REPORT ON THE RESEARCH OF ADEROUNMU ADEOLA OMOTAYO, UNESCO-ASM

TRAVEL GRANT RECIPIENT FOR 2002 AT THE DEPARTMENT OF IMMUNOLOGY,

STOCKHOLM UNIVERSITY, SWEDEN.

The research activity of Aderounmu Adeola Omotayo regarding the Travel Award of UNESCO-

ASM was completed in December 2002. During the period of the study, Adeola was able to learn a

few techniques that assisted immensely in his on going doctoral research at the University of Lagos

in Nigeria. In all, based on the report below, Adeola fulfilled the purpose for which the grant was

given.

REPORT 1.

SHORT TERM CULTIVATION OF PLASMODIUM FALCIPARUM IN A PLANT

BASED MEDIUMSummary

Short-term cultivation of P. falciparum, F32, was achieved in a medium containing plant exudate and mice

liver extract following earlier cultivation of the parasites in medium containing human serum. The

parasitaemia increased from 4.0% to 7.6% on day 4 and the addition of hypoxanthine (0.02-0.04µM) to the

new medium enhanced parasite growth as 9% parasitaemina was observed at 48 hours in a separate culture

well containing the new medium and hypoxanthine. All the asexual stages of the erythrocytic phase of the

parasite life cycle were seen throughout the duration of the cultivation. The results obtained in this study

probably represents the first successful attempt to cultivate P. falciparum in vitro in a plant-based medium

supplemented with animal extract. In addition, the essence of the ongoing research is to develop an

inexpensive malaria culture system that will enhance malaria research in poorer countries mostly afflicted

with the scourge of the infection of P. falciparum

Introduction

Cultivation of malaria parasites is an important tool for the understanding of parasite biology, biochemistry,

molecular biology, immunology and pharmacology (Ringwald et al., 1999). Since Trager and Jensen

established the in vitro culture of P.falciparum in 1976, the medium used has been complemented by the

addition of 10% human serum. The requirements for human serum pose technical limitations to the

application of this method (Asahi and Kanazawa, 1994; Asahi et al., 1996). For many reasons, it would be

advantageous to replace human serum in the culture medium. In malaria-endemic regions, local sera may

1

contain antimalarial drugs or immune factors that render them useless for culturing the parasites (Siddiqui

and Palmer, 1980; Divo and Jensen, 1982a; Jensen et al., 1982). A few reports however have shown that

African donors can support the growth of laboratory adapted strains of parasites and fresh isolates and that

acute phase homologous serum may be useful for the continuous in vitro culture of reference strain (Oduola

et al., 1992; Binh et al., 1997 and Ringwald et al., 1999).

It has generally been accepted that nonimmune human serum is required for optimal parasite growth.

However the requirement for a regular supply of nonimmune human serum entails difficulties in conducting

research in most of the African continent, where malaria transmission occurs at a high level throughout the

year. Nonimmune human type AB-positive serum is relatively scarce and expensive in countries where

malaria is not endemic (Divo and Jensen, 1982a; Ringwald et al., 1999).

Furthermore, serum from donors living in malaria-free areas differ considerably in their ability to support

parasite growth and therefore, it is recommended that several units of serum from different donors be pooled

together to reduce the batch-to-batch differences in the support of parasite growth (Divo and Jensen, 1982b;

Jensen, 1988). It cannot also be excluded that drugs not directed against the malaria parasites may

nevertheless influence the development of the parasites. More problems include blood type compatibility and

risks associated with the handling of infectious agents (Lingnau et al., 1994). In addition, any widely used

vaccine should not be grown in human serum when there is a real possibility of contamination with

infectious agents (Divo and Jensen, 1982a).

A number of successful attempts to replace the human serum components of the medium used for the in vitro

cultivation has been reported (Siddiqui and Richmond-Crum,1977; Ifediba and Vanderberg, 1980; Sax and

Rieckamn 1980; Divo and Jensen, 1982b; Willet and Canfield 1984; Asahi and Kanazawa 1994; Oduola et

al., 1985; Lingnau et al., 1993, 1994; Ofulla et al., 1993,1994). All outlined the obvious disadvantages of

drawing experimental data from parasites grown in serum supplemented medium due to batch-to-batch

invariabilities problems with availability and cost in some places and the probability of contracting viral

infections. Parallel comparisons of studies from different laboratories can therefore be easily standardised

once this important variable has been excluded. These investigators have used both commercial and defined

formulations to mimic the growth requirements supplied by the human serum though no thorough

identification has been carried out as to which factor(s) of human serum are necessary for growth (Ofulla et

al., 1993; Asahi et al., 1996; Binh et al., 1997 and Flores et al., 1997). The use of various animal sera and

serum factions as substitutes for human sera have had only limited success hence the attempts to find a

suitable substitute for human serum are continuing.

2

Materials and Methods

The following solutions were prepared and used in this experiment: glucose (0.2g/ml), saline (0.13g/ml),

PBS 1X, mice liver extracts (0.25g/ml PBX 1X), Jatropha curcas exudate (1% in distillled water-equivalent

to stock), hypoxanthine solution (0.05mg/ml). Because this was the first time we are doing this experiment,

we did a lot of conceptualisations. For example, we investigated the effect of plant exudate and its various

water dilutions on uninfected erythrocytes. The aims of these preliminary investigations were to ensure that:

the infected and uninfected erythrocytes would not agglutinate in the new media, there was no rapid

discolouration of the media in the incubator due to excess and/or abnormal concentration of the sap solution

and also to prevent the red cells in losing their form and function due to the concentration of the various

solutions. Sequel to these aims, 0.2ml washed uninfected blood was added to 1ml sap solution and diluted by

water and water plus glucose solution in various ratios.

The newly formulated medium

Based on the results of the investigations above, i.e the effect of water dilution (data not shown), the

following combinations were made to give a single batch of the newly formulated medium: 10 ml stock, 20

ml water, 2 ml glucose solution, 1 ml saline solution, 1 ml PBS 1X, 12ml liver extract, 15µl Gentamycin.

The pH of the new media is about 6.6 but usually adjusted to 7.4-7.6 using NAHCO 3 solution (0.002g/ml).

The complete new medium was stored at -210C.

The candle jar method of Trager and Jensen (1976) was adopted in these experiments in which 96 flat bottom

culture wells were used The parasitaemia was adjusted to 4% using blood washed thrice with Tris Hank

solution and suspended in PBS (1X). Five replicas were set up for each approach as shown in table 1. Prior

to this however, we made several attempts to cultivate the parasite in this plant based medium from lower

parasitaemia with limited success.

Table 1. The various formulations of the new media used in the cultivation of malaria parasites

showing hypoxanthine content (proportion and molarity values).

Culture Hypoxanthine content in 500µl Proportion Molarity (µM)

E new medium only - -

F new medium 15µl 3% 0.01

G new medium 30µl 6% 0.02

H new medium 50µl 10% 0.04

I new medium 100µl 20% 0.07

*MCM-HS malaria culture medium - -

*Malaria culture medium with 10% human serum (MCM-HS)

3

The total volume of each well of five replicas was 200µl of which 100µl was infected erythrocytes in PBS

(1X) solution. In all cases, the haematocrit is 4%. The culture wells were incubated at 37 oC after ensuring

low oxygen by burnt out candles. The gas phase in a candle jar has about 3% CO 2 and 15-17% O2. (Trager,

1987). The microtiter plates remained stationary at 37oC during the culture period, thus allowing the cells to

settle at the bottom. When the medium was changed, culture plate were carefully moved to the culture hood

area where the supernatant fluids were removed aseptically with sterilised Pastuer pipettes. At the same time

blood films were prepared for the evaluation of parasite growth and multiplication. Fresh media were then

placed as appropriate, the cells resuspended, and the culture plates returned to the incubator. These

approaches, as described by Siddiqui, (1979) are conventional.

