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Award Number: DAMD17-99-2-9025 


TITLE: The Associate Program on Ethnobiology, Socio-Economic 

Value Assessment and Community Based Conservation 


PRINCIPAL INVESTIGATOR: Maurice M. Iwu, Ph.D. 


CONTRACTING ORGANIZATION: Bioresources Development and 

Conservation Programme 
Silver Spring, Maryland 20902 


REPORT DATE: October 2002 


TYPE OF REPORT: Annual 


PREPARED FOR: U.S. Army Medical Research and Materiel Command 
Fort Detrick, Maryland 21702-5012 


DISTRIBUTION STATEMENT: Approved for Public Release; 

Distribution Unlimited 


The views, opinions and/or findings contained in this report are 
those of the author(s) and should not be construed as an official 
Department of the Army position, policy or decision unless so 
designated by other documentation. 


20030203 064 





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October 2002 Annual (1 Oct 01 - 30 Sep 02) 


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4. TITLE AND SUBTITLE 

The Associate Program on Ethnobiology, Socio-Economic Value 
Assessment and Community Based Conservation 

6 AUTHOR{S) 

Maurice M. Iwu, Ph.D. 


5. FUNDING NUMBERS 

DAMD17-99-2-9025 


7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 

Bioresources Development and Conservation 
Programme 

Silver Spring, Maryland 20902 

E-Mail: iwum@bioresources.org 


9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 

U.S. Army Medical Research and Materiel Command 
Fort Detrick, Maryland 21702-5012 


8. PERFORMING ORGANIZATION 
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13. ABSTRACT (Maximum 200 Words) 

During the reporting period, 206 plant samples were submitted for antimalarial screen out of which 19 plants showed activities 
against D-6 and W-2 strains of Plasmodium falciparum. In our priority list of potential antimalarial candidates, the following 
plants were bulk extracted: Picralima nitida, Spathodea campanulata, Uvaria chamae, Cryptolepis sanguinolenta, Glossocafyx 
brevipes, Cleistopholis patens , Leidobotrys staudtii, Pachypodanthium staudtii, Odyendyea gabonensis and Uapaca paludosa 
Eleven compounds were isolated from two plants - Penianthus longifolius and Homalium letestui that showed antimalarial and 
antitrypanosomal activities. Additional eight pure compounds were isolated from extracts of Glossocafyx brevipes that showed 
antitrypanocidal and antileishmanial activities. Fifty-two extracts were screened for antimicrobial activities out of which 60% 
showed very significant antibacterial activity and 7 of the extracts showed antifungal activity. Four antimicrobial compounds were 
also isolated from Peucedanum zenkeri and Araliopsis tabuoensis. Twenty-one compounds were synthesized using ciyptolepine as 
template to enhance their an timalari al and oral activity. Extracts were also processed for testing against HIV, cystic fibrosis, 
cancer and CNS. In collaboration with the Environmental Law Institute, Washington DC we are examining options for new 
approaches to the valuation of natural resources. A re-census of the 50ha Korup biodiversity plot was conducted with the 
Smithsonian Institution 


14. SUBJECT TERMS 

phytochemistry, anti-malaria, plant collection, fractionation, database, 
biodiversity, economic evaluation, medicinal plants, antiparasitic, 
bioassavs_ 


17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 
OF REPORT OF THIS PAGE OF ABSTRACT 

Unclassified Unclassified Unclassified 


NSN 7540-01-280-5500 


15. NUMBER OF PAGES 

_ 63_ 

16. PRICE CODE 


20. LIMITATION OF ABSTRACT 

Unlimited 


Standard Form 298 (Rev. 2-89) 

Prescribed by ANSI Std. Z39-18 
298-102 






















FOREWORD 


Opinions, interpretations, conclusions and recommendations are those of the author and are 
not necessarily endorsed by the U.S. Army. 

_X_ Where copyrighted material is quoted, permission has been obtained to use such 
material. 

_X_ Where material from documents designated for limited distribution is quoted, 
permission has been obtained to use the material. 

_X_ Citations of commercial organizations and trade names in this report do not constitute 
an official Department of Army endorsement or approval of the products or services of these 
organizations. 

N/A In conducting research using animals, the investigator(s) adhered to the "Guide for the 
Care and Use of Laboratory Animals," prepared by the Committee on Care and use of 
Laboratory Animals of the Institute of Laboratory Resources, national Research Council 
(NIH Publication No. 86-23, Revised 1985). 

N/A For the protection of human subjects, the investigator(s) adhered to policies of 
applicable Federal Law 45 CFR 46. 

N/A In conducting research utilizing recombinant DNA technology, the investigator(s) 
adhered to current guidelines promulgated by the National Institutes of Health. 

N/A In the conduct of research utilizing recombinant DNA, the investigator(s) adhered to the 
NIH Guidelines for Research Involving Recombinant DNA Molecules. 

N/A In the conduct of research involving hazardous organisms, the investigators) adhered to 
the CDC-NIH Guide for Biosafety in Microbiological and Biomedical Laboratories. 


//?'fAAA*/ 

PI - Signature Date 


/c 


3 



Table of Contents 


Cover.1 

SF 298. 

Introduction.5 

Body.6 

Key Research Accomplishments.6 


References 


60 









INTRODUCTION: 




The African ICBG, in general emphasizes three major goals: evaluation of rainforest plants from 
Nigeria and Cameroon as cures for parasitic diseases; research on forest dynamics to understand 
the effects of sustainable harvesting and cultivation of important medicinal plants; training of 
Cameroonians and Nigerians in natural products chemistry and tropical ecology. 

The ICBG project, jointly sponsored by the U S. National Institutes of Health, the National 
Science Foundation and the U.S. Department of Agriculture has the main focal point of establishing 
an integrated program for the discovery of biologically active plants for drug development and 
biodiversity conservation, while ensuring that source countries derive maximum benefits for their 
biological resources and their intellectual contribution. BDCP facilitates the drug discovery 
component of the ICBG and therefore serves as a link between the drug discovery part of the 
program, the biodiversity conservation component and the economic development projects. The aims 
of the Associate Programs administered by BDCP are: 

1 To conduct ethnobiological inventory of plants in the selected study areas; 

2. To guide the ICBG in its plant selection and collection strategies for drug discovery. Samples 
identified from ethnobiological inventory will be collected from biodiversity plots and from 
wild flora and screened for possible biological activity. 

3. To perform phytochemistry and preliminary bioassays on selected plants. 

4. To perform plant extraction, bioassay-guided isolation and structural elucidation, with 
research, training and infrastructure development being important components of each 
operation. 

5. To maintain and expand the database on African medicinal plants, which includes information 
on local names, traditional, uses, floristic data, possible constituents, conservation status, 
agronomic data and economic value. This involved the re-structuring and expansion of the 
existing AfricMed database to include data from other Associate Programs. This 
Computerized Information System of African Medicinal Plants (CISAMAP) will be linked to 
other regional databases. 

6. To conduct a socio-economic value assessment of the biological resources in the study area 
which seeks to: 

I) highlight the non-commercial value of forest products within the cultural/ religious 
context; 

II) quantify the economic value of biological resources for comparison with other land 
use options; 

III) place in priority order the production and marketing of biological resources in local 
markets to provide income for local residents; 

IV) provide baseline agronomic data for the formulation of a sustainable management 
plan for the forest resources; and 

V) train local natural resource managers and users at the National and Community 
levels to conduct economic and market research which will integrate the connection 
between conservation and development. The ICBG may organize rural farmers to 
cultivate, in fallow areas, certain plants of potential therapeutic value; 


5 


7. Assist in capacity building of West African scientists in the areas of ethnobiology, inventory 
and research management. Formal training will be organized in ethnobiological methods and 
field taxonomy and economic value assessment for local communities. 

BODY: 

KEY RESEARCH ACOMPLISHMENTS: 

1.1 PHYTOCHEMISTRY & PRELIMINARY BIOASSAYS 

Specific Aims: 

1. Evaluate the biological activity of plants used in African ethnomedicine. 

2. Isolate and characterize the bioactive constituents of these plants. 

3. Perform bulk extraction and fractionation of plant material for in vivo assays. 

4. Optimize lead compounds with significant potential for human health in Africa. 

5. Provide training for African scientists at all levels of drug development. 

Institutions: 

University of Dschang, Cameroon. 

International Center for Ethnomedicine and Drug Development, Nsukka, Nigeria. 

University of Buea, Cameroon 

University of Minnesota, Minneapolis, Minnesota. 

University of Mississippi 

Progress Report: 

This report summarizes the activities carried out by collaborating scientists at the participating 
institutions named above. All planned activities were actively pursued during the period covered 
by this report. Details are provided below: 

1.1.1 Phvtochemical Investigations. 

A. Extractions: 

As envisaged, a large number of small-scale and bulk extractions were carried out during the 
period covered by this report. Small-scale extractions were performed to provide new candidates 
for screening and to increase the size of the extract library, while bulk extractions were carried out 
on previously identified active extracts to provide material for isolation of active constituents and 
for in vivo bioassays. 

For small-scale extractions, the following plant parts were used wherever appropriate: stem, aerial 
parts, stem bark, leaves, fruit, seed and rhizome. Each sample was divided into two parts for 
extraction with methanol and methylene chloride following a standard procedure. A list of these 
extracts is provided in Table 1. 


6 



TABLE 1 List of Plants Extracted during the reporting Period. 
Methylene chloride extracts: 



Plant 

Plant part 

1) 

Paropsia guineensis 

leaves 

2) 

Synedrella nodiflora 

whole plant. 

3) 

Sterculia tragacantha 

aerial parts 

4) 

Momordica charantha 

leaves 

5) 

Mussaenda elegans 

leaves 

6) 

Macrosphyla longistyla 

leaves 

7) 

Chasmenthera dependens 

leaves 

8) 

Scoparia dulcis 

whole plant. 

9) 

Eclipta prostrate 

aerial parts. 

10) 

Morinda lucida 

leaves 

11) 

Erythrina senegalensis 

stem bark 

12) 

Brenania breyi 

leaves 

13) 

Schumaniophyton problematic 

leaves 

14) 

Osbeckie sp 

aerial parts. 

15) 

Chasmanthra dependens 

stem bark. 

16) 

Hoslundia opposta 

stem 

17) 

Dracaena mannii 

stem 

18) 

Paropsia guineensis 

stem 

19) 

Ficus exasperata 

stem. 

20) 

Crassocephalum sp 

whole plant. 

21) 

Olax subscorpoides 

leaves. 

22) 

Ehretia cymosa 

leaves. 

23) 

Ehretia cymosa 

stem. 

24) 

Berlina grandifolia 

leaves. 

25) 

Ritchea capparoides 

roots. 

26) 

Sphenocentrum jollyanum 

roots. 

27) 

Crescentia cujeta 

fruit juice. 

28) 

Ritchea capparoides 

roots 

29) 

Platycerium bifurcatum 

leaves. 

30) 

Hannoa klainiana 

leaves. 

31) 

Voacanga africana 

stem. 

32) 

Petersianthus macrocarpus 

leaves. 

33) 

Tabernataemontana pachysiphon 

leaves 

34) 

Funtumia elastica 

leaves. 

35) 

Pterygota macrocarpa 

leaves. 

36) 

Jatropha gossypiifolia 

leaves. 

Methanol extracts: 


37) 

Momordica charantha 

aerial parts. 



38) 

Paropsia guineensis 

leaves 

39) 

Synedrella nodiflora 

whole plant. 

40) 

Sterculia trangacantha 

leaves. 

41) 

Hoslundia oppoita 

stem. 

42) 

Paropsia guineensis 

stem. 

43) 

Dracaena mannii 

stem 

44) 

Berlina grandifolia 

leaves. 

45) 

Ritchea capparoides 

roots. 

46) 

Mormodica charantha 

fruits. 

47) 

Sphenocentrum jollyanum 

roots. 

48) 

Ritchea capparoides 

roots. 

49) 

Platycerium bifurcatum 

leaves. 

50) 

Tabernaemontana pachysiphon 

leaves. 

51) 

Ficus exasperata 

stem. 

52) 

Crassocephalum sp. 

Whole plant. 

53) 

Olax subscorpoides 

leaves. 

54) 

Ehretia cymosa 

leaves. 

55) 

Ehretia cymosa 

stem. 

56) 

Funtumia elastica 

leaves. 

57) 

Pterygota macrocarpa 

leaves. 

58) 

Jatropha gossypiifolia 

leaves. 

59) 

Garcina kola 

seeds. 

60) 

Zingiber officiales 

rhizomes. 

61) 

Ocimum gratissimum 

leaves. 

62) 

Afromamum melegueta 

seeds. 


Bulk extractions were carried out for the following plants: Picralima nitida, Enantia chlorantha, 
Spathodea campanulata, Synclisia scarbrida, Uvaria chamae, Cryptolepis sanguinolenta, 
Glossocalyx brevipes, Cleistopholis patens, Leidobotrys staudtii, Pachypodanthium staudtii, 
Odyendyea gabonensis and Uapaca palndosa. 

Moreover, previously identified extracts with activity against malaria such as Combretum 
dulchipelatum were fractionated and the fractions were submitted for biological testing in a bid to 
localize the active constituents. Other plants in this category include Dracaena mannii (CNS and 
antileishmanial activity), Cassytha filiformis (CNS activity), Triumfetta tomentosa (CNS activity), 
Scoparia dulcis (CNS) and Combretum dulchipelatum (antimicrobial). Detailed phytochemical 
investigations are proceeding on these compounds. 

B. Isolation & Characterization of Plant Secondary Metabolites: 

Bioassay-guided fractionation of above mentioned previously identified leads led to the isolation 
of several metabolites, whose identities have been determined in a number of cases. Further 
biological evaluation of the isolated compounds has confirmed the identities of the active 
constituents of these extracts. 


8 



Penianthus longifolius (Menispermaceae) 

The CH2C12-MeOH (1:1) extract of the stem bark is highly active against P. falciparum (D6: IC50 
= 350.066 ng/ml; W2: IC50 = 284.377 ng/ml). Repeated column chromatography of the extract led 
to the isolation of ten pure compounds designated PL1-PL10. PL1 and PL3 were subsequently 
identified as fats, while PL4 was identified as b-sitosterol glycoside. PL5 and PL7 which 
crystallized in different shapes in the same solvent are two different crystalline forms of the same 
compound - palmatine - PL6 and PL8 were identified as jatrorrhizine and vibroquercitol, 
respectively, while PL9 was found to be sucrose. PL10, a component isolated from the alkaloid 
fraction of the extract, was identified as N-methylcoridinium. The biological activities of the 
secondary metabolites are presented in Table 2 below. 


Figure 1: Secondary Metabolites Isolated from P. longifolius. 




PL10 : ammonium salt of N-methyl-corydine 


Table 2: Antiplasmodial activity of Compounds (PL1-7) isolated from Penianthm longifolius 

IC50 ng/mL 



D-6 

W-2 

PL1 

>5000 

> 5000 

PL2 

>5000 

>5000 

PL3 

> 5000 

>5000 

PL4 

> 5000 

>5000 

PL5 

24.4215 

37.3638 

PL6 

67.4211 

142.4276 

PL7 

26.6167 

37.7054 


9 



Homalium letestui (Flacourtiaceae). 

Phytochemical studies were initiated on this plant because the acetone extract of the stem bark 
displays significant antiplasmodial activity (W2: IC50 = 2564.11 ng/ml). Repeated column 
chromatography of this extract, combined with other separation techniques has so far yielded six 
constituents designated HL1-HL6. One compound, HL3, has been identified as 2-(hydroxymethyl) 
phenol. Efforts are currently underway to elucidate the structures of the other constitutents. 


Figure 2: Structure of HL3 Structures of the compounds isolated from Homalium letestui. 



HL3 

Glossocalyx brevipes (Monimiaceae) 

Extracts of this plant display both antiplasmodial and antileishmanial activity. Eight pure compounds 
(GBF1- GBF8) were isolated from the leaf extract while three (GBE2-GBE4) were obtained from the 
stem bark of this plant (Figure 4). Spectroscopic analysis led to the identification of seven compounds, 
including three alkaloids and four homogentisic acids. All four homogentisic acids (GBF1, GBF3, 
GBF5 & GBF6) and one alkaloid (GBF1) were identified as new compounds. Structure elucidation of 
the stem bark constituents is currently underway. 

Figure 3: Structures of the compounds (GBF1-3; GBF5-8) isolated from G. brevipes 



GBF1 


GBF3 


GBF5 







As shown in Table 3 below, some of the compounds exhibited antiplasmodial activity. 



IC50 ng/mL 

D-6 

W-2 

GBF1 

702.5863 

2125.7840 

GBF2 

> 5000 

>5000 

GBF3 

> 5000 

>5000 

GBF5 

1462.0020 

2552.9440 

GBE2 

> 5000 

> 5000 

GBE3 

1326.2950 

2373.7100 

GBE4 

1164.9080 

2367.5500 


Xymalos monospora (Monimiaceae): 

A re-examination of the alkaline extract of this plant yielded additional quantities of TZM4 and 
TZMHC1. Samples of these compounds have previously shown trypanocidal activity in vitro. The 
current effort was aimed at providing additional material for in vivo studies in animal models of 
trypanosomiasis. 


Hyptis suaveolens. 

Hyptis suaveolens is one of the plants identified as having the ability to correct the cystic fibrosis 
(CF) defect in the yeast two-hybrid assay. It is therefore desirable to isolate and identify the active 
constituents of this plant. A combination of silica gel based chromatographic techniques such as 
CC and Biotage Flash chromatography led to the isolation of the compound responsible for 
reversing the CF defect in the yeast-based screening assay. The method adopted in the isolation 
process is briefly described. Dried leaf (2.0 Kg) was extracted successively with petroleum ether, 
chloroform, methanol and water. Each extract was concentrated to dryness using the rotary 
evaporator. The petroleum ether extract was most active followed by the chloroform extract. 
About 8.20 g of the petroleum ether extract was chromatographed on a silica gel column eluted 
with Hexane: Ethyl acetate (9:1). A 50-ml fraction was collected and monitored by TLC using 
petroleum ether: ethyl acetate (3:1). Chromatographically similar fractions were pooled and 
assayed. Four of the 7 major fractions showed activity in the cystic fibrosis assay, with the most 
active fraction being Fr. 71 +72. Further fractionation of Fr. 71-72 was carried out. About 500 mg 
was re-chromatographed on Biotage Flash chromatography eluted with solvent system petroleum 
ether/ ethyl acetate (3:1). A total of 33 fractions (20 ml each) were collected and analyzed by TLC. 
As shown in Figure 3, seven subtractions were obtained. Fractions 1-6 gave the highest yield of 
96.5 mg. Thin layer chromatograms showed that fractions 7-18 contained a single component. The 
isolated compound has been submitted for further biological testing and structure elucidation is 
also underway. 


