International Journal of Veterinary Science and Medicine (2016) 4, 33–40

H O S T E D BY
Cairo University

International Journal of Veterinary Science and Medicine

www.vet.cu.edu.eg
www.sciencedirect.com

Full Length Article

In vivo ameliorative effects of methanol leaf extract

of Lawsonia inermis Linn on experimental
Trypanosoma congolense infection in Wistar rats
Abdullah Mohammad Tauheed a,*, Salisu Hashim Shittu a,
Mohammed Musa Suleiman a, Buhari Habibu b, Mohammed Umar Kawu b,
Patricia Ishaku Kobo a, Peter Ofemile Yusuf a

a
Department of Veterinary Pharmacology and Toxicology, Ahmadu Bello University, Zaria, Nigeria
b
Department of Veterinary Physiology, Ahmadu Bello University, Zaria, Nigeria

Received 1 June 2016; revised 19 October 2016; accepted 19 October 2016

Available online 10 December 2016

KEYWORDS Abstract The aim of this study was to investigate the ameliorative effect of Lawsonia inermis Linn
Antitrypanosomal; used traditionally against trypanosomosis. Twenty-five adult Wistar rats of both sex were individ-
Antioxidant; ually infected intraperitoneally (IP) with 106 Trypanosoma congolense per ml of blood. Following
Erythrocyte osmotic establishment of infection, the rats were randomly divided into five groups of 5 rats each. Rats
fragility; in groups I, II, and III were treated with 125, 250 and 500 mg/kg of the extract, respectively, while
Malondialdehyde; rats in groups IV and V were treated with 3.5 mg/kg and 2 ml/kg of diminazene aceturate (DM)
Phytochemistry once and physiological buffered saline, respectively. All treatments except DM were given orally
for 7 days IP. The antitrypanosomal effect of the plant was assessed by observing the level of
parasitaemia daily, packed cell volume (PCV) weekly, erythrocyte osmotic fragility (EOF) and
malondialdehyde (MDA) concentration on day 21. Phytochemical screening of the extract revealed
the presence of alkaloids, carbohydrates, triterpenes, steroids, cardiac glycosides, saponins, tannins
and flavonoids. The extract significantly (P < 0.05) reduced levels of parasitaemia at 250 mg/kg.
PCV was higher (P > 0.05) in extract treated groups but significantly higher (P < 0.05) in group
II at week 2 when compared to group V. Rats in group II had significantly lower values of EOF
and MDA when compared with groups IV and V. Thus, the leaf of L. inermis has in addition to
an antitrypanosomal effect against T. congolense in rats, an attenuating effect on the trypanosomo-
sis pathology probably mediated via protection of the erythrocyte membrane against trypanosome-
induced oxidative damage to the erythrocytes.
Ó 2016 Faculty of Veterinary Medicine, Cairo University. Production and hosting by Elsevier B.V. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding author.
E-mail address: mtauheed@abu.edu.ng (A.M. Tauheed).
Peer review under responsibility of Faculty of Veterinary Medicine, Cairo University.
http://dx.doi.org/10.1016/j.ijvsm.2016.10.005
2314-4599 Ó 2016 Faculty of Veterinary Medicine, Cairo University. Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
34 A.M. Tauheed et al.

