Development of the Stem of Jatropha Multifida Linn as a New Antimalarial Through Erythrocytes Test on Mus Musculus Infected by Plasmodium Berghei

Introduction : Jatropha multifida Linne is known as Betadin plant by the Bengkulunese. The stem extract of J. multifida has antibacterial activity. This study examined the potential of stem extract of J. multifida to be developed as an anti-malarial drugs through trials in M. musculus whose erythrocytes infected by Plasmodium berghei.Methods : Phytochemical test of the stem of J. multifida to test the flavonoids, alkaloids, tannins, saponins, terpenoids and steroids. The stem extract of J. multifida obtained by maceration with 96% ethanol. 30 M. musculus divided 6 groups, each consist of 5 animals. P0 is the negative control groups that were not infected by P berghei. P1 is the positive control groups that were only infected by P berghei, P2 is a comparison groups that were infected with P berghei and chloroquine, groups P3, P4, and P5 were infected by P berghei and treated with stem extract of J multifida with consecutive doses 0.028 g / kgbw, 0.056 g / kgbw and 0.084 g / kgbw. After 24 hours, the number of erythrocytes was observed with hemocytometer then was counted the number of uninfected erythrocytes by P berghei using a microscope at 1000x magnification.Results : The stem extract of J. multifida at doses of 0.028 g/kgbw, 0.056 g/kgbw and a dose of 0.084 g/kgbw were able to increase the number of uninfected erythrocytes by P berghei in M. musculus, respectively an average of 9.135million cell eritrosit/mm3, 7.618 millioncell eritrosit/mm3, and 9.856 millioncell eritrosit/mm3. The ability of stem extract of J. multifida in increasing the number of erythrocytes uninfected with P berghei was much higher than the malaria drug chloroquine diposphat. On one way ANOVA analysis of Fcount ( 13,2 ) > Ftable (2.76 ) , with (α = 0.05), there are noticeable differences in the provision of treatment. The increasing number of uninfected erythrocytes by P berghei was due to the content of flavanol glycosides in J multifida stem. Flavanol glycosides was expected to form Flvanolglycosides-heme complex that could inhibit the formation of P berghei parasites HemazoinConclusion : The stem extract of J. multifida could be developed as a potential anti-malarial drugs since it could increase the number of uninfected erythrocytes by P berghei in M. musculus. Its ability was much higher than the malaria drug chloroquine diposphat


INTRODUCTION
Malaria is a disease caused by the Plasmodium, a parasite which is transmitted by infected mosquito bites 1 . WHO reported that an approximately 300-500 million cases of malaria each year and 1.5-1.7 million people died because of that 2 . Indonesia is a malariaendemic areas with quite high fatal cases caused by malaria. Malaria patients in Bengkulu provincehas reached 122-130 per thousand of the population in each year and is increasing annually. An accurate diagnosis and treatment are two important principles in controlling malaria. There are many attempts were made to prevent and conquer the disease. The development of antimalarial drugs and models of effective therapy is still needed in conquering malaria.
Indonesia is one country that is known to have an abundance amount of biodiversity. There are about 30,000 species of plants and 1,260 species of them have been found which have medicinal savor.For example in Bengkulu Jatropha multifida Lin plant which is widely grown as an ornamental plant can be used as a new wound healing drug, so that the plant is better known by the public as a Betadin plant.The extract of J. multifidastem isproved to have a low bioactivity potential towards the shrimp larvae (Artemia salina Leach) but it has antibacterial activity (Salmonella typhi) which is characterized by the formation of limpid zone 3 .The study of J. multifidastem extract has also been provedto have the ability to increase the number of platelets in the Mus musculus 4 .
Because of the increasing threats against malaria, the purpose of this research is to develop a stem of J. multifida as an antimalarial.M. musculusinoculated withP. berghei is an animal model widely used for studying malaria pathogenesis 5 . J. multifidastem as an antimalarial drugs through trials was therefore performed in M. musculusin which the change number of erythrocytes infected with P. berghei was observed.

MATERIALS AND METHODS
Plant material and Experimental animals.The materials used are the J. multifida stems which were taken in Pematang Gubernur, Bengkulu and animal tested is male M. musculus Swiss strain, weighing 25-30 g, around 7-12 weeks old.Infected M. musculus males of P. bergheiwere imported from Health Research Center of Central Jakarta.