11 




































































a) mo oco mco os ms- o® m® o m mm o-^r m^r oco inn ocm iocn o 


303.13 


SU_2517_03#21- RT 0.30-0.36 AV:5 SB 6 0.01-0.09 NL: 7.55E5 
T + p Full ms £50.00, 



Figure 5 LC-MS of compound isolated from Hyptis suaveolens (above) 

Trichilia spp & Thuranthus africana 

Previous research by our team has shown that two constituents of these plants designated TS2 and 
PTA4 can correct the cystic fibrosis defect reconstruct in yeast. Additional quantities of these 
compounds were thus prepared for further examination of these promising compounds. 

Dracaena mannii 

Previously, we found that the fruit pulp extract of this compound displays significant 
antileishmanial activity. The extract was also found to bind monoamine transporters (see our 
2000/2001 Progress Report). Our subsequent work is thus aimed at isolating and identifying the 
secondary metabolites that are responsible for the observed activities. To this end, the first batch 
of bulk extracted material was used for dereplication of antileishmanial compounds. The methanol 
extract containing the highest concentration of saponins was fractionated by a combination of 
Sepahdex LH20, lobar and preparative TLC to yield mannispirostan A- a known antileishmanial 
compound. Further work on this plant is on going at the University of Buea with a focus on CNS 
activity. 

Isolation of Alstonine: Search for Alternative Sources. 

Previously, we have shown that alstonine, an indole alkaloid originally isolated from Alstonia 
boonei, is the active constituent of a Nigerian medicinal plant used for the treatment of 
schizophrenia. Recent studies by our collaborators reveal that alstonine is an atypical antipsychotic 
which displays a unique pharmacological profile (Elizabetsky et al., 1998). This alkaloid may thus 
represent a useful lead for a new class of antipsychotics agents. Therefore, we have launched a 


13 











detailed investigation of this compound. The current study was undertaken to identify alternative 
sources of alstonine for use in current and future pharmacological investigations. 

Working quantities of alstonine were isolated from the pericarp of Picralima nitida by a 
combination of column chromatography and preparative TLC. The methylene chloride extract of 
the pericarp of Picralima nitida was fractionated on silica gel 60 column. By means of 
comparative TLC, the fractions containing alstonine and the pure alstonine standard were matched 
leading to the identification and subsequent isolation of the alstonine from the pericap of Picralima 
nitida. By a similar procedure, alstonine was also isolated from the methanol extract of the root of 
Rauwolfia vomitoria. As a result of this study, we now have three sources of this potentially 
interesting compound. 

1.1.2 Preliminary Bioassavs 

1) Brine Shrimp Cytotoxicity Screening 

Thirty-three extracts were assayed during the project period. However, the results obtained were 
inconclusive probably because the eggs used were of questionable viability. As a result, these data 
have been set aside and the assays will be repeated. 

2) Antimicrobial Screening 

A total of 52 extracts consisting of various plant parts were screened for antimicrobial activities 
against four bacterial organisms (Bacillus subtilis, Staphlococcus aureus, E. coli, and 
Pseudomonas aeroginosa) and in some cases against one fungus (Candida albicans- a yeast). Out 
of the 52 extracts screened for antimicrobial activity, 31(60%) showed some activity against one or 
more bacterial organisms used in the study (Table 4). In addition, seven out of 30 extracts tested 
displayed antifungal activity. 

Table 4: Antimicrobial Activities of Selected Plant extracts 


Extracts and solvent Organisms 


1 . 

Dorstenia multiradita (whole plant) CH 3 OH 

Sa 

+ 

Bs 
' + 

Pa 

+ 

Ec 

Ca 

+ 

2. 

Dorstenia midtiradiata (whole plant) CH 2 CI 2 

+ 

+ 

+ 

+ 

4 - 

3. 

Portulaca oleracea (whole plant) 

CH 2 CI 2 

- 

+ 

+ 

+ 

+ 

4. 

Portulaca oleracea (whole plant) 

CH 3 OH 

- 

+ 

+ 

+ 

+ 

5. 

Guava leaves (upper fraction) 

H 2 0 

- 

+ 

- 

4 

- 

6 . 

Guava leaves (lower fraction) 

H 2 0 

4- 

+ 

- 

4- 

- 

7. 

Momordica charantha (aerial parts) 

CH 2 CI 2 

- 

+ 

+ 

- 

- 

8 . 

Combretum dulchipetalum CH 3 OH 

fr. 10-30 (i) 

+ 

- 

+ 

4 

- 

9. 

Combretum dulchipetalum fr. 

40-50 (ii) 

+ 

- 

- 

- 

- 

10 . 

Combretum dulchipetalum fraction 

60-80 (iii) 

+ 

- 

- 

- 

- 

11 

Combretum dulchipetalum fraction 

80-100 (iv) 

4 

4 - 

+ 

4 

- 


14 



12 . 

Dracaena manni stem CH 3 OH 

fr. 1-16 (i) 

+ 

+ 

+ 

+ 

+ 

13. 

Dracaena manni stem 

fr. 17-35 (ii) 

+ 

+ 

+ 

+ 

+ 

14 

Dracaena manni stem 

fr.36-60 (iii) 

+ 

+ 

+ 

+ 

+ 

15 

Eclipta prostrate (aerial part) 

CH 2 CI 2 

+ 

- 

+ 

- 

- 

16 

Scopora sp. (whole plant) 

CH 2 CI 2 

+ 

- 

- 

- 

- 

17 

Brenania brieyi (leaves) 

CH 2 C1 2 

+ 

- 

- 

- 

- 

18 

Erythrina senegalensis (root) 

ch 2 ci 2 

+ 

+ 

+ 

- 

- 

19 

Morinda lucida (leaves) 

ch 2 ci 2 

+ 

- 

- 

- 

- 

20 

Dorstenia multiradrata (whole plant) CH 2 CI 2 

+ 

+ 

- 

- 

- 

21 

Dorstenia multiradrata (whole plant)CH 30 H 

+ 

- 

- 

- 

- 

22 . 

Synedrella nodiflora (whole plant) 

ch 2 ci 2 

- 

- 

- 

- 

- 

23. 

Trema orientalis (leaves) 

CH 3 OH 

- 

- 

- 

- 

- 

24. 

Phyalis augidata (whole plant) 

CH 3 OH 

- 

- 

- 

- 

- 

25. 

Mitra carpus (whole plant) 

CH 2 C1 2 

- 

- 

- 

- 

- 

26. 

Morinda lucida (leaves) 

ch 2 ci 2 

- 

- 

- 

- 

- 

27. 

Crosophyta febrifuga (roots) 

h 2 o 

- 

- 

- 

- 

- 

28. 

Pterocarpus angloensis (leaves) 

ch 2 ci 2 

- 

- 

- 

- 

- 

29 

Euphorbiaa hirta (whole plant) 

ch 2 ci 2 

- 

- 

- 

- 

- 

30. 

Sterculia tragacantha (leaves) 

ch 2 ci 2 

- 

- 

- 

- 

- 

31. 

Osbeckie sp (aerial part) 

ch 2 ci 2 

- 

+ 

- 

- 


32. 

Synerdella nodiflora (whole part) 

ch 3 oh 

- 

+ 

+ 

- 


33. 

Paropsia guineensis (leaf) 

CH 30 H 

- 

- 

- 

+ 


34. 

Synclesic scabrida (Aerial parts) 

CH 30 H 

- 

+ 

- 

- 


35. 

Paropsia guineensis (stem) 

CH 2 C1 2 

+ 

- 

- 

- 


36 

Sterculea tragantha (leaf) 

ch 3 oh 

- 

+ 

+ 

- 


37. 

Hoslundia opposta (stem) 

CH 30 H 

+ 

+ 

- 

- 


38. 

Ficus exasperata (stem) 

CH 2 C1 2 

+ 

+ 

- 

+ 


39. 

Dracaena manni (stem) 

ch 2 ci 2 

+ 

+ 

- 

+ 


40. 

Hoshinda opposita (stem) 

ch 2 ci 2 

+ 

+ 

- 

+ 


41. 

Crassocephalum sp (whole part) 

ch 3 oh 

+ 

+ 

- 

+ 


42. 

Dorstenia multiradrata^ Whole part) CH 3 OH 

+ 

+ 

- 

+ 



15 



43 

Mussaenda elegans (leaves) 

CH 2 C1 2 

+ 

+ 

+ 


44. 

Dorstenia multiradrata (whole part) CH 3 OH 

+ 

+ 

+ 


The bacteria used for screening these extracts were 
resistant to Ciprofloxacin, Gentamicin and Ampicillin. 

isolated from surgical wounds. 

E.c B.s Sal. S.a 

They 

Shi 

45. 

Ficus exasperata (stem) 

ch 2 ci 2 

- 

- 

+ 

- 

46. 

Dorstenia midtiradrata (whole part) CH 3 OH 

- 

- 

“ 

- 

47. 

Crassocephalum sp (whole part) 

CH 3 OH 

- 

- 

+ 

- 

48. 

Dracaena manni (stem) CH2C12 

Fr. 17-35 

- 

- 

+ 

- 

49. 

Dracaena manni (stem) CH2C12 

Fr. 1-16 

- 

- 

+ 


50. 

Mussaenda elegans (stem) 

CH 3 OH 

- 

- 

- 


51. 

Morinda lucida (leaves) 

H 2 0 

- 

- 

- 


52. 

Voacanga africana (leaf) 

CH 3 OH 

- 

- 

- 



KEYS 

+ 


B. s 
S.a 
P.a 

C. a 

CH 2 C1 2 

ch 3 oh 


Clear zone of inhibition 
No zone of inhibition 
Bacillus subtilis 
Staphylococcus aureus 
Pseudomonas aerogenosa 
Candida albicans 
Methylene Chloride 
Methanol 


C. Anti- cancer and Anti- HIV Screening 

As part of the plan to start the anti-HIV screening activities at InterCEDD, Nsukka, Dr. Barrows 
(University of Utah) visited Nsukka, Nigeria in December 2001. A detailed plan of action was 
worked out between Dr. Barrows and the participants in the project at Nsukka. Items of equipment 
required for the project were identified. One of the Nigerian participants (Dr. K. Chah) will visit 
Dr. Barrows’ Lab at the University of Utah for hands-on training in anticancer and anti-HIV 
screening techniques. 

A total of 21 samples of higher plants were processed and submitted to the group at Southern 
Research Institute^ for antiviral testing. The twenty-one samples were chosen based on 
ethnomedical uses. 


16 



1.1.3 Training Activities 

A number of African scientists received training under the auspices of the I.CBG during the period 
of this report. Training in phytochemistry took place at four sites: University of Mississippi 
(National Center for Natural Products Research), University of Dschang (Laboratory of Organic 
Chemistry, Department of Chemistry), University of Buea (Department of Chemistry) and 
International Center for Ethnomedicine and Drug Development Nsukka (InterCEDD). Scientists 
received stipends and reagents for carrying out research project under the auspices of the ICBG. 
Details of the individual programs are provided below. 

University of Buea: Efforts at this center are largely devoted to the identification of centrally 
active agents. To this end, plants identified during the last report period as active in the CNS 
screen have been chosen for detailed phytochemical investigation. The following four students 
enrolled in the M. Sc. Program in Chemistry have been assigned to work on these plants: 

- Erambo Ayokosok; Monoamine Reuptake Inhibitors of Cassytha filiformis. 

- Edith Lepsia Fomunung: Neuroactive Constituents of Triumfetta tomentosa. 

- Ruth Loh Viboh: Modulators of Monoamine Function in Dracaena mannii. 

- Gregory Nkwe Nkepang; Monoamine Reuptake-Inhibiting Constituents of Scoparia dnlcis. 

An account of the follow-up work on these plants will be present in the next progress report. 

University of Dschang: Efforts at this center have been directed at the isolation and structural 
characterization of plants secondary metabolites. Compounds are then submitted to the various 
centers for biological evaluation. Training in phytochemistry is undertaken at both the 
undergraduate and graduate levels. During the period covered by this report, the ICBG funded the 
training of 8 students at the graduate level. Out of this group, 2 students graduated with a Ph D. in 
chemistry. Five students are currently enrolled in the Ph.D. program, and one of them has already 
passed the Ph D. candidacy exam. The names of the students and the titles of their projects are 
provided hereunder. 

- James Mbah: Title of Thesis: Antimalarial substances from Cameroonian medicinal plants: 
Reneilmia cinucinnata, Glossocalyx brevipes and Vernonia gninensis. 

Alembert Tchinda: Title of Ph.D. thesis: Contribution to the phytochemical study of some 
Cameroonian medicinal plants: Vernonia guinenis and Noeboutonia glabresens. 

- Currently enrolled in the Ph.D. program: 

- Christabel Tomla: Title of thesis project: Contribution to the phytochemical study of 
Aframomum species: Aframomum sulcatum. 

- Michel Tchimene: Title of thesis project: Anti-malarial metabolites from Khaya species. 

- Godfred Ayimele: Title of thesis: Contribution to the study of Aframomum species: 
Aframomum sceptrum. 

- Sob Tanemossu: Title of thesis project: Contribution to the study of Aframomum species: 
Aframomum letestuanum. 

- Virginie Ebessa: Title of thesis project: This is a new student. 

SimpliceTatsimo: Title of thesis project: Anti-malarial metabolites from Cameroonian 
medicinal plants: Peninathus longifolius. 


17 




Copies of the Ph D. thesis will be submitted to the ICBG Program Office in due course. 

INTERCEDD:_ICBG is currently supporting two students (Chioma Ezeobi and Deborah Adazika) 
undergoing M.Sc training at the University of Nigeria, Nsukka. 

University of Mississippi: Three graduate students enrolled in doctoral programs in Nigeria and 
Cameroon are working as Visiting Scholars at this site. The focus of their work is primarily to 
isolate and identify the active constituents of plants that were identified earlier in our bioassays as 
having anti-plasmodial and/or anti-protozoal activity. The names of the students and their projects 
and preliminary reports of their work are provided below. 

- Julius Ngunde (University of Buea, Cameroon); Antiparasitic Constituents of Selected 
Cameroonian Medicinal Plants. Studies focus on Peucedanum zenkeri, Glossocalyx 
brevipes, andEriosema glomerata. 

- Christopher Ezugwu (University of Nsukka, Nigeria); Phytochemical Studies and 
Biological Screening of Araliopsis tabouensis (Rutaceae) and Pachypodanthium staudtii 
(Annonaceae). 

- Odiri Onoruvwe (University of Jos, Nigeria); Isolation and Indentification of Antimicrobial 
and Antiprotozoal Constituents of Chasmanthera dependens, Dorstenia, multiradiata and 
Enantia chlorantha. 

A progress report covering the period from January 2002 to June 2002 is presented below. 

A. Topic: Phytochemical Studies and Biological Screening of Glossocalyx Brevipes and 
Peucedanum Zenkeri- Julius Ngunde 

Introduction 

Glossocalyx brevipes is a small tree, which grows in the equatorial forest of West and Central 
Africa. Our interest in G. brevipes was aroused by evidence of antileishmanial activity in 
preliminary bioassays. Its leaves are edible. Peucedanum zenkeri is a herb found in the West- 
Central African equatorial forest. Some cattle breeders use the leaf as a tobacco substitute. 
Members of this genus are widely used in Chinese folk medicine as expectorant, antitussive, 
antipyretic and stomachic. Coumarins isolated from Peucedanum genera possess some 
pharmacological properties. No work has yet been carried out on Peucedanum zenkeri. The reason 
to begin with Peucedanum zenkeri seed was its high activity observed in primary bioassays. 

Mm 

Isolate and characterize the compounds present in those plants. 

- Screen and evaluate the biological activities of the pure compounds. 

Methodology 

Collection of Plant Material: 

Glossocalyx brevipes was collected in November 2001 from the neighborhood of the Korup 
Project headquarters near the river banks of a secondary forest in Mundemba, Ndian Division in 
the South West Province of Cameroon, by Dr Chuyong George an ICBG Botanist. Dr. Claire 
Wirmum, Director of Medicinal Foods and Plants Bamenda, and an ethnobotanist of the ICBG 


18 



Project collected Peucedatmm zenkeri in November 2001 in the forest of Babanki village of the 
North West Province of Cameroon. 


Extraction 

20 g samples of air-dried and ground plant material of the leaf and stem bark of G. brevipes were 
extracted with MeOH. The crude extracts were partitioned with Hexane, EtOAc, CHCI 3 and 
MeOH respectively. 20 g samples of air-dried and ground material of the seed, leaf and stem of 
both the cultivated and wild P. zenkeri were extracted with MeOH. All fifteen samples were 
submitted for biological screening. The extraction process continued with the bulk extraction of 
the leaf and stem bark of G. brevipes and the cultivated seed of P. zenkeri. 

Results 

Primary Biological Screening. 

The following extracts showed activity from the results of the primary biological screening 
(antimalarial and antiprotozoal): 

G. brevipes leaf: Hexane and EtOAc fractions 

G. brevipes stem bark: Hexane and EtOAc fractions 

P. zenkeri cultivated: seed extract (generally 100 % activity) 

P. zenkeri wild: seed extract (generally 100 % activity) 

Isolation of the Major Constituents of the Seed of P. zenkerL 

The Hexane fraction was chromatographed on a column packed with silica gel. Now in its final 
phase, the column has so far yielded four coumarins: Umbelliprenin, Imperatorin, Bergapten and 
Isopimpinellin, and some fatty acids. All four have some documented pharmacological properties. 
Up to 2.0 g of imperatorin was isolated, and a similar quantity is expected from the CHCI 3 
fraction. All compounds isolated are re-submitted for activity testing. 