1. Introduction cancer and rheumatoid arthritis [17–21]. Since the search for
a vaccine against trypanosomosis remains elusive for now
Trypanosomosis is a chronic debilitating haemoparasitic dis- [22], there is an urgent need to find more efficacious drugs, par-
ease of man and domestic animals characterised by para- ticularly, from traditional medicinal plants to combat the men-
sitaemia, pyrexia, anaemia, loss of condition, reduced ace of the disease. Thus, the aim of this study was to elucidate
productivity and death in some cases [1,2]. The disease accounts the antitrypanosomal property of L. inermis against experi-
for over 3 million livestock and 55,000 human deaths, respec- mental Trypanosoma congolense infection in Wistar rats.
tively, annually [3,4]. It has negative impact on food production
and economic growth in many parts of the world, particularly, 2. Materials and methods
sub-Saharan Africa, with estimated direct annual losses to pro-
ducers and consumers exceeding US$1 million [5,6]. 2.1. Plant collection and identification
Oxidative stress, defined as imbalance between free radical
generation and free radical scavenging, has been postulated to Fresh leaves of L. inermis were collected from the main campus
play a pivotal role in the pathogenesis of trypanosomosis [7,8]. of Ahmadu Bello University, Zaria, Nigeria. The leaves, flow-
Free radicals cause lipoperoxidation in the body and an ers and seeds of the plants were sent to the Herbarium, Depart-
increase in the production of these radicals leads to overpro- ment of Biological Sciences, Ahmadu Bello University Zaria,
duction of malondialdehyde (MDA). MDA is one of the best Nigeria for identification. A specimen voucher Number
known degradation products of lipid peroxidation due to free (10461) was assigned to the plant. The leaves were dried to a
radicals and therefore, can be used as a marker of cell mem- constant weight at room temperature in the laboratory. The
brane injury due to lipid peroxidation. The polyunsaturated dried sample was ground into powder using mortar and pestle
fatty acids of the erythrocyte membrane are the main target and kept in a polythene bag until required.
for free radical lipoperoxidation [9,10]. By-products of this
lipid peroxidation have been reported to produce changes in 2.2. Plant extraction, concentration and phytochemical
the structures and functions of cell membrane, leading to screening of the extract
decreased membrane fluidity, increased membrane permeabil-
ity, inactivation of membrane-bound enzymes and loss of Three hundred grams of the powdered leaf of L. inermis was
essential fatty acids [9,11]. The end result of these changes cold extracted in a percolator using 900 ml of methanol as sol-
on the surface of erythrocytes is increased erythrocyte osmotic vent. The mixture was allowed to stand in the percolator for
fragility [12]. This may ultimately lead to anaemia and poor 72 h. Thereafter, the liquid extract was drained into a clean
distribution of nutrient in the body. Control of anaemia in bottle. The extracted powder was rinsed off with 100 ml of
trypanosome-infected cattle is more important for survival, fresh solvent and added to the initial solution collected. The
weight gain and productivity than control of parasitaemia liquid extract was concentrated to dryness over water bath.
[13]. Oxidative stress may be harmful to both the host and
the trypanosome, but it is mainly due to a defensive reaction 2.3. Phytochemical screening
of the host (or vector) against the presence of the trypanosome.
Both the host and the parasite dispose of an antioxidant
defence system. However, the system of the mammalian host Phytochemical screening to detect the presence of alkaloids,
(having glutathione as the major intracellular antioxidant) is flavonoids, saponins, tannins, glycosides, triterpenes and ster-
different from that of the trypanosomes (having trypanothione oids on the extract was carried out as described by Trease and
as the main defence system against oxidative stress). Evans [23].
Currently, only three trypanocides are available for con-
trolling tsetse-transmitted trypanosomosis in domestic rumi- 2.4. Experimental animals
nants. These are isometamidium and homidium which have
both prophylactic and therapeutic effects, and diminazene ace- Wistar rats of both sexes weighing between 90 and 100 g were
turate which has only therapeutic properties. It is estimated obtained from Nigerian Institute for Trypanosomiasis
that 35 million doses of these drugs are used in Africa annually Research (NITR), Kaduna. The animals were kept for
for the control of the disease [14]. The toxicity associated with 2 months in the animal house before the commencement of
the use of these drugs and the resistance developed against the experiment. They were housed in clean plastic cages with
them by trypanosomes are some of the problems associated wood shavings as beddings. The beddings were changed twice
with the trypanosomosis. Drug-screening activities from plants in a week. The rats were fed on standard rat feed and given
have started decades back, and an emerging number of studies access to clean water ad libitum. The ethical approval for the
have now been developed and reported so far to discover drugs use of rats was obtained from Ethical Committee on Animal
from medicinal plants that can help to combat trypanosomo- Use and Care, Ahmadu Bello University, Zaria.
sis. The efforts have been geared towards medicinal plants with
significant trypanocidal effect but fewer side effects. 2.5. Test organism
Lawsonia inermis (Henna) is a branched glaborous shrub or
a small tree (2–6 m high) belonging to the family Lythraceae T. congolense was obtained from the Department of Vector
[15]. It is widely used for traditional and prophetic medicine and Parasite, Nigerian Institute for Trypanosomiasis and
in Africa, Asia, and Middle East [16]. The plant is reputed Onchocerciasis Research (NITR), Kaduna. The parasite was
to be used for treatment of plethora of diseases, such as try- maintained in the laboratory by continuous passage in rats
panosomosis, malaria, fungal, viral, and bacterial infections; until required. Each cycle of passage was done when
In vivo ameliorative effects of methanol leaf extract of Lawsonia inermis Linn 35