RESULTS

The dilution of the stock sap solution (1% v/v) by twice volume of water was found to be best suitable for

the erythrocytes in view of the objectives analysed earlier. It is yet to be determined if the glucose fraction of

the media gave optimal growth. Growth of the parasites that have been coincubated in 96 wells plates was

monitored for 5 days. The media were changed daily but no subcultivation was done. There had been many

previous attempts to culture the parasite at various start parasitaemia. An example is shown in table 2 where

at day 4 only the parasites in MCM-HS showed appreciable growth. The parasitaemia was 8% (growth

rate=1.6% with respect to start parasitaemia). It was however possible to observe substantial increase in

parasitaemia in the various cultures (E-I) with a start parasitaemia of 4%. Table 3 and figures 1 and 2 showed

the results obtained. The mean growth rates of the parasites on day 5 are shown in Table 4. Growth rates in

these experiments are expressed as percentage increase in parasitaemia over a specified period of time

(formula given below). For MCM-HS, the culture was discontinued after day 3 due to decreased in number

of parasitised erythrocytes. Since no subcultivation was carried out, the probable accumulation of lactic acid

and other metabolites known to inhibit growth cannot be ruled out.

Growth rate= percentage parasitaemia-start percentage parasitaemia

Day(s) in culture

Where 24 hours represents one day in culture and 120 hours is 5 days in culture.

Table 3. The percentage parasitaemia obtained at 4% start parasitaemia in new media and MCM-HS.

Time Percentage parasitaemia

4

E F G H I MCM-HS

0 4.0 4.0 4.0 4.0 4.0 4.0

24 5.4 (+1.4) 4.8 (+0.8) 3.9 (-0.1) 7.5 (+3.5) 3.7 (-0.3) 8.3 (+4.3)

48 5.0 (+0.5) 4.0 (0.0) 9.0 (+2.5) 5.0 (+0.5) 6.0 (+1.0) 15.0 (+5.5)

72 6.0 (+0.6) 6.7 (+0.9) 8.5 (+1.5) 5.5 (+0.5) 3.8 (-0.1) 12.0(+2.7)

96 7.6 (+0.9) 5.9 (+0.5) 8.3 (+1.1) 6.1 (+0.5) 7.4 (+0.9) **

120 7.1 (+0.6) 6.2 (+0.4) 8.0 (+0.8) 6.5 (+0.5) 7.5 (+0.7) **

Molarity of hypoxanthine: F=0.01µM, G =O.O2µM, H=0.04µM, I=0.07µM. Growth rate wrt start

parasitaemia in parenthesis.

** not determined.

5

0

5

10

15

20

Perc

enta

ge p

aras

itaem

ia

0 24 48 72 96 120

Time in culture(hours)

Hypoxanthine content

MCM-HS -

I O.O7µM

H 0.04µM

G 0.02µM

F 0.01µM

E -

Figure 1. P. falciparum : the percentage parasitaemiaobserved in new media (E-I) and MCM-HS.

DISCUSSION

Plasmodium falciparum malaria is responsible for millions of deaths worldwide annually (Gatton and Cheng,

2002). The availability of a method for the continuous cultivation of P.falciparum described by Trager and

Jensen (1976) has stimulated multiple and varied laboratory investigations on the most virulent of the human

parasites. However the ease with which P.falciparum parasites adapt to culture by this method of Trager-

Jensen is variable (Chin and Collins, 1980). According to WHO, (1977), there has always been variation in

the ease with which isolates or cultured parasites can be maintained, moreso in a new medium.This

variability in the ease of adapting to culture conditions is a common experience shared by investigators using

the Trager-Jensen method to establish strains of falciparum parasites. In one experiment, it was observed that

only 2 out of 15 isolates grew well upon culturing while the remainder strains required an adaptive period of

1-2 months before growth rates greater than 10 fold per 96 hours were reached. During the period of no

apparent growth, the muliplication rate was seldom more than 2-fold every 96 hours and parasites were

difficult to detect even in thick smears (Chin and Collins, 1980). These investigators considered a strain as

adapted when the multiplication rate of the parasites exceeds 10-fold every 96 hours. Domarle et al.,(1997)

found 4 out of 19 field samples adapted to culture conditions.

In this study, several preliminary trials were conducted until P. falciparum F32 strains was succesfully

cultured in vitro for a limited number of days during which no subculturing was carried out. These results are

basis for ongoing further investigations and optimisations. No previous attempts have been made to use

plants extracts for the in vitro cultivation of P.falciparum except one report in which coconut extract was

6

00.5

11.5

22.5

3

3.54

4.5

Perc

enta

ge m

ean

grow

th r

ate

E F G H I MCM-HScultures

Figure 3. The mean growth rate observed at day 5 for new media and day 3 for MCM-HS

growth rate

used (Renapurkar and Sutar, 1989). However the sap of Jatropha curcas used here as a basal medium has

been shown to support the growth of Trypanosoma cruzi (Fagbenro-Beyioku and Jagun, 1994-unpublished).

It is imperative to state that, since it takes substantial time for malaria parasites to adapt to a new growth

medium, it will seem almost unlikely that the parasites would survive long enough to proliferate at a start

parasitaemia of ≤1%. This can explain why a parasitaemia of 4% was later used in this study. Indeed at 48

hours, a parasitaemia of 9% was observed in one of the culture wells containing hypoxanthine, 0.02µM. Sax

and Rieckmann, (1980) suggested that the cultivation of parasites in a new culture medium should be

pursued if comparable growth is not achieved immediately after changing from one serum (medium) to

another.

Lingnau et al.,(1994) reported that continuous cultivation of various P.falciparum strain was possible with

serum-free medium in Nutridoma-SR supplemented RPMI medium. However, the growth in the serum

control was better and the reason for this was unclear. The ability of one serum-free formulation to support

parasite growth also varies from strain to strain (Flores et al.,1997). For example, Nutridoma SR (N-SR;

Boehringer Mannheim) has been shown to substitute serum in culturing of geographically distinct strains of

malaria. Two of the test strains grew comparatively as well as the serum-grown controls, whereas another

reached half the growth rate of control cultures (Lingnau et al.,1994). Likewise, other groups have

substituted serum with a serum-defined lipid cholesterol-rich mixture and bovine albumin, with differing

effects on parasite development (Asahi and Kanazawa, 1994; Ofulla et al., 1993).

The beneficial effects of adding glucose and hypoxanthine to parasite culture have previously been described

(Zolg et al.,1982; Ofulla et al., 1993). Optimal glucose concentration in serum free media seems to be

beneficial to parasites considering the fact that a parasitised erythrocytes uses 26 times more glucose than an

unparasitised red blood cell (Jensen et al., 1983). Divo and Jensen, (1982b) tested various supplements,

individually and in various supplements, with dialysed human serum and found that only hypoxanthine

contributed to increased parasite growth. Hypoxanthine which is important for the formation of nucleic acid,

was used in these experiments to investigate its role as a growth-promoting factor. At 0.05mg/ml and 0.02-

0.04µM, it was found to promote the mean growth of the parasites compared to the media in which

hypoxanthine was absent. However, there was no statistical significant difference between the growth

observed in the media when used alone and when various percentages of hypoxanthine was added. The

success of the various media that have been used for the cultivation of P. falciparum may be hinged on the

presence of hypoxanthine ranging from 30 to 375µM in the culture media (Divo and Jensen, 1982b; Asahi et

al., 1996) although Ofulla et al.,(1993) described that RPMI 1640 supplemented with Bovine Albumin

Serum (BSA) sustained growth of the parasites for a long-term without added hypoxanthine. Obviously,

there are various other supplements other than hypoxanthine that can place developmental demands on the

parasites (Asahi et al., 1996).

