Immunomodulatory and in vivo Antiplasmodial Activities of Propolis Extracts

The purpose of this research was to evaluate the immunomodulator and antiplasmodial activities of Indonesian propolis extracts. This research utilized not only hypersensitivity reaction which measures the humoral immunity by SRBC-immunized mice but also the activity and capacity of peritoneum macrophage phagocytosis in Plasmodium berghei-infected mice. The parasitaemia number was calculated using blood smear method every day for 4 day after the mice had been infected P. berghei to identify the antiplasmodial activity. The research results reveal that propolis hydroalcoholic solution (PHS) has a strong immunomodulatory activity but weak antiplasmodial activity.


INTRODUCTION
mechanism of humoral immunity in eliminating Propolis (bee glue) is a dark-coloured resinous antimalarial antibody is strongly suspected to play substance collected by bees from poplar buds and other important role in immunity. The aim of this study was to plants and used to seal their hives [1]. More than 180 evaluate the antiplasmodial and immunomodulatory propolis constituents have been identified by gas activities of propolis. chromatography-mass spectrometry (GC-MS). These compounds can be grouped as follows: free aromatic MATERIALS AND METHODS acids; flavonoids; benzyl, methylbutenyl, phenylethyl, cinnamyl and other esters of these acids; chalcones and Propolis Hydroalcoholic Solution: Propolis was produced dihydrochalcones; terpenoids and others such as sugars, by Cibubur honeybees from the apiary located on Lawang ketones, alcohols [2,3]. Although in small quantities, (East Java, Indonesia). A 30% propolis ethanolic solution these compounds can be very important to propolis was prepared. A week later, this solution was filtered and activity [4].
used to prepare a 10% propolis hydroalcoholic solution It has been used in folk medicine since ancient times (PHS). and its now known to be a natural medicine with antibacterial, antifungal, antitumoral, antioxidative, Animals: Thirty male BALB/c mice weighing immunomodulatory and other beneficial activities [5]. The approximately 25-30 g aged between 6 and 8 weeks old effect of propolis on the immune system has also been were used for propolis treatment in vivo. The mice investigated by some authors, who showed its ability to infected with 0.1 mL of the P. berghei suspension at a activate macrophages [6,7] and stimulate antibody concentration of 10 parasite per mouse on day 0. The production by SRBC-immunized mice [8]. The exact drug was administered orally for 4 days from day 0 to 3 at plasmodium is not completely understood. However, an 7 doses 25, 50 and 100 mg/kgBW in the experimental group.

RESULTS AND DISCUSSION
The control group was given the solven in equal volume for the same duration [9]. During the experimental period, The functional immune response occurs when the the animals were housed under standard laboratory parasites undergo asexual erythrocitic phase. As soon as conditions with ad libitum water and balanced food.
the parasites enter the red-blood cells, the antibody can No of Parasitaemia Analysis: The methods of Giemsa the research results shown on Table 1, we can see that the Blood Smear was used to count the number of the group of mice PHS-administered in the dosage of 100 parasites. The blood in the periphery was taken from the mg/kg BW has higher IgG concentration than those in the tails of mice and prepared to a sample of thin and thick dosage of 25 and 50 mg/kg BW. With regards to the smeared blood method with Giemsa coloring. The number humoral immune response, the ethanolic extract of of parasitemia was calculated by determining the propolis (500 ìg/mouse) increases the antibody percentage of red blood cells infected by P. berghei in production in sheep red blood cells (SRBC)-immunized 5000 red blood cells [10].