parasitaemia was in the range of 35–40 parasites per field. For rat in a heparinized capillary tube and centrifuged the tube
several passages, about 3 ml of blood was obtained from an at 3000g for 10 min, where after the PCV was measured
infected rat by cardiac puncture after light chloroform anaes- using a haematocrit reader. At the end of the experiment
thesia into a 5 ml syringe and emptied into a vial containing (day 21), the animals were sacrificed by severing the jugular
9 ml of physiological buffered saline (PBS). About 1
106 par- vein. At that occasion blood was collected from each rat
asite in 0.2 ml blood/PBS solution was injected intraperi- and the PCV determined.
toneally (IP) into a rat previously unexposed to
trypanosomal infection. Parasitaemia was monitored daily 2.11. Determination of erythrocyte osmotic fragility (EOF)
using the method of Herbert and Lumsden [24].
The EOF test was carried out as described by Faulkner and
2.6. Inoculation of experimental animals
King [26]. Briefly, 5 ml of different concentrations (0.0%,
0.1%, 0.3%, 0.5%, 0.7% and 0.9%) of buffered sodium chlo-
For inoculation of experimental rats, blood was obtained from ride pH 7.4 was poured into labelled test tubes and 0.02 ml of
a donor rat at peak parasitaemia by sacrificing the rat via jugu- blood collected at day 21 from each rat was added into the
lar venisection after light chloroform anaesthesia into a vial respective test tubes. The contents were mixed gently and
containing PBS. About 1
106 parasite in 0.2 ml blood/PBS allowed to stand for 30 min at room temperature. The test
solution was injected IP into a rat previously unexposed to try- tubes were centrifuged at 2000g for 5 min. Thereafter, 4 ml
panosomal infection. of the supernatant was transferred to a clean glass cuvette
and the absorbance measured at 540 nm wavelength with spec-
2.7. Determination of median lethal dose 50 (LD50)
trophotometer (Spectronic 20, Bausch and Lomb, USA). The
percentage haemolysis for each sample was calculated as:
The median lethal dose (LD50) was determined as described by
Lorke [25]. In the initial phase, 9 rats of both sexes were ran- % Haemolysis ¼ optical density of test solution=
domly divided into three groups of 3 rats each. Groups 1, 2 optical density of standard solution
and 3 were treated IP with crude methanol extract leaf extract ðdistiled waterÞ
100:
of L. inermis at 10, 100 and 1000 mg/kg, respectively. The rats
were then observed over 48 h for signs of toxicity and mortal-
ity. In the second phase of the study, 3 rats for each extract 2.12. Determination of malondialdehyde (MDA)
were assigned into 3 groups of 1 rat each. The animals were
individually administered 2, 6, and 8 mg/kg of crude methanol Serum concentrations of MDA were measured as described by
leaf extract of L. inermis. The LD50 was then computed as geo- Draper and Hadley [27]. Briefly, 0.5 ml of serum from each
metric mean of highest dose that did not kill the rat and lowest infected rat was pipetted into a centrifuge tube and 2.5 ml of
dose that killed the rat. 100 g/L trichloroacetic acid (TCA) solution was added to it
and then placed in boiling water bath for 15 min. Thereafter,
2.8. Experimental grouping it was cooled in tap water and centrifuged at 1000g for
10 min. Then 2 ml of the supernatant was taken and added
Twenty-five adult Wistar rats of both sexes were individually to 1 ml of 6.7 g/L thiobarbituric acid (TBA) in a test tube
infected, IP, with 106 of T. congolense per ml of blood. Imme- and placed in boiling water bath for 15 min. It was then cooled
diately after the establishment of infection, the rats were ran- in tap water and its absorbance was measured at 532 nm with
domly divided into five groups of 5 rats each. Rats in groups spectrophotometer (spectrumlab 23A China). One ml of 10%
I, II, and III were treated with crude methanol extract of the TCA and 1 ml of 0.67% TBA was used as the blank. The
leaf of L. inermis at 500, 250 and 125 mg/kg, respectively, intensity of the pink pigment formed from MDA-TBA con-
per os for 7 consecutive days. Similarly, rats in group V were densation was measured by spectrometer and indicates the
given PBS 2 ml/kg for 7-day; while rats in group IV were trea- extent of lipid peroxidation.
ted with diminazene aceturate 3.5 mg/kg IP once.
2.13. Statistical analysis
2.9. Determination of parasitaemia in experimental rats
Values obtained were expressed as mean ± standard error of
The treated rats were observed daily for development of para- mean (±SEM). Analysis of variance (ANOVA) was used fol-
sitaemia throughout the duration of the experiment. Para- lowed by Tukey’s post hoc test for multiple comparisons of
sitaemia was assessed using wet mount method. A drop of groups using GraphPad Prism version 5.03. Values of
blood was collected on a clean microscope slide from the tail P < 0.05 were considered significant.
of each rat, covered with a cover slip and observed for para-
sitaemia under light microscope at
400 magnification. The 3. Results
number of parasites per field was converted to the number of
parasites per ml of blood using a standard chart [24]. 3.1. Phytochemical screening

2.10. Determination of packed cell volume (PCV) The crude methanol extract of L. inermis was positive for
the presence of alkaloids, carbohydrates, triterpenes and
The PCV of rats from all the groups was determined on steroids, cardiac glycosides, saponin glycosides, tannins and
days 8 and 15 by collecting blood from tail vein of each flavanoids.
36 A.M. Tauheed et al.

