Abstract: The aim of this study was to asses the active fraction of the stem bark of G. parvifolia Miq to P. berghei on total red cells of mice and the number of intraperitoneal macrophage phagocytocide latex after infected by P. berghei. Three doses of active fraction of G. parvifolia at the dose of 25, 50, 100 and 200 mg kg-1 b.wt., respectively were used in this study and chloroquine was used as the positive control, while distilled water was used as a negative control. Phagocyte activity was measured by the ability of mice peritoneal macrophages to phagocytize latex particles in vitro. The result of this research indicated that at the dose of 200 mg/kg/day could reduce the number of parasite in the blood up to 54.90%. From the results indicated that the reduction of the number of parasite which circulated in the blood occurs at the doses of 50 and 100 mg/kg/day, although its activity is much lower than malaria drug of chloroquine. Macrophages activity (87.19%) increased at the dose of 200 mg/kg/day, compared to the control group (52.99%), these indicated that the fraction of G. parvifolia induced immune response. The increase of the phagocyte activity of the macrophage was stimulated by the active fractions of G. parvifolia (dose dependent) which was given to the P. berghei-infected mice.
INTRODUCTION
Malaria remains one of the most infamous widespread diseases in the world. It is estimated that there are 300to 500million clinical cases of malaria every year and 1.7to 2.5million deaths (WHO, 1996). Recently, interest in plants as the potential sources of antiparasitic drugs was encouraged by the isolation of artemisinin from Artemisia annua, which was traditionally used in Chinese medicine (Dua et al., 2004).
Macrophages as the trigger in the cellular immune system play a role in the eradication of plasmodium, mainly in the erythrocyte stage. After the macrophages are activated by gamma interferon (IFN γ) produced by native T, they change into phagocyte to do the phagocytosis, to eliminate the parasites (Abbas et al., 1994; Yaneto et al., 2003). The macrophage phagocytosis activities could be activated with the administration of imunostimulant drugs, not only in the form of vaccine but also certain natural chemicals (Subramaniam et al., 2000). A pharmacological screening of n-hexane, ethylacetate and n-butanol extracts of the stem bark of G. parvifolia Miq (Clusiaceae) exhibited significant activity against the drug-resistant clone of P. falciparum (FCR-3) and the sensitive clone of P. falciparum (3D7) (Soesanto et al., 2007). In this study, the effect of the Garcinia parvifolia (active fraction) on the evaluation of experimental P. berghei infection and on the activation of infected macrophage in vitro was assessed.
MATERIALS AND METHODS
Preparation of Extracts
The stem barks of G. parvifolia Miq were collected in Nang Kalis
Village, Borneo, Indonesia, June 2005 and were identified by comparison
with authentic specimens at the Herbarium Bogoriense, Research Center
for Biology, Indonesian Institute of Science and specimen samples were
preserved. The stem bark was dried, powdered and macerated with dengan
n-hexane (1, 5 Lx3), using rotavapor to dry and evaporate to solvent,
to obtain 9 g dry extract. After that, The n-hexane dry extract was fractionated
with methanol and then separated with a centrifuge (5°C, around 10
min), to obtain the extract in the methanol as the solvent (7.2 g) (fraction
A). The active fraction in 2% gum acacia was pulverized into a fine paste
and prepared into a suspension of the desired concentration with 2% gum
acacia for oral administration.
Animals
Male Swiss albino mice, 4-6 weeks' old, from the Litbangkes were used.
The animals were divided into 5 groups of 50 mice each. The mice were
infected with 0.1 mL of the P. berghei suspension at a concentration
of 1.0x10 7 parasite per mouse on day 0. The drug was administered
intraperitoneally for 4 days from day 0 to 3 at doses 25, 50, 100 and
200 mg kg-1 in the experimental group. The control group was
given the solvent in equal volume for the same duration (Peters,
1987). During the experimental period, the animals were housed under standard
laboratory conditions with ad libitum water and balanced food.
No. of Parasitaemia Analysis
The method of Giemsa Blood Smear was used to count the number of the
parasites. The blood in the periphery was taken from the tails of mice
and prepared to a sample of thin-and-thick-smeared-blood method with Giemsa
coloring. The number of parasitemia was calculated by determining the
percentage of red-blood cells infected by P. berghei in 1000 red-blood
cell (Markell et al., 1987).
Macrophage of Phagocytocyte Analysis
The test of non-specific phagocytosis activity was conducted in vitro,
in reference to Leijh et al. (1986), using latex particles with
the diameter of 3 m. The latex particles were resuspended in PBS to obtain
the concentration of 2.5x107 mL-1. Peritoneum macrophages,
cultured a day before, were washed twice in the RPMI medium and then added
with latex suspension of 200 μL/well and incubated in a CO2
5% incubator, 37°C, for about 60 min. After that, the cells were washed
with PBS 3X to remove the unphagocytosysed particles, dried in the room
temperature and fixated with absolute methanol. Once dried, the cells
attached to the cover slip were colored with Giemsa 20%. The percentage
of cells phagocytizing latex particles and the number of phagocytized
latex particles were counted from 100 cells, using a light microscope
with the zoom of 400x (Leijh et al., 1986).
Acute Toxicity Assay
The LD50 of G. parvifolia (active fraction) was determined
in mice using the Lorke method (Lorke, 1983). Fifty Swiss mice of both
sexes weighing 20-25 g were divided randomly into 5 groups of 10 animal
each (5 males and 5 females). The tested Extracts were prepared to suitable
dose levels in water solution. The test groups received a single dose
intraperitoneally of the extracts at different concentrations. The control
group was treated with distilled water, used in all extracts testing.