Figure 6 Structures of the compounds isolated from P. zenkeri 



19 




B. Phytochemical studies and Biological screening of AraHopsis tabouensis (Rutaceae) and 
Pachypodanthium staudtii (Annonaceae) - Ezugwu Christopher 

Introduction 

AraHopsis tabouensis Aubrev. Et Pellegr (Rutaceae) and Pachypodanthium staudtii Engl. & Diels 
(Annonaceae) are trees, which grow in the tropical forest of West and Central Africa. Various 
parts are used to remedy many ailments in African folk medicines. Previous work shows that the 
plants are rich in quinoline alkaloids and other secondary metabolites having several biological 
activities. 

Aim 

• To isolate, and determine the structures of the compounds present in the plant; 

• Screen and evaluate the biological activities of the pure compounds. 

Methodology 

Collection of Plant Material: 

The plants were collected from Owai, Cross River State, Nigeria on the 6 th of December 2001 and 
identified by Mr. Ozioko, a taxonomist at the Bioresources Development and Conservation 
Program at Nsukka. Voucher specimens are deposited at the B.D.C.P Herbarium. 

Preparation of the plant material: 

2 kg stem bark of A. tabouensis , 350 g stem bark and 930 g stem of P. staudtii were extracted at 
room temperature with methanol (yield: 340 g, 16 g and 30 g, respectively). 60 g of the methanolic 
extract of A. tabouensis was suspended in 1.0 N HC1 ( 1 L) and then ammonia was added to solution 
and partitioned with chloroform to obtain crude alkaloid extract. The alkaloid extract ( 8.0 g) was 
chromatographed on silica and eluted with increasing amount of MeOH in CHCI 3 . Identical 
fractions were pooled and further purification was done at small scale using centrifugal 
chromatography (Chromatotron), gel filtration (Sephadex LH- 20 ) and preparative TLC (Silica 

gel). 

Results 

Biological Screening 

Hexane extract of A. tabouensis showed activity against C. albicans and C. neoformans while both 
EtOAc and Hexane extract of A. tabouensis showed activity against Plasmodium falciparium. 

Phvtochemical studies 

Six compounds were isolated from A. tabuoensis and re-submitted for biological testing. The 
structures of the 5 compounds have so far been elucidated: 3 indolequinazoline alkaloids, a 
quinolone alkaloid and a coumarin shown below. We are in the process of isolating other 
constituents of A. tabuoensis. 


20 



C. Isolation and Identification Of Antimicrobial and Antiprotozoal Constituents Of 
Chasmathera dependens, Dorstenia multiradiata and Enantia chlorantha. - Odiri 
Onoruvwe 

The current project aims at isolating and identifying the anti microbial and anti protozoal 
constituents in three selected medicinal plants vis: Chasmanthera dependens Hochst. 
(Menispermaceae), Dorstenia multiradiata (Moracea) and Enantia chlorantha Oliv. 
(Annonaceae). In Nigeria, each of these plants is used for the treatment of different disease 
conditions, some of which suggest possible antimicrobial activities. E. chlorantha is widely used 
to treat malaria fever. (Oliver, 1960; Irvine, 1961; Ajali, 2002). The preliminary investigation 
involved solvent-solvent fractionation of the cold methanol extract of each plant sample (20-25 g) 
to yield hexanes, chloroform, ethyl acetate and water fractions. The extracts and fractions were 
sent for in vitro bioassay screening. Larger scale extraction of each plant part in a gradient fashion 
using solvents of different polarities, and chromatographic analyses of active extracts/fractions are 
currently being carried out. Resultant column fractions have been sent for further bioassay to test 
for anti microbial and anti malarial activities. 


The in-vitro bioassay screening showed that the methanol extracts and, chloroform and ethyl 
acetate fractions of D. multiradiata and E. chlorantha have significant antimalarial antimicrobial 
activities. Some of the significantly active extracts and fractions showed no cytotoxic effects, 
while others were not significantly toxic. The result of the anti-leishmanial bioassay testing is 
being awaited. 


21 






Further studies will focus on the isolation of the active constituents from the column fractions as 
well as non-active chemical compounds. Using spectroscopic analysis and other methods, 
structural elucidation of isolated compounds for their identification will be carried out. The data 
generated from the present study will contribute to further information on these plants for 
ethnomedicine applications and databank for the shorter-term development of phyto-medicines and 
the more regular conventional medications. 


2. ANTIMALARIAL DRUG DEVELOPMENT 

In continuation of work previously carried out, we aimed to conduct chemical optimization of a 
number of natural product based ICBG compounds with promising antimalarial activities, whilst 
improving their pharmacokinetic properties. These include certain active constituents of 
Cryptolepis sanguinolenta and selected protoberberine alkaloids. Preparative scale isolation or the 
synthesis of already identified compounds for in vivo studies will also be undertaken. In addition 
chromatrographic methods will be developed for the separation and isolation of the active 
constituents of Picralima nitida and related indole alkaloids. 

This report also describes a series of in vitro studies performed as part of the AP-3 Antimalarial 
Drug Development effort. Studies were conducted with purified fractions and isolated pure 
compounds. The procedure used measures the ability of the extracts/fractions/compounds to inhibit 
the incorporation of [G- 3 H] hypoxanthine into the malaria parasites. Details of the protocol are 
described by Desjardins et al 1979 and Milhous et al 1985. Similar tests were performed using 
chloroquine and mefloquine as control standard drugs. We also examined the ability of 
extracts/fractions/compounds from our data bank to inhibit Plasmodium falciparum MRK and PK5 
kinases. The results indicate that two compounds out of the 24 samples screened inhibited the 
enzyme MRK at 2.5ng/ml representing novel chemotype of compounds that preferentially inhibit 
Cyclin-Dependent Protein Kinases (CDKs). 

2.1 Chemical Optimization of ICBG Lead Compounds with Antimalarial Potential. 

A. Separation and isolation of the active constituents of Picralima nitida. 

Using a combination of column chromatography (with silica) and preparative thin layer 
chromatography (silica gel plates) techniques, compound isolation from seed, stem bark and 
pericap (polar and non-polar) extracts have been performed. Over 20 constituents have been 
separated and are being analysed using mass spectrometry. Once they have been identified, any 
new constituent will undergo in vivo animal studies for activity and further investigation. 

This has been a particularly laborious task considering each extract contains several (>8) 
constituents. However many of these are the same and are likely to be well characterized. The 
polar extracts were especially difficult to separate and purify. 

Picralima nitida compound isolation from seed extract 

Column chromatography: on silica, starting with 60% MeOH: 40% ACN with a gradual increase 
in polarity. Isolated 9 fractions, 3 contained more than one compound and underwent further 
isolation with preparative TLC to produce 5 additional compounds. 


22 



Picrglima nitidg compound isolation from stem bark ('CH 9 Q 9 fraction') 

Column chromatography: on silica, starting with 40% MeOH: 60% CH 2 CI 2 with a gradual increase 
in polarity. Isolated 1 compound (however, an extremely polar substituent could not be isolated). 

Picrglimg nitida compound isolation from pericap (CH 7 CI 7 fraction) 

Column chromatography: on silica, starting with 60% MeOH: 40% CH 2 CI 2 with a gradual increase 
in polarity. Isolated 3 fractions, 1 contained more than one compound and underwent further 
isolation with preparative TLC resulting in 3 additional compounds. 

Picrglima nitida compound isolation from pericap (MeOH fraction! 

Column chromatography: on silica, starting with 60% MeOH: 40% CH 2 CI 2 with a gradual increase 
in polarity (extract contained a waxy insoluble substance which was not placed on column). 
Isolated 4 fractions, 1 contained more than one compound and underwent further isolation with 
preparative TLC to afford 3 additional compounds. 

B. Synthesis of cryptolepine analogues. 

Cryptolepine (Fig. 8(1)) isolated from Cryptolepis sanguinolenta, has long been employed as an 
antimalarial agent in traditional West African medicine. Several other alkaloids have also been 
characterized from this plant (Paulo et al, 1995). Its activity is 64.8 and 145.9 pg/ml against D6 
and W2 strains respectively. The antimalarial activity is known of several cryptolepine analogues 
that have been synthesized (Bierer, 1997) and tested against chloroquine-sensitive (D6) and 
chloroquine-resistant (W2) strains of Plasmodium falciparum. The three analogues with the 
greatest antimalarial activity (Fig. 8 (2-4)) have been used as a basis for further analogue design. It 
is planned that these compounds undergo further chemical optimization in order to enhance 
antimalarial and oral activity. 



Fig.8: Structures of cryptolepine and previously synthesized analogues. 


23 



Two main synthetic routes have been employed for the preparation of further analogues. The first 
is based upon the Holt and Petrow(Holt etal, 1947) procedure (Fig. 9), which has been chosen due 
to its brevity and high yields. Other synthetic routes are longer and yields are significantly lower. 
Other analogues were prepared according to the route depicted in Fig. 10 (Bierer, 1997). Overall, 
these synthetic pathways have been time consuming but straightforward and minor problems have 
been overcome by modifications based upon other well-documented procedures (Bierer et al, 
1998; Fan & Ablordeppy, 1997; Yang et al, 1999; De et al, 1997). To date, 10 analogues (Fig. 10) 
have been synthesized and a few more are planned. They are currently undergoing antimalarial and 
pharmacokinetic testing. 






Fig.9: Scheme depicting synthetic pathway via the Holt & Petrow method. 


24 



C. Synthetic Procedures: 

Quindoline-ll-carboxylic acid 

Isatin (4.25g, 28.6 mmol) in cooled aqueous KOH (25g in 115mL H 2 O) was added under nitrogen, 
to indolyl acetate (5g, 28.6 mmol) with shaking. The mixture was left to stir vigorously for 3 days 
at room temperature. The reaction mixture was diluted with H 2 O (65mL) and air was bubbled 
through while heating for 20mins between 75-80°C. The reaction mixture was filtered hot and the 
filtrate diluted with 200 mL ethanol. HC1 (1M) was added until precipitate formed (~ pH4), which 
was subsequently filtered and washed with hot H 2 0 followed by ethanol. Dried orange powder 
(96.1%) in vacuum oven for 1 day, m.p: 321-326°C. ‘HNMR (DMSO-de) 8 11.45 (s, 1H, NH), 
9.10 (d, 1H), 8.40 (d, 1H), 8.24 (d, 1H), 7.77 (d, 1H), 7.67 (m, 4H), 7.35 (t, 1H). 

5.10- Dimethylquindoline-ll-carboxylate 

A mixture of Quindoline-ll-carboxylic acid (4g, 13.5 mmol) in DMF (40mL), KOH (2.9g, 0.05 
mol), BaO (8.1g, 0.05mol) and methyl iodide (llmL, 0.17mol) was stirred at room temperature for 
48h. The reaction mixture was then partitioned between CHCI 3 (lOOmL) and H 2 O (150mL). The 
organic layer was washed with H 2 0 (2x75mL), dried with Na2S0 4 and evaporated. 
Recrystallization from hexanes afforded yellow crystals (85.5%), m.p: 122.5-123.5°C. NMR 
(DMSO-d 6 ) 5 8.40 (d, 1H), 8.25 (d, 1H), 8.00 (d, 1H), 7.70 (m, 1H), 7.38 (t, 1H), 4.20 (s, 3H), 
3.81 (s, 3H). 

5.10- Dimethyl-ll-(methoxycarbonyl)quindolinium iodide 

Stirred 5,10-Dimethylquindoline-l 1-carboxylate (0.6g, 2 mmol) in methyl iodide (5mL, 50 mmol) 
for 48h at room temperature. Evaporated excess methyl iodide in vacuo and recrystallized from 
methanol/diethyl ether to yield an orange solid (96.7%), m.p: 255.3-257.1°C. *H NMR (DMSO-de) 
8 8.92 (dd, 2H), 8.35 (d, 1H), 8.24 (t, 1H), 8.08 (t, 2H), 8.04 (t, 1H), 7.68 (t, 1H), 5.10 (s, 3H), 4.33 
(s, 3H), 4.02 (s, 3H). 

5.10- Dimethyl-1 l-(ethoxycarbonyl)quindolinium iodide 

10-Dimethylquindoline-ll-carboxylate (0.3g, 1 mmol) was refluxed in EtOH (lOmL) for 1.5h. 
Filtered resulting precipitate (89.7%), m.p: 243-245°C. ] H NMR (DMSO-de) 8 9.08 (d, 2H), 8.55 
(d, 1H), 8.34 (d, 1H), 8.08 (t, 1H), 7.99 (t, 2H), 7.68 (d, 1H), 7.49 (t, 1H), 4.85 (t, 2H), 4.06 (q, 
3H). 

5.10- Dimethyl-l l-(butyloxycarbonyl)quindolinium iodide 

10-Dimethylquindoline-l 1-carboxylate (0.3g, 1 mmol) was refluxed in BuOH (lOmL) for 1.5h. 
Filtered resulting precipitate (72.6%), m.p: 238-239°C. 'H NMR (DMSO-d 6 ) 9.04 (d, 1H), 8.35 (d, 
1H), 8.28 (d, 1H), 8.07 (d, 1H), 7.77 (m, 4H), 7.42 (m, 1H), 4.15 (s, 3H), 3.78 (s, 3H), 3.60 (t, 2H), 
1.41 (m, 2H), 1.25, (m, 2H), 0.95 (t, 3H). 

5,10-Dimethyl-l l-(benzyIoxycarbonyl)quindolinium iodide 

10-Dimethylquindoline-l 1-carboxylate (lg, 3.4 mmol) was refluxed in benzyl alcohol (30mL) for 
2h. Allowed to cool and evaporated excess alcohol, recrystallized from acetone (%), m.p: 211- 
212°C. ] H NMR (DMSO-d(>) 8 9.11 (d, 1H), 8.35 (d, 1H), 8.21 (d, 1H), 8.04 (d, 1H), 7.57 (d, 1H), 
7.52 (d, 1H), 7.37-7.16 (m, 4H), 5.41 (s, 2H), 4.14(s, 3H), 3.99 (s, 3H). 


25 



11-HydroxymethyIquindoIinium hydrochloride 

5.10- Dimethyl-1 l-(methoxycarbonyl)quindolinium iodide (0.59g, 1.9 mmol) treated with LiAlH 4 
in THF (1M) (lOmL) under reflux for 45mins. Allowed to cool, then dissolve in EtOAc (~40mL). 
Filtered the precipitate and concentrated the filtrate. Column chromatography on silica 
(ACN:MeOH/ 70:30). Combined fractions, concentrated and acidified to produce an 
orange/yellow solid (36.7%), m.p: >280°C. l H NMR (DMSO-d 6 ) 5 9.60 (s, 1H), 8.90 (dd, 2H), 
8.50 (d, 1H), 8.19 (t, 1H), 8.13 (m, 3H), 7.60 (t, 1H), 5.08 (s, 3H). 

5.10- Dimethyl-ll-(carbonyl chloride)quindoline 

5.10- Dimethylquindoline-l 1-carboxylate (3.3g, 8.8 mmol) refluxed in SOCI2 for 2h. Evaporated 
excess SOCI2 in vacuo to give a bright red solid. Used without any further purification, m.p: 228- 
229.5°C. : H NMR (DMSO-d 6 ) 5 9.10 (d, 1H), 8.57 (d, 1H), 8.42 (d, 1H), 7.75 (m, 3H), 7.37 (t, 
1H), 4.19 (s, 3H), 3.81 (s, 3H). 

Quindoline 

Quindoline-11-carboxylic acid (2g, 7.6 mmol) was refluxed in diphenyl ether (40mL) for 6h at 
250°C allowed reaction mixture to cool then diluted with 25mL petroleum ether. Filtered mustard 
coloured precipitate (63.5%), m.p: 248-249.5°C. *H NMR (DMSO-d 6 ) 6 11.53 (s, 1H, NH), 8.35 
(d, 2H), 8.22 (d, 1H), 8.13 (d, 1H), 7.60 (m, 4H), 7.35 (t, 1H). 

5-(4-MethyI)-benzylquindolinium hydrochloride 

Quindoline (1.03g, 4.7 mmol), 4-methyl benzyl bromide (4mL, 16 mmol) and CHCI3 (5mL) was 
left to stir in a sealed tube for 48h at 140°C. Dissolved reaction mixture in CHCI3 (20mL) after 
cooling followed by ether (lOOmL). Filtered precipitate and washed with ether. Dissolved mustard 
coloured product in minimal CHCI3 and purified with column chromatography; CHCUMeOH 
(2%) on silica. Collected fractions and acidified (32.7%), m.p: 227.5-230°C. l H NMR (DMSO-d6) 
5 11.51 (s, 1H, NH), 9.10 (d, 2H), 8.92 (d, 1H), 8.75 (d, 1H), 8.54 (d, 1H), 8.48 (t, 2H), 8.28 (d, 
1H), 7.81 (m, 8H), 7.40 (t, 2H), 7.13 (d, 2H), 7.06 (d, 1H), 5.83 (s, 3H). 

5-(4-Nitro)-benzylquindolinium hydrochloride 

Quindoline (lg, 4.6 mmol) and 4-nitro benzyl bromide (2.5g, 11.6 mmol) in CHCI3 (5mL) was left 
to stir in a sealed tube for 48h at 140°C. Dissolved reaction mixture in minimal CHCI3 (20mL) 
after cooling and diluted with ether (lOOmL). Filtered precipitate and washed with ether. Dissolved 
yellow coloured product in minimal CHCI3 and purified with column chromatography; 
CHCUMeOH (98:2) on silica. Collected fractions and acidified (22.3%), m.p: 243.5-245°C 
(decomp). *H NMR (DMSO-d 6 ) 6 13.08 (s, 1H, NH), 9.51 (s, 1H), 8.72 (d, 1H), 8.53 (d, 1H), 8.33 
(m, 4H), 7.97 (m, 4H), 7.53 (t, 1H), 6.98 (s, 2H). 

