Prophylactic Effect of Grapefruit Juice Against Plasmodium berghei berghei Infection in Mice
Grapefruit juice was assessed for chemoprophylactic
activity against Chloroquine-sensitive Plasmodium berghei berghei
infection in mice. A standard inoculum of 1x106 infected erythrocytes
was used to assess the prophylactic effect of grapefruit juice (15 mL
kg-1) and this was compared with the prophylactic effect of
high dose Ascorbic acid (150 mg mL-1 or 2.25 g kg-1).
The result of the experiment showed that grapefruit juice and high dose
vitamin C significantly delayed the establishment of parasitaemia compared
with the control group. Furthermore, grapefruit juice and ascorbic acid
prolonged the mean survival time of the mice with corresponding decreases
in mean peak percentage parasitaemia, respectively. Grapefruit juice however
demonstrated a stronger chemoprophylactic activity than ascorbic acid
(p<0.05). These effects were however lower than the standard prophylactic
drug (Pyrimethamine-1.2 mg kg-1). Regular intake of grape juice
may protect against malaria infection. Further studies are necessary to
elucidate possible mechanisms involved.
Grapefruit is the fruit of the citrus plant. Its botanical name is Citrus
paradisi, family Rutaceae. It is believed to be native to Jamaica.
This fruit is similar to the orange fruit (Citrus sinensis) but
differs by being bigger than the orange fruit. Other differences are the
thicker and smoother rind and the bitter taste of grapefruit juice.
Grapefruit, has very useful medicinal values. It is high in vitamin C
and potassium (Mahan and Stump, 2000). It also contains bioflavonoids
and other phytochemicals that protect against cancer and heart disease
(Turner et al., 2004).
Ascorbic Acid (AA), the reduced form of vitamin C, is an important cofactor
in several enzyme reactions and plays a pivotal role in the defense against
oxidative stress (Levine, 1986; Gambhir et al., 1997); whereas
the oxidized form, Dehydroascorbic Acid (DHAA) is considered a marker
of oxidative stress (Ayori et al., 2000).
Vitamin C, an antioxidant vitamin, present in grape juice prevents the
free radical damage that triggers the inflammatory cascade and is therefore
also associated with reduced severity of inflammatory conditions, such
as asthma, osteoarthritis and rheumatoid arthritis (Kurl et al.,
There has been claims that consistent intake of grape juice which is
high in vitamin C content will protect the individual against malaria
infection (Hassan et al., 2004). We and others have demonstrated
the prophylactic activity of other antioxidant vitamin preparations like
vitamin A and Cod liver oil (Oreagba and Ashorobi, 2006; Awodele et
al., 2006; Levander et al., 1989).
It has also been shown that Plasmodium falciparum infection could
compromise the level of ascorbic acid, the reduced form of vitamin C (Isamah
and Asagba, 2003).
These accounts suggest that grapefruit, an excellence source of vitamin
C, may protect against malaria infection however scanty information is
available to articulate this viewpoint.
The aim of this study is therefore to investigate the chemoprophylactic
effect of grapefruit juice against Plasmodium berghei infection
MATERIALS AND METHODS
The parasite: The NK65 strain of Plasmodium berghei berghei
used in this study was obtained from Dr. G.O. Ademowo of the Chemotherapy
Research Laboratory, Institute of Advance Medical Research and Training,
(IMRAT) College of Medicine University of Ibadan Ibadan Nigeria and maintained
in mice by weekly passaging into fresh mice. Each mouse was inoculated
intraperitoneally with 0.1 mL of infected blood containing about 1x107
Plasmodium berghei berghei parasitized red blood cells obtained
from a donor mouse having about 60% parasitaemia.
Thin blood films were made by collecting blood from the tail, this was
stained with Geimsa stain and the percentage parasitaemia was determined
by counting the number of parasitized red blood cells out of 1000 blood
cells in 10 random microscopic fields.
The animal grouping and treatments: The repository activity of
grapefruit juice was evaluated using the method described by Peters (1965).
Male Swiss albino mice, weighing 20-25 g kept under good ventilation
and balanced diet, were used in this study. Animals were subdivided into
five groups of 10 each. Group 1 (control) received distilled water only.
Group 2 received high dose vitamin C (150 mg mL-1) given 3
times daily for 7 days before parasite inoculation. Group 3 received grapefruit
juice at a dose of 15 mL kg-1 three times daily for 7 days
before inoculating with malaria parasite. Group 4 received Pyrimethamine
(1.2 mg kg-1 day-1) once daily for 7 days. Administration
was by means of the oral cannular.
On the 8th day, the treated mice were passaged with Plasmodium berghei
parasite. Microscopic examination of the blood film was made to determine
percentage parasitaemia 72 h post inoculation. The average suppression
of parasitaemia was calculated in comparison to control as shown below:
The result of the experiment showed that pretreatment of mice with grapefruit
juice for 7 days delayed the establishment of parasitaemia compared with
the control. On day 3, average parasitaemia of mice pretreated with distilled
water was 5.21% while that of the mice pretreated with grapefruit juice
was 2.22%, a difference of 2.99% (Table 1). Mice pre-treated
with high dose vitamin C (150 mg mL-1) recorded an average
parasitaemia of 4.81% which showed mild prophylactic activity when compared
with the control group (Table 1).