3.2. Acute toxicity 25

a a a

Mean survival rate (Days)

20
The crude methanol leaf extract of L. inermis did not show
b b
any sign of toxicity or mortality even at highest dose of 15
5000 mg/kg.
10
3.3. Effect of treatment on the level of parasitaemia
5

The effect of treatment on the level of parasitaemia in all the

animals treated is shown in Fig. 1. From day six onwards, 0

mean daily parasitaemia was significantly lower (P < 0.05) MLELI 500 MLELI 250 MLELI 125 DA 3.5 mg/kg PBS 2 ml/kg
mg/kg mg/kg mg/kg
in rats treated with 250 mg/kg of the extract than rats admin-
istered PBS. However, rats treated with 500 and 125 mg/kg of Fig. 2 Effect of treatment with extract of the leaf Lawsonia
the extract treated did not show significant suppression of inermis on mean survival periods of experimental rats. a,bMeans
parasitaemia. Nonetheless, diminazene treated group cleared with different superscript letters are statistically significant
parasites from the blood 24 h post-treatment until day 13 when (P < 0.05). MLELI = Methanol leaf extract of L. inermis;
parasitic relapse occurred. DA = Diminazene aceturate; PBS = Physiologic buffered phos-
phate saline.
3.4. Survival periods of rats treated with leaf extract of
L. inermis
3.5. Effect of treatment with L. inermis extract on mean weekly
The effect of treatment with the methanol leaf extract of packed cell volume (PCV)
L. inermis (MLELI) on the survival periods of rats is shown
in the Fig. 2. Rats treated with 500 and 250 mg/kg MLELI The PCV was higher in all the extract treated groups at weeks
had significantly (P < 0.05) longer survival periods than the 1 and 2 than PBS treated group (Fig. 3). The PCV of rats trea-
rats treated with 125 mg/kg LLELI and PBS. Mortality was ted with 250 mg/kg of the extract at week 2 was significantly
first noticed on days 14 and 12 in the MLELI 125 mg/kg higher (P < 0.05) than the PCV of rats in PBS group. Also,
and PBS groups, respectively, culminating to the death of all among the groups treated with the leaf extract, rats treated
the rats in MLELI 125 mg/kg and PBS on day 18. with 250 mg/kg of the extract had the highest PCV at weeks

180000000

160000000

140000000
Mean daily parasitaemia/ml of blood

120000000

100000000 MLELI 500mg/kg

MLELI 250 mg/kg
80000000 MLELI 125 mg/kg
DA 3.5 mg/kg
60000000 PBS 2 ml/kg

40000000

20000000

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time/day

Fig. 1 Effect of methanol extract of the leaf of L. inermis on the level of parasitaemia in rats infected with T. congolense.
MLELI = Methanol leaf extract of L. inermis; DA = Diminazene aceturate; PBS = Physiologic buffered phosphate saline.
In vivo ameliorative effects of methanol leaf extract of Lawsonia inermis Linn 37

60
Mean weekly PCV
b b
50

Packed cell volume (%)

40 a
MLELI 500 mg/kg
MLELI 250mg/kg
30
MLELI 125 mg/kg

20 DA 3.5 mg/kg
PBS 2 ml/kg
10

0
1 2 3

Fig. 3 Effect of treatment with extract of the leaf Lawsonia inermis on mean weekly PCV of experimental rats. a,bMeans with different
superscript letters are statistically significant (P < 0.05). MLELI = Methanol leaf extract of L. inermis; DA = Diminazene aceturate;
PBS = Physiologic buffered phosphate saline.

120
b
bb
100
b
Erythrocyte osmotic fragility (%)

a
80

CMLELI 500 mg/kg

60
CMLELI 250 mg/kg
a
40 D.A. 3.5 mg/kg
PBS 2 mg/kg
20

0
0 0.1 0.3 0.5 0.7 0.9
-20
NaCl concentrations (%)

Fig. 4 Effect of Lawsonia inermis extract on EOF of treated rats experimentally infected with T. congolense. a,bMeans with different
superscript letters are statistically significant (P < 0.05). MLELI = Methanol leaf extract of L. inermis; DA = Diminazene aceturate;
PBS = Physiologic buffered phosphate saline.

1 and 2, though not statistically significant (P > 0.05) when 3.7. Effect of L. inermis extract on serum malondialdehyde
compared to other extract treated groups. (MDA) concentrations of treated rats

3.6. Effect of L. inermis extract on erythrocyte osmotic fragility The effect of treatment on serum MDA concentration is
(EOF) of treated rats shown in Fig. 5. There was an increase in the serum MDA con-
centration of rats in the control (PBS) group when compared
The ameliorative effect of the crude methanol extract of L. to extract treated groups. The serum MDA concentration of
inermis on experimental T. congolense infection is shown in rats treated with 250 mg/kg of the extract was significantly
Fig. 4. The percentage EOF of extract treated groups was sig- lower (P < 0.05) than in rats treated with PBS. Furthermore,
nificantly lower (P < 0.05) at 0.1% and 0.3% NaCl concentra- serum concentration of rats treated with 250 mg/kg group was
tions when compared to PBS group. While rats treated with lower than that of rats treated with 500 mg/kg and diminazene
500 mg/kg had lowest EOF at 0.3% NaCl concentration, rats 3.5 mg/kg groups though not statistically significant
treated with 250 mg/kg of extract had the lowest EOF at 0.1% (P > 0.05). However, the serum concentration of MDA in
NaCl concentration. However, diminazene (3.5 mg/kg) treated diminazene aceturate treated group is lower (P > 0.05) than
rats have significantly higher (P < 0.05) values of EOF when the PBS treated group.
compared with extract treated groups.
38 A.M. Tauheed et al.