7

The exact content/constitution of the plant 'milky exudate' used in this study remains to be elucidated but it is

known that 100g of the seed contains 6.6g water, 18.2g protein, 38.0g fat, 33.5g total carbohydrate, 15.5g

fiber and 4.5g ash (Duke and Atchley, 1984). It has been reported that albumin may be one of the major

factors in the growth-promoting influence of serum. The mammalian liver is the source of albumin, a water-

soluble protein. Liver extracts also contain many vitamins and minerals including iron. If it could be possible

to use fractions of the liver extracts without autoclaving, it may be possible to obtain better parasite growth.

The results of this study will be of practical and research value and may also help to unravel the precise role

of serum in malaria culture medium. F32 strain is commonly used in laboratory-based malaria research and a

serum-free medium compatible to their growth requirements, as well as other strains of Plamodium

falciparum, will be advantageous for standardisation of experimental data. In addition, to verify that that

cells have not lost their physiological and/or metabolic functions, it will be interesting to perform drug

sensitivity and metabolic assays on them when the medium is better established.

References

Asahi, H and Kanazawa, T (1994). Continuous cultivation of intra-erythrocytic P.falciparum in a serum-free

medium with the use of a growth promoting factor. Parasitology 109: 397-401.

Asahi, H., Kanazawa, T., Kajihara, Y., Takahashi, K and Takahashi, T (1996). Hypoxanthine: a low

molecular weight factor essential for the growth of erythrocytic P. falciparum in a serum-free medium.

Parasitology 113: 19-23

Binh, V.Q., Luty A.J.F and Kremsner P.G (1997). Differential effects of human serum and cells on growth of

P. falciparum adapted to serum-free in vitro culture conditions. American Journal of Tropical Medicine and

Hygiene 57(5): 594-600.

Chin, W and Colins, W.E (1980). Comparative studies of three strains of P.falciparum isolated by the culture

method of Trager and Jensen. American Journal of Tropical Medicine and Hygiene 29(6): 1143-1146.

Divo, A.A and Jensen, J.B (1982a). Studies on serum requirements for the cultivation of P. falciparum 2.

Animal sera. Bulletin of the World Health Organisation 60: 565-569.

Divo, A.A and Jensen, J.B (1982b). Studies on serum requirements for the cultivation of P. falciparum 2.

Medium enrichment. Bulletin of the World Health Organisation 60: 571-575

Flores, M.V.C., Berger-Eiszele, S.M and Stewart, T.S (1997). Long-term cultivation of Plasmodium

falciparum with commercial non-serum supplements. Parasitology Research 83: 734-736

Domarle, O., Ntoumi, F., Belleoud, D., Sael, A., Georger, A.J and Millet, P (1997). P. falciparum: adaptation

in vitro of isolates from symptomatic individuals in Gabon: Polymerase chain reaction typing and evaluation

8

of chloroquine susceptibility. Transaction of the Royal Society of Tropical Medicine and Hygiene 91: 208-

209.

Duke, J.A and Atchley, A.A (1984). Proximate analysis. In: Christie, B.R (ed.). The handbook of plant

science in agriculture. CRC Press, Inc., Boca Raton, FL.

Gatton, M.L and Cheng, Q. (2002). Evaluation of the pyrogenic threshold for Plasmodium falciparum

malaria in naive individuals. American Journal of Tropical Medicne and Hygiene 66(5): 467-473

Ifediba, T and Vanderberg, J.P (1980). Peptones and calf serum as replacements for human serum in the

cultivation of P falciparum. Journal of Parasitology 66: 236-239.

Jensen, J.B (1988). In vitro cultivation of malaria parasites: erythrocytic stages, p 307-320. In W.H

Wernsdorfer and I.A McGregor(ed.), Malaria. Principles and practice of malariology. Churchill Livingstone,

Edinburgh, United Kingdom.

Jensen, J.B., Boland, M.T., Hayes, M and Akoda, M.A. (1982). Plasmodium falciparum: a rapid assay for in

vitro inhibition due to human serum from residents of malarious areas. Experimental Parasitology 54: 416-

424

Jensen, M .D., Conley, M., Helstowski L.D (1983). Culture of P. falciparum. The role of PH, glucose and

lactate. Journal of Parasitology 69: 1060-1067.

Lingnau, A., Margos, G., Maier, W.A and Seitz, H.M (1993). Serum-free cultivation of Plasmodium

falciparum gametocytes. Parasitology Research 109: 378-384.

Lingnau, A., Margos, G., Maier, W.A and Seitz, H.M (1994). Serum-free cultivation of several P.falciparum

strains. Parasitology Research 80: 84-86.

Oduola, A.M.J., Alexander, B.M., Weatherly, N.F., Bowdre, J.H and Desjardins, R.E (1985). Use of non-

human plasma for the in vitro cultivation and antimalarial drug susceptibility testing of P.falciparum.

American Journal of Tropical Medicine and Hygiene 34(2): 209-215.

Oduola, A.M.J., Ogundahunsi, O.A.T., Salako, L.A (1992). Continuous cultivation and drug susceptibility

testing of P. falciparum in a malaria endemic area. Journal of Protozoology 39: 605-608.

Ofulla, A.V.O., Okoye, V.C.N., Khan B., Githure J.I., Roberts, C.R., Johnson, A.J and Martin S.K (1993).

Cultivation of P.falciparum in a serum-free medium. American Journal of Tropical Meidicne and Hygiene

49: 335-340.

Ofulla, AV.O., Orago, A.S., Githure J.I., Burans, J.P Aleman, G.M., Johnson, A.J and Martin, S.K (1994).

Determination of fifty percent inhibitory concentration (IC50) of antimalarial drugs against P.falciparum

parasites in a serum-free medium. American Journal of Tropical Medicine and Hygiene 51: 214-218.

Renapurkar, D.M and Sutar, N.K (1989). Coconut water and the cultivation of Plasmodium in vitro.

Transaction of the Royal Society of Tropical Medicine and Hygiene 83: 720.

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Ringwald P., Meche, F.S., Bickii, J and Leonardo, K.B (1999). In vitro culture and drug sensitivity assay of

P. falciparum with nonserum substitute and acute-phase sera. Journal of Clinical Microbiology 37: 700-705

Sax, L.J and Rieckmann, K.H (1980). Use of rabbit serum in the cultivation of P.falciparum. Journal of

Parasitology 66: 621-624.

Siddiqui, A.B (1979). Continuous in vitro cultivation of Plasmodium falciparum in human erythrocytes:

description of a simple technique to obtain high yields of parasites. Practical Tissue Culture Applications,

267-277. Academic Press, Inc.

Siddiqui, W.A and Richmond-Crum, S.M (1977). Fatty acid-free bovine albumin as plasma replacement for

the in vitro cultivation of P. falciparum. Journal of Parasitology 63: 583-584.

Siddiqui, W.A and Palmer, K.L (1980). Propagation of malaria parasites in vitro. Advances in cell culture

1:183-212.

Trager,W (1987). The cultivation of Plasmodium falciparum: applications in basic and applied research on

malaria. Annals of Tropical Medicine and Parasitology 81(5): 511-529.

Trager, W and Jensen, J.B (1976). Human malaria parasites in continuous culture. Science 193: 673-675.

Willet, G.P and Canfield, C.J (1984). Plasmodium falciparum: continuous cultivation of erythrocyte stages in

plasma-free culture medium. Experimental Parasitology 57: 76-80.

