ANTI-TRYPANOSOMA ACTIVITY OF ETHANOLIC EXTRACT OF NEEM LEAF ( Azadirachta indica ) ON Trypanosoma evansi IN RATS ( Rattus norvegicus )

The aim of this study was to determine the effect of neem leaf extract (Azadirachta indica) on parasitemia of rats infected with Trypanosoma evansi (T. evansi) Aceh local isolate. A total of 24 male rats aged three months were used in this study and randomly divided into six treatment groups equally. The negative control group (K0) without T. evansi infection and neem leaf extract, the positive control group (K1) was infected with T. evansi but no neem leaf extract given, group K2, K3, K4, and K5 were infected with 5x10 T. evansi and were given neem leaf extract after patent infection with dose of 50, 100, 400, and 800 mg/kg BW respectively. The extract was given orally for three consecutive days. On the fourth day, rat blood was drawn for parasitemia examination. The results showed that no T. evansi detected in rats in negative control group (K0), while parasitemia in group K1; K2; K3; K4; and K5 was 12,295 x10/mL; 10,495 x10/mL; 9,360 x10/mL; 5,080x10/mL; and 2,398x10/mL of blood, respectively. Percentage of inhibition of parasitemia in K2, K3, K4, and K5 reached 14.64, 23.78, 58.68, and 80.50%, respectively. Based on the result of the study, neem leaf extract of 800 mg/kg BW gave the highest reduction of parasitemia in rats infected with T. evansi. ____________________________________________________________________________________________________________________


INTRODUCTION
Animal trypanosomiasis (surra) is a deadly, fly-born parasitic disease for some animal species caused by the blood protozoan Trypanosoma evansi (T.evansi) (Luckins et al., 1992).The parasite that has the widest host range and geographical distribution is transmitted by the bite of infected horse flies (Tabanus spp.) and stable flies (Stomoxys spp.) (Bawm, 2010).The host includes wildlife such as deer, elephant, rhinoceros, and tapir; and domestically high economic value animals such as cattle, horse, and buffalo (Vellayan et al., 2004;Adrian et al., 2010).This disease has become a major obstacle to livestock industry and economic developments and thus become important priority for biomedical and public agencies, agricultural sector and the scientist in some countries (WHO, 2001;Aksoy, 2003).
Clinical manifestation of surra in animals are varied greatly depending upon isolates of the parasite and animal species infected (Luckins, 1996;Desquesnes et al., 2013).As consequences the disease is not only multispecies but also polymorphic (Desquesnes et al., 2013).
Chemotherapy and chemoprophylaxis are still the main way in controling animal trypanosomiasis.Currently, there are six coumpounds available for treatmen of animal trypanosomiasis (diminazene azeturate, isometamedium chloride, homidium bromide/ homidium chloride, quinapyramine sulphate/sulphate chloride, suramine sodium, and melarsomine dihydrochloride).However, there are increasing evidences that drug resistance were observed in some countries (Giordani et al., 2016).For instance, Tsegaye et al. (2015) reported that resistance of trypanosome to anti-trypanosomal drugs occurs in 21 African countries and multiple resistances occur in 10 African countries.Resistance of trypanosomes to anti-trypanosomal drugs was also reported in other countries.In China, resistance of some isolates of T. evansi was observed to Suramin and Antrycide (Zhou et al., 2004).In Indonesia, some T. evansi isolates are resistance to isometamedium and some are resistance to diminaze azeturate (Sukanto et al., 1987).These condition sparked research toward new trypanosomacide to anticipate total resistance.Last few decades, research toward new drug has been focused on plants based drugs and neem (Azadirachta indica, A. indica) is one of herb that has been extensively explored for its potency (Kardiman and Dhalimi, 2003).Previous studies have shown that neem has antimalarial activity (Setiawan, 2009;Isa et al., 2012).Trypanosoma evansi and plasmodium are both blood parasite that share the same class in taxonomy, therefore, it is assumed that neem has antitrypanosoma activity.In this article, we describe the potency of ethanol extracted neem leaves in reducing parasitemia level in rats.

Trypanosoma evansi
Trypanosoma evansi used in this study was a local isolate obtain from whole blood of buffalo in Aceh Besar, Aceh Province, Indonesia.The isolate has been cryopreserved in liquid nitrogen.

Neem (Azadirachta indica)
Sufficient numbers of neem leaves were taken from the field and air dried.The leaves were macerated and ethanol extracted.Maceration was done 3 x 24 hours and the solution was filtrated.The filtrate was then evaporated to eliminate solvent (BPOM, 2000).

