J.Exp. Life Sci. Vol. 8 No. 3, 2018 ISSN. 2087-2852 E-ISSN. 2338-1655 177 Immunostimulant Activity of Neem Leaf Ethanol Fraction on Tilapia (Putri et al) Immunostimulant Activity of Neem Leaf (A. indica A. Juss) Ethanol Fraction on Tilapia (Oreochromis niloticus) Rika Putri 1* , Sri Andayani 2 , Ating Yuniarti 2 1 Master Program of Aquaculture, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Malang, Indonesia 2 Department of Aquatic Resources Management, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Malang, Indonesia Abstract Intensive cultivation of Tilapia with high stock density and continuous artificial feeding will decrease the water quality and increase the growth of pathogenic bacteria. That situation led to the fish stressed so that the fish become the target of bacterial pathogen attacks. Alternatively, the environmental friendly treatment using herbal immunostimulant to solve that problem is required. The herbal in this study used neem leaf (Azadirachta indica A. Juss). The aim of this study was to evaluate ethanol fraction of neem leaf on immunostimulant activity of Oreochromis niloticus. In this study, the extraction method used maceration. Fractionation methods were using Thin Layer Chromatography (TLC) and Column Chromatography (CC). The biological component was analyzed using Phytochemical, FTIR and GC-MS methods. TLC test showed that ethyl acetate: n-hexane (1:1, v/v) result in brightest luminescence colors on the plates of silica gel under UV light (365 nm). The chromatography column from the ethanol extract of neem leaf (A. indica A. Juss) showed that separation of the fraction was done based on the formation of color, which obtained 5 (five) types of fractions. The results of the phytochemical test for the fraction 4 of neem leaf contains two compounds, i.e. flavonoids (+) and triterpenoids (++). The FTIR test showed that fraction 4 of neem leaves contain a class of terpenoid and flavonoid compounds. GCMS test results on fraction 4 of the neem leaves (A. indica A. Juss) obtained the high peak, which has 4 terpenoid compounds, 1 compound of flavonoid, and 1 type of lauric acid. The total leukocyte of the fish test increased after treated with fraction 4 of neem leaf (A. indica A. Juss) compared with control on day 7 after injection, i.e. 25.29 x 10 4 cells.mL -1 . Keywords: fraction, leukocyte, Neem Leaves, Tilapia, Phytochemicals. INTRODUCTION 1 Production targets of tilapia fish are still below the expectation. Production target of the fish at 2016 was 1,882,200 tons while it only realized for 1,187,812 tons, and the target until 2019 is 2,500,600 tons.year -1 [1]. To meet the target of production, cultivation was done intensively with the application of high stock density and artificial feeding continuously. As the effects, the quality of culture media will decrease due to the accumulation of organic material on the bottom of the ponds, which is favorable for the growth of pathogenic bacteria. With the bad condition of the cultivation media, the fish become stress so that the fish become the target of bacterial pathogen attacks that grow fast in the maintenance media resulting in the failure for reaching the production target [2,3,4]. Treatment is the last alternative in cultivation activities. Thus before the fish attacked by the disease, the maintenance activities were given immunostimulants with the aim to increase the  Correspondence address: Rika Putri Email : rikaputri_bppptegal@yahoo.com Address : Faculty of Fisheries and Marine Sciences, University of Brawijaya, Veteran Malang 65145. fish body's endurance if there is a situation that exceeds the normal limit of the environment. Alternative ingredients that are environmentally friendly and safe for health for immuno- stimulants are using plant herbal ingredients [2,5- 9]. One of the potential herbal ingredients as an immunostimulant for fish is neem leaves. This is because neem leaves have been widely used to overcome the attack of pathogenic bacteria. Neem leaves are used as an antimicrobial because the extract of neem leaf can inhibit the growth of pathogenic bacteria both for gram- positive and gram-negative bacteria [10,11,12]. Terpenoids or triterpenoids increase the total leukocytes of carp that attacked by Koi Herpes Virus (KHV) virus as a body defense because terpenoids have inhibitory activity against bacteria, fungi, virus, and protozoa [13]. The increase in total leukocytes in the fraction-4 treatment of the neem leaves makes the immune response more readily available to detect any attack of foreign substances entering the fish body. An increase in the number of leukocytes will make the phagocytosis process in the antigen will be better and increased the antibodies [14].