5-(2-Methyl)-propylquindolinium hydrochloride 

Quindoline (0.5g, 2.3 mmol) and l-bromo-2-methylpropane (2ml, 18 mmol) in DMF (3mL) was 
left to stir in a sealed tube for 48h at 140°C. Diluted reaction mixture with ether (100mL). Filtered 
precipitate and washed with ether. Dissolved dark orange product in minimal CHCI3 and purified 
with column chromatography; CHCUMeOH (98:2) on silica. Collected fractions and acidified 
(50%), m.p: 141-14,2%:. 'H NMR (DMSO-d 6 ) 6 11.53 (s, 1H, NH), 9.12 (d, 1H), 8.40 (d, 1H), 
8.25 (d, 1H), 7.70 (d, 1H), 7.67 (m, 4H), 7.35 (t, 1H), 5.60 (t, 2H), 2.11 (d, 1H), 1.55 (m, 3H), 1.27 
(m, 3H). 


26 



5-hexylquindoIinium hydrochloride 

Quindoline (0.5g, 2.3 mmol) and bromohexane (2mL, 14.3 mmol) in DMF (3mL) was left to stir 
in a sealed tube for 48h at 140°C. Diluted reaction mixture with ether (lOOmL). Filtered precipitate 
and washed with ether. Dissolved dark orange product in minimal CHC1 3 and purified with column 
chromatography; CHCl 3 :MeOH (98:2) on silica. Collected fractions and acidified (42.3%), m.p: 
129-130.5°C. *H NMR (DMSO-d 6 ) 5 11.53 (s, 1H, NH), 8.97 (d, 1H), 8.40 (d, 1H), 8.29 (d, 1H) 
7.70 (d, 1H), 7.67 (m, 4H), 7.36 (t, 1H), 4.55 (t, 2H), 2.08 (m, 2H), 1.91 (m, 4H), 1.64 (m, 2H),’ 
1.17 (m,3H). 


2-Bromoacetamido-5-fluorobenzoic acid 

2-amino-5-fluorobenzoic acid (2g, 12.8 mmol) in anhydrous DMF (5mL) and anhydrous dioxane 
(5mL) placed in a sealed flask and cooled to 0°C, bromoacetyl bromide (1.25mL, 13 mmol) was 
added slowly over 30mins (ensuring that temperature is maintained below 1°C). Left to stir 
overnight at room temperature. Added H 2 0 (40mL), filtered and washed the resulting white 
precipitate with H 2 0 (3x5mL) (93.2%), m.p: 196.2-196.9°C. ‘H NMR (DMSO-d 6 ) 5 11.39 (s, 1H, 
NH), 8.17 (dd, 1H), 7.40 (dd, 1H), 7.13 (m, 1H), 4.00 (s, 2H). 

5-FIuoro-2-[(N-phenylamino) acetamido]benzoic acid 

2-Bromoacetamido-5-fluorobenzoic acid (5g, 18 mmol) and aniline (4mL, 44 mmol) in dry DMF 
(30mL) was refluxed at 120°C for 30h. After cooling, the reaction mixture was poured on to ice 
water (~200mL) and aq. KOH (5%) was added to adjust the pH to 10-11. Extracted with CH 2 C1 2 
(3xl00mL), then separated and acidified aq. layer with cone. HC1. The resulting white crystals 
were filtered (25.2%), m.p: 182-183°C. 'HNMR (DMSO-d 6 ) 5 11.84 (s, 1H, NH), 8.73 (dd, 1H), 
7.62 (dd, 1H), 7.45 (m, 1H), 7.11 (m, 2H), 6.65 (m, 3H), 3.91 (s, 2H). 

2-FIuoro-l 1-quindolone 

5-Fluoro-2-[(N-phenylamino) acetamidojbenzoic acid (1.3g, 4.5 mmol) in PPA (45g) was heated 
at 130°C for 2h. Poured into crushed ice (~250mL), added saturated KOH to neutralize and 
extracted with EtOAc (2x300mL). Washed EtOAc fractions with water followed by brine. Dried 
and evaporated solvent. Purified via column chromatography using EtOHMeOH (5:1) to produce 
a dark green product (70.4%), m.p: >300°C. ‘H NMR (DMSO-d 6 ) 6 12.65 (s, 1H, NH), 11.70 (s, 
1H, NH), 8.20 (d, 1H), 8.05 (dd, 1H), 7.77 (dd, 1H), 7.60 (t, 1H), 7.53 (m, 2H), 7.21 (t, 1H). 

2-Fluoro-l 1-chloroquindoline 

2-Fluoro-l 1-quindolone ( 0 . 8 g, 3.2 mmol) refluxed in POCI 3 (15mL) for 2 h. Poured into ice water 
and neutralized with saturated KOH solution. Extracted with EtOAc (3xlOOmL), and washed the 
combined EtOAc fractions with water followed by brine, dried and concentrated solvent. 
Purification via column chromatography on silica, with EtOAc:Hexanes (1:6) produced a yellow 
solid (22.2%). 

2-Bromoacetamido-6-chlorobenzoic acid 

2-amino-6-chlorobenzoic acid (5g, 29 mmol) in anhydrous DMF (15mL) and anhydrous dioxane 
(15mL) placed in a sealed flask and cooled to 0°C, bromoacetyl bromide (4.4mL, 48.5 mmol) was 
added slowly over 20mins (ensuring that temperature is maintained below 1°C). Left to stir 


27 



overnight at room temperature. Diluted with cooled H 2 O (lOOmL), filtered and washed the 
resulting white precipitate with H 2 0 (3xl2mL) (97.4%), m.p: 125-127°C. *H NMR (DMSO-de) 6 
10.08 (s, 1H, NH), 7.55 (d, 1H), 7.40 (t, 1H), 7.34 (d, 1H), 4.09 (s, 2H). 

6-Chloro-2-[(N-phenylamino) acetamido]benzoic acid 

2-Bromoacetamido-6-chlorobenzoic acid (lOg, 17 mmol) and aniline (4.3mL, 46 mmol) in dry 
DMF (30mL) was refluxed at 100°C for 5h. After cooling to room temperature, the reaction 
mixture was poured on to ice water (~125mL) and aq. KOH (5%) was added to adjust the pH to 9. 
The resulting milky solution was extracted with CH 2 CI 2 (3x70mL), then separated and acidified 
(pH 3) aq. layer with cone. HC1 and extracted with EtOAc (4x70mL). Combined the EtOAc 
washings, dried and evaporated. Recrystallized from EtOAc and hexanes (30.6%), m.p: 159- 
161°C. l H NMR (DMSO-d 6 ) 6 9.91(s, 1H, C0 2 H), 7.95 (d, 1H), 7.41 (t, 1H), 7.28 (d, 1H), 7.12 (t, 
2H), 6.57 (t, 3H), 3.85 (s, 2H). 

1-Chloro-l l-quindolone 

6-Chloro-2-[(N-phenylamino) acetamidojbenzoic acid (1.53g, 4.9 mmol) was heated with PPA 
(16g) at 120°C for lh. Upon cooling to 60°C, it was stirred for 15min and treated with crushed ice. 
NaHC 03 was added and the solid filtered. This was washed with hot water (200mL), cold water 
(50mL) and then dried in a vacuum oven. Dissolved the crude compound in minimal DMSO and 
diluted with H 2 0. Filtered the resulting dark yellow precipitate and dried overnight in a vacuum 
oven (90.8%), m.p: >305°C. *H NMR (DMSO-d 6 ) 8 12.49 (s, 1H, NH), 11.61 (s, 1H, NH), 8.18 (d, 
1H), 7.67 (d, 1H), 7.45 (m, 3H), 7.17 (m, 2H). 

1-Chloro-l 1-chloroquindoline 

1-Chloro-l l-quindolone (1.2g, 4.5 mmol) was refluxed for 2h in POCI 3 (20mL). Allowed to cool 
to room temperature then poured into ice and neutralized with saturated KOH solution. Extracted 
with EtOAc (3xl00mL), and washed the combined EtOAc fractions with water followed by brine, 
dried and evaporated solvent. Purification via column chromatography on silica, with 
EtOAc:Hexanes (1:6) produced a yellow solid (92.2%), m.p: 265°C (decomp). *H NMR (DMSO- 
d«) 8 11.82 (s, 1H, NH), 8.38 (d, 1H), 8.20 (dd, 1H), 7.66 (m, 4H), 7.39 (dd, 1H). 

l-Chloro-5-methyl-l 1-chloroquindolnium hydrochloride 

1-Chloro-l 1-quindoline (lg, 3.5 mmol) in anhydrous toluene (60mL) was added to methyl triflate 
(1.5mL) and stirred at room temperature for 1 day. Filtered orange/yellow precipitate, then washed 
with ether (89.7%), m.p: 254-256°C (decomp). ! H NMR (DMSO-d 6 ) 8 13.15 (s, 1H, NH), 8.82 
(dd, 1H), 8.30 (s, 1H), 8.13 (d, 1H), 8.10 (d, 1H), 7.92 (d, 1H), 7.85 (d, 1H), 7.49 (d, 1H). 


28 





The next generation of cryptolepine analogues takes pharmacokinetic parameters into greater 
consideration in order to achieve favorable ADME (absorption, distribution, metabolism and 
excretion). Thus, removal, addition, conversion of substituents and other structural modifications 
have been carried out. However, the same basic structure is maintained in order to avoid loss of 
activity. Analogue design will be facilitated if the pharmacophore of the lead compound can be 
identified. The pro-drug rationale is behind a number of those that have been synthesized and the 
use of bioisosteric replacement (thus maintaining electron density) is also employed. All analogues 
previously tested possessed the tetracyclic structure. Whether this is required for activity, is yet to 
be determined. In addition, on the basis of previous data, it appears that a completely delocalised 
structure is favored for activity. The planar aromatic structure is destroyed once the free base is 
formed. It is predicted that there will also be a reduction in activity. This also requires further 
investigation. One other route yet to be explored is the combination of cryptolepine with 
established antimalarials, which are becoming increasingly ineffective due to the emergence of 
resistance. This may prove to be a particularly successful route in the design of a cryptolepine 
analogue with antimalarial activity. With the identification of the pharmacophore that is essential 
for the activity of the cryptolepine analogues against drug-resistant P. falciparum, this class of 
compounds represents a new chemotype in the ongoing fight against malaria. 

D. Future work 

Upon completion of the preliminary antimalarial and pharmacokinetic testing, there will be 
continued optimization and characterization of identified lead compounds. In addition, the mode of 
action of these analogues is yet to be satisfactorily established. If it could be, this would aid the 
development of future analogues of this class of compounds. The spectroscopic characterization 
(UV, IR, MS, NMR, 13 C-NMR) of active constituents of Cryptolepis sanguinolenta will also be 
beneficial. In parallel, another approach yet to be undertaken is the synthesis of C-9 and C-3 alkyl 
derivatives of protoberberine alkaloids with improved pharmacokinetic properties. Finally, pilot 
scale isolation of indole alkaloids from Picralima nitida is planned for in vivo antimalarial 
bioassays. 


2.1.2 Whole Cell Assays (Plasmodium. Jalcipanim} 

A total of 206 plants samples used in traditional medicine for the treatment of different forms of 
malaria were submitted for in vitro testing against Plasmodium falciparum at the Division of 
Experimental Therapeutics, Walter Reed Army Institute of Research. So far, we have received 
results for 110 samples (Tables 6A-C) while the data for the remaining 96 samples are being 
expected. Separation and purification of the extracts of that revealed noteworthy effects in 
antimalarial bioassays and led to the isolation of a total of 22 compounds. The pure compounds 
were isolated mainly from the following five plant species: Glossocalys brevipes, Penianthus 
longifolius and Homalium letestui, Picralima nitida and Khaya anthotheca. A list of these samples 
and their in vitro IC 50 values against two clones of Plasmodium falciparum, one sensitive to 
chloroquine (D6) and one chloroquine-resistant (W2) are shown in Table 6A-C. Extracts are 
designated Inactive (IC50 > 50, 000 ng/ml). Weak to Moderately Active (IC50 = 5,000 - 50,000 
ng/ml) or Highly Active (IC50 < 5,000 ng/ml). Out of the 206 plant samples submitted for 
antimalarial screening (Table 6A-C), 19 plant species revealed noteworthy activity against D-6 or 
W-2 strains Plasmodium falciparum . 


30 



Table 5A: 


Summary of the Antiplasmodial Activity Extracts /Fractions/ Compounds showing (IC 50 < 
3 pg/ml) 


Plant Sample 

Code 

Lab No 

Target 


IC 50 

(ng/ml) 

Control 

Chloroquine 


D 6 

= 

2.656 

Control 

Chloroquine 


W2 

= 

81.777 

Euphorbia poinsonii 

EPA 

SU-2056 

D 6 

= 

2968.422 




W2 

= 

1542.585 

Anogeissus leiocarpus 

ALE 3 

SU-2061 

D 6 

> 

2500 




W2 

= 

2951.42 




W2 


2162.125 

Renealmia porypus 

PR A A 

SU-2081 

D 6 

= 

2899.03 




W2 

= 

1664.396 

Penianthus longifolius 

PLE 

SU-2086 

D 6 

= 

350.066 




W2 

= 

284.377 

Penianthus longifolium Stem bark PL5 

SU-2116 

D 6 

= 

24.4215 




W2 

= 

37.3638 

Penianthus longifolium Stem bark PL 6 

SU-2117 

D 6 

= 

67.4211 




W2 

= 

142.4276 

Penianthus longifolium Stem bark PL7 

SU-2118 

D 6 

= 

26.6167 




W2 

= 

37.7054 

Glossocalyx brevipes Leaves 

GBM1 

SU-2119 

D 6 

= 

702.5863 




W2 

= 

2125.7839 


GBM5 

SU -2122 

D 6 

= 

1462.002 




W2 

= 

2552.9441 

Glossocalyx brevipes 

MTG3 

SU-2124 

D 6 

= 

1326.2953 




W2 

= 

2373.7095 

Glossocalyx brevipes 

MTG4 

SU-2125 

D 6 

= 

1164.9077 



SU-2125 

W2 

= 

2367.5496 

Aspilia africana (Aerialparts) 

CH2CL2 

SU-2175 

D 6 

= 

2145.6838 


CH2CL2 

SU-2175 

W2 

= 

2856.7156 

Combretum dulchipetalum (Roots) MeOH 

SU-2178 

D6 


2000 



SU-2178 

W2 

= 

1908.5365 

Hymenocardia acida (Leaves) 

CH2C12 

SU-2153 

D6 

= 

1949.9795 



SU-2153 

W2 

= 

950.7654 

Jatropha curcas (Leaves) 

CH2C12 

SU-2166 

D6 

= 

2636.8037 



SU-2166 

W2 

= 

1327.6487 

Hyptis suaveolens (Leaves) 

PET ETHER 

SU-2158 

D6 

= 

201.2735 



SU-2158 

W2 

= 

158.0374 


31 


Table 5A contd. 

Code 

Lab No 

Target 


IC 50 IC* 


Plant Sample 







Guarea thompsonii (Stem Bark) 

CH2C12 

SU-2148 

D6 

= 

2545.72 




SU-2148 

W2 

= 

777.8114 


Penianthus longifolius 


SU-2194 

D6 

= 

184.4256 




SU-2194 

W2 

= 

248.106 


Renealmia porypus 


SU-2191 

D6 

= 

2671.4805 




SU-2191 

W2 

= 

1261.1901 


Renealmia porypus 


SU-2192 

D6 

= 

1562.7423 




SU-2192 

W2 

= 

1156.9641 


Melian excelsa (Stem Bark) 

CH2C12 

SU-2151 

D6 

=s 

793.9665 



CH2C12 

SU-2151 

W2 

= 

283.7151 


Hyptis senegalensisfr. 53-57 

Leaves 

SU-2252 

D6 


109.5586 

245.9536 

Hyptis senegalensis fr. 53-57 

Leaves 

SU-2252 

W2 


190.2197 

321.3354 

Hyptis senegalensis fr. 62-64 

Leaves 

SU-2254 

W2 

= 

276.8698 

433.6004 



SU-2254 

D6 

= 

139.7729 

437.1471 

Hyptis senegalensis fr. 65-67 

Leaves 

SU-2255 

D6 

= 

65.0873 

169.262 



SU-2255 

W2 

= 

118.1469 

170.5544 

Hyptis senegalensis fr. 68-69 

Leaves 

SU-2256 

W2 

= 

145.6978 

240.4132 



SU-2256 

D6 

= 

119.3595 

251.7279 

Hyptis senegalensis fr. 70 

Leaves 

SU-2257 

W2 

=s 

427.5711 

670.7832 



SU-2257 

D6 

= 

307.6728 

716.6232 

Dicrocephala integrifolia (C) 

Whole plant 

SU-2292 

W2 

= 

2978.639 

3267.491 



SU-2292 

D6 

= 

2935.179 

3531.798 

Eriosema glomerata (M) 

Stem/Leaves 

SU-2325 

W2 

= 

1282.807 

1656.092 



SU-2325 

D6 

= 

1364.024 

1777.928 

Pittosprum viridiflorum (C) 

Bark 

SU-2336 

W2 

= 

1397.6 

2147.834 



SU-2336 

D6 

= 

1035.728 

3142.469 

Pittosprum viridiflorum (M) 

Bark 

SU-2337 

W2 

= 

2577.767 

2913.585 



SU-2337 

D6 

= 

2094.092 

3124.385 

Baillonella toxisperma (C) 

Bark 

SU-2343 

W2 

= 

2850.672 

3640.399 



SU-2343 

D6 

= 

2430.021 

7570.06 

Triumfetta heudoletti (C) 

H 

SU-2352 

D6 

— 

2824.606 

3634.512 


If 

SU-2352 

W2 

= 

3011.981 

3712.39 

Aframonum pruinosum (C ) 


SU-2362 

D6 

= 

2109.006 

6710.301 



SU-2362 

W2 

= 

2745.24 

929489.8 


The most active plant samples with IC 50 less than 3.0 ug/ml against two strains of Plasmodium 
falciparum D6 and W2 are shown in Table 5A. The table indicates that nineteen plant species 
showed remarkable antiplasmodial activity. The most active compound was isolated from stem 
bark of Penianthus longifolium (MENISPERMACEAE) with IC 50 values (D6= 24.42 and W2 
=37.36) while the most active extract was obtained from the petroleum ether extract of the leaves 
of Hyptis suaveolerts (LAMIACEAE) gave IC 50 values (D6 = 201.27, W2 = 158.04). Bioassay- 
guided fractionation of the H. suaveolens extract also yielded fractions with lower IC 50 when 


32 



compared with the extract. Other extracts with promising antplasmodial activity include Eriosema 
glomeratci, Pittosprum viridiflorum, Renealmia porypus , Melian excelsa and Hymenocardia 
acida. 