0.4 have overwhelmed the host body antioxidant defence system

a
Malondialdehyde concentraon (nmol)

0.35 resulting in loss of integrity of erythrocyte membrane and their

subsequent lysis. Similarly, Umar et al. [37] earlier reported
0.3 b
that T. bruei brucei infection altered the host’s antioxidant
0.25 defence against free radicals. In this study, rats treated with
0.2 L. inermis extract at 250 mg/kg ameliorated oxidative stress
as observed by significantly lower values of EOF. This implied
0.15
that the membrane of erythrocytes was protected from the
0.1 deleterious effects of the trypanosome on the erythrocyte
0.05 membrane. Antioxidants stabilize free radicals and ameliorate
free-radical-induced damage to the cells [38]. Anaemia in try-
0
CMLELI 500 CMLELI 250 D.A. 3.5 PBS 2 ml/kg panosomosis has been attributed to injury to the membrane
of erythrocytes resulting from either the lashing effect of the
Fig. 5 Effect of Lawsonia inermis extract on serum MDA of flagellum of trypanosomes on the erythrocyte membrane
treated rats experimentally infected with T. congolense. a,bMeans [39,40]; or cleavage of sialic acid of the erythrocyte membrane
with different superscript letters are statistically significant by the sialidase of trypanosome [41]. The leaf extract of
(P < 0.05). MLELI = Methanol leaf extract of L. inermis; L. inermis has shown to protect the erythrocyte membrane
DA = Diminazene aceturate; PBS = Physiologic buffered phos- against trypanosome-induced-EOF and therefore, may
phate saline. ameliorate anaemia associated with trypanosome infection in
animals. Since anaemia but not level of parasitaemia in the
4. Discussion trypanosome-infected animals is primarily responsible for
decrease in the productivity of such animals and loss of their
condition [13,42], it is possible that the leaf of L. inermis could
The absence of any apparent toxic signs in rats treated with the be used to improve productivity of trypanosome-infected
extract even at 5000 mg/kg showed that the leaf extract of animals.
L. inermis is relatively safe. Any substance that is not toxic One of the end products of lipid peroxidation is MDA,
to animals at a dose of up to 5000 mg/kg is relatively safe which is measured by concentration of MDA in body tissues
for use as therapeutic agent [25]. According to the World or serum [8]. These products of lipid peroxidation produce
Health Organisation acute toxicity rankings [28], the extract changes in the structures and functions of the cell membrane,
could be described as unlikely to be associated with hazard leading to decreased membrane fluidity, increased membrane
when administered as a drug. This may underscore the reason permeability, inactivation of membrane-bound enzymes and
why traditional herbalists use a wide range of doses of decoc- loss of essential fatty acids [9,11]. The end result of these
tion and infusion made from the plant. changes on the surface of erythrocytes is increased erythrocyte
The L. inermis extract used in this study was able to sup- osmotic fragility [12]. The increase in the concentration of
press parasite growth. Surprisingly, the activity of the extract MDA in PBS group suggests an increase in the production
was higher at the dose of 250 mg/kg when compared with of free radicals by T. congolense, resulting from imbalance
the dose of 500 mg/kg. This agrees with the finding of Das between radical generating and radical scavenging activities.
et al. [29], who reported better efficacy of extract of Argemone This observation agrees with the findings of earlier workers
mexicana at lower dose than at higher dose. This effect may [7,36,37]. The reduction in the concentration of MDA in
perhaps be attributed to the antioxidant action of the extract. L. inermis extract treated groups shows that the plant was able
It has been shown that antioxidants prevent the deleterious to ameliorate lipid peroxidation due to T. congolense infection.
effects of Trypanosoma brucei [30]. Also, antioxidant action This observation further corroborates the results of EOF,
is maintained at certain dose above which it becomes a pro- which is an indirect index of measuring oxidative stress on
oxidant with resultant lower pharmacological action and dele- erythrocyte membrane. It follows that methanol leaf extract
terious effect on the animal host. This may explain the poor of L. inermis enhances antioxidant defence against free
antitrypanosomal effect observed at the higher dose of radicals. Leaf of L. inermis has been reported to have an
500 mg/kg in this study. Bouayed and Bohn [31] and Martins antioxidant effect against oxidative damage comparable to
[32] observed that at higher doses, an antioxidant becomes vitamin C [43]. Furthermore, reduced EOF and lipid peroxida-
pro-oxidant thereby disrupting redox balance. Optimal doses tion caused by the leaf extract of L. inermis lend credence to
of antioxidants are crucial for maintaining homeostasis higher PCV values obtained in the extract treated groups,
[33,34]. Besides, better antitrypanosomal effect at 250 mg/kg probably resulting from the protective effect of the extract
than 500 mg/kg suggests that the former is the optimal dose on the erythrocyte membrane against oxidative stress.
which conforms to the drug-receptor occupation theory. This The higher values of EOF and MDA in the diminazene ace-
theory states that drugs give their highest efficacy at full recep- turate (DA) treated group compared to the lower values in the
tor occupation and no further effect is observed with further 250 mg/kg extract treated group show that DA does not have a
increase in dose [35]. Therefore, 250 mg/kg of the extract protective effect on the membrane of erythrocytes and proba-
may be the optimal dose with full receptor occupation and also bly lacks antioxidant effect. DA achieves its trypanocidal effect
optimal dose for its antioxidant effect. by binding to kinetoplast deoxyribonucleic acid (kDNA) and
Trypanosoma brucei brucei has been shown to induce ery- induces irreversible loss of the kDAN [44,45]. On the other
throcyte osmotic fragility (EOF) [36]. The increase in EOF in hand, the lower value of MDA in the DA treated group when
the infected untreated group in the present study could be compared with the PBS treated group may be a result of
attributed partly to generation of free radicals which could clearance of parasites from the blood of the treated rats, which
In vivo ameliorative effects of methanol leaf extract of Lawsonia inermis Linn 39

