Journal of Ethnopharmacology 111 (2007) 537540

Antimalaria activity of ethanolic extract of Tetrapleura tetraptera fruit

Jude E. Okokon a, , Aniekan E. Udokpoh b , Bassey S. Antia c
aPharmacology and Toxicology Department, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria
b Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria
c Department of Chemistry, University of Uyo, Uyo, Nigeria

Received 18 September 2006; received in revised form 8 December 2006; accepted 19 December 2006
Available online 28 December 2006

Abstract
The in vivo antiplasmodial activity of the ethanol fruit extract of Tetrapleura tetraptera used as spice and in the treatment of various ailment
in Niger Delta region of Nigeria was evaluated in Plasmodium berghei infected mice. Tetrapleura tetraptera (300900 mg/kg day) exhibited
significant (P < 0.05) blood schizonticidal activity both in 4-day early infection test and in established infection with a considerable mean survival
time comparable to that of the standard drug, chloroquine, 5 mg/kg day. The fruit extract possesses significant (P < 0.05) antiplasmodial activity
with may have contributed to the immune status of the Nigerians against malaria in addition to its nutritive value.
2007 Published by Elsevier Ireland Ltd.

Keywords: Antiplasmodial; Antimalarial; Tetrapleura tetraptera; Plasmodium berghei; Malaria

1. Introduction 2. Materials and methods

Tetrapleura tetraptera (Taub) (family fabaceae) is a peren- 2.1. Plant material

nial tree that is naturally distributed over a large part of tropical
Africa, especially in the rain forest belt of West, Central and
East Africa. The four winged fruit with a fragrant, character-
istically pungent aromatic odour is use in folkloric medicine
for the treatment of various diseases and as spice in the
preparation of varieties of white soups by Efiks and Ibibios
of Niger Delta region of Nigeria. The fruits which possess
insect repellent property have been reported to be nutritional,
molluscicidal, anticonvulsant, analgesic, antiinflammatory and
antidiabetic (Adewunmi and Sofowora, 1980; Adewunmi and
Marquis, 1981, 1987; Adewunmi, 1984; Adesina and Reisch,
1985; Nwaiwu and Akah, 1986; Ojewole and Adewunmi, 2004;
Ojewole, 2005). The present study was designed to evaluate the
claims by some traditional health practitioners from Ibibio tribe
in Niger Delta region of Nigeria that teas and decoctions of the
Tetrapleura tetraptera fruits are useful remedies for malarial
fever.
Fresh, ripe fruits of Tetrapleura tetraptera were procured at
Uyo main market, Uyo Akwa Ibom State of Nigeria in June,
2006 and authenticated by Dr Margaret Bassey, a taxonomist
in the Department of Botany, University of Uyo, Uyo, Nigeria.
A voucher specimen has been deposited in the Faculty of Phar-
macy Hebarium, University of Uyo, Uyo (UUH 321). The plant
materials were air dried at room temperature and then powdered.
2.2. Preparation of extract
The dried and powdered fruit of Tetrapleura tetraptera
(<1 kg) was exhaustively macerated in 70% ethanol for 72 h.
The liquid extract obtained was concentrated in vaccum at 40 C.
The yield was 0.48%. The extract was stored in a refrigerator
at 4 C until used for experiment reported in this study. The dry
ethanolic extract was dissolved in distilled water to make the
stock solution from which the various doses administered were
prepared for use by serial dilution.

2.3. Animals

Corresponding author. Tel.: +234 802 3453678. Albino swiss mice (2128 g) of either sex were obtained from
E-mail address: judeefiom@yahoo.com (J.E. Okokon). the University of Uyo animal house. They were maintained on

0378-8741/$ see front matter 2007 Published by Elsevier Ireland Ltd.

