ARTICLE IN PRESS

Phytomedicine 13 (2006) 295298

www.elsevier.de/phymed

SHORT COMMUNICATION

In vitro cytotoxicity studies of 20 plants used in

Nigerian antimalarial ethnomedicine
E.O. Ajaiyeobaa,, O.O. Abiodunb, M.O. Faladec, N.O. Ogbolea, J.S. Ashidia,
C.T. Happid, D.O. Akinboyec
a

Faculty of Pharmacy, Department of Pharmacognosy, University of Ibadan, Ibadan, Nigeria

Department of Pharmacology, University of Ibadan, Ibadan, Nigeria
c
Department of Zoology, University of Ibadan, Ibadan, Nigeria
d
Malaria Research Group, Institute of Advanced Medical Research & Training, University of Ibadan, Ibadan, Nigeria
b

Abstract
Twenty plants identied and selected from Southwest and Middle belt Nigerian antimalarial ethnopharmacology
were evaluated for in vitro cytotoxicity using the brine shrimp lethality assay. The methanol extracts of 20 plant
samples from 11 plant families were subjected to the assay. Of the studied plants, Lippia multiflora and Morinda lucida
bark were found to be cytotoxic, with LC50 values of 1.1 and 2.6 mg/ml, respectively. The least toxic plant extract was
Bridelia micrantha (LC50 value 49.0
106 mg/ml). Most of the plants were found to be relatively non-toxic.
r 2005 Elsevier GmbH. All rights reserved.
Keywords: Cytotoxicity; Plants; Nigeria; Antimalarial ethnomedicine

Introduction
Ethnomedicine has no doubt played a central role in
the search for and development of new drugs (Kirby,
1997; Heinrich, 2000). Tropical rainforest plants are
known to have higher concentrations and a greater
diversity of chemical defenses than plants from any
other biome and are also a potential source of new
medicines (Balick et al., 1996). It is well-known that in
ethnopharmacology and natural products chemistry, the
modes of preparation and administration of herbal
preparations are often crucial variables in determining
efcacy in pharmacological evaluations (Lewis et al.,
1998).

Corresponding author. Tel.: +234 2 8101100 04;

fax: +234 2 8103043.
E-mail address: edajaiye@yahoo.com (E.O. Ajaiyeoba).

0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2005.01.015

Simple bioassays developed for screening plant

extracts to detect plant compounds with relevant
biological activities may be used as a guide for
fractionating plant extracts. The eggs of the brine
shrimp, Artemia salina, have been used in the Brine
Shrimp lethality (BSL) assay, a simple bench top
bioassay which has yielded good results. A. salina, with
same purine metabolism as that of mammalian cells, has
been shown to have a good correlation with antitumor
activity, although drugs that require metabolic activation in the liver may not be detected by A. salina. The
DNA-dependent RNA polymerases of A. salina are also
similar to the mammalian type (McLaughlin, 1991; Solis
et al., 1993).
An ethnographic evaluation of ethnomedicine in
Nigeria revealed that many herbal remedies have been
used traditionally for treatment of febrile illnesses
(Ajaiyeoba et al., 2003). These plants are taken orally
without any consideration of the toxicity of components

ARTICLE IN PRESS
296

E.O. Ajaiyeoba et al. / Phytomedicine 13 (2006) 295298

in such plants. As part of our interest in the efcacy and

safety of Nigerian antimalarial traditional herbal
remedies, we have evaluated 20 plants for in vitro brine
shrimp cytotoxicitiy using the BSL assay. This was done
in an attempt to evaluate efcacious plants for toxic
properties and prioritize plants that are found bioactive
in the BSL assay, with the main objective of selecting
plants with impressive antiplasmodial selectivity.