Zolg J.W., MacLeod, A.J., Dickson, I.H and Scaife J.G (1982). Plasmodium falciparum: modifications of

the in vitro culture conditions improving parasite yield. Journal of Parasitology 69: 1060- 1067

10

REPORT 2

THE ANTIMALARIAL ACTIVITY OF THE CRUDE ORGANIC EXTRACTS OF FOUR

COMMONLY USED NIGERIAN MEDICINAL PLANTS.

Summary

The organic solvent extracts from four Nigerian medicinal plants belonging to different families have been

screened for antimalarial activity against P.falciparum. Based on this form of extraction, three of the extracts

derived from Cymbopogon giganteus, Enantia chlorantha and Morinda lucida showed significant activity

against the parasite. Though the greatest activity was observed in E.chlorantha with 68.9% inhibition at

500µg/ml, the findings in this study support the ethnomedicinal use of these traditional plants for the

treatment of fever and malaria.

Introduction.

Malaria is today a disease of poverty and undeveloped countries but it remains an important health problem

globally. In the last decade, the prevalence of malaria has been escalating at an alarming rate, especially in

Africa. An estimated 300 to 500 million cases each year cause 1.5 to 2.7 million deaths, more than 90% in

children under 5 years of age in Africa (Good, 2001; Sachs and Malaney, 2002).

Recently the malaria situation has deteriorated and mortality from malaria is probably increasing in the

whole of sub-saharan Africa. Some of the reasons for this are: drug resistance to most antimalarial drugs,

insecticide resistance in mosquitoes, war and civil disturbances, environmental changes, climatic changes,

travel and population increase. The main problem for malaria control, at present, is the antimalarial drug

resistance, especially of Plasmodium falciparum, the most deadly malaria parasite (Krettli, 2001). Another

important reason for the persistence of malaria in Africa is the presence of the vector, Anopheles gambiae,

although social and economic factors are also worth mentioning. The female A. gambiae feeds preferentially

on humans and is long-lived, making it particularly effective at transmitting malaria from one person to

another. This makes the task of interrupting transmission daunting because the measure of malaria

transmission intensity, the Entomological Inoculation Rate (EIR) is high and exceeds 1 000 in several parts

of sub-Saharan Africa. EIR is the product of the vector biting rates times the proportion of sporozoite-

infected mosquitoes (Sagara et al., 2002). It measures the frequency with which an individual is bitten by an

infectious mosquito (Greenwood and Mutabingwa, 2002).

11

A major problem with the demise of inexpensive drugs such as chloroquine and sulphadoxine/pyrimethamine

is that the newer drugs cost 7-60 times as much (A. Rietveld, cited in Olliaro et al., 1996). According to

Okochi et al., (1999), one of the factors that draws back the rate of development of the health care system in

Nigeria and probably other developing countries is the prohibitive high cost of importing drugs and

producing new ones. Meanwhile as the levels of resistance to chloroquine and mefloquine continue to rise,

the future for antimalarial treatment with existing drugs look increasingly bleak (Foley and Tilley, 1998).

Artemisinin and related drugs are being used successfully in Southeast Asia and parts of Africa, but

recrudescence after artemisinin treatment is a major problem and resistance to this drug is also appearing

(Meshnick et al., 1996). With this increasing level of chloroquine resistance and fears of toxicity and

decreased efficacy of sulphadoxine-pyrimethamine, there is an urgent need for an affordable, effective and

safe alternative to chloroquine.

Medicinal plants, since times immemorial, have been used in virtually all cultures as a source of medicine

(Hoareau and Dasilva, 1999). Traditional plants play an important role in medical system in Nigeria and

plant materials remain an important resource to combat serious diseases in the world. Pharmacognostic

investigations of plants are carried out to find novel drugs or templates for the development of new

therapeutic agents. Since many drugs, e.g quinine and artemisinin were isolated from plants and because of

the increased resistance of many pathogens, e.g malaria parasites, towards established drugs, investigation of

the chemical compounds within traditional plants is necessary, (Phillipson, 1991).

New antimalarial drugs and approaches to overcome parasite resistance are needed to deal with the

expanding problem of drug resistance which continues to challenge malaria control efforts based on early

diagnosis and treatments. Only a limited number of antimalarial drugs are currently at an advanced stage of

clinical development. In line with this, there is a renewed interest in plant products since the identification of

sesquiterpene lactone artemisinin (quighaosu). An attractive option for poor countries is the exploitation of

the possible therapeutic effects of their local herbsour local herbs.

Materials and Methods

The medicinal plants used in this experiment are Azadirachta indica, Enantia chlorantha, Cymbopogon

giganteus and Morinda lucida.

Table 1. The medicinal plants and the various parts used.

Plant Parts used

E. chlorantha bark

M. lucida bark and leaves

C giganteus leaves

A. indica stem and leaves

12

Preparation of extracts

About 10g of each dried powdered extract was dissolved in 50ml alcohol (95%) for 7 days. The alcohol was

allowed to evaporate at room temperature. 10ml PBS (1X) was added to 10mg of each extract to make

1mg/ml. The aqueous extracts were sterilised by passing through Acrodisc Syringe filter (0.2µm) to obtain

clear filterate used for the inhibitory assay in culture wells.

Culture technique.

P. falciparum, F32, that have been cultured continuously according to the methods of Trager and Jensen

(1976) were used for this investigation. The parasites were exposed to a range of different concentrations of

the four extracts in corning microtest (96) culture wells at 1-2% parasitaemia. Each extract concentration has

a replicate and serial dilutions of 1:2 (500µg/ml), 1:4 (250µg/ml], 1:8 (125µg/ml), 1:16 (62.5µg/ml) and 1:32

(31.3µg/ml). The number of parasitized cells per 40 000 cells were counted by fluorescent microscopy after

24 hours in culture incubated at 37o C.

Fluorescent microscopy

At 24 hours, the cells were harvested and put in 4ml centrifuge tubes, washed thrice with Tris-Hank solution.

Tris-hank solution was prepared by adding 2.11g Tris HCl, 0.2g Tris base and 7.88g NACl in 1000ml

distilled water. This soluton (Tris) was mixed with same volume of Hanks solution, hence the name Tris-

Hank solution (0.15M, pH 7.2). The harvested cells were resuspended in 400µl Tris-Hank solution and fixed

by Glutardialdehyde, GDA (4%) unto multitest slides coated with buffer. In 8 wells, with approximately 200

cells/field, an estimated 40 000 cells was counted in 25 fields for each well using high-power fluorescent

microscope lens with oil immersion, and the percentage growth inhibition with respect to the control was

determined by simple arithmetic calculation. This approach is one of the in vitro antiplasmodial tests for

detecting antiplasmodial activity of plant extracts in the erythrocytic stage of malaria parasites (Rasoanaivo

et al., 2003[In Press])

Results

The organic extracts of 4 medicinal plants at 5 different concentrations were tested for 24hours only in

unsynchronised cultures of MCM-HS at1-2% parasitaemia. The untreated culture served as the control. The

basic measurement of antimalarial activity used in this study was a reduction in the number of parasitised

cells in the test cultures compared to the negative control at 24 hours of incubation. Table ?? shows the

percentage inhibition± SEM. Figures 1-5 depicts the outcomes of the inhibition assay at the various

concentration administered. The dose-dependent antimalarial activity of the organic extracts is shown in

figure ??