Table 5B: List of Plants Displaying Moderate to High Antiplasmodial Activity 


Plant Name 

Ancistrocladus barteri (Ancistrocladaceae) 
Enantia chlorantha (Annonaceae) 
Pachypodanthium staudtii (Annonaceae) 
Xylopia aethiopica (Annonaceae) 

Uapaca palndosa (Euphorbiaceae) 
Amphimas pterocarpoides (Fabaceae) 
Eiythrophleum suaveolens 
Sida acuta (Malvaceae) 

Gnetum africanum (Gnetaceae) 

Xymalos baillon (Monimiaceae) 
Glossocalyx brevipes (Monomiaceae) 
Xymalos baillon (Monimiaceae) 
Zanthoxylum bungei (Rutaceae) 

Eugenia uniflora (Myrsinaceae) 

Alchormea cordifolia (Euphorbiaceae) 
Fagara Lemairei (Rubiaceae) 


Plant Part/Solvent 

Activity 

Activity 


(ug/ml)-D6 

(ug/ml)-W2 

Stem Bark (MeOH) 

457.25 

553.42 

Stem Bark (MeOH) 

133.93 

121.65 

Unkown (MeOH) 

126.75 

138.60 

Seed (Hexane) 

124.88 

7955.10 

Bark (MeOH) 

111.02 

194.93 

Root (exudate) 

1235.87 

>5,000 

SK (pet ether) 

>50.000 

1820.83 

Leaf (Hexane) 

143.828 

13,978.12 

Leaf (MeOH) 

626.06 

7093.16 

Bark (CH2C12) 

1846.90 

3400 

Unknown (CH2C12) 

762.27 

2361.14 

Bark (CH2C12) 

1846.90 

3400 

Unknown (CH2C12-EtOH) 131.31 

180.43 

Leaf (MeOH) 

2352.77 

1220.77 

Leaf (Hexane) 

846.49 

970.16 

Stem Bark (MeOH) 

1697.82 

1786.35 


2.1.3 Molecular Target-Based Assays: Inhibitors of Plasmodial Cyclin-Dependent Protein 
Kinases (CDKs) 

Effective inhibitors of Plasmodium falciparum CDKs can be identified by screening the ICBG 
plant database. This may lead to the discovery of a new class of anti-malarials. The CDKs project 
was based on previous work in the Department of Parasitology, WRAIR where these CDKs were 
characterized as potential antimalarial drug targets. 

Background: 

New drug discovery efforts in malaria today focus on target based drug screens to identify 
inhibitors of key enzymes vital to the survival of the organism. This study was undertaken to 
identify novel inhibitors of the malarial parasite that target a family of key enzymes. 

Control of cellular growth and differentiation is highly regulated in all organisms in response to 
several intra- and extra-cellular signals. Cellular division ensures the propagation of the next 
generation of daughter cells that maintain the genetic integrity of the organism (Heuvel & Harlow, 
1993). Cell growth is dependent on developmental cues as well as metabolic resources to ensure 
the proper timing of cell division. In some cases cellular transformation occurs when mutations 


33 



occur in the cell cycle regulatory mechanisms resulting in uncontrolled proliferation (Hall & 
Peters, 1996; Hunter & Pines, 1994). At the heart of the cell cycle machinery, Cyclin Dependent 
protein Kinases (CDKs) are the key regulators that ensure the cellular division occurs in a well 
ordered fashion (Heuvel et al, 1993). 

CDKs are highly conserved in many eukaryotic organisms from yeast to man. In fact, yeast CDKs 
are able to restore normal cell cycle control in mammalian cells which contain mutations in several 
homologous CDKs (Lee & Nurse, 1987). This demonstrates that the CDKs not only share 
sequence homology but also they are functionally compatible among different species. The 
conservation of CDKs and their regulatory function implies that control of cellular division has 
evolved as a mechanism critical to the viability of the organism. In fact, this mechanism of cell 
division control relies heavily on the activity of the various CDKs (Grana & Reddy, 1995; Heuvel 
etal , 1993). 

CDK activity is regulated by phosphorylation and association with regulatory proteins. Full 
activation of a CDK requires phosphorylation on a conserved threonine residue in a loop called the 
T loop and the association of a cyclin subunit (Morgan, 1995). Monomeric CDK lacks kinase 
activity due to several structural constraints. Without a cyclin subunit, substrates are denied access 
to the active site and key residues involved in the transfer of phosphate from ATP to the substrate 
are misaligned. The current model suggests that upon cyclin binding, the T loop moves away from 
the active site, allowing ATP to bind (Jeffery et al , 1995). Phosphorylation of the threonine residue 
within the T loop locks the CDK into its most active conformation. Additionally, conformation 
changes induced by cyclin binding orient the g-phosphate of ATP and facilitate the 
phosphotransfer reaction. Once active, the CDK phosphorylates proteins required for cell cycle 
progression. To ensure that cellular progression occurs in a sequential fashion, CDKs become 
inactivated by the targeted degradation of the cyclin subunit and the association of inhibitory 
proteins (Willems et al, 1996; Lee et al, 1987). In this type of sequential activation and 
deactivation, the cell cycle progresses so those critical events are completed before the cell 
commits to another round of proliferation. 

In light of the CDKs role in cellular proliferation, they have become attractive drug targets for 
cancer therapy and the development of antifungal compounds (Meijer, 1996; Meijer et al, 1997; 
Saul & Battistutta, 1998). Several drug discovery efforts, which aim at either inhibiting the CDK 
directly or interfering with regulatory mechanisms required for CDK activation, have been 
reported. From these efforts, 6 classes of CDK inhibitors have been identified with one class of 
compounds now in clinical trials. Furthermore, inhibition of the cell cycle is widely considered as 
a new approach toward treatment for diseases caused by unregulated cell proliferation, including 
cancer (Schulze-Gahmen et al, 1996). Taken advantage of these developments and targeting 
Plasmodium CDKs may lead to the identification of a new class of anti-malarial compounds. 

CDKs are highly conserved among eukaryotic species and, not surprisingly, several CDKs have 
been isolated from Plasmodium (Doerig et al, 1995; Ross-MacDonald et al, 1994; Vinkenoog et 
al, 1998). Unfortunately, the biochemical mechanism of regulation and the cellular role of these 
CDKs are not fully understood. A CDK from Plasmodium falciparum, known as Pfmrk, shares 
significant sequence homology with human CDK7 (46% identical, 62% similar) (Li et al, 1996; 
Waters et al, 2000). In mammalian cells, CDK7 and its cyclin H subunit function as the CDK 


34 



activating kinase (CAK), which phosphorylates the conserved threonine residue within the T loop 
of several CDKs (Fesquet et al, 1993). In addition to its role in CDK activation, CDK7 associates 
with the TFIIH transcription factor and regulates transcription and DNA repair (Serizawa et al, 
1995). In this regard, CDK7 integrates cell cycle control with gene expression. It is possible that 
Pfmrk has a similar role in Plasmodium falciparum and this warrants further investigation of its 
regulatory and functional role. Homology with human CDK7 suggests that Pfmrk is in the position 
to regulate the other Plasmodium CDKs involved in cell growth and development. 

Pfmrk functions at the extreme end of the cell signaling pathway, thus making it an attractive drug 
target. Based on homologous systems, inhibiting Pfmrk activity would shut down the cell cycle 
machinery resulting in the death of the parasite. This approach to antimalarial drug discovery is 
fairly new as it represents the first time that an approach has been undertaken to directly inhibit the 
machinery responsible for cell growth within the malaria parasite. CDK inhibitors currently under 
investigation include flavopiridol, olomoucine, roscovitine, puvalanol B, the dihydroindolo[3,2- 
d][l]benzazepinone kenpaullone, indirubin-3 -monoxime and novel diaminothiazoles such as 
AG12275. The anticancer therapeutic potential of CDK inhibitors has been demonstrated in 
preclinical studies, and Phases I and II clinical trials in cancer patients are currently underway 
(Buolamwini, 2000). 

Therefore, screening ICBG plant database for identification and characterization of effective 
compounds that inhibit the Plasmodium falciparum CDKs may lead to the discovery of a new 
class of antimalarials. 

Methodology 

Expression and Purification of Plasmodium falciparum CDKs was carried out by CPT Waters, 
Department of Parasitology, WRAIR (Waters et al , 2000; Buolamwini, 2000) 

Kinase assays by autoradiography: 

Purified Pfmrk (0.01 mg) was assayed in a 15ul kinase reaction containing kinase buffer (50 mM 
Tris-HCl pH 7.5, 10 mM MgC12, and 1 mM DTT), 10 mg of Histone HI (Upstate Biotechnology), 
1 mg Pfcycl, and 5 mCi [gamma-32P] ATP (Amersham). Reaction mixtures were incubated at 30 
C for 15 minutes and stopped by the addition of SDS sample buffer, boiled, and resolved by 12% 
SDS-PAGE. Proteins were transferred to PVDF membrane, Coomassie stained, and exposed to 
film for autoradiography. PfPK5 and PfPK6 assays were performed in the same manner as above 
except that ribonucleotide reductase was used as substrate with PfPK6. 

High throughput kinase assay: 

The high throughput assay uses a 96 well microtiter plates that contains p81 phospho-cellulose 
filter paper in the bottom of each well to capture the phosphorylated substrate. These plates are 
used successfully in industry to assay for kinase inhibitors. This system includes Microtiter plates 
(Whatman Inc ), an automated sampler processor (Biomek Inc), microtiter plate shaking incubator, 
and a Top Count microtiter plate scintillation counter (Packard). The Biomek liquid handler has 
been programmed to pipet reagents required for the kinase reaction into each well of the microtiter 
plate. Total reaction volume per well in the working plate is 50 ml and includes kinase reaction 
buffer (50 mM Tris-HCl pH 7.5, 10 mM MgC12, and 1 mM DTT), 10 mg of substrate, 1 mg 


35 



Pfcycl, and 5 mCi [gamma-32P] ATP (Amersham). Prior to each assay, the Biomek serially 
dilutes drugs in a master drug plate and then transfers the diluted drugs to the working plates 
containing the components above. The last reagent added to the working plate is the [gamma-32P] 
ATP, which initiates the reaction. The working plate is then incubated for 30 minutes at 37 C. 
Following the incubation each well is washed 4 times with 250 ml of 1% phosphoric acid and then 
placed in the Top Count Scintallation counter to be measured for kinase activity. Each reaction is 
performed in triplicate to increase the accuracy of the assay. Several controls are included on each 
plate to include, background in the absence of kinase and kinase in the absence of drug to measure 
maximal activity. Based on these controls, the Top Count automatically computes the percent 
inhibition of each drug and reports it in a spreadsheet format. 

The drug screen was conducted in two separate phases. Phase I consists of a pre-screen that 
measures inhibition of compounds to select those compounds demonstrating inhibition less than 50 
mM. During phase II, selected compounds in phase I were evaluate to determine IC50 of those 
compounds. Such an approach eliminated the time and resources needed to evaluate compounds 
that do not make the minimum inhibition cut off. Compounds that were identified as effective 
inhibitor of a given Plasmodium CDK were evaluated against two other plasmodial CDKs. 

A total of 24 ICBG samples consisting of extracts, purified fraction and compounds were screened 
against Plasmodium CDKs (MRK and PK5). The IC 50 was recorded in ng/ml as shown in Table 6 . 

Results & Discussion: 

We examined the ability of these samples to inhibit Plasmodium falciparum MRK and PK5 
kinases and found that the IC 50 determined were in the low nanogram/ml range for 29% of the 
samples tested. Although both MRK and PK5 were inhibited to varying degrees, MRK is more 
susceptible to inhibition by our samples than PK5. As shown in Table 6, out of the 24 samples 
tested SU2243 (a diterpene isolated from Aframomum datiiellii and SU-2258 (a 
hexahydroxyflavone) were among the first five lead compounds encountered in this assay. Both 
samples inhibited the enzyme MRK at 2.5ng/ml. Similarly the two samples also inhibited PK5 
(IC 50 , 40ng/ul). About 50% of the samples selected for this assay inhibited MRK. Although the 
nature of the inhibition remains unclear at this time, the results of this study represent the 
discovery of two compounds (SU2243 and SU-2258) that could serve as leads for new classes of 
CDK inhibitors. 

Another class of compounds that significantly inhibited MRK and PK5 are the biflavonoids 
isolated from Garcinia kola seeds (Iwu, 1978; Okunji el al ., 2002). Working up the organic extract 
from 10.Og of G. kola , yielded four major compounds in sufficient amounts and purity (>98%). 
The compounds were identified by comparison of their spectral data and Rf values with those of 
the authentic isolates as 3", 3’”, 4’, 5,5”, 7,7”-heptahydroxy-4"'-methoxy-3,8"-biflavanone 
(Kolaflavanone); ",4',4'",5,5",7,7"-heptahydroxy-3,8"-biflavone (GB1); 7"-0-alpha-D- 

glucopyranosyloxy- 3",4',4"',5,5",7-hexahydroxy-3,8"-biflavanone. (GB1 glucoside) and 
3",3"',4',4"’,5,5",7,7"'-octahydroxy-3,8"-biflavanone (GB2). GB1 was the most active biflavanone 
(IC50, 5ng/ul) followed by kolaflavones and GB-2. The structures of these compounds are shown 
in Figure 11. In subsequent studies, we intend to compare the antiplasmodial activity of these 


36 


compounds with their activities in the CDK assay. Such a comparison may reveal a correlation 
between the two assays. 


Table 6 Plant Samples/Compounds Tested against MRK and PK5 Kinase Assay 


ID Number 

Extracts/Compounds 

MRK ICso 

ng/ul 

PK5 

ng/ul 

SU-2243 

Labda-8( 17), 12-diene-15,16-dial 

2.5 

>40 

SU-2244 

Ajmalicine Hydrocholoride 

>20 

>40 

SU-2245 

Alstonine Tetrahydro 

80 

>40 

SU-2246 

Asculin 

80 

>40 

SU-2247 

BN 87724 

>40 

>40 

SU-2248 

BN 81553 

40 

40 

SU-2249 

Cinchonine 

>80 

>40 

SU-2250 

Colchicine 

>80 

>40 

SU-2251 

Curcumine 

5 

>40 

SU-2252 

Eserine Sulfate 

>20 

>40 

SU-2253 

Dracaena manii CMH(2:2:1) 