In addition, pretreatment of mice with grapefruit juice prolonged the
average lifespan of mice, extending it by 6 days from 7 days in the control
group to 13 days in the group pre-treated with grapefruit juice. The corresponding
mean percentage parasitaemia were 37.5 and 23.08%, respectively. This
was also the case with the group pre-treated with high dose vitamin C
(150 mg mL-1) which recorded a Mean Survival Time (MST) of
11 days (an extension of 4 days) and a Mean Percentage Parasitaemia (MPP)
of 28.08% (Table 2).
||Prophylactic effect of grapefruit juice and vitamin
C against P. berghei infection 3 days post inoculation
|Data expressed as mean ± SEM for 10 animals per
group df 4.45 *p<0.05 when compared to control; *: Equal volumes
(0.3 mL) of distilled water and drugs were used
||Effect of grape juice and vitamin C on survival time
during P. berghei infection
|MST: Mean Survival Time, MPP: Corresponding Mean Percentage
Parasitaemia, *: Equal volumes (0.3 mL) of distilled water and drugs
On day 5, it was noticed that while the stage of development of the parasite
in the control group were mostly schizonts, those of the mice pretreated
with grapefruit juice and vitamin C had a prolonged trophozoite stage
indicating a delay in their growth rate.
Malaria parasites are highly susceptible to alterations in redox equilibrium
of its system and environment and this offers great opportunities for
chemotherapy and chemoprophylaxis. In the present study, we examined the
chemoprophylactic effects of grapefruit juice, which is rich in ascorbic
acid, against Plasmodium berghei infection in mice. Grapefruit
juice indeed delayed the onset of parasitaemia as shown in our study indicating
chemoprophylactic activity. This delay was similar to but stronger than
that produced by pretreatment with high dose vitamin C alone indicating
a form of synergistic interaction between vitamin C and other components
of grapefruit juice like lycopene and bioflavonoids present in grapefruit
juice. Incidentally a recent study suggests that higher plasma concentrations
of lycopene at enrollment was significantly associated with clearance
of malaria parasitemia by day 3 in young children (Amy et al.,
Although we did not study the antiplasmodial effect of grapefruit juice
on established infection, this observation is nonetheless consistent with
the study by Marva et al. (1992) which showed that Ascorbic acid
caused stage-dependent destructive effects on the in vitro development
of Plasmodium falciparum especially the advanced forms of the parasite.
Normally, as an antioxidant, ascorbic acid will help neutralize peroxides
and other oxygen free radicals. However, in large doses ascorbic acid
can change behavior and act like a pro-oxidant. The possible mechanism
of action for the pro-oxidant effect of vitamin C has been well postulated.
During the growth of P. falciparum the infected erythrocytes release
increasing levels of iron-containing structures that are redox-active,
vitamin C interacts with these structures by reducing iron and oxygen
thereby inducing the conversion of hydrogen peroxide to yield highly reactive
hydroxyl radicals (Udenfriend et al. 1954). It is speculated that
hydroxyl radicals generated in this fashion is responsible for the pro-oxidant
effect of vitamin C (Winter et al., 1997).
Another possible mechanism which may compliment the above could be through
the immunomodulatory effect of vitamin C and some other phytochemicals
present in the juice e.g., bioflavonoids and coumarins, studies have shown
that ascorbic acid appears to stimulate humoral immunity through increased
antibody synthesis particularly IgG, IgA and IgM types (Ansari et al.,
1998). It also activate the macrophages which produce Reactive Oxygen
Species (ROS) (Mohan et al., 1994) as part of the host immune reaction
against malarial parasites (Adelekan and Thurnham, 1998), a mechanism
similar to that of vitamin A chemoprophylaxis against P. falciparum
infection (Serghides and Kain, 2002) in non-immune host which in this
case include present study animals.
Stocker et al. (1986) have shown that Ascorbic acid is taken up
readily by P. vinckei parasitized red blood cells when administered
and the uptake was increased in proportion to the rate of malarial infection.
This may also be applicable to our findings which showed protection against
P. berghei parasite by ascorbic acid and grapefruit juice.
The greater prophylactic effect of grapefruit juice compared with ascorbic
acid may be associated with a higher rate of uptake into the parasitized
erythrocytes. This mechanism may also account for the delay in the development
of the parasite from the trophozoite stage to the schizont stage delay
in the group that received grapefruit juice.
One important limitation of this study was the absence of a separate
group of mice placed on continuous administration of grapefruit juice
even after parasite inoculation to determine the possibility of a permanent
prophylactic effect. Since there is an increase in the concentration of
plasma Thiobarbituric Acid Reactive Substances (TBARS) in malaria patients
(Prasannachandra et al., 2006) and Malondialdehyde (MDA), in the
blood of mice infected with P. berghei (Golenser and Chevion, 1989),
a look at the chemoprophylactic effect of grapefruit juice on the level
of these lipid peroxidation products would also be useful for future studies.
The results of this study has demonstrated the repository activity of
grapefruit juice against P. berghei infection in mice. This confirms
its use in ethnomedicine as a malaria prophylactic. Further studies are
necessary to elucidate possible mechanisms involved.
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