Phytochemical test
Flavonoids, a total of 4 g of fresh J. multifida stems were cut into small pieces, then boiled in a beaker glass containing 30 ml of 96% technical ethanol using a water bath.After that, filtering had done in hot conditions.The filtrate was concentrated by half, then added 1 drop of concentrated HCl 6 M and magnesium powder 6 .
Alkaloids, as much as 50 mg of powdered J. multifida stemswas put into a test tube and diluted with 10 ml of HCl and then filtered.The filtrate was tested with Mayer reagents 7 , Wagner reagents and Dragendroff eagents 6 . Tannin, as much as 500 mg of J. multifida stems was added 50 ml of distilled water, boiled for 15 minutes, after being cold down, dripped iron (III) chloride reagent, greenish black coloration indicates the presence of tannin.
Saponins, as much as 50 mg of powdered J. multifidastems were put into a test tube and were added 20 mL of distilled water, the mixture was shaken for 15 minutes. The formation offoam layer as high as 2 cm indicates saponin 8 . Terpenoids and steroids, as much as 0.5 g of J. multifidastems were put into a test tube and were added 2 mL glacial acetic, then were added 3 mL of concentrated H 2 SO 4 .Green to blue coloration indicates a positive steroid and brownish red to purple indicates positif terpenoids 7 .

Preparation of J. multifidastem extract
J. multifida rods were cleaned, and subsequently were cut into small pieces. This wasthen dried aerated with no direct sunlight. This followed by finely grinded and macerated using 96% ethanol for 7 days. The maceration results were filtered. The filtrate obtained was evaporated using a rotary evaporator.

Plasmodium berghei cultivation
P. berghei transferred from M. musculus which has been infected by taking blood from the heart with 2.5 mL injection syringe that had previously been filled with the anti-coagulant heparin of 0.5 mL. Blood was injected into healthy M. musculus with volume ± 0 , 2 mL intraperitoneally.
Then, parasitemia of M. musculus was examined on blood-thin smear. This followed by fixation using absolute methanol, and then flooded with 10% Giemsa solution for 45 minutes. After that the blood-thin smears were washed and dried. This was then examined under a light microscope with a magnification of 100x with oil emersi given. Parasitemia was then calculated in percentage 9 . After parasitemia reached about 30-40%, the blood of M. musculus can be used as a source of P. berghei inoculum of experimental animals.

Animals assay
M. musculus as experimental animals were purchased from Bandung Institute of Technology and infected M musculus were provided by laboratory testing of malaria Litbang Kesehatan Jakarta. Before the treatment, M. musculus were adapted for aweek in Kebun Biologi, University of Bengkulu. Thirty of M. musculusthen divided equally in 6 groups. The group was inoculated with P berghei and treated either with chloroquine, or different dose of J. multifida as shown in Table 1. After 24 hours, the erythrocyteswas counted by using a hemocytometer. The procedure mentioned as following. Mice tails poked with a sterile knife, first drop of blood was discarded, and the next drop was sucked by hemocytometer to the extent of 0.5 or 1. Suction of diluting solution until the number 101, the suspension then was shaken until completely homogeneous (solution becomes to red in the tube). The cover glass wasplaced on the correct place in such way avoiding the cover glass from falling behind. Blood was dripped on the edge of the cover glass and then was counted for the number of infected and uninfected erythrocyte within ± 1000 erythrocyte representation (with a microscope magnification 1000x). The infected -uninfected erythrocytes were determined manually at several fields. Parasitemiapercentagewas the number of infected erythrocytes divided by the total number of erythrocytes multiplied by 100% 10 .

RESULT
Based on the phytochemical test, J. multifida stems contain flavonoids, alkaloids, tannins and saponins, test results are presented in Table 2.  Based on the phytochemical test, it is known that J. multifidastem extracts contain secondary metabolites of the flavonoid, saponins, tannins, and alkaloids.
J. multifidastem ethanol extract has a great activity in increasing the number of uninfected erythrocytes towardsM. musculus which are infected with P berghei, the result ofthe increasing in non-infected erythrocytes can be seen in Table 3.
The difference in the condition of uninfected erythrocytes and infected erythrocytes under the microscope can be seen in Figure 1.