>10 

>40 

SU-2254 

Garcinia Kola LH-20 ff. 15 

10 

>40 

SU-2255 

Garcinia Kola LH-20 fr. 16 

5 

20 

SU-2256 

Garcinia Kola LH-20 fr. 20 

10 

>40 

SU-2257 

Garcinia Kola LH-20 fr. 23-25 

20 

40 

SU-2258 

Myricetin 

<2.5 

40 

SU-2259 

Lupeol 

>10 

>40 

SU-2260 

Malvin 

>10 

>40 

SU-2261 

Naringin 

>40 

>40 

SU-2262 

Palmatine Chloride 

>40 

>40 

SU-2263 

Morin 

10 

>40 

SU-2264 

Rauwolscine Hydrochloride 

>40 

>40 

SU-2265 

Solamargine 

>40 

>40 

SU-2266 

Ursolic Acid w/ HPLC 

>10 

40 




mm 


Labda-8(17), 12-diene- 15,16-dial 


Myncetin 


CH = CH -c = CH-CO-CH = CH 





Malvin 



COOH 


ursolic acid 



)H O 
Morin 


Curcumin 



Lupeol 



OH O 


Rj R2 R3 R4 R5 

GB1 H OH H H H 

GB1 a- T'-O- glue os de H H Glc H H 

GB2 H OH H OH H 

Kolafl avononc H OH H H CH 3 


Figure 11 Compounds Active against MRK Kinase at > 5ng/ml 




Table 6A 

Antimalarial activity of plant extracts against P. falciparum in vitro 


Plant Sample Code Lab No 

WRAIR No 

Target ng/ml 

<=> 

IC 50 

Control 


Chloroquine 

D6 

1000 

= 

2.656 

Control 


Chloroquine 

W2 

1000 

= 

81.777 

Homalium letestui HL1 

SU-2052 

BP22782 

D6 

5000 

> 

5000 



BP22782 

W2 

5000 

= 

2564.11 

Schefflera Abo 

SU-2053 

BP22791 

D6 

5000 

> 

5000 



BP22791 

W2 

5000 

> 

5000 

Khaya anthotheca TKA 

SU-2054 

BP22808 

D6 

5000 

> 

5000 



BP22808 

W2 

5000 

= 

1468.094 

Aframomum su lea turn ADH 

SU-2055 

BP22817 

D6 

5000 

> 

5000 



BP22817 

W2 

5000 

> 

5000 

Euphorbia poinsonii EPA 

SU-2056 

BP22826 

D6 

5000 

= 

2968.422 



BP22826 

W2 

5000 

= 

1542.585 

Euphorbia kinnii EKA 

SU-2057 

BP22835 

D6 

5000 

> 

5000 



BP22835 

W2 

5000 


2588.85 

Euphorbia eutorrofillaEEA 

SU-2058 

BP22844 

D6 

5000 

> 

5000 



BP22844 

W2 

5000 

= 

2034.394 

Anogeissus leiocarpus ALE1 SU-2059 

BP22853 

D6 

5000 

> 

5000 



BP22853 

W2 

5000 

> 

5000 

Anogeissus leiocarpus ALE2 SU-2060 

BP22862 

D6 

5000 

> 

5000 



BP22862 

W2 

5000 

> 

5000 

Anogeissus leiocarpus ALE3 

SU-2061 

BP22871 

D6 

5000 

> 

2500 



BP22871 

W2 

5000 

= 

2951.42 

Lannea acida LPA 

SU-2062 

BP22880 

D6 

5000 

> 

5000 



BP22880 

W2 

5000 

> 

5000 

Inula klingii IK 

SU-2063 

BP22899 

D6 

5000 

> 

5000 



BP22899 

W2 

5000 

> 

5000 

Terminalia superba TST 

SU-2064 

BP22906 

D6 

5000 

> 

5000 



BP22906 

W2 

5000 

> 

5000 

Terminalia glaucecens TGC1 SU-2065 

BP22915 

D6 

5000 

> 

5000 



BP22915 

W2 

5000 

= 

2854.179 

Terminalia glaucecens TGC2 SU-2066 

BP22924 

D6 

5000 

> 

5000 



BP22924 

W2 

5000 

= 

2620.585 

Terminalia superba TST2 

SU-2067 

BP22933 

D6 

5000 

> 

5000 



BP22933 

W2 

5000 

> 

5000 

Aframomum sceptrum ASS 

SU-2068 

BP22942 

D6 

5000 

> 

5000 



BP22942 

W2 

5000 

> 

5000 

Garcinia kola GSP 

SU-2069 

BP22951 

D6 

5000 

> 

5000 



BP22951 

W2 

5000 

> 

5000 

Ellophobia spp ELS 1 

SU-2070 

BP22960 

D6 

5000 

> 

5000 



BP22960 

W2 

5000 

> 

5000 



Table 6A contd 

Plant sample Code Lab No 

WRAIR No 

Target ng/ml 


IC 50 

Ellophobia spp ELS2 SU-2071 

BP22979 

D6 

5000 

> 

5000 

BP22979 

W2 

5000 

> 

5000 

Anisopus mannii ANIM SU-2072 

BP22988 

D6 

5000 

> 

5000 

BP22988 

W2 

5000 

> 

5000 

Combretum glutinosum CCG SU-2073 

BP22997 

D6 

5000 

> 

5000 


BP22997 

W2 

5000 

> 

5000 

Combretum aculeatum CCA SU-2074 

BP23001 

D6 

5000 

> 

5000 


BP23001 

W2 

5000 

> 

5000 

Pteleopsis hylodendronMPH 1 SU-2075 

BP23010 

D6 

5000 

> 

5000 


BP23010 

W2 

5000 

> 

5000 

Pteleopsis hylodendron MPH2 SU-2076 

BP23029 

D6 

5000 

> 

5000 


BP23029 

W2 

5000 

> 

5000 

Pteleopsis hylodendron MPH3 SU-2077 

BP23038 

D6 

5000 

> 

5000 


BP23038 

W2 

5000 

> 

5000 

Pteleopsis hylodendron MPH4 SU-2078 

BP23047 

D6 

5000 

> 

5000 


BP23047 

W2 

5000 

> 

5000 

Vi tellaria paradoxa PVA SU-2079 

BP23056 

D6 

5000 

> 

5000 


BP23056 

W2 

5000 

> 

5000 

Renealmia porypus PREA SU-2080 

BP23065 

D6 

5000 

> 

5000 


BP23065 

W2 

5000 

= 

2162.125 

Renealmia porypus PRAA SU-2081 

BP23074 

D6 

5000 

= 

2899.03 


BP23074 

W2 

5000 

~ 

1664.396 

Albizia ferruginea AAF SU-2082 

BP23083 

D6 

5000 

> 

5000 


BP23083 

W2 

5000 

> 

5000 

Marantochloa purpurea MPA SU-2083 

BP23092 

D6 

5000 

> 

5000 


BP23092 

W2 

5000 

> 

5000 

Marantochloa purpurea MPAC SU-2084 

BP23109 

D6 

5000 

> 

5000 


BP23109 

W2 

5000 

> 

5000 

Penianthus longifolius PLA SU-2085 

BP23118 

D6 

5000 

> 

5000 


BP23118 

W2 

5000 

> 

5000 

Penianthus longifolius PLE SU-2086 

BP23127 

D6 

5000 

= 

350.066 


BP23127 

W2 

5000 

— 

284.377 

Crotalaria incana CIA SU-2087 

BP23136 

D6 

5000 

> 

5000 

Control 

Mefloquine 

D6 

250 

= 

7.827 


Mefloquine 

W2 

250 

= 

1.385 



Table 6B Antimalarial activity of plant extracts/Fractions/Compounds against/*, falciparum 





Synonym 

Target 

<=> 

IC50 

Penianthus longifolium 

Stem bark 

PL1 SU-2113 

BP79030 

D6 

> 

5000 




BP79030 

W2 

> 

5000 

Penianthus longifolium 

Stem bark 

PL3 SU-2114 

BP79049 

D6 

> 

5000 




BP79049 

W2 

> 

5000 

Penianthus longifolium 

Stem bark 

PL4 SU-2115 

BP79058 

D6 

> 

5000 




BP79058 

W2 

> 

5000 

Penianthus longifolium 

Stem bark 

PL5 SU-2116 

BP79067 

D6 

= 

24.4215 




BP79067 

W2 

= 

37.3638 

Penianthus longifolium 

Stem bark 

PL6 SU-2117 

BP79076 

D6 

= 

67.4211 




BP79076 

W2 

= 


142.4276 







Penianthus longifolium 

Stem bark 

PL7 SU-2118 

BP79085 

D6 

= 

26.6167 




BP79085 

W2 

= 

37.7054 

Glossocalyx brevipes Leaves 

GBM1 SU-2119 

BP79094 

D6 



702.5863 










BP79094 

W2 

= 

2125.7839 

Glossocalyx brevipes " 


GBM2 SU-2120 

BP79101 

D6 

> 

5000 




BP79101 

W2 

> 

5000 

Glossocalyx brevipes " 


GBM3 SU-2121 

BP79110 

D6 

> 

5000 




BP79110 

W2 

> 

5000 

Glossocalyx brevipes " 


GBM5 SU-2122 

BP79129 

D6 

= 


1462.002 










BP79129 

W2 

= 

2552.9441 

Glossocalyx brevipes \ 

Stembark 

MTG2 SU-2123 

BP79138 

D6 

> 

5000 




BP79138 

W2 

> 

5000 

Glossocalyx brevipes " 


MTG3 SU-2124 

BP79147 

D6 

= 

1326.2953 




BP79147 

W2 

= 


2373.7095 







Glossocalyx brevipes " 


MTG4 SU-2125 

BP79156 

D6 

= 


1164.9077 










BP79156 

W2 

= 


2367.5496 







Uapaca paludosa Stembark 

Betulinic SU-2126 

BP79165 

D6 

> 

5000 



acid 

BP79165 

W2 

> 

5000 

Khaya anthotheca 


TKA1 SU-2127 

BP79174 

D6 

> 

5000 




BP79174 

W2 

> 

5000 

Khaya anthotheca 


TKA2 SU-2128 

BP79183 

D6 

> 

5000 




BP79183 

W2 

> 

5000 

Khaya anthotheca 


TKA4 SU-2129 

BP79192 

D6 

> 

5000 




BP79192 

W2 

> 

5000 

Khaya anthotheca 


TKA6 SU-2130 

BP79209 

D6 

> 

5000 




BP79209 

W2 

> 

5000 


41 


Control 

MEFLOQUINE 

D6 

11.6708 

Control 

MEFLOQUINE 

W2 = 

4.7754 

Control 

CHLOROQUINE 

D6 

4.7697 

Control 

145.3013 

CHLOROQUINE 

W2 = 



Table 6C Antimalarial activity of plant extracts against P. falciparum in vitro 

PLANT NAME 

Plant 

Lab No. 

Plasmodium 

IC 50 


Extract 

SU-NO 

Target <=> 

(ng) 

Asystasia gangetica 

H20 

SU-2141 

D6 

> 

12500 

Asystasia gangetica 

H20 

SU-2141 

W2 

> 

12500 

Asystasia gangetica 

CH2CL2 

SU-2155 

D6 


8406.8447 

Asystasia gangetica 

CH2CL2 

SU-2155 

W2 

= 

4802.1255 

Pupalia lappacea 

H20 

SU-2143 

D6 

> 

12500 

Pupalia lappacea 

H20 

SU-2143 

W2 

= 

7500 

Lannea acida 


SU-2186 

D6 


12502.6768 

Lannea acida 


SU-2186 

W2 

= 

8313.3281 

Uvaria chamae 

CH2C12 

SU-2146 

D6 

= 

14644.3213 

Uvaria chamae 

CH2C12 

SU-2146 

W2 

= 

7454.4888 

Uvaria chamae 

MeOH 

SU-2147 

D6 

= 

8608.335 

Uvaria chamae 

MeOH 

SU-2147 

W2 

= 

4745.4888 

Holarrhena floribunda (Leaves) 

MeOH 

SU-2164 

D6 

= 

10775.6084 

Holarrhena floribunda (Leaves) 

MeOH 

SU-2164 

W2 

— 

5175.6792 

Holarrhena floribunda (Leaves) 

CH2C12 

SU-2165 

D6 

= 

8504.6807 

Holarrhena floribunda (Leaves) 

CH2C12 

SU-2165 

W2 

= 

3975.2646 

Picralima nitida (Seed) 

CHC13 

SU-2160 

D6 

> 

12500 

Picralima nitida (Seed) 

CHC13 

SU-2160 

W2 

— 

2163.3948 

Picralima nitida (Seed) 

HEXANE 

SU-2161 

D6 

> 

25000 

Picralima nitida (Seed) 

HEXANE 

SU-2161 

W2 

= 

7101.5361 

Culcasia scanders (whole plant) 

CH2CL2 

SU-2157 

D6 

= 

3816.0396 

Culcasia scanders (whole plant) 

CH2CL2 

SU-2157 

W2 

— 

1777.4214 

Anisopus mannii 


SU-2189 

D6 

> 

25000 

Anisopus mannii 


SU-2189 

W2 

> 

25000 

Aspilia africana (Aerial parts) 

CH2CL2 

SU-2175 

D6 

— 

2145.6838 

Aspilia africana (Aerial parts) 

CH2CL2 

SU-2175 

W2 

= 

2856.7156 

Rilchiea capparoides (Roots) 

MeOH 

SU-2168 

D6 

> 

25000 

Ritchiea capparoides (Roots) 

MeOH 

SU-2168 

W2 

> 

25000 

Ritchiea capparoides (Roots) 

CH2C12 

SU-2169 

D6 

> 

25000 

Ritchiea capparoides (Roots) 

CH2C12 

SU-2169 

W2 

> 

25000 

Combretum dulchipetalum (Roots) 

CH2CL2 

SU-2176 

D6 


15000 

Combreturn dulchipetalum (Roots) 

CH2CL2 

SU-2176 

W2 

= 

6021.9927 

Combretum dulchipetalum (Roots) 

H20 

SU-2177 

D6 

> 

12500 


42 



Combretum dulchipetalum (Roots) 

H20 

SU-2177 

W2 = 

6292.667 

Combretum dulchipetalum (Roots) 

MeOH 

SU-2178 

D6 

2000 

Combretum dulchipetalum (Roots) 

MeOH 

SU-2178 

W2 = 

1908.5365 

Combretum didchipetalum (Leaves) 

H20 

SU-2179 

D6 

9000 

Combretum dulchipetalum (Leaves) 

H20 

SU-2179 

W2 = 

6084.103 

Combretum didchipetalum (Leaves) 

CH2CL2 

SU-2180 

D6 

5750.7998 


Table 6C contd. 


PLANT NAME 

Plant 

Lab No. 

Plasmodium 

IC 50 


Extract 

SU-NO 

Target <=> 

(ng) 

Combretum dulchipetalum (Leaves) 

CH2CL2 

SU-2180 

W2 

= 

2345.4836 

Anogeissus leiocarpus 


SU-2185 

D6 

= 

9733.2979 

Anogeissus leiocarpus 


SU-2185 

W2 

= 

7081.4946 

Terminalia glaucecens 


SU-2187 

D6 

= 

3049.1604 

Terminalia glaucecens 


SU-2187 

W2 

= 

2159.6104 

Terminalia glaucecens 


SU-2188 

D6 

= 

3371.874 

Terminalia glaucecens 


SU-2188 

W2 

= 

2986.4358 

Combretum glutinosum 


SU-2190 

D6 

= 

5221.4712 

Combretum glutinosum 


SU-2190 

W2 

= 

4761.0923 

Hymenocardia acida (Leaves) 

CH2C12 

SU-2153 

D6 


1949.9795 

Hymenocardia acida (Leaves) 

CH2C12 

SU-2153 

W2 

= 

950.7654 

Jatropha curcas (Leaves) 

CH2C12 

SU-2166 

D6 

= 

2636.8037 

Jatropha curcas (Leaves) 

CH2C12 

SU-2166 

W2 

= 

1327.6487 

Jatropha curcas (Stem) 

CH2C12 

SU-2167 

D6 

— 

6687.3477 

Jatropha curcas (Stem) 

CH2C12 

SU-2167 

W2 

= 

3105.2563 

Hymenocardia acida (Leaves) 

MeOH 

SU-2170 

D6 

> 

12500 

Hymenocardia acida (Leaves) 

MeOH 

SU-2170 

W2 

= 

6405.7739 

Hymenocardia acida (Leaves) 

CH2C12 

SU-2171 

D6 

= 

3422.4402 

Hymenocardia acida (Leaves) 

CH2C12 

SU-2171 

W2 

= 

1326.8647 

Phyllanthus amarus (Aerial parts) 

CH2CL2 

SU-2181 

D6 

= 

4237.1372 

Phyllanthus amarus (Aerial parts) 

CH2CL2 

SU-2181 

W2 

= 

1916.8948 

Euphorbia kinnii 


SU-2183 

D6 

= 

4591.0288 

Euphorbia kinnii 


SU-2183 

W2 

= 

2168.1262 

Euphorbia eutorrofilla 


SU-2184 

D6 

= 

10225.6299 

Euphorbia eutorrofilla 


SU-2184 

W2 

= 

5375.6167 

Cassia siamea 

CH2C12 

SU-2145 

D6 

= 

7671.6602 

Cassia siamea 

CH2C12 

SU-2145 

W2 

= 

3608.8835 

Homaltium letestui 


SU-2182 

D6 

= 

6517.5439 

Homaltium letestui 


SU-2182 

W2 

= 

3507.427 

Solenostemon monostachyus (Leaves)CH2C12 

SU-2152 

D6 

= 

5584.895 

Solenostemon monostachyus (Leaves)CH2C12 

SU-2152 

W2 

= 

2381.5771 

Hyptis suaveolens (Leaves) 

PET ETHER 

SU-2158 

D6 

= 

201.2735 

Hyptis suaveolens (Leaves) 

PET ETHER 

SU-2158 

W2 

= 

158.0374 

Hyptis suaveolens (Leaves) 

CHC13 

SU-2159 

D6 

= 

4999.1265 

Hyptis suaveolens (Leaves) 

CHC13 

SU-2159 

W2 

= 

2727.4133 


43 



Solenostemon monostachyus (Leaves) CH2C12 

SU-2172 

D6 


8000 

Solenostemon monostachyus (Leaves)CH2C12 

SU-2172 

W2 

= 

4072.9399 

Solenostemon monostachyus (W/P) 

H20 

SU-2173 

D6 

> 

12500 

Solenostemon monostachyus (W/P) 

H20 

SU-2173 

W2 

> 

12500 

Cassythafiliformis (Whole plant) 

MeOH 

SU-2162 

D6 

= 

12723.3242 

Cassytha filiformis (Whole plant) 

MeOH 

SU-2162 

W2 

= 

9248.9561 

Cassytha filiformis (Whole plant) 

CH2C12 

SU-2163 

D6 

= 

6658.1289 

Cassytha filiformis (Whole plant) 

CH2C12 

SU-2163 

W2 

= 

4074.1946 

Table 6C contd. 

PLANT NAME 

Plant 

Lab No. 

Plasmodium 

IC 50 


Extract 

SU-NO 

Target <=> 

(ng) 

Guarea thompsonii (Stem Bark) 

CH2C12 

SU-2148 

D6 

= 

2545.72 

Guarea thompsonii (Stem Bark) 

CH2C12 

SU-2148 

W2 

= 

777.8114 

Guarea thompsonii (Stem Bark) 

MeOH 

SU-2149 

D6 

= 

11644.6729 

Guarea thompsonii (Stem Bark) 

MeOH 

SU-2149 

W2 

— 

8278.5352 

Guarea thompsonii (Stem Bark) 

H20 

SU-2150 

D6 

> 

12500 

Guarea thompsonii (Stem Bark) 

H20 

SU-2150 

W2 

> 

12500 

Penianthus longifolius 


SU-2193 

D6 

= 

9503.3965 

Penianthus longifolius 


SU-2193 

W2 

= 

12001.1133 

Penianthus longifolius 


SU-2194 

D6 

= 

184.4256 

Penianthus longifolius 


SU-2194 

W2 

= 

248.106 

Prunus africana 

CH2C12 

SU-2174 

D6 

> 

25000 

Prunus africana 

CH2C12 

SU-2174 

W2 

= 

10396.1895 

Waltheria indica 

MeOH 

SU-2142 

D6 

=: 

3445.9602 

Waltheria indica 

MeOH 

SU-2142 

W2 

- 

3582.8086 

Grewia cissoides 

H20 

SU-2144 

D6 

= 

3607.4185 

Grewia cissoides 

H20 

SU-2144 

W2 

= 

2663.1753 

Premna quadrifolia 

CH2CL2 

SU-2140 

D6 

= 

9909.5068 

Premna quadrifolia 

CH2CL2 

SU-2140 

W2 

= 

5528.3281 

Renealmia porypus 


SU-2191 

D6 

= 

2671.4805 

Renealmia porypus 


SU-2191 

W2 

= 

1261.1901 

Renealmia porypus 


SU-2192 

D6 

= 

1562.7423 

Renealmia porypus 


SU-2192 

W2 

= 

1156.9641 

Melian excelsa (Stem Bark) 

CH2C12 

SU-2151 

D6 

= 

793.9665 

Melian excelsa (Stem Bark) 

CH2C12 

SU-2151 

W2 

= 

283.7151 

Aspienium bulbiferum (whole plant)CH2CL2 

SU-2154 

D6 

= 

13132.3945 

Aspienium bulbiferum (whole plant)CH2CL2 

SU-2154 

W2 

= 

8553.8164 

Goyania longpelara (Leaves/Stems)CH2CL2 

SU-2156 

D6 

= 

14360.4893 

Goyania longpelara (Leaves/Stems)CH2CL2 

SU-2156 

W2 

= 

7266.1099 


2.4 Training: 

Brian Harris, a high school student, interested in medicinal plants, worked at WRAIR utilizing 
ICBG/BDCP plant* samples.. The student learned techniques in extraction, chromatographic 
fractionation and isolation of bioactive compounds from African plants. The Science & 


44 


Engineering Apprentice Program (SEAP) summer program supported the student for a period of 8 
weeks. The SEAP summer program is operated in conjunction with Department of Defense (DOD) 
laboratories and the George Washington University. Its purpose is to give academically talented 
students, ranging from Middle School through College, a hands-on experience in a scientific 
laboratory under the guidance of a mentor. The program lasts 8 weeks (8 hours per day - 5 days 
per week). Students are required to work the entire 8-week period and to produce a poster and a 
paper to document their work. The program offers students a unique and positive experience in 
their fields of interest, thus encouraging them to pursue careers in science and engineering. BDCP 
provided all the necessary plant materials as well as mentoring in the area of natural products. In 
addition. Dr. Foluke Fakorede, a post-doc, joined us to conduct chemical optimization of 
protoberberine alkaloids. The work is aimed at improving the pharmacokinetic properties of 
selected antimalarial compounds as well as conducting preparative scale isolation or synthesis of 
already identified compounds for in vivo animal studies. We also hired a technician, Ogo 
Nwabukwu, to provide technical laboratory assistance on the isolation of preparative quantities of 
active compounds from medicinal plants used in our antimalarial studies and also to assist in the 
processing of natural product samples for shipment to extramural laboratories for additional 
bioassays. 