Materials and methods

Plant collection and authentication
The plant materials were collected based on information from ethnobotanical surveys of plants used in the
treatment of fevers in Southwest (SW) and Middle belt
(MB) Nigeria. The plants were collected from Oyo and
Otu (SW), in January 2000, and from Kastina-Ala and
Gboko in Benue State (MB) in October 2000. Plants
were authenticated by plant taxonomists at the Forestry
Research Institute of Nigeria (FRIN), Ibadan, and the
Department of Botany, University of Ibadan (UI),
where voucher specimens were deposited.

Plant extraction
The plant materials were air-dried and powdered with
a hammer mill and extracted into 90% aqueous
methanol at room temperature (29 1C) by maceration
for 72 h. After removal of solvent, percentage yields
were calculated and plant extracts were stored in the
refrigerator until use.

Brine shrimp lethality assay

Brine shrimp eggs (A. salina) were obtained from Prof
A. Ogundaini, Department of Pharmaceutical Chemistry, Obafemi Awolowo University (OAU), Ile-Ife. They
were hatched in natural seawater obtained from the Bar
Beach, Ikoyi, Lagos, Nigeria, and incubated for 48 h in
3.8 g/l seawater. After hatching, the nauplii were
collected and treated with selected concentrations (ve
dilutions, 105000 mg/ml) of plant extracts and chloroquine phosphate, (a reference antimalarial drug, also
obtained from Sigma Chemical Co., UK). The method
used was that of McLaughlin (1991).

Data analysis
The 50% lethal concentration (LC50 value) at 95%
condence interval was calculated for each plant
methanol extract, using a non-linear regression curve,
using the Graph pad prism statistical software.

Results
The 20 plants screened in the BSL assay, their full
names and the percentage yield of the methanol extracts
are listed in Table 1. Yields of extracts ranged from
4.2% to 14.4%. The plant materials, arranged in
alphabetical order, with respect to plant families,
consisted of 11 families. Leaf samples accounted for
65% (13) of the samples, while stem bark was 30%. In
only one of the listed plants (Lippia multiflora) were the
aerial parts (leave and young stems) used. The plants
studied were identied from the SW and MB zones of
Nigeria. Fifty-ve percent (11) of the plants identied
were found solely in SW, 25% (ve) in MB and 20% in
both ethnobotanies. In a few cases, either leaf or stem
bark of the same plant was mentioned as a single herbal
ingredient in the regions. Table 1 also shows the in vitro
cytotoxicity of the study plants in the BSL assay,
expressed as LC50 values, alongside the condence limits
at 95% interval. The cytotoxicity values ranged from 1.1
to 986,663 mg/ml.

Discussion
Most of these plants have not yet been assessed for in
vitro cytotoxicity, apart from Gossypol, a major
constituent of Gossypium sp., and Tithonia diversifolia,
which have shown cytotoxic properties (Coyle et al.,
1994; Wu et al., 2001). A lot of work has been reported
on the phytochemical and other biological properties of
many of the listed plants. These include sesquiterpene
lactones as well as an artemesinin acid analog from T.
diversifolia (Kuo and Chen, 1998; Bordoloi et al., 1996)
and avonoids from Cajanus cajan and Erythrina sp. An
antifungal compound, Cajanin, had also been reported
from C. cajan, in addition to stilbene derivatives from
the leaves (Fomum et al., 1994; Ohwaki et al., 1993;
Waffo et al., 2000). Morinda lucida stem bark, a known
traditional antimalarial in the West African subregion,
has been reported to contain anthraquinones and an
unidentied antineoplastic agent (Koumaglo et al.,
1992; Durodola, 1974). The presence of the antineoplastic compound in the stem bark of M. lucida might
have been responsible for the observed cytotoxicity.
Cassia siamea is reported to contain benzopyranone
derivatives, anthraquinones and bianthraquinones
(Teeyapant et al., 1998; Ingkaninam et al., 2000; Lu et
al., 2001; Koyama et al., 2001). Prenylated isoavonoids
have been isolated from Enantia chlorantha and
chromones have been isolated from Piliostigma thonningii (Ibewuike et al., 1996; Oh et al., 1999).
M. lucida stem bark and leaves were two of the herbal
ingredients mentioned in the ethnomedicine of SW
Nigeria. It is interesting to note that while the leaves

ARTICLE IN PRESS
E.O. Ajaiyeoba et al. / Phytomedicine 13 (2006) 295298

Table 1.