precludes trypanosome-induced lipoperoxidation (oxidative [5] Swallow BM. Impacts of African trypanosomiasis on African
damage) of the membrane of erythrocytes. This agrees with agriculture. International Livestock Research Institute 1999:1–46.
the finding of Kobo et al. [36] who reported lower values of [6] Kristjanson PM, Swallow BM, Rowland GJ, Krusoka RL,
MDA in T. brucei brucei infected rats treated with diminazene Belew PP. Measuring the cost of animal African
trypanosomiasis, the potential benefit of control and returns to
aceturate.
research. Agricult Syst 1999;59:79–98.
The antitrypanosomal effect of L. inermis vis-à-vis its [7] Eze JI, Anene BM, Chukwu CC. Determination of serum and
antioxidant property observed in the present study could be organ malondialdehyde (MDA) concentration, a lipid
attributable to the presence of one or combined effects of peroxidation index in Trypanosoma brucei - infected rats.
secondary metabolites detected. Antitrypanosomal effects of Comp Clin Pathol 2008;17:67–72.
plant extracts are associated with the presence of one or more [8] Umar IA, Toma I, Akombum CA, Nnaji CJ, Mahdi MA,
biologically active ingredients [17,46]. Phytochemical con- Gidado A, et al. The role of intraperitoneally administered
stituents such as flavonoids and phenols are known to possess vitamin C during Trypanosoma congolense infection of rabbits.
good antioxidant activities [47]. Afri J Biotechnol 2010;9:5224–8.
The antitrypanosomal activities of leaf of L. inermis [9] Ambali SF, Abubakar M, Shittu M, Yaqub LS, Anafi SB,
Abdullahi A. Chorpyrifos-induced alteration of haematological
observed in this study are not corroborated by the study of
parameters in Wistar rats: ameriolative effect of zinc. Res J Env
Wucherokke et al. [48]. This discrepancy could be attributed Toxicol 2010;4:55–66.
to differences in plant age/developmental stage, season and [10] Wahab AA, Mabrouk MA, Ayo JO, Ambali SF, Shittu M,
plant collection sites between both studies. In this study, the Adenkola AY, et al. Effects of co-administration of antioxidants
plant was collected in Zaria, Nigeria, which falls within the on erythrocyte osmotic fragility of Wistar rats during the hot-fry
northern Guinea Savannah zone of Nigeria. Age, species, season. Eur J Sci Res 2010;46:73–9.
location, season of collection of plant and plant part affect [11] Kolanjiappan K, Manoharan S, Kayalvizhi M. Measurement of
the pharmacological activity of plant extracts [49]. Plants erythrocyte lipids, lipid peroxidation, antioxidants and osmotic
cultivated under different environmental conditions produce fragility in cervical cancer patients. Clin Chim Acta
different phytochemical profiles or different amounts of indi- 2002;326:143–9.
[12] Saluja PS, Gupta SL, Malhotra DV, Ambawat HK. Status of
vidual components [50].
plasma malondialdehyde in experimental T. annulata infection
in crossbred bovine calves. Ind Vet 1999;76:379–781.
5. Conclusion [13] Naessens J. Bovine trypanotolerance: a natural ability to
prevent severe anaemia and haemophagocytic syndrome? Int J
The results of this study validate the traditional use of the leaf Parasitol 2006;36:521–8.