3. PLANT COLLECTION, ETHNOBOTANICAL STUDIES, AND ECONOMIC VALUATION 
During this project-reporting period (September, 2001-June, 2002), we expanded the 
ethnobotanical studies and socio-economic evaluation of plant species to new areas. With the 
support of McArthur Foundation, we are in the process of conducting socio-economic valuation of 
two plant species in two communities in the Niger Delta. 

3.1 Ethnobotanical studies: 

The analysis of our ethnobotanical data in Cameroon has been completed. The Nigerian 
ethnobotanical studies, which cover a more heterogeneous population than Cameroon, have been 
continued with an increased emphasis on the Niger Delta region. 

3.2 Plant Collection and Processing: 

The Herbarium unit at InterCEDD collected 35 various plant parts (see Table 7) based on their use 
in ethnomedicine and dried them for extraction. Each plant was identified and a voucher specimen 
was prepared and deposited in the herbarium. Twenty other plants are currently being processed. 

Table 7: The plant parts collected by InterCEDD 



Plant 

Parts 

Family 

1 . 

Hymenodictyon pachyaniha 

stem 

Rubiaceae 

2. 

Crescentia macrocarpus 

leaves 

Bignomcaceae 

3. 

Petersianthus macrocarpus 


Lecythidaceae 

4. 

Afromomum melegueta 

fruits 

Zingiberaceae 

5. 

Ocimum gratissium 

leaves 

Lamiaceae 

6. 

Vernonia colorata 

leaves 

Asteraceae 

7. 

Vernonia colorata 

stem 

Asteraceae 

8. 

Protea madeiensis 

leaves 

Proteaceae 

9. 

Protea madeiensis 

stem 

Proteaceae 

10. 

Vitex simplicefolia 

leaves 

Verbenaceae 


45 



11. 

Funtumia elasticci 

leaves 

Apocynaceae 

12. 

Jatropha gossypiifolia 

leaves 

Euphorbiaceae 

13. 

Jatropha gossypiifolia 

stem 

Euphorbiaceae 

14. 

Pterygota macrocarpa 

leaves 

Sterculiaceae 

15 

Ocimum virdis 

leaves 

Lamiaceae 

16. 

Ficus umbrella 

leaves 

Moraceae 

17. 

Ficus umbrella 

stem 

Moraceae 

18 

A canthospermum hispidium 

aerial parts 

Asteraceaae 

19. 

Haemanthus multiflorus 

aerial parts 

Liliaceae 

20 

Stemonocloleus micranthus 

stem bark 

Caesalpmiaiaceae 

21. 

Uvaria anglolensis 

leaves 

Annonaceae 

22. 

Uvaria anglolensis 

stem 

Annonaceae 

23. 

Schumanniophyton problematicum 

leaves 


24. 

Brenania brieyi 

leaves 


25. 

Cleistopholis patens 

leaves 


26. 

Borreria venticilata 

aerial parts 


27. 

Hannao klaineana 

stem bark 


28. 

Hannao klaineana 

leaves 


29. 

Berlinia grandifolia 

leaves 


30. 

Pennisetum purpureum 

whole plant 


31. 

Ehretia cymosa 

leaves 


32. 

Ehretia cymosa 

stem 


33. 

Corchorus oli torus 

leaves 


34. 

Corchorus ollitorus 

stem 


35. 

Momordica charantha 

aerial parts 



3.3 Socio-economic Value Assessment 
A) Summary 

Although a number of laws exist that seek to protect the environment or promote sustainable 
development in Nigeria, national efforts to implement these have been poorly coordinated between 
bodies executing the legislation. The result is that there is a significant gap between policy 
formulation and implementation leading to duplications and fragmented approach to 
environmental economics. 

In recognition of this constraint, our approach is based on adaptive management, which provides a 
framework to systematically extrapolate from lessons in project implementation to the 
development or execution of new projects (Fig 12). Such a framework directly addresses the lack 
of synergy in environmental management in the country as well as facilitates the full engagement 
of all stakeholders in project implementation. 


46 




Fig 12: EVA framework 


As the ICBG program ends its fourth year, the 
economic valuation program is establishing vertical 
and horizontal linkages, following a triple-helix 
model involving the scientific community, policy 
makers and the private sector (Fig 13). A fundamental 
aspect of this model is the recognition of the role of 
people and their local communities as the nexus of 
biodiversity use and management. This incremental 
approach expands the participatory base of 
biodiversity policy, sustainable use and management. 



Fig 13: EVA Triple-helix model 


B. Policy feedback: 

i. Natural Resource Valuation Project: 

Implementation of the economic value assessment program in Nigeria has established that 
imbalances in the law and practice of environmental valuation are central to the crisis the 
communities and ecosystems of the Niger Delta now face, as well as in the greater Nigerian 
environment. The indigenous value structure reflected by a reliance on NTFPs is not adequately 
captured by existing valuation and compensation laws in Nigeria. Recognition of the value of 
NTFPs could lead to their increased use as a tool in social and economic development, as well as 
to the development of a more complete understanding of the Nigerian environment. Additionally, 
more effective valuation practices could reduce conflict and civil strife due to inadequate 
compensation for damage wrought to the sources of food, water, and livelihoods of communities 
throughout the Niger Delta, as well as elsewhere in Nigeria. 

Oil and gas extraction, development activities, and environmental accidents continue unabated in 
the Niger Delta; although the responsible decision-makers currently take into account only a small 
fraction of the full economic value of affected species and ecosystems. To respond to this growing 


47 





































































































































































































threat to both natural and human communities, the Bioresources Development and Conservation 
Programme (BDCP) and the Environmental Law Institute (ELI) seek to develop and advance 
approaches to ensure that natural resource decision-making incorporates the full economic value of 
natural ecosystems and their services. This work will contribute directly to the development of an 
integrated management plan for biodiversity conservation in the Niger Delta. 


Specific Objectives 

a. ) The first objective is to establish the full economic value of one or two sample species in the 
Niger Delta that lie outside of official trade statistics and the gross national product (GNP), but at 
the same time play significant roles in the livelihood activities of local communities and their 
small producers. To accomplish this objective, partners will first establish, on the basis of existing 
models, an appropriate valuation methodology. They will then apply the methodology to selected 
species through the implementation of in-depth, on-the-ground case studies. 

b. ) The second objective is to develop and present recommendations for improving the legal 
framework governing natural resource valuation and natural resource damage assessment in 
Nigeria. These recommendations will be based upon a broad review of valuation literature, 
analysis of relevant legal frameworks in other countries, and interviews with legal specialists in the 
Niger Delta. 

Activities 


To satisfy the project goal and objectives, the BDCP and ELI will work in close partnership to 
carry out two sets of integrated activities: 

Valuing Sample Species 

To meet the objective of establishing the full economic value of natural resources, two sample 
species in the Niger Delta will be evaluated. This work will be based upon the BDCP’s own 
extensive experience and research in developing valuation methodologies, and also upon 
consultation with other professionals conducting valuation activities in West Africa and elsewhere. 

Methodology 

To collect information on wild resource use, researchers have generally combined a variety of 
conventional data gathering techniques, including questionnaire-based surveys, market surveys, 
time and motion studies, notebooks recorded by community members, etc. The most reliable of 
these methods require a large amount of time and resources, but yield fairly precise data sets for 
extensive statistical analysis. However in many instances less detailed information is required 
and/or less time is available; for example, in many project assessments or planning situations. In 
such cases, there is potential for making greater use of participatory assessment techniques, such as 
participatory rural appraisal (PRA). Emphasizing the importance of local people's understanding of 
their environment, participatory research methods can be very appropriate for addressing the often 
hidden' nature oOyild resource use. The project seeks to develop a methodology, which uses a 
range of research approaches, including PRA and conventional social and biological science 


48 



techniques. The origins of PRA lie partly in the techniques devised to undertake farming systems 
research and this illustrates the conceptual link between the values that have been sought by PRA 
and the values, which are sought by conventional research techniques. 

Household Survey/ Participatory Research Techniques 

In terms of methodology, this study concentrated on the use of participatory research techniques. 
The aim of this section is to provide an introduction to the concepts of participatory research and 
highlight their relevance for valuing NTFPs (Table 8). 


1. The Value of Indigenous Knowledge. 

Participatory research has both a philosophical and practical interest in indigenous knowledge. Many 
people who use participatory research tools do so because they believe that research should be at least 
partly a participatory process, which involves, rather than exploits, the population being studied. In 
practice, this means that the views and opinions of local informants are paramount and should be 
understood. Researchers need to listen carefully to what informants have to say, and make an effort to 
understand the issues that confront them. (Schoonmaker-Freudenberger and Gueye, 1990) 

2. Offsetting Bias. 

Because PRA does not usually involve interviewing large samples of people, it is not possible to rely on 
numbers alone to guarantee a diversity of views. Special efforts have to be made to ensure that a range 
of opinions is represented and that all aspects of an issue are understood. In some situations, it is 
impossible to eliminate bias. Here participatory research acknowledges this and attempts to manage the 
effects of bias on the study. (Schoonmaker-Freudenberger and Gueye, 1990). This is achieved through 
explicit awareness of the context in which information is gathered (Pretty, 1993). Participatory 
researchers should be vigilant for sources of bias. Where bias exists, participatory research 
acknowledges this and seeks to understand the limits, which it imposes on research findings. 

3. Triangulation. 

Examining an issue from only one angle incorporates serious bias into the analysis. Therefore, 
participatory research looks at the same issue from different angles (Mettrick, 1993): 

i) PRA uses a variety of information gathering techniques: key interviews, group discussions, secondary 
sources such as aerial photographs, transects, direct observation in addition to the participatoiy research 
tools. 

ii) PRA uses a variety of units of observation: individuals, households, farms, and communities. 

iii) PRA varies the composition of research teams so that their different perspectives can be brought to 
bear. 


Table 8: Participatory Research Concepts 

The use of participatory research techniques to assess the value of natural resources in the Niger 
Delta, and as a basis for reform of valuation standards and practice in Nigeria has both conceptual 
and practical advantages. Conceptually, the hidden nature of wild resources warrants an approach 
that emphasizes local-level knowledge and experience. Furthermore, certain participatory research 
tools, such as wealth ranking and seasonal calendars, are ideally suited to investigating the 
complex economic issues of value to whom?' and when do they occur?’ From a practical 
perspective, household and community-level values are a useful contribution to the community 
awareness aspect of the project. 


49 




Returns to Labor 

Using information from a variety of sources, the returns to labor for harvesting and selling two 
NTFPs will be calculated. The returns to labor are simply the revenues less any relevant fixed or 
variable costs per unit of time devoted to the activity. These estimates provide a rough indication 
of the relative value of these resources since the return to labor captures both the value of the 
resource and the opportunity cost of labor. Where the opportunity cost of labor, and hence the 
actual economic value, are too difficult to assess, returns to labor are useful for understanding the 
economic importance of an activity only in the context of other available opportunities for people's 
time. A higher return to labor represents a greater incentive to engage in that activity, although 
opportunities may be limited due to social, cultural or other economic factors. 

For all NTFPs, the groups of villagers harvesting them will be identified through a semi-structured 
interview with the village head and cross-checked with household heads and informants from the 
groups in question. The prevailing agricultural wage within villages is an interesting benchmark 
against which to compare the returns to labor. The wage paid for agricultural work will vary 
depending on the amount of effort required for the task and the abilities of the laborer. 

Market Values 

Information from a variety of sources, including previous studies, will be combined to assess the 
gross market value of non-timber forest products harvested. 

Where possible, estimates will be obtained of the number of people involved in each activity. The 
market value is the total amount of resource harvested, whether marketed or not, valued using 
market prices from markets in the local communities to be sampled. The market value provides a 
rough indication of the importance of each resource to the local economy, in terms of its overall 
scale and in comparison to other activities. This calculation would be the first step in calculating 
the economic value. The estimated financial value of agricultural production will be provided for 
comparative purposes. 

Estimates of the aggregate amount of harvesting for the various wild resources will be calculated 
using information about the number of people engaging in each activity. This will be estimated 
through a pile-of-stones discussion with a group of men and then crosschecked with village and 
ward heads, as well as individual informants involved in other interviews/discussions. 

Changes in Resource Availability 

A description of the values generated by resources at one point in time says little about their 
sustainability. It is helpful therefore, to ascertain whether any of the resources, particularly those 
with high values, appear to be declining in abundance, due to harvesting or other changes in their 
availability. In the survey, attempts will be made to determine the extent to which ecosystem 
changes affect the supply of economically important resources. 

In this study, changes in the availability and harvesting of resources over time will be investigated 
primarily by means of historical matrices and the results will be more qualitative, involving 
relative changes. In the absence of archival records or other secondary sources, this is the only 
available means to assess initially the changing state of resources. Any indications of scarcity 


50 



could provide the basis for considering a more in-depth study, including the use of biological 
sampling techniques. 

Sampling Plan 

Based on a two-stage methodology, the team will consider a list of 10 species or products that 
include both NTFPs and a few agricultural products. 

[i] Identification of species of local importance according to: 

• Economic criteria 

• Social/cultural criteria 

• Spiritual value criteria 

[ii] Then, the team will score each species based on the selection criteria, starting out with “cross¬ 
cutting” criteria: 

• Importance of species / products to local communities and small producers; 

• Potential to contribute to conservation and sustainable development (livelihoods) by 
mitigating threats to species; and 

• Potential for generating alternative income, providing communities with improved 
livelihoods. 

[iii] The next steps will include the following: 

Further and more intensive marketing research on the two highest ranked species, resulting in 
“product packets” with comprehensive data on: 

• Prices, 

• Markets and companies, 

• Research on local trade networks 

• Socio-economic, ecological, and cultural features of product use and trade 

Combination of [iii] with the above “product packets” will form a comprehensive informational 
and technical basis from which to make informed decisions. Comparative (and competitive 
analysis) will be conducted for the selected species analysis) will be conducted for the selected 
species / products and allow decisions based on relative benefits and costs. 

The number of communities to be sampled will be limited to two, due to constraints in time and 
resources. The number of households to be sampled in the two communities will be one hundred. 
A semi-structured questionnaire will be used to conduct interviews with village heads. Other 
interviews will be conducted in groups, using PRA techniques. 

ii. National Biodiversity Strategy and Action Plan (NBSAP) Project 

Background: 

Biodiversity is often misunderstood and in the minds of many, it is inexhaustible. Recent 
developments further worsen the case, as sustainable use is not practiced. The natural ecosystems 


51 



in Nigeria used to host a high degree of biodiversity which is poorly researched and largely 
undocumented. The exact number of wild plants and animal species occurring in the country 
including endemic species are not known. Yet the biotic species are constantly being used by rural 
communities to sustain their livelihoods. This use includes for food, medicine, culture and other 
auxiliary uses that are now being better recognized. Unfortunately, at the extreme, fiscal policies 
aimed at national development are instruments used to further deplete the nation’s biodiversity 
through urbanization, road construction, oil extraction and other exploitation processes. Traditional 
farming systems and uncontrolled harvesting of biological resources have led to the loss of the 
biodiversity in the country. In the circumstances, it is becoming extremely difficult to adequately 
design programs for biodiversity conservation in the absence of realistic data as many taxa of flora 
are still not documented and some have been lost due to extreme misuse. 

It was on the basis of the foregoing that Nigeria signed the convention on biological diversity 
(CBD) in 1992 during the Earth Summit, to among others design an international pathway for the 
conservation and utilization of biodiversity. A draft National Action Plan for biodiversity 
conservation was prepared earlier, but this seemingly desk report was found to be deficient in the 
inclusion of data generated through participatory rural appraisal, a process which brings people 
into the development process. 

In order to implement Article 6 and 8 of the CBD, the Federal Ministry of Environment has 
engaged the services of six consultants to fill the gaps identified in the first version of the 
biodiversity strategy and action plan through participatory approach (PRA) at the grassroots, with 
BDCP as the lead civil society institution. The country was divided into four zones vis: North- 
East, North-West, South-East and South-West for data collection. The series of data generated was 
reviewed at workshops held at the zonal level. The zonal workshops were designed for 
stakeholders in each zone to participate in reviewing the reports of consultants in order to get a 
truly representative document for each zone. In turn, the zonal reports will form a working 
document for the first National Workshop, which is designed to collate the views of all 
stakeholders under a cross sectional approach. 