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
a

297

Brine shrimp lethality activities of 20 plants from Nigerian antimalarial ethnomedicine

Plant familya

Plant/Drugb

%Yield

LC50 value (mg/ml)

95% Interval

Anacardiaceae (A)
Anacardiaceae (A)
Annonaceae (B)
Annonaceae (A,B)
Asteraceae (A)
Combretaceae (B)
Euphorbiaceae (B)
Fabaceae (A)
Fabaceae (A)
Fabaceae (A)
Fabaceae (A)
Malvaceae (A)
Malvaceae (A)
Malvaceae (A)
Myrtaceae (A)
Papilionaceae (B)
Rubiaceae (A,B)
Rubiaceae (A,B)
Rubiaceae (A,B)
Verbanaceae (A)
Chloroquine diphosphate

Mangifera indica L. (sb)

M. indica L. (l)
Annona senegalensis Rolyns &Gh (l)
Enantia chlorantha Oliv. (sb)
Tithonia diversifolia A.Gray (l)
Terminalia latifolia Engl. (l)
Bridelia micrantha Benth. (l)
Cajanus cajan Millsp. (l)
Cassia siamea Lam.(sb)
C. siamea Lam. (l)
Piliostigma thonnigii Schum (l)
Gossypium arboreum L. (l)
G. barbadense L. (l)
G. hirsitum L. (l)
Psidium guajava L. (sb)
Pericopsis elata Harms (l)
Morinda lucida Benth. (sb)
M. lucida Benth. (l)
Nauclea latifolia S.M. (sb)
Lippia multiflora Moldenke (ap)

9.4
14.2
12.8
7.8
8.9
14.4
5.1
4.1
11.5
2.8
11.5
6.2
12.3
11.0
12.6
10.9
7.5
9.7
6.1
9.8

2456.0
3079.1
6811.0
214.3
2304
272.9
4 90,000
988.5
808.8
8232.2
7958.0
94.1
3585.0
257.2
707.2
601.8
2.6
383.9
9368.0
1.1
449.1

26.6228840
347.627284
18.925010
42.71077
117.745159
2.530326

200.94863
480.81361
1412.047981
41,000,000
32.5272
1003.012819
23.42823
273.51829
162.22233
0.88.4
59.42481
223.570115

79.4-2539

A, Southwest ethnobotany; B, Middle belt ethnobotany.

sb, bark; l, Leaf; ap, aerial part.

were relatively non-toxic (LC50 value 383.9 mg/ml),

the bark extract was extremely toxic with an LC50 value
of 2.6 mg/ml. This conrms the earlier report of a
Nigerian stem bark sample that displayed antineoplastic
properties (Durodola, 1974). Similarly, C. siamea bark
and leaf samples were identied from the ethnomedicine
and the bark extract was ten-fold more active than the
leaf extract, as shown in Table 1. Constituents from the
bark extracts have been found to be mainly benzopyranones and anthraquinones, while stilbene derivatives
have been reported from the leaves (Teeyapant et al.,
1998; Ingkaninam et al., 2000). The presence of the
stilbene derivatives might account for the observed
cytotoxcity of the stem bark extract of C. siamea.
This is the rst report of cytotoxicity from constituents of L. multiflora (Verbenaceae), and the most toxic
of the 20 plants studied (LC50 value 1.1 mg/ml). L.
multiflora is a traditional antimalarial, hypotensive,
antiseptic and diuretic, and it is also known as bush
tea, a popular breakfast beverage (Pelissier et al., 1994).
Carvacrol isolated in the hexane leaf extract showed
antimicrobial activity (Kunle et al., 2003). The essential
oil from the leaves has demonstrated pediculocidal and
scabicidal properties against body lice, head and scabies
mites in a randomized, controlled clinical trial. The
main components in the leaf oil were terpineol, a-pinene
and b-pinene (Oladimeji et al., 2000). The components
of the essential oil of L. multiflora leaves, which include
(Z)-tagetone and (E)- tagetone, could be responsible for