of L. inermis in treating trypanosomosis. The ability of the leaf [14] Geerts S, Holmes PH, Diall O, Eisler MC. African bovine
trypanosomiasis: the problem of drug resistance. Trends
of L. inermis to enhance erythrocyte membrane integrity and
Parasitol 2001;17:25–8.
maintain a high erythrocyte count in trypanosome-infected [15] Orwa C, Mutua A, Kindt R, Jamnadass R, Anthony S.
rats can be exploited to reduce anaemia and thus, improve pro- Agroforestree Database: a tree reference and selection guide
ductivity of trypanosome-ridden animals in trypanosome- version 4.0 2009; <http://www.worldagroforestry.org/sites/
endemic areas. treedbs/treedatabases.asp>.
[16] Ali NAA, Julich WD, Kusnick C, Lindequist U. Screening of
Funding Yemeni medicinal plants for antibacterial and cytotoxic
activities. J Ethnopharmacol 2001;74:173–9.
[17] Atawodi SE, Ameh DA, Ibrahim S, Andrew JN, Nzelibe HC,
This research did not receive any specific grant from funding Onyike E, et al. Indigenous knowledge system for treatment of
agencies in the public, commercial, or not-for-profit sectors. trypanosomiasis in Kaduna state of Nigeria. J Ethnopharmacol
2002;79:279–82.
Acknowledgement [18] Mikhaeil BR, Badria FA, Maatooq GT, Amer MMA.
Antioxidant and immunomodulatory constituents of henna
We are grateful to L. Usman and H. Garba of the Department leaves. Z Naturforsch C 2004;59:468–76.
[19] Endrini S, Rahmat A, Ismail P, Taufiq- Yap YH. Comparing of
of Parasitology and Entomology, Faculty of Veterinary Med-
the cytotoxicity properties and mechanism of Lawsonia inermis
icine, Ahmadu Bello University, Zaria for technical assistance.
and Strobilanthes crispus extract against several cancer cell lines.
J Med Sci 2007;7:1098–102.
References [20] Zumrutdal ME, Ozaslan M, Tuzcu M, Kalender ME, Daglıoglu
K, Akova A, et al. Effect of Lawsonia inermis treatment on mice
[1] Murray M, Morrison WI, Whitelaw DD. Host susceptibility to with sarcoma. Afr J Biotechnol 2008;7:2781–6.
African trypanosomiasis: trypanotolerance. Adv Parasitol [21] Syamsudin I, Winarno H. The effects of Inai (Lawsonia inermis)
1983;21:1–68. leave extract on blood sugar level: An Experimental Study. Res J
[2] Nwosu CO, Ikeme MM. Parasitaemia and clinical Pharmacol 2008;2:20–3.
manifestations in Trypanosoma brucei infected dogs. Rev Elev [22] Aldhous P. Fighting parasites on a shoe string. Science
Med Vet Pays Trop 1992;45:273–7. 1994;264:1857–9.
[3] Abenga JN, Enwezor FNC, Lawani FAG, Ezebuiro C, Sule J, [23] Trease GE, Evans WC. Drugs of biological origin. In:
David KM. Prevalence of trypanosomiasis in trade cattle at Pharmacognosy. United Kingdom: Ballire Tindal; 1983.
slaughter in Kaduna. Nig J Parasitol 2002;23:107–10. [24] Herbert WJ, Lumsden WH. Trypanosoma brucei: a rapid
[4] Mulumba K. Socio-economic and cultural factors in the ‘‘matching” method for estimating the host’s parasitemia. Exp
research and control of trypanosomiasis: paat technical and Parasitol 1976;40:427–31.
scientific series, 4. Italy: Food and Agriculture Organization of [25] Lorke D. A new approach to practical acute toxicity testing.
the United Nations (FAO); 2003. Arch Toxicol 1983;54:275–87.
40 A.M. Tauheed et al.