13

Table 3. The percentage inhibition±SEM of the organic extracts of the medicinal plants

Extracts concentration (µg/ml) Percentage inhibition (%) ±SEM

AZ

500 41.3±5.7

250 41.3±4.7

125 37.9±2.1

62.5 24.1±7.4

31.3 20.7±3.6

CG

500 58.6±1.5

250 55.2±2.2

125 51.7±3.9

62.5 41.4±3.3

31.3 37.9±4.0

ML

500 65.5±1.4

250 48.3±4.7

125 37.9±4.2

62.5 31.0±5.0

31.3 27.6±2.5

EC

500 68.9±2.7

250 65.5±2.5

125 62.1±2.3

62.5 55.2±1.4

31.3 48.3±3.6

AZ = Azadirachta indica, CG = Cymbopogon giganteus, ML= Morinda lucida, EC = Enantia chlorantha

14

15

0102030405060708090

100

Extract concentration of 250µg/mll0

102030405060708090

100

Perc

enta

ge p

aras

itae

mia

Extract concentration of 500µg/ml

0102030405060708090

100

Extract concentration of 62.5µg/ml

0102030405060708090

100

Perc

enta

ge in

hibi

tion

Extract concentration of 125µg/ml

010

2030

4050

607080

90100

Perc

enta

ge in

hibi

tion

Extract concentration of 31.3µg/ml

Enantia chlorantha

Morinda lucida

Cymbopogon giganteus

Azadirachta indica.

Figure 1.The percentage inhibition of the organicextracts at different extract concentrations

10

20

30

40

50

60

70

80

90

100

50025012562.531.3

Concentration of extracts (µg/ml)

E. chlorantha

M. lucida

C. giganteus

A. indica

Keys

Discussion

In sub-saharan Africa where malaria is endemic and in other parts of the world, plants are extensively used

for treating periodic fevers and malaria. The spread of multi drug-resistant P. falciparum has highlighted the

urgent need to develop new antimalarial drugs, preferably inexpensive drugs that are affordable for

developing countries, where malaria is prevalent (Miller, 1992; Vial, 1996). About 75% of the population in

Africa do not have direct access to chemical treatment, such as chloroquine, but they have access to

traditional medicine for treating fevers. Treatment with these remedies has suffered a number of deficiencies;

diagnosis is often a problem, identification of plant extracts may be insecure and the chemical content of

extracts may vary considerably (Azas et al., 2002).

In this study, four crude organic extracts obtained from medicinal plants used in Nigerian folk medicine for

the treatment of fever and malaria were tested in vitro against P.falciparum. The most active extract was

obtained from E. chlorantha that showed appreciable inhibition of the parasites at all the concentrations used

in the study. Enantia chlorantha (bark and leaf) which is used for sore treatment, fevers, coughs, vulnerary

ulcer, haemostatic and febrifuge by traditional healers contains alkaloids, lignin, saponins and tannins (Gill

and Akinwunmi, 1986). For M. lucida, dose-dependent inhibitory outcomes were marked. Awe and Makinde,

(1997) reported the dose-dependent and seasonal variation in the activity of M. lucida using both in vitro and

in vivo techniques. M. lucida was reported to contain anthraquinones which showed in vitro activity against

P.falciparum and also possess antifungal properties. Morinda lucida is used locally in the treatment of yellow

fever and jaundice (Guido et al., 1995). The inhibition shown by C. giganteus can be said to be remarkable

because the plants is usually boiled with a mixture of certain other plants in Nigeria for prophylaxis or

traditional chemotherapy of malaria. Occasionally, a few people take it alone. The relatively lower inhibition

observed for the organic extract of Azadirachta indica in this study may correlates earlier findings that A.

indica functions more as an antipyretic than as a schizonticidal agent in malaria therapy (Okpaniyi and

Ezeuku, 1981). It is not clear why the inhibition was 41.3% at 250µg/ml and 500µg/ml. However, because of

the complex nature of biological systems, no kind of test can be expected to function perfectly (Rasoanaivo

et al., 2003[In press])

It is noteworthy that whenever they have been studied, antimalarials such as chloroquine and quinine have

not been found to have useful antipyretic properties (Krishna et al., 1995) contrary to dogma. Reasons to

treat fever (using antipyretics) in malaria include making the patients more comfortable, minimising

metabolic stress of infection and perhaps reducing the risk of convulsions and neurological sequelae in

children (Newton and Krishna, 1998). Treatment with antipyretic agents such as A.indica would lead to early

relief of fever and pyrexia to eliminate the parasite thereby helping the body's immune system. Fever is a

host response associated with schizont rupture and is the most common clinical manifestation of malaria

(Gatton and Cheng, 2002).

16

Kimbi et al., (1998) reported the chemosuppressive and prophylactic activities in mice of the medicinal herbs

used in this present study. Their results showed that the boiled water extracts of C.giganteus and

E.chlorantha have good potentials against chloroquine-resistant P.yoeli nigeriensis both as schizonticidal and

prophylactic agents when compared to artemether. Also, in their study, very little antimalarial activity was

reported for A.indica and M. lucida in the mice. However, Makinde and Obih (1985) reported that the boiled

water extract of A.indica showed schizonticidal activity against chloroquine-sensitive P.berghei. It is

therefore possible that the strain of the parasites or the species accounted for the differences observed. In

addition it is not uncommon that some plants which are popularly used to treat fever or malaria in some areas

may be found to be inactive or toxic in mice (Kimbi et al.,1998; Krettli et al.,2001). One plausible

explanation is the unsuitability of the in vivo rodent malaria models to demonstrate the expected activity.

Truly, no in vitro drug sensitivity test can entirely mimic the in vivo situation, but these in vitro methods

should ideally utilise both uniform drug exposure and a test medium that approximates the in vivo milieu

(Sixsmith et al., 1984). Additional in vivo models may be needed to adequately evaluate these antimalarial

plants (Dow et al., 1999).

Conclusion

Despite the considerable progress in malaria control over the past decade, malaria remains a disease of

priority, particularly in Africa where about 90% of clinical case occur. One of the greatest challenges facing

malaria control worldwide is the spread and intensification of parasite resistance to antimalarial drugs. The

limited number of such drugs has led to increasing difficulties in the development of antimalarial drug policy

and adequate disease management (WHO, 2000).

Medicine, in several developing countries, using local traditions and beliefs, is still the mainstay of health

care (Hoareau and Dasilva, 1999). Africa is a rich source of medicinal plants yet a relatively small number of

drugs against malaria are available today. Although new drugs have appeared in the last 20 years, including

atovaquone, malarone, halofantrine, mefloquine, proguanil, artemisinin derivatives and co-artemether, new

and affordable drugs as well as better formulations are needed (Persidis, 2000). Likewise, the relatively high

cost of new drugs is a major obstacle to their use in resource-poor settings where the burden of malaria is

greatest (Olliaro et al., 1996; Goodman et al., 2000)

Drug resistance to malaria has become one of the most significant threats to human health and the search for

new and effective drugs is urgent (Lisgarten et al., 2002). One of the key challenges in the fight against

malaria is not just to develop effective and safe treatments, but also to make sure they are available to local

governments and people at a price that will allow widespread use. New antimalarials are also needed because

resistance has rapidly been building up against existing treatments. In addition the dilemma that now faces

malaria control authorities arise as a result of the global resistance prevailing against the two most commonly

17

used antimalarial drugs, chloroquine and the antifolate sulphadoxine/pyrimethamine. The challenge ahead

lies in determining the best alternative therapies for use now, the best prospect for drug development,

regulatory approval and use in short term and the establishment of mechanisms and projects to ensure that

improved drugs are sustainably discovered and developed into the future. Continued and sustainable

improvements in antimalarial medicines research and development are essential for the world's future ability

to treat and control malaria (Ridley, 2002).