The LD50 was expressed as the 50% lethal dose, which corresponds
to the dose leading 50% deaths, 14 days after extract administration.
Statistical Analysis
The results are expressed as mean±SEM. The group differences were
analysed in each week using non-parametric analysis of variance (one-way
ANOVA), followed by the Tukey test or Kruskal-Wallis test and then by
the Dunns test, for multiple comparisons. The level of significance used
was 0.01.
RESULTS
The in vivo Effect of G. parvifolia (Active Fraction)
on Phagocytosis
The drug showed a dose dependent stimulation of phagocytosis of opsonized
Plasmodium. At an optimum dose (200 mg kg-1) the phagocytosis
was 87.19%, compared to 52.99% in the control mice. The treatment also
increased the activity and capacity of macrophages in the peritoneal cavity
(Table 1).
The in vivo Antimalarial Activity of G. parvifolia (Active
Fraction)
The procedure followed is the classical 4 day suppressive test of Peters
(1987) where the tested compound is administered during four days to the
malarial mice. With a dose of 200 mg/kg/day, parasitaemia is reduced by
54.90% (Fig. 1). Three mice out of five survived more
than 20 days, while the other four mice (treated with 0.9% saline solution)
died after 4 days (Lorke, 1983).
Acute Toxicity Assay
Daily feeding for 15 days with G. parvifolia (active fraction)
2,000, 4,000 and 8,000 mg kg-1 did not result in any change
in general behaviour of the animals. Body temperature and state of the
excretion were also not influenced by the drug treatment. The weights
of some organs (liver, kidneys and spleen) as well as food and water intake
were not significantly altered by the drug administration.
| Table 1: | Effect of G. parvifolia Miq stem bark (active fraction) treatment to mice on phagocytosis by peritoneal macrophages |
| Values are mean±SD n = 5, *p<0.01, **p< 0.001 | |
| Fig. 1: | Effect Garcinia parvifolia Miq (active fraction) on established infection |
DISCUSSION
This study shows for the first time that the stem bark of G. parvifolia Miq extracts have in vitro and in vivo antimalarial activity (Soesanto et al., 2007). Since macrophages have a major role in stimulating immune function against malarial infection. Other studies are still in progress in this laboratory to identify the effect of the orally-administered active fraction on the activity and the capacity of P. berghei-infected macrophages.
The in vivo Effect of G. parvifolia (Active Fraction) on
Phagocytosis
The phagocytosis response includes the phagocytosis activity (the number
of active phagocyt in 100 phagocyt cells) and phagocyt capacity (the number
of phagocytized plasmodium in 50 active phagocyte cells). The 87.19% increase
of activity and phagocytosis capacity of phagocyt cells in the dose group
of 200 mg/kg/day might be caused by the chemical components in the active
fraction stimulating the lymphocyte cells to mature and self-divide into
lymphocyte B and T producing limfokin (cytocin/interleucine) to keep macrophage
active. Not only can the activated macrophage produce lisozyme enzyme
and the complements, but also increase their capacity to kill through
the phagocytosis process on the plasmodium. In the control group, the
activity of phagocyt cells was low because the phagocyt cells were not
induced.
The in vivo Antimalarial Activity of G. parvifolia (Active Fraction)
In Fig. 1, the parasitemia deterrent of active fraction of 200 mg/kg/day dose (54.9%) is still lower than chloroquine of 5 mg kg-1 dose (88.35%). The ED50 (effective dose 50) is 74.54±10 mg kg-1 BW. The antimalaria activity of the active fraction could be caused by the chemical components in the stem bark of G. parvifolia. Although a number of xanthones from genus Garcinia have been isolated and tested for their antimalaria activity, none of these studies involved G. parvifolia. Likhitwitayawuid et al. (1998a) isolated five xanthones from the bark of G. cowa and evaluated for their antimalaria activity. The results showed that cowaxanthone was the most active against P. falciparum with an IC50 of 1.5 μg mL-1 (Likhitwitayawuid et al., 1998a). The others xanthones from G. dulcis, were also evaluated for their antimalaria activity. Among five xanthones tested, garcinia xanthone exhibited the most active with an IC50 of 0.96 μg mL-1 (Likhitwitayawuid et al., 1998b). It was suggested that antimalaria activity of stem bark extracts of G. parvifolia was due to its xanthones constituents.
Four mice died in the control group on the second day. The death was influenced by the passage stage or the parasite transfer process from a mouse to another. The greater the passage stage, the greater the virulence. The administration of the active fraction seemed to prolong the life of the mice proportional to the administered dose. Perhaps this is because the active fraction have the effect of antiplasmodium as well as increasing the immune system
Acute Toxicity Assay
The limit dose of 8 g kg-1 did not cause any mortality or any signs of acute toxicity in any of the ten mice tested in the short term (i.e., 24 h) and long term (i.e., 14 days) observatory period. No mortality was also observed when varyng doses 2000, 4000 and 8000 mg kg-1 of G. parvifolia (active fraction) were administered orally. The results of the acute toxicity study indicated that the LD50 of G. parvifolia (active fraction) is greater than 8000 mg kg-1 oral.
The present results suggest that the control of the infection may be due to the activation of the cellular response in the animals treated with active fraction. When the macrophages are activated, they may have additional effects on P. berghei, through the release of pro-inflammatory cytokines. The primary target of most of the immunomodulatory compound is believed to be the macrophages which play a key role in the generation of an immune response (Fenichel and Chiriges, 1984).