doi:10.1016/j.jep.2006.12.030
538 J.E. Okokon et al. / Journal of Ethnopharmacology 111 (2007) 537540
standard animal pellets and water ad libitum. Permission and
approval for animal studies were obtained from the College of
Health Sciences Animal Ethics committee, University of Uyo.
2.4. Parasite inoculation
The chloroquine-sensitive Plasmodium berghei berghei was
obtained from National Institute of Medical Research, Lagos,
Nigeria and maintained in mice. The inoculum consisted of
5 107 Plasmodium berghei berghei parasitized erythrocytes
per millilitre. This was prepared by determining both the per-
centage parasitaemia and the erythrocytes count of the donor
mouse and diluting the blood with isotonic saline in proportions
indicated by both determinations. Each mouse was inoculated
on day 0, intraperitoneally, with 0.2 mL of infected blood con-
taining about 1 107 Plasmodium berghei beghei parasitized
red blood cells.
2.5. Determination of LD50
The LD50 of the extract was determined using albino mice
by intraperitoneal (i.p.) route using the method of Lorke (1983).
2.6. Evaluation of schizontocidal activity on early infection
(4-day test)
Schizontocidal activity of the extract was evaluated using the
method described by Knight and Peters (1980). Each mouse
was inoculated on the first day (day 0), intraperitoneally, with
0.2 ml of infected blood containing about 1 107 Plasmod-
ium berghei berghei parasitized erythrocytes. The animals were
divided into five groups of five mice each and orally adminis-
tered, shortly after inoculation with 300, 600 and 900 mg/kg day
doses of the Tetrapleura tetraptera fruit extract (TTE), chloro-
quine 5 mg/kg day and an equivalent volume of distilled water
(negative control) for four consecutive days (days 03). On the
fifth day (day 4), thin films were made from the tail blood of each
mouse and the parasitaemia level was determined by counting
the number of parasitised erythrocytes out of 200 erythrocytes
in random fields of the microscope. Average percentage chemo-
suppression was calculated as 100((A B)/A), where A is the
average percentage parasitaemia in the negative control group
and B, average percentage parasitaemia in the test group.
2.7. Evaluation of schizontocidal activity on established
infection (curative or Rane test)
Evaluation of curative potential of the extract was done using
a method similar to that described by Ryley and Peters (1970).
The mice were injected intraperitoneally with standard inoculum
of 1 107 Plasmodium berghei berghei infected erythrocytes
on the first day (day 0). Seventy-two hours later, the mice were
divided into five groups of five mice each. The groups were orally
administered with Tetrapleura tetraptera fruit extract (TTE)
(300, 600 and 900 mg/kg day), chloroquine (5 mg/kg) was given
to the positive control group and an equal volume of distilled
water to the negative control group. The drug/extract was given
once daily for 5 days. Thin films stained with Giemsa stain were
prepared from tail blood of each mouse daily for 5 days to mon-
itor the parasitaemia level. The mean survival time (MST) for
each group was determined arithmetically by finding the aver-
age survival time (days) of the mice (post-inoculation) in each
group over a period of 28 days (days 027). The parasitaemia
level of the animals that survived after the 28 days of study was
determined from the thin film prepared from tail blood of the
animals.
2.8. Statistical analysis
Data obtained from the study were analyzed statistically
using Students test and values of P < 0.05 were considered
significant.
3. Results
3.1. Acute toxicity
The extract (10005000 mg/kg) produced physical signs of
toxicity such as writhing, gasping, palpitation, decreased respi-
ratory rate, body and limb bone and death depending on the dose.
All the mice treated with 4000 mg/kg dose of the extract and
above died. The i.p. LD50 of the extract in mice was calculated
to be 3240.37 mg/kg.
3.2. Four-day test
Ethanolic fruit extract of Tetrapleura tetraptera (TTE), pro-
duced a dose dependent chemosuppressive effect at various
doses employed in this study. The chemosuppression were
59.63, 69.49 and 76.37% for 300, 600 and 900 mg/kg day doses,
respectively. The chemosupression produced by the extract were
significant (P < 0.05) compared to control and comparable to that
of the standard drug (chloroquine 5 mg/kg day) with a chemo-
suppression of 88.23% (Table 1).
3.3. Curative test
On established infection, it was observed that there was a
daily increase in parasitaemia of the control group. However,
there was a daily reduction in the parasitaemia levels of the
extract treated group as well as that of positive control (chloro-
quine).
On day 7, the average percentage parasitaemia for the groups
were 12.0, 10.5, 9.8, 8.0 and 82.0% for 300, 600, 900 mg/kg day
of the extract, chloroquine and control groups, respectively
(Fig. 1). The mean survival time (MST) of the extract treated
groups were significantly (P < 0.05) longer than that of control
and was comparable to that of the standard drug, chloroquine.
The values are given in Table 2.The mice that were treated
with chloroquine and those treated with the highest dose of the
extract (TTE), (900 mg/kg) survived throughout the duration of
the study (28 days). The chloroquine treated mice had their blood
cleared of the parasite, while the mice treated with 900 mg/kg of
the extract had a mean percentage parasitaemia of 21.3 13.5%.
 J.E. Okokon et al. / Journal of Ethnopharmacology 111 (2007) 537540 539