Recently, progress in chemical analysis presented evidence that complex molecules elaborated by natural

organisms could hardly be synthesized by chemical processes. Moreover, the resistance of P. falciparum to

chemical treatment still remained important (Azas et al., 2002). Hence, natural products isolated from plants

used in traditional medicine, which have potent antiplasmodial action in vitro, represesents potential sources

of new antimalarial drugs (Wright and Phillipson, 1990; Gasquet et al., 1993). Plant materials remain an

important resource to combat serious diseases in the world (Tshibangu et al., 2002) and pharmacognostic

investigations of plants are carried out to find novel drugs or templates for development of new therapeutic

agents (König, 1992). It had been advocated that direct crude drug formulation of the herbs following

toxicological absolution may not only produce dosage forms faster but will also lead to cheaper and more

affordable drugs for the communities that need them (Elujoba, 1998). Also, there is a belief that these

medicines are safe because they are natural (Sofowora, 1993; Willcox et al., 2003. [In press])

The results in this study lend some credence to the use of the active species in traditional medicine in the

treatment of fever and malaria although the potencies of these active extracts would have to be tested and

compared to those of the standard drug test. In addition it is suggested that E. chlorantha and the other three

medicinal plants used in this study which are very popular in Nigerian rural and urban centres are potential

sources of antimalarial agents and should therefore be the subject of further research to study their active

principles or consituents. Finally, since the new concept in malaria chemotherapy is towards combination

therapy, it would be worthwhile to test the effect of the combination of a potent extract such as E.chlorantha

and a chemosuppressive and antipyretic substance like Azadirachta indica. Such plants will be helpful in

Africa where many people cannot afford expensive drugs.

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clinical efficacy of traditional antimalarials. In: Willcox, M.L., Bodeker, G., Rasoanaivo, P (Eds.) Traditional

Medicine, Medicinal Plants and Malaria. London: Taylor and Francis.

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20

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REPORT 3

THE SYNERGISTIC PROPERTIES AND STAGE SPECIFICITY OF ANTIMALARIAL PLANTS

COMMONLY USED IN NIGERIA, WEST AFRICA

Summary

The combination effects of a promising antimalarial plant, Enantia chlorantha and three others commonly

used for traditional treatment of malaria were tested in vitro and evaluated. The results showed that lower

dosages of the aqueous extracts of these plants could be combined to obtain higher efficacy of antimalarial

and fever treatments. In determining the mode of activity, virtually all the extracts inhibited re-invasion of

erythrocytes showing that they possess mechanisms that interfere with the biochemistry of the parasite in

vitro. This study supports the malaria chemoprophylactic concept of using drugs combination and also

reveals the need to adopt such a concept in prospective bid to develop new drugs against P. falciparum

malaria.

21

Introduction

Malaria is still one of the most widespread parasitic disease in the world with an estimated incidence of 300-

500 million clinical cases each year and a corresponding mortality of more than 1.5 million deaths per year

(WHO, 1996a). The vast majority of deaths occur in Africa, south of the Sahara where malaria also presents

major obstacles to social and economic development. Malaria is endemic in Nigeria and the population at

highest risk includes children, pregnant women and the non-immune. Not less than 25% of infant deaths and

20% of maternal mortality cases in Nigeria are attributed to malaria. In addition, more than 65% of Nigeria's

population of 100-120 million people experiences at least one attack of malaria each year (Aderounmu,

1999, M.Sc thesis).

In most African countries, chloroquine remains the only drug recommended by health authorities for the

first-line treatment of uncomplicated malaria. It is also the only antimalarial frequently kept at home and

used for self-treatment (Baird et al., 2002; Kofoed et al., 2002; Thomas et al., 2002; Trape et al., 2002). The

continued use of chloroquine is understandable since the drug is cheap, always available and only associated

with rather minor side effects (Kofoed et al., 2002). Also patients treated with this drug improve clinically,

even though they remain parasitaemic after treatment (Trape et al., 2002). Meanwhile the spread of

chloroquine-resistant P. falciparum through sub-Saharan Africa has become a major obstacle for malaria

control (Dorsey et al., 2000; Kofoed et al., 2002) and chloroquine resistance has been linked to malaria-

specific mortality (Trape et al., 1998).

The dramatic resurgence of malaria in recent decades has renewed interest in the possibility of a vaccine or

perhaps several vaccines to prevent infection or to limit pathology due to malaria (Long, 1993; Good et al.,

1998). In malaria holoendemic areas, clinically protective immunity is acquired during the first 5-10 years of

life so that most of the severe disease in these areas is found in young children. As the parasite develops in

the host erythrocytes, a number of changes takes place including modification of the cell membrane to form

knob-like structures and increasing the rigidity of the cell alterations in metabolite transport and the insertion

of a number of parasite-derived proteins into the surface of the infected erythrocyte membrane. The

importance of these changes is not fully understood but the development of immunity to malaria is thought to

involve responses to the malarial antigens expressed at the surface of infected erythrocytes (Marsh et al.,

1989;: Bull et al., 1999). Until recently these antigens were poorly defined in molecular terms but a

combination of genomic and biological research has begun to address this important area of research, (Craig

and Scherf, 2001).

22

For historical and operational reasons, organised antimalarial vector-control campaigns are absent in most

sub-Saharan African countries. Malaria transmission intensities, therefore, are typically one or two orders of

magnitude greater than those that occur in most other malaria-endemic regions of the world. (Alles et al.,

1998). An understanding of the epidemiology of parasite diversity is thus important both in relation to the

ability of parasites to escape antimalarial drugs as well as the immune system, including future vaccines

(Farnett et al., 2002). The WHO (1993) had stated that the mainstay of malaria control remains early,

effective treatment of clinical cases. Even though the effectiveness of this policy is likely to decrease as

resistance to many available and affordable antimalarial increases (Schellenberg et al., 2002), it is almost

certain that chemotherapy will remain the hallmark of malaria control especially in the absence of a malaria

vaccine.

Materials and methods

Plants combination

The plant extracts were combined as follows:

Enantia chlorantha + Azadirachta indica (E+A)

Enantia chlorantha + Morinda lucida (E+M)

Enantia chlorantha + Cymbopogon giganteus (E+C)

Plant extracts

In this experiment, the aqueous boiled water extracts of the plants were used. For the extraction, about 10g

of each dried powdered extract was boiled in 50ml water until all the water evaporated. The extracts were

thereafter dried by heat application. 10ml PBS (1X) was added to 10mg of each extract making 1mg/ml.

These extracts in their PBS solutions were sterilized by passing them through syringe filters (0.2µm]. Equal

amounts of each organic extracts were mixed and diluted with RPMI 1640 solution, were necessary, to

achieve the desired concentrations in the culture wells.

In vitro inhibition assay for extract combination.

Serial double dilutions of the extracts were made in quadruplicate in 96 well micro titer plates. The extract

solutions were diluted with RPMI 1640 to achieve the required concentration to be tested in culture. Each

well contained 100µl of the diluted extract and 100µl of parasitized red blood cells at 4% haematocrit in

23

MCM (1-2% parasitaemia, non-synchronised). The final combined extract concentrations tested ranged from

31.3-500µg/ml. The micortiter culture plates were incubated at 37oC in the candle jar. At 24 hours, fresh

media with extracts were added and the experiment was terminated at 48 hours. After the incubation period,

parasitized red blood cells were collected in 4ml centrifuge tubes, washed thrice with Tris- Hank solution and

resuspended in 400µL Tris-Hank. The harvested cells were fixed unto 8 well multitest slide using buffer coat

using GDA . Approximately 40 000 cells were counted under fluorescent microscopy after staining with

dilute acridine orange.