Experimental Design
This study used a completely randomize design with six treatment groups and four replications each.Here, 24 male rats (Rattus norvegicus) aged three months old were equally assigned into six groups as the following: K0 was untreated rats (negative control) whereas K1 (positive control), K2, K3, K4, and K5 were rats infected with 5x10 4 T. evansi and treated with 0, 50, 100, 400, and 800 mg/kg BW neem leaves extracts, respectively.

Anti-trypanosomal Test
Trypanosoma evansi isolate was thawed at room temperature, diluted in 100 µl of phosphate buffer saline glucose (PBSG), and intraperitoneally injected into a rat.Parasitemia was checked every two days using improved Neubauer counting chamber for leucocyte.When parasitemia reached 10 7 -10 8 /mL, blood samples were collected and used for the infection step.Here, rats were individually injected with 5x10 4 T. evansi and given neem leaves extracts orally for three consecutive days after patent infection.On day 4, blood samples were collected and the numbers of parasites were microscopically determined using improved Neubauer chamber for leukocyte and counted using the formula below.
Numer of parasite/mL = A x B x 10 4 A = number of parasites counted B = dilution factor

RESULTS AND DISCUSSION
Levels of parasitemia in T. evansi infected rats treated with different doses of A. indica leaf extracts are presented in Table 1.While rats from negative (uninfected) control showed no parasitemia, infected rats given ethanolic extract of the medicinal plant of 0 mg/kg BW (K1), 50 mg/kg BW (K2), 100 mg/kg BW (K3), 400 mg/kg BW (K4) and 800 mg/kg BW (K5) had parasitemia 12.30x10 3 /mL, 10.5x10 3 /mL, 9.4x10 3 /mL, 5.1x10 3 /mL, and 2.4x10 3 /mL, respectively.Parasitemia suppression caused by the respective dose of the extracts on the growth of T. evansi in vivo was 14.6, 23.9, 56.7, and 80.5%, respectively.Reduced parasitemia in T. evansi infected rats given increasing doses of ethanolic extracts of A. indica (Figure 1) indicated a dose-dependent antitrypanosomal activity of the extract.This finding was in agreement with result reported by Raphael et al. (2009) when evaluating chemo-preventive effect of methanolic extracts of A. indica on T. brucei in dogs.When given at higher doses, the extract even showed comparable to or better efficacy than the well-known commercially available trypanocidal drug suramin.
Anti-trypanosomal potential of A. indica plant is supported by better bioavaiability and lower toxicity of the whole extract (Aggarwal et al., 2011).In contrast, lower parasitemia level of T. evansi of rats administrated with higher doses of A. indica showed toxicity effect of the extract on the parasite.The toxicity was one of mechanisms responsible for better antitrypanosoma effects of some medicinal plants against T. cruzi (Teixeira et al., 2014).
The ability of A. indica extract to reduce or kill trypanosome parasites and other apicomplexan blood protozoan such as plasmodium (Momoh et al., 2015) and Leishmania (Teixeira et al., 2014) can be attributed to high phytochemical contents of the extract.Methanolic extract of A. indica contains some secondary metabolites like tannins, saponins, flavonoids, and glycoside (Momoh et al., 2015).The occurrences of these secondary metabolites were identified by other researchers who found alkaloid, terpenoid, kuinolid, phenolic, flavonoid substances in the neem plant extract (Karira et al., 2004;Puspitasari et al., 2009).The neem leaves extract also contains tannin and saponin (Puspitasari et al., 2009).Tasdemir et al. (2006) added that flavonoid had activity as growth inhibitor for T. brucei at trypomastigote stage.Ekaningtias (2014) supported that terpenoid compound is able to inhibit ATP production that is crucial in development of T. evansi.
These compounds might be available in other parts of the plants since all parts of the A. indica plant are useful and have been used in treatment of diseases ranging from tooth decay, swollen liver, ulcers, dysentery, diarrhea, malaria, and other bacterial infections (Allameh et al., 2002;Mossini et al., 2004).

CONCLUSIONS
Rat that survived from T. evansi infection after administration of various doses of ethanol extracted neem leaves proved that the neem leaves have antitrypanosomal activity.It was also proven that the higher the dose the higher percentage of antitrypanosomal activity.

Table 1 .Figure 1 .
Figure 1.Parasitemia from Trypanosoma evansi infected rats treated with various doses of neem leaves extract