Transactions of the Royal Society of Tropical Medicine and Hygiene (2004) 98, 435437

An antimalarial extract from neem leaves

is antiretroviral
I.J. Udeinya a,1 , A.U. Mbah b , C.P. Chijioke b , E.N. Shu b,*
a

Department of Pharmacology, Howard University College of Medicine, Washington DC, USA

Department of Pharmacology and Therapeutics, College of Medicine, University of Nigeria,
Enugu Campus, P.M.B. 01129, Enugu, Nigeria

Received 3 June 2003 ; received in revised form 25 October 2003; accepted 30 October 2003

KEYWORDS
Malaria;
Cancer;
HIV;
Neem leaf extract;
Antiretrovirals;
Nigeria

Summary An acetonewater neem leaf extract with antimalarial activity was evaluated in vitro at 5 g/ml for inhibition of adhesion of malaria parasite-infected erythrocytes and cancer cells to endothelial cells, and at 10 g/ml for protection of
lymphocytes against invasion by HIV. The extract was also evaluated in 10 patients
with HIV/AIDS at 1000 mg daily for 30 d. The mean binding of infected erythrocytes
and cancer cells per endothelial cell was 15 and 11 respectively in the absence of the
extract, and 0 and 2 respectively in with the extract. In the absence and presence of
the extract, 0% and 75%, respectively, of lymphocytes were protected. In the treated
patients, haemoglobin concentration, mean CD4+ cell count and erythrocyte sedimentation rate, which were initially 9.8 g/dl, 126 cells/l and 90 mm/h respectively, improved to 12.1 g/dl, 241 cells/l and 49 mm/h. Mean bodyweight and platelet count,
initially 57 kg and 328 103 /mm3 respectively, increased to 60 kg and 359 103 /mm3 .
No adverse effects were observed during the study. The extract showed antiretroviral
activity with a mechanism of action that may involve inhibition of cytoadhesion. The
results may help in the development of novel antiretroviral and antimalarial drugs.
2004 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All
rights reserved.

1. Introduction
The neem tree (Azadirachta indica A. Juss) has provided various medicinal preparations in many parts
of the world for centuries. Antimalarial preparations remain one of the earliest and most widely
used in Africa (Ekanem, 1971). Udeinya (1993)
showed that an acetonewater extract of neem
* Corresponding author. Tel.: +234-42-259569;
fax: +234-42-259569.
E-mail address: enshu1@yahoo.com (E.N. Shu).
1 Present address: ROCITUS Institute of Research, 34 Valley
Crescent, Independence Layout, Enugu, Nigeria.

was heat stable and caused complete cessation of

morphological development of malaria parasites,
hence preventing maturation beyond the ring stage.
Adhesion of infected erythrocytes to the endothelium is a key factor in the pathogenesis of severe Plasmodium falciparum malaria (MacPherson
et al., 1985). Cytoadhesion also plays a major role
in the pathogenicity of other diseases including
cancer metastasis, and bacterial and viral infections (Clapham and McNight, 2002; Schifferli and
Beachey, 1988; Thomas et al., 1998).
We studied the effect of an acetonewater neem
leaf extract on the cytoadhesion of malaria-infected
erythrocytes (ITG 2F6 strain) and of a cancer cell

0035-9203/$ see front matter 2004 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.trstmh.2003.10.016

436

I.J. Udeinya et al.

line (CCRL 1593. U-937) to human umbilical vein

endothelial cells (EC-FP5). We also studied its effect on the invasion of a human lymphocyte cell
line (CEM-IW) by HIV and on HIV-related conditions
in 10 volunteers with HIV/AIDS.

after, with treatment of any opportunistic infections. After the treatment period, the volunteers
were again followed up (through bi-weekly home
visits) for 1 month to monitor their post-treatment
conditions.

2. Materials and methods

3. Results

The extract was prepared as previously described

(Udeinya, 1993) and diluted to the desired concentrations with RPMI-1640 media for the in vitro
studies. In the malaria-infected erythrocytes and
cancer cell experiments, co-cultures containing the
adherent cells and target endothelial cells were
pre-incubated with the extract (5 g/ml) or without (control) for 60 min followed by adhesion assay as previously described (Udeinya et al., 1985).
At the end of the assay period, non-adhered cells
were removed and those attached to the endothelial (target) cells were Giemsa-stained and counted
by light microscopy.
In the HIV invasion experiments, monolayers of
the lymphocytes (target cells) were co-cultured
with the virus in the presence of the extract
(10 g/ml) or its absence (control) for 2 h then
washed and further cultured without virus or extract for 24 h. The percentage of the target cells
infected was determined by indirect immunouorescence. Control cultures of endothelial cells and
lymphocytes with extract were maintained for 24 h
to determine possible toxic effects of the extract.
Ethical clearance and informed consent were
obtained for the clinical study. In these experiments, 1000 mg of the extract in gelatin capsules
was administered daily by mouth to 10 ambulatory
HIV-positive volunteers for 30 d, immediately following completion of diagnostic and pre-treatment
laboratory investigations. No other antiretroviral
drug was administered to the volunteers during
this study. The volunteers (four males and six females with a mean age of 37 years) were recruited
at the outpatient clinic of the University of Nigeria Teaching Hospital, Enugu, Nigeria. The patients
were treated and laboratory tests carried out in the
same hospital. Antiretroviral-naive patients with
conrmed HIV, CD4+ cell count <200 cells/l and
who had, or had had, one or more AIDS-dening
symptoms such as fever with generalized lymphadenopathy, prolonged diarrhoea, skin rash,
weight loss, oral thrush, cough and anorexia were
included in the study. Patients aged <18 years,
pregnant women and patients who had received
antiretrovirals in the past were excluded. Clinical
and laboratory evaluations were performed before
commencement of treatment and bi-weekly, there-