Stage specificty experiment:

The parasites in culture were synchronised twice at 4-h interval using sorbitol lysis described previously. At

24hours after first synchronised, the culture was diluted to a parasitaemia of 1% and immediately used in

the set up (96 wells, flat bottom micro-titer plates) for the stage specificity experiment. Each of the extract

(uncombined) was used at a single concentration of 500µg/ml in 6 replicas. This was achieved by adding

100µl of infected (synchronised) culture and 100µl of each extract. The control was untreated and with the

same number of replicas. At specific intervals (18-42h) after treatment, the cells were harvested and fixed in

multitest slides using buffer coating and GDA. At 24 h after first treatment (and after second fixation), the

media were changed in the remaining replicas meaning that fresh MCM-HS was added in the control and

fresh extracts in the tested wells. The first harvest and fixation was at 18 hours and the last one was at 42

hours after first treatment or 54 hours after first synchronisation

Results

The synergistic properties and stage specificity antimalarial activities of the aqueous extracts have been

examined. Tables 1 and 2 and figures 1-5 showed the outcome of the synergistic effects of combining

E.chlorantha and each of the other three antimalarial plants namely, Azadirachta indica, Cypbopogon

giganteus and Morinda lucida at different combined concentrations. Chloroquine was used in this

experiment to compare the efficacy of these extracts. The stage specificity experiment was performed using

only a single concentration of the aqueous extract (500µg|ml) in their uncombined forms. Table 3 showed the

inhibition from 18 to 42 hours after first treatment. The experiment was conducted using six replicas; the

second treatment was carried out after harvesting of the cells for 24hour fixation. Hence it was possible,

using this approach to compare the efficacy of the different forms of the extracts at 500µg/ml, i.e organic and

aqueous extracts as shown in figure 6. Figure 7 shows the chemosuppressive and antiplasmodial effects of

the antimalarial plants on the blood stages of the parasites while figure 8 shows their increase in inhibitory

activity over a 42hour period.

24

Table 2. The final concentration and percentage inhibition±SEM of the various extract combination

Final extract concentration (µg/ml) Percentage inhibition ± SEM

E+A

500 85.2 ± 3.1

250 78.8 ± 3.6

125 47.8 ± 5.6

62.5 24.2 ± 6.9

31.3 17.2 ± 6.2

E+C

500 86.2 ± 1.6

250 70.2 ± 5.1

125 47.8 ± 8.6

62.5 37.7 ± 4.1

31.3 32.7 ± 7.8

E+M

500 71.7 ± 4.6

250 63.0 ± 8.2

125 53.9 ± 9.9

62.5 52.9 ±3.4

31.3 45.8 ± 7.3

CQ*

500 80.1 ± 6.7

250 73.7 ± 3.1

125 68.4 ± 6.1

62.5 63.0 ± 5.4

31.3 55.9±5.77

E+A= E. chlorantha and A. indica: E+C= E.chlorantha and C.giganteus: E+M= E.chlorantha

and M. lucida. CQ = chloroquine *not combined

25

26

0

1020

30

4050

60

7080

90

100

Perc

enta

ge in

hibi

tion

500µg/ml

Figure 1. The synergistic antimalarial activity of aqueous extracts of E.chlorantha and the other medicinal plants

0

10

20

30

40

50

60

70

80

90

100

250µg/ml.

CQ* CQ*

0

10

20

30

40

50

60

70

80

90

100

Perc

enta

ge in

hibi

tion

125µg/ml

CQ*

0

10

20

30

40

50

60

70

80

90

100

62.5µg/ml

.

Chloroquine, uncombined

E.chlorantha+M.lucida

E.chlorantha+C.giganteus

E. chlorantha+A. indica

CQ*

0

10

20

30

40

50

60

70

80

90

100

Perc

enta

ge in

hibi

tion

31.3µg/ml

.

CQ*

keys

27

05

101520253035404550556065707580859095

100

Perc

enta

ge in

hibi

tion

Figure 6.Comparative inhibitory activity of the different forms of extracts at 500µg/ml (24hours only)

A.indica, aqueous extract

A.indica, organic extract

C.giganteus, aqueous extract

C.giganteus, organic extract

M.lucida, aqueous extract

M.lucida, organic extract

E.chlorantha, aqueous extract

E.chlorantha, organic extract.

Keys

Table 3. Stage specificity: The inhibition of parasite growth by the aqueous extracts of the 4 medicinal

plants

Time (hours)

After 1st Sync. After treatment Percentage inhibition (%)

EC ML CG A1

30 18 36.0 32.0 36.0 40.0

36 24* 54.0 60.0 52.1 75.4

42 30 75.4 67.7 73.8 79.6

48 36 78.4 79.6 83.8 77.4

54 42 91.7 84.2 92.5 95.0

EC=E.chlorantha, ML=M.lucida, CG=C.giganteus, AI=A.indica. *2nd treatment after fixation of second

replicas.

28

29

05

101520253035404550

# of

par

asit

es/2

0 00

0 ce

lls

18 24 30 36 42

Time after Ist treatment(h)

(a)E.chlorantha

Figure 7(a)-(e). The stage specificity of the aqueous extracts of thevarious medicinal extracts.

05

101520253035404550

# of

par

asit

es/2

0 00

0 ce

lls

18 24 30 36 42

Time after 1st treatment(h)

(b)M.lucida

05

101520253035404550

# of

par

asit

es/2

0 00

0 ce

lls

18 24 30 36 42Time after 1st treatment(h)

(c) C. giganteus

05

101520253035404550

# of

par

asit

es/2

0 00

0 ce

lls

18 24 30 36 42

Time after 1st treatment(h).

(d) A.indica

0102030405060708090

100110120130140150

# of

par

asit

es/ 2

0 00

0 ce

lls

18 24 30 36 42

Time(h)

(e)Untreated culture(control)

SCHIZONTS

TROPHOZOITES

RINGS

KEYS

7. DISCUSSION

Drug resistance is a serious global problem making treatment increasingly difficult and the main obstacle to

malaria control is the emergence of drug resistant strains of P.falciparum. The strategy of drugs combination

couple with early detection and confirmed diagnosis represent the only way forward in the chemotherapy of

malaria (Nosten and Brasseur, 2002). This will prevent or delay the emergence and spread of drug resistance

and also interrupt the transmission of P. falciparum.

30

0 10 20 30 40 50 60 70 80 90 100

Figure 8. Stage specificity: Antimalarial activity of aqueous extracts at 500µg/mlshowing increasing inhibition over a period of time in test cultures

18

24

30

36

42

Tim

e in

cul

ture

aft

er 1

st tr

eatm

ent

Percentage inhibition

A.indica

C. giganteus

M.lucida

E.chlorantha

In this study, the synergistic antimalarial activity / combination effects of boiled water extracts of four

medicinal plants was observed at 48 hours. The antimalarial activity of the organic extracts of these plants

has been previously described. Treatment was repeated at 24hours after first treatment and the medium in the

control was also replenished. All the combinations used showed significant but dose dependent inhibitory

activity (Figures 1-5). These data are interesting because the combination effects or synergism permits the

quantity of each extract to be reduced with higher efficacy which is comparable to the use of chloroquine

alone. However at 31.3-125µg/ml, chloroquine showed a higher inhibitory activity than all the combinations

of extracts used. Perhaps, at suboptimal levels, the extracts failed to show synergism even though the final

extracts concentrations equates that of chloroquine. Since, the extractions are crude, their activities might

have be devoid of the enhance efficacy that their isolated active principle (as in chloroquine) would have

probably exerted at same concentrations. Nevertheless, considering the spread of resistance to chloroquine,

which is the cheapest available antimalarial drug to date, the combination of active principles in drug

research approaches using plant extracts or natural products will go a long way in tackling issues related new

and cheap drugs that will improve cure rates, delay emergence of resistance and reduce transmission.