[26] Faulkner WR, King JW. Manual of clinical laboratory [39] Saror DI. Aspects of the anaemia of acute bovine
procedures. Cleveland, OH: Chemical Rubber Company; 1970. trypanosomiasis. In: Proc first Natl Conf Tsetse
[27] Draper HH, Hadley M. Malondialdehyde determination as Trypanosomiasis Res. p. 12–4.
index of lipid peoxidation. Met Enzymol 1990;186:421–31. [40] Igbokwe IO, Esievo KAN, Saror DI, Obagaiye OK. Increased
[28] World Health Organisation. PAN pesticides database. San susceptibility of erythrocytes to in vitro peroxidation in acute
Francisco: Pesticide Action Network, North America; 2001. Trypanosoma brucei infection of mice. Vet Parasitol
[29] Das PK, Pillai S, Kar D, Pardhan D, Sahoo S. Pharmacological 1994;55:279–86.
efficacy of Argemone mexicana plant extract, against [41] Esievo KAN. In-vitro production of neuraminidase (sialidase)
cysteamine-induced duodenal ulceration in rats. Ind J Med Sci by Trypanosoma vivax. In: Proceedings of the 16th meeting of
2011;65:92–9. the International Scientific Council for Trypanosomiasis.
[30] Eghianruwa IK. The effect of supplemental antioxidants Nairobi, Kenya. Organization of African Unity Scientific,
vitamin C and dimethyl sulfoxide on weight gain and survival Technical and Research and Control; 1979. p. 205–10.
in T. brucei infected and diminazene treated rats. Vet Arhiv [42] Trail JCM, d’Ieteren GDM, Feron A, Kakiese O, Mulungo M,
2012;2:519. Pelo M. Effect of trypanosome infection, control of
[31] Bouayed J, Bohn T. Exogenous antioxidants-double-edged parasitaemia and control of anaemia development on
swords in cellular redox state: health beneficial effects at productivity of N’Dama cattle. Acta Trop 1991;48:37–45.
physiologic doses versus deleterious effects at high doses. Oxid [43] Al-Demegh MA. Evaluation of the antioxidant activity effect of
Med Cell Longev 2010;3:228–37. Henna (Lawsonia inermis linn) leaves and or vitamin c in rats.
[32] Martins LA, Coelho BP, Behr G, Pettenuzzo LF, Souza IC, Life Sci J 2014;3:234–41.
Moreira JC, et al. Resveratrol induces pro-oxidant effects and [44] Brack C, Delain E, Riou G, Festy B. Molecular organization of
time-dependent resistance to cytotoxicity in activated hepatic the kinetoplast DNA of Trypanosoma cruzi treated with berenil,
stellate cells. Cell Biochem Biophys 2014;68:247–57. a DNA interacting drug. J Ultrastruct Res 1972;39:568–79.
[33] Ratnam DV, Ankola DD, Bhardwaj V, Sahana DK, Kumar [45] Gonzalez VM, Perez JM, Alonso C. The berenil ligand directs
MN. Role of antioxidants in prophylaxis and therapy: a the DNA binding of the cytotoxic drug Pt-berenil. J Inorg
pharmaceutical perspective. J Control Release Biochem 1997;68:283–7.
2006;113:189–207. [46] Mbaya AW, Nwosu OC, Onyeyili PA. Toxicity and
[34] Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser antitrypanosomal effects of Butryospermum paradoxum
J. Free radicals and antioxidants in normal physiological (Sapotaceae) stem bark in rats infected with Trypanosoma
functions and human disease. Int J Biochem Cell Biol brucei and Trypanosoma congolense. J Ethnopharmacol
2007;39:44–84. 2007;111:26–530.
[35] Pugh DM. Qualitative and quantitative aspects of drug action. [47] Zargar M, Azizah AH, Roheeyati AM, Fatimah AB, Jahanshiri
In: Brander GC, Pugh DM, Bywater RJ, Jenkkins WL, editors. F, Pak-Dek MS. Bioactive compounds and antioxidant activity
Veterinary applied pharmacology and of different extracts from Vitex negundo leaf. J Med Plants Res
therapeutics. London: Bailliére Tindall; 1991. p. 9–27. 2011;5:2525–32.
[36] Kobo PI, Ayo JO, Aluwong T, Zezi AU, Maikai V, Ambali SF. [48] Wurochekke AU, Chechet G, Nok AJ. In vitro and in vivo
Flavonoid mixture ameliorates increase in erythrocyte osmotic antitrypanosomal activity of the leaf of Lawsonia inermis against
fragility and malondialdehyde concentration induced by Trypanosoma brucei brucei infection in mice. J Med Sci
Trypanosoma brucei-infection in Wistar rats. Res Vet Sci 2004;4:236–9.
2014;96:139–42. [49] Raskin I, Ribnicky DM, Komarnytsky S, Ilic N, Poulev A,
[37] Umar IA, Ogenyi E, Okodaso D, Kimeng E, Stanecheva GI, Borisjuk N. Plants and human health in the twenty-first century.
Omage JJ, et al. Amelioration of anaemia and organ damage by Trends Biotechnol 2002;20:522–31.
combined intraperitoneal administration of vitamin A and C to [50] Stevens LH, Stoopen GM, Elbers IJW, Molthoff JW, Bakker
Trypanosoma brucei brucei infected rats. Afr J Biotechnol HAC, Lommen A, et al. Effect of climate conditions and plant
2007;6:2083–6. developmental stage on the stability of antibodies expressed in
[38] Georgiva NV. Oxidative stress as a factor of disrupted ecological transgenic tobacco. Plant Physiol 2000;124:173–82.
oxidation balance in biological system – a review. Bulg J Vet
Med 2005;8:1–11.