In the infected erythrocyte and cancer cell adhesion assays, the mean binding per target cell was 15
and 11 respectively in the absence of the extract,
and 0 and 2 respectively in the presence of the extract (Figure 1). The binding in the presence of the
extract was signicantly lower (P < 0.01) than the
binding in the absence of the extract in the two
cell types. In the HIV invasion assay, the target cells
were all invaded by HIV resulting in 100% infection
and, hence, zero protection in the absence of the
extract and 75% of the cells were protected in the
presence of the extract. The protection in the presence of the extract was signicant (P < 0.01) compared with protection in the absence of the extract
(Figure 1). In control cultures of target cells with
the extract, the growth of endothelial cells and lymphocytes was normal compared with the growth in
cultures without the extract.
The changes in laboratory parameters in the
HIV/AIDS volunteers before treatment and after
30 d treatment are shown in Table 1. The changes
were statistically signicant for haemoglobin, CD4+
cell count and erythrocyte sedimentation rate
(ESR). The changes in the values for bodyweight
and platelet count were impressive considering the
short time of the study. AIDS-related conditions
before treatment (as stated above) were completely resolved within the treatment period and
remained so during the follow-up. There were no
adverse/side effects related to the extract during the treatment and follow-up period, as shown

Figure 1 Adhesion to cultured human endothelial cells

by malaria-infected erythrocytes (A) and metastatic cancer cells (B); and protection (C) of lymphocytes cultured
with HIV, in the absence and presence of neem extract.

Antimalarial neem extract and HIV/AIDS

437

Table 1 Values of laboratory parameters in 10 patients with HIV/AIDS before and after 30 days treatment with
neem extract
Parameter
Bodyweight (kg)
Haemoglobin (g/dl)
Platelet count 103 /mm3
CD4+ cells/l
Erythrocyte sedimentation rate (mm/h)

Before treatment
Mean SD (range)
57
9.8
328
126
90

by absence of drug-related changes in liver function tests, full blood count, urinalysis, and central
nervous system and cardiac function monitoring.

8 (4076)
2 (6.613.5)
40 (284360)
45 (30195)
32 (26132)

After treatment
Mean SD (range)

P-value

60
12.1
359
241
49

>0.05
<0.05
>0.05
<0.01
<0.01

9 (4580)
1.5 (1015)
45 (301421)
60 (216340)
28 (1494)

vide encouragement for further studies to develop

neem-based antiretroviral as well as antimalarial
agents.
Conicts of interest statement

4. Discussion
The results of the adhesion assays showed that
the acetonewater neem leaf extract possessed
anti-cytoadhesion activity. The activity appears
to be broad spectrum as it inhibited adhesion of
malaria-infected erythrocytes as well as that of
cancer cells and prevented the invasion of human
lymphocytes by the HIV, hence protecting the target cells.
In the HIV/AIDS volunteers, the depression in
CD4+ cell counts, bodyweight and haemoglobin
caused by HIV infection was signicantly reversed
by the extract. Depression in these parameters,
especially the CD4+ cell counts, is widely related
to the status and progression of the HIV infection,
while antiretroviral activity is identied and assessed by reversal of such depression (Kalin, 1992).
Hence, on the basis of the protection of target cells
in vitro, reversal of the HIV-induced immune decit
(CD4+ cell depression) and facilitated clearance of
AIDS-related conditions, this extract can be identied and classied as an antiretroviral agent with
a possible mechanism of action involving inhibition
of viral invasion of the host cells.
In conclusion, this study provided evidence for
the presence of antiretroviral activity in the neem
leaf extract previously shown to have antimalarial
activity (Udeinya, 1993). Evidence was also provided to show that the extract possessed broad
spectrum anticytoadhesion activity which may be
related to the mechanism of its antiretroviral activity. The complete absence of adverse side effects
coupled with a novel mechanism of action pro-

The authors have no conicts of interest concerning

the work reported in this paper.

Acknowledgements
This work was supported partly by the USAID. Ted
Miller and Peter L. Malkin also provided material
and moral support during the study.

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