the observed cytotoxicity. Orally, the use of L. multiflora

as an antimalarial aqueous infusion, used in the Yoruba
ethnomedicine, in SW Nigeria, does not seem to be
cytotoxic. This seems to be reinforced by its use as bush
tea in most parts of Africa (Abegaz et al., 1998). Caution
should be exercised in the use of the other plant
preparations until exhaustive phytochemical and bioassay-guided fractionation of the components have been
achieved. To determine the safety of the plant, extensive
toxicological studies in vitro and in vivo animal assays,
must be undertaken before drug development. The
selectivity of these extracts to malaria parasites in
relation to their cytotoxic properties is being investigated in detail in order to ascertain their suitability as
antimalarial drugs.
The results reported here not only provide an insight
into the toxic nature of some of the extracts used
traditionally for the prevention and treatment of
malaria in tropical Africa, but also provided an
opportunity for the selection of bioactive extracts for
initial fractionation and further studies in antimalarial
assays.

Acknowledgments
The authors are thankful to WHO/TDR/MIM
Africa for nancial support of part of this work and

ARTICLE IN PRESS
298

E.O. Ajaiyeoba et al. / Phytomedicine 13 (2006) 295298

to Prof A. Ogundaini, OAU, Ile-Ife, for the provision of

brine shrimp eggs.

References
Abegaz, B.M., Connoly, J.D., Nkunya, M.H.H., 1998. African
genetic resources for the development of pharmaceuticals
and agrochemicals. In: Proceedings of the Seventh Symposium on Natural Ptroducts, NAPRECA, Dar-Es-Salaam,
Tanzania.
Ajaiyeoba, E.O., Oladepo, O., Fawole, O.I., Bolaji, O.M.,
Akinboye, D.O., Ogundahunsi, O.A.T., Falade, C.O.,
Gbotosho, G.O., Itiola, O.A., Happi, T.C., Ebong, O.O.,
Ononiwu, I.M., Osowole, O.S., Oduola, O.O., Ashidi, J.S.,
Oduola, A.M.J., 2003. Cultural categorization of febrile
illnesses in correlation with herbal remedies for treatment in
Southwestern Nigeria. J. Ethnopharmacol. 85, 179185.
Balick, M.J., Elizabetsky, E., Laird, S.A., 1996. Medicinal
Resources of the Tropical Rain Forest. Columbia University Press, New York.
Bordoloi, M., Barua, N.C., Ghosi, A.C., 1996. An artemesinic
acid analogue from Tithoniadiversifolia. Phytochemistry 41,
557559.
Coyle, T., Levante, S., Shetler, M., Wineld, J., 1994. In vitro
and in vivo cytotoxicity of gossypol against central nervous
system tumor cell lines. J. Neuro Oncol. 19, 2535.
Durodola, J.I., 1974. Antineoplastic property of a crystalline
compound extracted from Morinda lucida. Planta Med. 26,
208211.
Fomum, Z.T., Koch, M., Seguin, E., Tillequin, F., Wandji, H.,
1994. Two isoavones from Erythrina senegalensis. Phytochemistry 35, 245248.
Heinrich, M., 2000. Ethnobotany and its role in drug
development. Phytother. Res. 14, 478488.
Ibewuike, J.C., Ogundaini, A.O., Ogungbamila, F.O., Martin,
M.T., Gallard, J.F., Bohlin, L., Pais, Y., 1996. Piliostigmin,
a 2-phenoxychromone and c-methyl avanols from Piliostigma thonningii. Phytochemistry 43, 687690.
Ingkaninam, K., Ijzerman, A.P., Verpoorte, R., 2000. Luteolin, a compound with adenosine A receptor binding activity
and chromone and dihydronaphthalenone constituents
from Senna siamea. J. Nat. Prod. 63, 315317.
Kirby, G.C., 1997. Plants as sources of antimalarial components. Trop. Doctor 27, 711.
Koumaglo, K., Gbeassor, M., Nikabu, O., De Souza, C.,
Werner, W., 1992. Effects of three compounds extracted
from Morinda lucida. Planta Med. 58, 533534.
Koyama, J., Morita, I., Tagahara, K., Aqil, M., 2001.
Bianthraquinones from Cassia siamea. Phytochemistry 56,
849851.