Macrophage of Phagocytocyte Analysis:
The test of non-system play a role in the eradication of plasmodium, specific phagocytosys activity was conducted in vitro, in mainly in the erithrocyt stage. After the macrophages are reference to Leijh et al. (1986), using latex particles with activated by gamma interferon (IFN ã) produced by native the diameter of 3 m. The latex particles were resuspended T, they change into phagocyte to do the phagocytosys, in PBS to obtain the concentration of 2.5 x 10 / ml.
to eliminate the parasites [14, 15]. The macrophage 7 Peritoneum macrophages, cultured a day before, were phagocytosys activities could be activated with the washed twice in the RPMI medium and then added with administration of imunostimulant drugs, not only in the latex suspension of 200 ìl/well and incubated in a CO 5% form of vaccine but also certain natural chemicals [16]. 2 incubator, 37 C, for about 60 minutes. After that, the cells The primary target of most of the immunomodulatory o were washed with PBS 3X to remove the compound is believed to be the macrophages which play unphagocytosysed particles, dried in the room a key role in the generation of an immune response [17]. temperature and fixated with absolute methanol. Once The phagocitosys response includes the phagocytosis 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 [11].
Hypersensitivity Reaction Which Measures the Humoral Immunity: The PHS (in doses of 25, 50 and 100 mg/kg, body weight) was administered to the animals (test group) orally for five days and the vehicle was administered to the control animals. Each group consisted of five mice. The mice were immunised by injecting 0,5 mL of SRBCs intraperitoneally (i.p) on the day of the immunisation. Blood samples were collected by retro-orbital puncture on the tenth day after immunisation. Antibody levels were determined by the hemagglutination technique [12]. The antibody titer was determined by a two-fold serial dilution of one volume (100 µL) of serum and one volume (100 µL) of 0,1% SRBCs in BSA in saline was added and the tubes were mixed thoroughly. They were allowed to settle at room temperature for about 60-90 min until the control tube showed a negative pattern. The value of the highest serum dilution showing visible hemaglutination was taken as the antibody titer. be detected using conventional serology method. From Macrophages as the trigger in the cellular immune     % parasitemia (mean ± SD) on the following day after treatment activity (the number of active phagocyt in 100 phagocyt immunostimulatory effect produced by the PHS cells) and phagocyt capacity (the number of phagocytized containing flavonoids, may be due to cell mediated and plasmodium in 50 active phagocyt cells). The humoral antibody mediated immune response. phagocytosis activity and capacity of propolis extract in As the mice were infected by P. berghei, the the dosage of 100 mg/kg BW/day is higher than those in parasitaemia increase (Table 3) since the body immune the dosage of 50 and 25 mg/kg BW might be caused by response was not quite perfect and the parasites were still the chemical components in the active fraction stimulating phagocytosized slowly mainly in lymph. A lot of infected the limphocyt cells to mature and self-divide into erythrocytes were found in lymph and phagocytosis by lymphocyt B and T producing limfokin macrofage. The phagocytosis on IgG-sensitized cells and (cytocin/interleucine) to keep macrophage active. Not C3b-attached cells by the lymphatic macrofages of only can the activated macrophage produce lisozyme infected mice was higher than that of normal mice. enzym and the complements, but also increase their Plasmodium berghei is a synchronized parasite target capacity to kill through the phagocytosys process on the erythrocytes infected by young parasites might not cause plasmodium. In the control group, the activity of the change of erythrocyte membrane surface. Lymph phagocyt cells was low because the phagocyt cells were macrofages activated by malarial infection may not induced. phagocytosize those. The factor increasing infected-Lymph as one of the secondary organs also plays erythrocytes phagocytosis is macrofage activation. Free important role in supporting body defense. By parasites and full-grown erythrocytes are targets of transferring lymphatic cells from P berghei-immune mice phagocytosis because they express surface antigens to recipients, all parasites were eradicated relatively fast.

Dose (mg Kg No of Mice -----------------------------------------------------------------------------------------------------------
against serum [21]. The Table 3 reveals that PHS Under the P. berghei-infected condition, the lymph sizes administered showed weaker antiplasmodial activity than of the mice in negative control group were larger than chloroquine dose. On the other hand, Dantas et al. (2006) those in normal mice and PHS-administered mice. The investigated that effects of Bulgarian propolis (25 and 50 increase of lymph weight in the mice of control negative mg/kg) in the experimental model of Trypanosoma cruzimice was probably due to the increase of erythropoietic infected Swiss mice, verifying that this bee product led to activity in the lymph. During malarial infection the number a decrease in parasitemia and showed no hepatic or renal of infected erythrocytes multiplies 8 times every 48 h and toxic effect [22]. Propolis hydroalcoholic solution (PHS) all of them will be destroyed when the schizonts breaks showed more immunostimulant activity than out. The lost of these numerous erytrocytes triggers the antiplasmodial activity, proved by the increase of IgG and bone marrow to produce new ones [18]. the macrofage phagocytosis activity and capacity in the Recent reports indicate that several types of dosages of 25, 50 and 100 mg/kg BW. The antiplasmodial flavonols stimulate human perpheral blood leukocyte activity of PHS was due to the mice immunity increase so proliferation. They significantly increase the activity of that they lived longer. helper T cells, cytokines, interleukine 2, -interferon and macrophages and are thereby useful in the treatment of