WORKSHOP OBJECTIVES 

The overall objective of the workshops was to involve stakeholders at the grassroots under a 
participatory approach to review as well as collect current data on the role and status of 
biodiversity in the country. The specific objectives were to: 

i. Document the perceptions, aspirations, and the problems of biodiversity utilization 
and conservation at the community level; 

ii. Examine the effects of traditional practices and how these can be improved upon to 
conserve biodiversity. 

iii. Assess the role and uses of biodiversity, identify trends in land use, and determine 
indigenous conservation strategies and human condition index in the use of wild 
plants and wild animals in rural communities. 

iv. Create a general awareness about the existing opportunities and gains that would 
accrue to communities under a collective support system for biodiversity 
conservation. . 


52 



v. Use the data collected from the workshops to develop programs for efficient and 
effective conservation of biodiversity in the country. 

WORKSHOP METHODOLOGY 

I. The presentations at the zonal and national workshops were through in-house delivery of 
reports by Consultants. 

These were followed by group discussions on issues raised. Participants asked questions 
while consultants provided answers. Audiovisual materials were used to facilitate 
participants’ understanding of the main issues raised during the workshop. 

II. Participation at the workshop involved the following stakeholders: 

i. Representatives of communities from the three senatorial districts of participating 
States in each zone. 

ii. Directors of Forestry, Agriculture, Public Health and Environment in each State or 
their representatives. 

iii. Representative of a Research Institute or institutions of higher learning in the State 
and the academia in general. 

iv. Representative of the private sector. 

v. NGOs, CBOs and members. 

vi. The Technical Committee of the project. 

vii. The workshop consultants (the lead consultants, the national consultants). 

III Participants at the workshops were constituted into three working groups to brainstorm and 
discuss some key issues related to the workshop. The topics discusses were earlier 
identified by the National Consultant. These include: 

a) Sustainable utilization and incentive structure for biodiversity conservation. 

b) Traditional conservation practices 

c) Conservation issues in the zone 

IV The workshop program in each zone and at the national level included opening ceremony 
and presentation of reports. Each presentation was followed by plenary discussions that 
examined and addressed critical issues in the report and vital biodiversity conservation 
issues. 

After the discussions by the three working groups, they were collapsed to form two other 
groups on the workshop recommendations and resolutions respectively, at the Zonal levels. 

This process, though hectic was valuable in reviewing and collecting more data to 
actually fill the gaps in the available document in the conservation status and needs of 
Nigeria’s biodiversity. 

iii. Nigeria Biotrade Program 

The Biotrade program is an integrated approach towards trade biodiversity conservation and 

sustainable development, initiated by the United Nations Conference on Trade and Development 

(UNCTAD) in November 1996 to provide technical and financial support to enable developing 

countries to maximize the potential economic value of their biological resources. 


53 


The BIOTRADE Initiative seeks to enhance the capability of 
developing countries to produce value-added products and 
services from biodiversity for both domestic and international 
markets (Fig 14). It is an integrated program consisting of three 
complementary components: the BIOTRADE country 
program; market research and policy analysis; and Internet 
services: 

Like many developing countries, Nigeria is endowed with rich 
and highly diverse biological resources. These resources 
provide a wide range of products and services, such as 
watershed protection, carbon sequestration, eco-tourism, and 
products derived from bioprospecting, intermediate products 
(e.g. natural dyes, colorants, oils, biochemicals compounds, 
medicinal extracts) and final products (e.g. timber, handicrafts, 
nuts, fruits, perfumes, medicines). Many of these products are 
collected for subsistence use. Some of them have served as an 
important source of innovation for the pharmaceutical, biotechnology, cosmetic or agrochemical 
industries. 

The potential economic value of biodiversity should be translated into tangible economic benefits 
for populations whose livelihood depends on biodiversity. One of the ways to achieve this is by 
taking advantage of the new investment and trade opportunities that are emerging for biodiversity- 
based products and services. Interest for these products is on the rise because of the intensifying 
search of industries for recyclable products, the globally emerging biotechnology industry, and 
shifts in consumer behavior in industrialized countries and urban areas of developing countries. 

It is our opinion that if Nigeria is able to seize these opportunities, biodiversity could be turned 
into an engine for growth and sustainable development. 

To achieve this objective, we have proposed the initiation of a Biotrade-Nigeria program in 
collaboration with the Federal Ministry of Agriculture and Rural Development (FMARD) with 
UNCTAD and BDCP as technical partners. 

Responding to the Challenge 

BDCP/ICBG members recently represented Nigeria at the G-15 Meeting on “Trade and 
Investment in Biodiversity Products and Services, Intellectual Property Rights and Traditional 
Knowledge” in Caracas, Venezuela, 3-4 April 2002, where initial contact was made with 
UNCTAD representatives. 

Establishing a BIOTRADE program in Nigeria requires a focal point in government with the 
requisite mandate for food and related products. The FMARD best meets the institutional capacity 
and capability for this program. Accordingly, the BDCP is currently exploring strategies for 
implementing this initiative with FMARD by examining the following: 

• Technical requirements for initiating a Biotrade program; 


liThe 


IfFig 14: The world market o] 
^products and services derived from|| 
feiibiological resources is estimated to|f 
[|fbe more than US$900b per annum|| 
world market for herbaf 
in 1997 amounted toil: 
||US$ 16.5b. The organic food and|| 
|ibeverage market for Japan, the US|| 
|§and EU is approximately US$20b for§| 
||2000. The market for natural 
^coloring and flavoring materials iM 
Ifestimated at US$150m worldwide I 
|§High demand exists for fresh and|| 

I llprocessed fruits and vegetables.!; 
!Among developing countries, India|| 
^captures 2.5% of the global marketfl 
jlffor herbal products. It is estimated!!] 
nfthat Cameroon exports US$150“^ 
Hyearly in medicinal plants. 


»»>»»»» 


54 



• Legal and policy frameworks; 

• Identification of all relevant stakeholders; 

• Cooperation agreements (including initiation of MOU) 

• Timeline and modalities for implementation. 

The BIOTRADE country programs are the most comprehensive part of the Initiative. They 
identify opportunities and constraints for sustainable resource development in each country, 
focusing on bio-business development, bio-partnerships, sustainable use, conservation, and 
benefit-sharing incentives. Country program are managed by national focal points with long 
standing experience in the area of sustainable development and strong links with other national 
organizations. For example, the focal point in Colombia is the Humboldt Institute, and in Peru, the 
National Environmental Council (CONAM), while Guinee Ecologie, an NGO, is the Guinean 
focal point and Centre beninois pur le developpement durable (CBDD) is the lead agency in Benin. 

The BIOTRADE Initiative thus aspires to fully integrate the private sector, government agencies, 
local and indigenous communities, and other relevant players in a mutually beneficial framework. 
It develops concrete partnerships with governments, private sector, international organizations and 
NGOs. 


Suggestions on the focus of future long-term assessments: 

Development on the agricultural and pharmaceutical sectors relies immensely on the access and 
utilization of new genetic resources for innovations that ultimately lead to new products. Recent 
developments in biotechnology are not only increasing demand in these sectors, but are also 
opening up new applications and markets for genetic resources. This has led to rapidly increasing 
demand for genetic resources, many of which are found in tropical forests. 

It is important to understand the value placed by various stakeholders in the various forms of the 
exploitation of NTFPs. Given that the benefits (and costs) of exploitation are complex, interrelated 
and global, it is equally important to adopt a systematic methodology in establishing these benefits 
and costs flows of NTFPs. At the moment, many studies (including the pilot study by BDCP in 
1997) used cross-sectional data to value NTFPs. The experience is that rarely have researchers 
embarked on time-series data in their economic valuation of biological diversity in general, and 
NTFPs in particular. 

Given the preference for market-based policies around the world, and the theory, that for any 
resource to be properly managed, the cost of using the resource needs to reflect the values that 
society places on it. There is need to adopt reliable methodologies that would provide a reliable 
basis for policy action in respect of natural endowments. 

We believe that generation of time-series data carried continuously and over a period of years will 
not only address relevant issues, but will also fill data gaps inherent in cross-sectional data, and 
therefore add to existing data pool. 


55 



1. Objectives ofthe study 

1. Evaluate exploitation and uses of specific NTFPs in the communities over a period of 12 
months, in the first instance. This will be used to establish the basis for subsequent time 
series study. 

2. Determine market and non-market values of the species 

3. Determine values that indigenes place on the NTFPs and forests in general 

4. Assess market structure and marketing channels or specific NTFPs locally and/or across 
border. 

2. Methodology and nature of data 

The first phase consists of data generation to fill data gaps in the two communities (Imo and 
Ebonyi) where the study was carried out in 1997. Data gaps for 1998 and 1999 may be filled 
through projections, while 2000 and 2001 data could be established through memory recall, using 
household data for specific products. 

Subsequently, multiple visit approach will be conducted in 6 communities. We are proposing 
Taraba, Cross River, Abia and Rivers states, in addition to the two former states (Imo and Ebonyi). 

A semi-structured questionnaire will be designed to elicit information from households in the 
following areas: products/plant species usually collected, revenue generated from such products, 
gender issues with respect to access and use of products, knowledge levels of NTFPs, marketable 
and non-marketable value of plant species, market structure of specific products. 

Data on three specific forest products, including bush mango, Gnetum sp. and Afromomum sp, will 
be collected with respect to product type, usual period of collection, quality and price of product. 

3. Time Frame 

Data collection will be for one year in the first instance, with plans to continue during the second 
year. Specific data will be collected on weekly basis. This implies four entries in a month. 

4. Enumerators 

We propose to employ one enumerator for each community. Thus, there will be 6 enumerators for 
the six states. Contact persons will be used in states where BDCP has already established some. 

5. Sampling size 

For more in-depth study, we propose 15 households per state, totaling 90 in all. We may need to 
segregate between households with medicinal knowledge (medicine men) and households without 
plant knowledge on the one hand, and female headed and male-headed on the other. Data will also 
be obtained from local medicine men. 

6. Data Management 

Data collection and entry will be regular. To facilitate this, we need to hire the services of a data 
analyst. Quarterly evaluation and reporting of data will be done to establish trends, identify lapses 
and gaps and modify data collection, if need be. 


56 





3.4 Computerized Information System for African Medicinal and Aromatic Plants 
(CISAMAP) 

This report is organized as follows. In section 1, we give review of work done during the current of 
this past year that is year 4 (September 30, 2001 - September 30, 2002) of ICBG program. Section 
2 provides plans for year 5 (September 30, 2002 - September 30, 2003). Section 3 presents future 
work plan, sustainability plan and wrap up of the current program. 

In the next section, we present the progress on the database application development, the 
Interactive CD-ROM creation and the dynamic web site development. For each segment, we will 
outline the status at the end of last year (September 30, 2000 - September 29, 2001) and work 
performed this year (September 30, 2001 - September 29, 2002). 

3.4.1 Database Application Development 

1.1. Situation at the end of last year 

Last year, we had proposed to unify the four databases (AFRICMED, ICBG-WRAIR, BIOMON 
and KFDP) in one ORACLE database called CISAMAP as shown in figure 15. Now, a user can 
choose to work with the whole database, or just one of its subset (a view). For example, if one is 
interested only in information related to medicinal plants, the view ICBG-WRAIR will be 
projected. For someone desiring only information related to chemical and biological aspect of 
plants, BIOMON view would be projected. The view KFDP will be projected if only demographic 
or geographical information is needed. 



Figure 15: New Database System architecture 


This structure allows any group working on a similar theme to import our system with few or no 
modifications. To reach this general structure, which increases the range of users of the system, we 
had to consider information related to: 

Medicinal plants (here stands for a medicinal plant or an animal since part of animal are 
also used in herbal medicine). 


57 









- Potions or herbal medicines (natural medicine), and 

- Diseases as represented by traditional healers that treats them. 

At the end of last year, we had finished conceiving and designing the ORACLE database entity 
tables. The coding and implementation using ORACLE SQL were quite advanced and we were 
waiting for the ORACLE System to progress normally. We had provided a detail description of the 
database structure in our last year report. 

1.2 Work performed this year 

To implement an ORACLE database there are four important phases: 

a. Phase 1 (Definition of objectives): In this phase the objective is defined and the type of 
information to be contained in the database is determined. This task was performed last 
year. 

b. Phase2 (Definition of database schemas) : A Database schema is a collection of structures 
of tables, triggers, indexes, views, stored procedures, clusters, programmer defined objects 
(new types of data), tablespaces, partitions, etc. Phase 2 is the main phase in creating an 
ORACLE database. It is here that all objects that will be manipulated in the database are 
defined. These definitions necessitate the following steps: 

• Definition of tablespaces : A tablespace logically regroups data on a specific subject. One or 
more physical file(s) can be associated to each tablespace. 

• Definition of tables : After defining the tablespaces, the definition of tables follows by 
defining the entities of the database. Each entity corresponds to a table of the database. 

• Definition of users : Different users of the database are defined with appropriate rights. 

• Definition of relations : Different links existing between the data and between the tables are 
determined. If necessary, new fields are added to tables or new tables are created to clarify 
the links in order to satisfy the integrity constraints and data coherence. 

• Definition of indexes : New structures that will permit rapid access to various element of 
information are created. 

c. Phased (Creation of sequences): Theses sequences of numbers are used to populate 
primary keys of tables in the database. At this phase, the sequences are generated. 

d. Phase4 (Verification and Finishing): This fourth phase consists of testing the behavior of 
the database with real data. If necessary, the database is updated as well as the database 
schemas. 

Sixty percent (60%) of phase 1 and phase 2 were accomplished last year. By the end of 
December 2001, we had completed the two phases. At the end of March, 2002, we were at 
70% through phase 3. Today, we have completed phase 3 and are at 90% through phase 4. 
At this level of phase 4 (last phase of database system), we are in the process of installing 
the system (database and its interface: MEDITRA SOFTWARE) at designated sites to start 
the validation process. 


2. User Interface (MEDITRA SOFTWARE) 

2.1 Situation last year 

Last year we also proposed a unified approach in specifying and modeling the database user 
interface that we named « MEDITRA SOFTWARE ». From that model, a user who wants to deal 
only with plants fosits botanical proprieties for example will only receive two subsections of the 
interface: the subsection related to “source organs” and the one related to “Administration”. The 


58 


interface was specified bilingual (French and English versions). A database populated using any of 
the two languages can be managed or consulted in either language. We had provided a detailed 
description of the specification of the system interface. At the end of last year 40% of the coding 
was done. 

2.2 Work performed this year 

Now, MEDITRA SOFTWARE system is fully implemented and is operational. Being application 
software and not a standardized software like Microsoft word, users must be trained to know how 
to use it properly and efficiently. This process will commence on completion of installation. As we 
mentioned in our last year report, MEDITRA SOFTWARE is designed to be used by several 
specialists in ethnobiology. It is thus necessary, when installing the software, to adapt it to the 
specific needs of each user by removing irrelevant information or procedures, or by adding new 
information or procedures not present. We have done 40% of the write up of user manual and 
technical manual. 

3. Interactive CD ROM 

3.1 Situation at the end of last year 

As in the case of database organization, we had used a demo version to start developing a 
prototype with no video since we were still waiting for the video acquisition tool. The entire 
sequencing specification of the proposed CD-ROM was presented last year. 

3.2 Work performed this year 

We started the work with Authorware Professional. More than 90% of all the links specified are 
implemented. We are in the final stage of filling in all the multimedia data to check that the links 
implemented are functioning properly. So far, we have integrated textual data on Cameroon and a 
videotape on ICBG program in Nigeria. Additional video and audio data on Cameroon has been 
received to populate the Interactive CD-ROM. One of the key data is table of plants with their 
images, textual description of their uses and possibly video clip demonstrations of these uses. 

In the mean time, we propose to combine video clips derived from a digitalized video film on 
Cameroonian traditional medicine, which is in color, and the black and white video clips from the 
videotape of ICBG, to fill the Interactive CD-ROM. The Interactive CD-ROM is bilingual 
(English and French) and is composed of four parts: ETHNOMEDICINE, ETHNOBOTANY, 
REGION, and REFERENCE. Presently, 80% of all the links implemented are working. 

4. Online Sen’er 

4.1 Situation at the end of last year 

We were asked to implement a Dynamic Web Site allowing consultation and updating of the 
database from any Internet Navigator such as Netscape Communicator or Internet Explorer. The 
Tools required for that task is Oracle WebDB. We were not able to start this activity because the 
demo version of Oracle WebDB in our possession could only run with ORACLE 8i.7 or later 
version that we did not have. At that time we were working with a demo version of 8i.5. 


59 



m 


4 


4.2 Work performed this year 

Now that we have the correct version of Oracle (9i), we have assigned the two students we 
recruited on this part of the project. They started the work in July. We plan to combine Oracle 
WebDB and DREAMWAVER to produce attractive pages. 

5. Conclusion 

The database system, we have implemented, as well as its user interface will allow any 
organization working on a similar topic, to import our structure to generate its own database. The 
system comprises two components: an empty database and MEDITRA SOFTWARE (the interface 
to the database). MEDITRA SOFTWARE provides tools needed to populate an empty database. 
Work on the Interactive CD-ROM is progressing slowly because mounting video clips with Adobe 
Premiere is time consuming. 80% of links specified and implemented are working properly with 
the already filled in multimedia. The insufficiency in human resources forced us to postpone the 
starting date of the dynamic web site coding. 


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Peer Reviewed Articles: 


1. Iwu MM. Antihepatoxic constituents of Garcinia kola seeds. Experientia. 1985, 41(5): 
699-700. 

2. DD. Chris O. Okunji, Tantalia A. Ware, Rickey P. Hicks, Maurice M. Iwu, David J. 
Skanchy, Capillary Electrophoresis Determination of Biflavanones from Garcinia kola in 
Three Traditional African Medicinal Formulations, Planta med 2002; 68: 440-444. 



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