Kunle, O., Okogun, J., Egamana, E., Emojevwe, E., Shok, M.,
2003. Antimicrobial activity of various extracts and
carvacrol from Lippia multiflora leaf extract. Phytomedicine
10, 5961.
Kuo, Y.H., Chen, C.H., 1998. Sesquiterpenes from the leaves
of Tithonia diversifolia. J. Nat. Prod. 61, 827828.
Lewis, W., Mutchler, D., Castro, N., Elvin-Lewis, M.,
Farnsworth, N., 1998. Ethnomedicine, Chemistry and
Biological Activity of South American Plants. Chapman
& Hall, London.
Lu, T., Yi, Y., Mao, C., Zhou, D., Xu, Q., Tang, H., Zhang,
S., 2001. Studies on the anthraquinones of Cassia siamea.
Yaoxue Xuebao 36, 547548.
McLaughlin, J.L., 1991. Crown gall tumors on Potato disc and
brine shrimp lethality. Two simple bioassays for higher
plant screening and fractionation. In: Hostettmann, K.
(Ed.), Assays for Bioactivity, vol. 1. Academic Press,
London.
Oh, W.K., Lee, H.S., Ahn, S.C., Ahn, J.S., Mbafor, J.T.,
Wandji, J., Fomum, Z.T., Chang, H.K., Kim, Y.H., 1999.
Prenylated isofalvonoids from Erythrina senegalensis. Phytochemistry 51, 11471150.
Ohwaki, Y., Ogino, J., Shibano, J., 1993. 3-Hydroxy-5methoxystilbene-2-carboxylic acid, a phytotoxic compound isolated from methanolic extract of pigeon pea
(Cajanus cajan Millsp.) leaves. Soil Sci. Plant Nutr. 39,
5561.
Oladimeji, R.A., Oradiya, O.O., Ogunniyi, T.A., Adewunmi,
T.A., 2000. Pediculocidal and scabicidal properties of
Lippia multiflora oil. J. Ethnopharmacol. 72, 305311.
Pelissier, Y., Marion, C., Casadebag, J., Milhau, M., Kone,
D., Loukou, G., Nanga, Y., Besseiere, J.M., 1994. A
chemical, bacteriological, toxicological and clinical study of
the essential oil of Lippia multiflora Mold. (Verbenaceae). J.
Essen. Oil Res. 6, 623630.
Solis, P.N., Wright, C.W., Anderson, M., Gupta, M.P.,
Phillipson, J.D., 1993. A microwell cytotoxicitiy assay
using Artemia salina (brine shrimp). Planta Med. 26,
250252.
Teeyapant, R., Srikun, O., Wray, V., Witte, L., 1998. Chemical
investigation of anhydrobarakol from Cassia siamea.
Fitoterapia 69, 475476.
Waffo, A.K., Azebaze, G.A., Nkengfack, A.E., Fomum,
Z.T., Meyer, M., Bodo, B., van Heerden, F.R., 2000.
Indicanines B and C, two isoavonoid derivatives from
the root bark of Erythrina indica. Phytochemistry 53,
981985.
Wu, T.S., Shi, L.S., Kuo, P.C., Leu, Y.L., Meei, J., Wu, P.N.,
Wu, Y.C., Iou, S.C., Chen, Y.P., Hsien, C., 2001. Cytotoxic
principles from leaves of Tithonia diversifolia. Chin.
Pharmaceut. J. 53, 217223.