Table 1
Antiplasmodial activity of Tetrapleura tetraptera during 4-day test
Drug/extract Dose (mg/kg day) Average (%) parasitaemia Average (%) suppression

Tetrapleura 300 17.6 3.85* 59.63

tetraptera extract 600 13.33 4.49* 69.49
900 10.3 3.85* 76.37
Chloroquine (standard) 5 5.13 0.38* 88.23
Distilled water (control) 0.2 ml 43.6 3.16

Data are expressed as mean S.D for five animals per group. P < 0.05 when compared to control.

activity of the ethanolic fruit extract of Tetrapleura tetraptera.

Fig. 1. Effect of Tetrapleura tetraptera on established infection (curative test).
4. Discussion
The results show that the ethanolic fruit extract of Tetrapleura
tetraptera is slightly toxic as shown in its LD50 value of
3240.37 mg/kg (Homburger, 1989) and also possesses a sig-
nificant (P < 0.05) antiplasmodial activity as evident from the
chemosuppression obtained during the 4-day early infection test
.The fruits extract also exhibited a significant curative effect
during established infection comparable to that of the standard
drug, chloroquine (5 mg/kg day) as demonstrated in the mean
survival time of the mice in the extract and chloroquine treated
groups. Tetrapleura tetraptera fruits have been reported to con-
tain many chemical compounds such as triterpenoid glycoside
(aridanin), coumarin (scopoletin), flavonoids and other pheno-
lic compounds (Adewunmi and Sofowora, 1980; Adewunmi
and Marquis, 1981, 1987; Adewunmi, 1984; Adesina and
Reisch, 1985; Maillard et al., 1992). Antiplasmodial screening
of plants have implicated terpenes and flavonoids in this activity
(Philipson and Wright, 1991; Christensen and Kharazmi, 2001).
Since the fruits extract of Tetrapleura tetraptera contains triter-
penoid and flavonoids, it is very likely that these compounds
might have contributed largely to the observed antiplasmodial
Table 2
Mean survival time of mice receiving various doses of ethanolic fruit extract of
Tetrapleura tetraptera
Drug/extract Dose (mg/kg day) Mean survival time (day)
Tetrapleura 300 16.3 0.47*
tetraptera extract 600 21.0 1.63*
900 28.0 0.00*
Chloroquine (standard) 5 28.0 0.00*
Distilled water (control) 0.2 ml 9.81 1.32
Data are expressed as mean S.D for five animals per group. P < 0.05 when
compared to control.
Moreso, the observed antiplasmodial activity of the fruits extract
might have contributed to the low parasitaemia rate reported for
adults in parts of the Niger Delta region of Nigeria (Calabar
area) (Ezedinachi et al., 1992; Okokon and Ezedinachi, 2002)
as most of their foods are spiced with the fruits of Tetrapleura
tetraptera. Diets are known to be medicinally important as plants
consumed as food are ingested in relatively large amounts and
more regularly than those of same plants used in rituals or for
cosmetics (Etkin and Ross, 1991; Etkin, 1994).
We conclude that Tetrapleura tetraptera fruits extract possess
antiplasmodial activity and the results of this study justifies and
confirms the traditional usage of this plant as malarial remedy.
Acknowledgement
The authors are grateful to Mr. Nsikan Malachy for his tech-
nical assistance.
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