Drugs with different mechanisms of actions may enhance their respective efficacies and extend their

therapeutic life span. This is a major public health consideration for developing countries whose needs for

effective (and new) drugs is pressing (Taylor et al.,2001). In traditional settings, healers normally use

decoctions to cure patients. Currents trends in malaria chemotherapy suggest a drift towards drug

combination therapy. Drug combination approach has been adopted in cancer chemotherapy and in the

treatment of infection with human immunodeficiency virus, tuberculosis, leprosy and Pseudomonas infection

(Tjitra et al., 2001). In malaria, the combination of artesunate and mefloquine is highly effective against

multi-drug resistant P. falciparum (Nosten et al.,2000). Even though no toxicity test has yet been performed,

the general and traditional use of these plants in rural and urban areas across West Africa suggest that no

highly toxic components are present in these extracts. Pre-clinical toxicity testing is only required for new

medicinal herbal products which contain herbs with no traditional history of use (WHO1996b; 1998). It

would be desirable to determine the IC50 of the active principles, which obviously would be more active,

when isolated.

Usually, the end point for assessing drug sensitivity is parasite reinvasion of erythrocytes (Trigg, 1985).

Inhibition of parasite invasion is due either to inhibition of the release of progeny merozoites or to inhibition

of merozoite invasion of erythrocytes. The level of parasitaemia after merozoite release from erythrocytes

containing schizont-stage parasites and subsequent invasion of new erythrocytes was less in all test cultures

(with extracts) than in the untreated (control) cultures. Figure 7(a)-(e) showed the chemosuppressive and

antiplasmodial effects of the aqueous extracts of the various medicinal plants. Using an untreated culture as

the control, it was possible to show that the culture adpated parasites were sensitive to the plant extracts and

31

chloroquine because of the inhibition of parasite re-invasion and appreciable degree of schizonticidal effects

as evident in test cultures A.indica and E.chlorantha at 42hours. In all test cultures, growth suppression was

pronounced as shown by the increase in inhibition over time (figure 8). This stage specificity experiment

provided an opportunity to make a comparative analysis of the efficacy of the plant extracts at 24 hours with

respect to the organic extract used in the previous experiment. It was observed that all the medicinal plants

have higher inhibitory activity as organic extracts at same concentration of 500µg/ml except A.indica that

showed that showed a remarkable inhibition of 75.4% compared to 41.3% as organic extract. It is possible

that the solubility of the active ingredients of these plants differ based on the mechanisms of extraction.

Another possible factor that may have influenced this variability is that the culture was synchronised in the

stage-specificity experiment.

Investigations have been carried out on A.indica with a view to finding some scientific evidence for its use in

traditional medicine. This plant has been shown to possess a steam-volatile, oily constituent in trace

amounts, which showed one component common to leaf, stem and root bark when examined

chromatographically (Sofowora, 1978-unpublished work cited in Sofowora, 1993). Ekanem (1978),

Aladesanmi et al., (1988) and Kimbi et al., (1998) have reported the antimalarial effect of this plants on both

chloroquine sensitive and multidrug resistant strains of P.falciparum. In this study, even though all stages of

the parasites were still found at 42 hours (inhibition 95%), the schizonticidal effect of A.indica was marked

(figure 7d)

Morinda lucida showed a seemingly comparative but higher inhibition as organic over aqueous extracts. This

correlated with the earlier findings that organic extract of M.lucida demonstrated higher antimalarial

(schizoticidal) activity than the aqueous extract (Awe and Makinde, 1997). This plant is popularly known for

its use against fever within the West Africa communities because of its schizonticidal activity (Irvine 1962;

Burkill, 1985) and a number of compounds have been isolated from it including anthraquinones (Adesogan,

1979). At 42hhours in this experiment M.lucida showed parasite inhibition of 84.2% and was more

chemosuppressive than schizonticidal when compared to the control culture (figures 7b & e). Awe and

Makinde (1997) suggested that the organic fraction of M.lucida could be further studied in order to isolate

the antimalarial active principle in it as a promising medicinal plant.

The leaves of C. giganteus contains volatile oil, hesperidin bitter principles, and is used as flavouring agent,

stimulant and anti-pyretic (Tyler et al., 1988). This plant, generally regarded as a fever-reducing herb, is also

antiplasmodic as shown here. It has schizonticidal effect and had shown 92.5% inhibition on chloroquine

sensitive P.falciparum at 42 hours (figure 7c). It has also been shown to have good potentials against

chloroquine-resistant P.yoelii nigeriensis both as schizonticidal and prophylactic agents even when compared

to artemether (Kimbi et al., 1998).

32

E.chlorantha showed a 91.7% inhibition of parasite growth at 42 hours (figure7a) and higher schizonticidal

effects was observed compared to M.lucida and C.giganteus. This plant also contains alkaloid., which are

antiprotozoal even at low concentrations. Like C.giganteus, it has also been reported to have good potentials

against chloroquine-resistant rodent malaria parasite in vivo. In rural, periurban and urban settings of West

Africa, it is not uncommon that E.chlorantha is usually administered as alcoholic tinctures. The finding here

that it has a higher antimalarial activity as an organic extracts supports its common traditional form of usage.

Also, most other medicinal plants have specific ways in which they are utilised in traditional prophylaxis and

chemotherapy of malaria. These observations are important in understanding how traditional healers have

safely administered treatment successfully from one generation to another. Their roles and contributions

would remain crucial in the knowledge of extracting or isolating the active substances embedded in

medicinal plants.

Conclusions

A global resurgence of malaria began around 1970 and continues with gaining momentum. (Krogstad, 1996).

The dominant factors responsible are the proliferation of resistance to standard antimalarial drugs and the

social reluctance to apply residual insecticides (Baird, 2000). DDT and chloroquine eradicated malaria or

controlled malaria across vast reaches of the globe and the loss of these weapons without practical

alternatives has left those areas susceptible to encroachment and reestablishment of endemic malaria. The

most worrisome aspect of the encroachment of endemic malaria is the lack of infrastructure and implements

with which to gain control (Barcus et al., 2002).

As one of the most important parasitic diseases of mankind, malaria causes considerable morbidity and

mortality in the developing world and there is a clear need for new treatments, particularly as resistance to

available drugs is increasing (Olliaro and Trigg, 1995). The life span of currently used antimalarial drugs are

been extended by combination therapy. However, it is crucial that this implementation is accompanied by

close monitoring of the impact, including malaria-specific and all-cause mortality, side effects of drugs,

treatment seeking behaviour and parasite sensitivity to drugs (Trape et al., 2002). Nevertheless, new efficient

antimalarial treatment, in particular drugs combination, which are well-tolerated and simple to use are

urgently needed (Lefevre et al., 2001).

Herbal remedies for malaria are already in popular use in developing countries especially due to lack of

access to effective, cheap, safe and user-friendly medicines and many (of these herbal medicines) have been

shown to have antiplasmodial activities in experimental studies (Willcox et al., 2003[In press]). The plants

used in this study in form of aqueous extracts remain effective as antimalarial treatments for fever and

malaria. The combination of two or more of the extract leads to higher efficiency in terms of antimalarial

activity while at the same time reducing the dosage of each extract required to achieve such. With due

33

consideration to dosage administration to minimise toxicity and other side effects, these plants are effective

for combination therapy, especially in the forms for which they are used locally. Subsequent to the high

population, a high number of deaths occur from malaria in Nigeria. The actions to be taken include

prevention efforts and research; search for new drugs or drug discovery ought to be an integral part of the

research and development especially as the clues for an effective and licensed vaccines remain elusive.

Artemisinin (qinghaosu) had been in use in China for many centuries before the Chinese scientists isolated

the active principle from the plant, Artemisia annua, in 1972 (Ringwald et al., 1999). There are numerous

traditional antimalarial plants in Nigeria and West Africa as a whole. It will not be out of context to suggest

that a few of these plants will provide alternatives and posible replacements for the current antimalarial in

routine use. Conclusively therefore, in view of the problems of inappropriate administration of dosage and

insecurity due to toxicity, it is important that more chemotherapeutic research against malaria parasites be

investigated especially with the commonly used plants known for their antiparasitic activities.

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