside C

240

V S w ' V Y N i l Y V Y

Ui

? A = R 8 : s â § 3 %R s % s Â ofs ? Rx: « ; A s a s i & i i P §& z z z g Biz 9 ? s a s s s : ; £ s s s s s s s s s r s s % 3 g ai ai g a i a i g R a g g * * %%

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 co 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

01 • r

1

•M . / -,Vt'w : 4

.

r # \ • •

J

Spectrum 56. ROESY NMR spectrum of picramnioside 0

243

93028111 x l Bgd=0 16-NQ V-87 8 ^ 8 « 8 88 80 1 2 -2 5 8 E hBpH=8 I= 4 .2 v Hm=980 T IC = 150942800 RV Rcnt= SysSTEHDEFPRE 1 E l 1B0OEGC PT= 8 ° C a llC R L

HMR: 27732000HHSS: 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 .

213224

252

150 208 258

Spectrum 57. Mass spectrum of aloe-emodin

244

C u rrin t D t t i P i r iM t i r i NAME p i l lEXPNO 10PROCNO 1

p lo t p ii M i t i r i

12.600 ppa

0.32500 ppa/ca 130.04430 Hz/ca

Spectrum 58. 'H NMR spectrum of aloe-emodin

245

93S E 1253# ! x l BpH=0 1 = 4 ,5 v H#: PflFR E l

23-A U G -93 89 39^ T IC = 2 7 9 6 5 ie B 8 RV

1 8 1

9 5 .

9 8 .

8 5 .

88

75

78

6 5 .

68.5 5 .

5 8 .

4 5 .

4 8 .

3 5 .

3 8 .

2 5 .

2 8 .

1 5 .

1 8 .

5 .

8

89

83

llii

94

188 2R8-SE E l*RcntULSOP SysDCI

PT=8® CalBCICRL

197

i i# 4uiI L

181

165

HHR:HRSS:

16836088256

256

8 226 238

58

128 148 168 188 288 228 248 268 288

Spectrum 59. Mass spectrum of aloe-emodianthrone

308

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

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