DISCUSSION
J. multifida stem extract after evaporated with a rotary evaporator was insoluble in distilled water because of the possible presence of many sugar groups which are disconnected at the time of evaporation. Therefore, for the importance of oral administration by gavage using the tool on the M. musculus, J. multifida stem extract dissolved in olive oil. The administration of J. multifida stem extract on M. musculus, each in treatment 1 (P3) with a dose of 0.028 g/kgbw and treatment 3 (P5) with a dose of 0.084 g/kgbw, continuously, are able to increase the average number of uninfected erythrocytes to 9.135 million erythrocytes cells/mm 3 and 9.856 million uninfected erythrocytes cells/mm 3 which closer to normal erythrocytes in M. musculus, whereas treatment 2 (P4), the administration of J. multifida stem extract with a dose 0.056 g/kgbw was able to increase the average number of uninfected erythrocytes around to 7.618 million erythrocytes cells/mm 3 . J multifida stem extract has the higher ability to increase uninfected erythrocyte than the malaria drug chloroquine diphospate. Full results of the increasing in non-infected erythrocytes can be seen in Table 3.
Conditions of uninfected and infected erythrocytes under the microscope showed clear differences. Infected erythrocytes have different forms compared to normal erythrocytes. The difference can be seen in Figure 1.
P. berghei is widely used as an inducer of malaria in M. musculus to study the molecular basis of antimalarial for having similarities with P. falciparum.P. bergheiparasite is transmitted by the Anopheles mosquito and are able infect the liver after entering the blood vessels.Parasite growth requires food taken from the erythrocyte cytoplasm, the parasite digests hemoglobin,byproduct of heme degradation of hemoglobin is a substance that is toxic to parasit 11,12,13 .Hemeis a compound [IX ferriprotoporphyrin FP, FP FE (II)],This compound then detoxified by the parasite itself into hemozoin 1,13,14 .Hemozoin is a synthesis that is unique to the malaria parasite, which is regarded as a polymer compound, which is associated with a unit of Fe (III) PPIX or known to the polymer compound of heme.The development of research at this time was hemozoin is a cyclic dimer compounds of Fe (III) PPIX in which the propionate group of each molecule Fe (III) PPIX binds coordination with Fe (III) as the central atom, whereas dimers linked through hydrogen bonding of the acid groups propionat 12, 15 .
Chloroquine shown to inhibit heme detoxification into hemozoin by inhibiting polymerization of hematin through-oxo bonds into dimers, so that hemozoin formation is not occured.This process is also thought to be other molecular targets in the development of antimalaria 11, 16 .Many have written evidence that antimalarial drugs like chloroquine acts forming a complex with heme [FP Fe (II)] and haematin or aqua complex [ferriprotoporphyrin IX, Fe (III) FP], which is derived from the proteolysis of hemoglobin by parasit 13 .

A B B
J. multifida stem extract has the ability to increase the number of uninfected erythrocytes by P berghei in M. Musculus.It is possibly that flavonol glycosides of J. multifida stem extract inhibits heme detoxification.If the flavonoid glycoside compound is not present then P berghei is able to detoxify the heme by turning it into hemazoin that are not toxic to P berghei.The presence of flavonol glycosides compounds is able to inhibit heme detoxification by forming flavonolglycosideshemecomplexes. This was subsequently unable the formation of P berghei hemazoin.A mixture of unreacted heme and flavonolglycosides-hemecomplexes is a toxic compound for P berghei which then reduce the ability of P berghei to infect erythrocytes.The formation of complex compounds flavonolglycosidesheme estimated through such means as presented in Figure 3. Administering stem extract of J. multifida in M. musculus is able to increase the number of uninfected erythrocytes ability even higher than the malaria drug chloroquine diposphat for comparison (see again Table 3)

CONCLUSION
J. multifida stem extract at a dose of 0.028 g/kgbw, 0.056 g/kgbw, and a dose of 0.084 g/kg bwis able to increase the number of uninfected erythrocytes on M. musculus infected by P berghei.The ability of stem extract of J. multifida in increasing the number of erythrocytes infected with P berghei is not much higher than the malaria drug chloroquine diposphat.Based on this study, the ethanol stem extract of J. multifida can be developed as a potential new anti-malarial.

SUGGESTION
Research needs to be conducted in vitro to determine the ability of the active compounds on stem J. multifida: the flavonol glycosides in inhibiting P berghei so it will be able to obtain more detailed information will be the ability of the stem extract of J. multifida as an antimalarial.Identification of flavonol glycosides compounds need to be completed using 13C NMR spectroscopy and 2D NMR to ensure the truth of the alleged structure identified flavonol glycosides.