2. Classification and morphology of Phyllanthus niruri L.

Phyllanthus niruri L. (Figure 1) belongs to the Euphorbiaceae family, a plant that is distributed in tropical and subtropical regions. In Malay this plant is called dukong anak, in China it is known as zhu zi cao, in Brazil it is called chanca Piedra and in southern India it is called Bhumyamalaki [10]. This plant lives on land to an altitude of 1000 m DPL, in rocky places, damp places such as along waterways, or among grass and shrubs [Dalimarta]. The classification of Phyllanthus niruri L. is as follows:

Kingdom: Plantae

Division: Spermatophyta

Sub Divisi: Angisopermae

Class Order: Dicotyledonae

Family: Dicotyledonae

Genus: Euphorbiaceae

Species: Euphorbiaceae

: Phyllantus niruri L. [14 Kardinan, 2004]

Phyllanthus niruri L. is a wild herb plant, originating from Asia and has now reached the African, Australian and American continents, having light green or dark green stems. Each branch or twig consists of 8-25 leaves. The leaves are green, measuring 0.5-2 x 0.25-0.5 cm [14]. It has white male and female flowers. Male flowers appear under the leaf axils, while female flowers appear above the leaf axils [15]. The ripe meniran anthers will burst longitudinally [14].

3. The content and benefits of Phyllanthus niruri L.

In general, the Phyllanthus niruri L. plant contains several complete chemical compounds, so it is very beneficial for health. P. niruri contains alkaloids, flavonoids, tannins, saponins, steroids, phenols and terpenoids [16, 17, 18, 19]. Triterpenoid compounds contain components of lupeol acetate and betasitosterol [20]. Apart from containing bioactive compounds of flavonoids, tannins, alkaloids, steroids, P. niruri also contains molecules of lignans, phyllanthin, hypophyllanthin, glycosides, ellagitannins, triterpenes, phenyl propanoids, ricinolic acid, niruriside, phyltetralin, essential oils, anthraquinones and arbutin [21, 22, 23, 24, 25]. Components of flavoniod compounds such as quercetin are mostly found in the leaves, while the stem contains niruri components, niruritenin, rutin. Lignin compounds such as phyllanthin and hypophyllanthin are present in all parts of the plant [26]. Quantitatively, the Phyllanthus niruri L plant contains a total phenol equivalent to 28.05 g of gallic acid in 1 mg of plant extract, while the content of flavonoids in 1 mg of plant extract is equivalent to 61.41 g of quercetin, while the filantin content in 70% P niruri ethanol extract of 0.864% [16]. Traditionally this plant has been widely used by people in tropical countries to treat various types of diseases including intestinal infections, kidney stones [27, 28], chronic liver disease, diabetes and preventing hepatitis B virus infection [28, 29], asthma, gonorrhea, bronchitis, and syphilis [30, 31] and boosting the immune system [11]. The ability of P. niruri to treat various diseases is due to its pharmacological properties, including as an immunomodulator, antiviral, antibacterial, diuretic, anti-hyperglycemic and hepatoprotector [10, 12]. In Indonesia, this plant is used to boost the immune system. This plant extract has been shown to be an immunomodulator that can increase blood peripheral proliferative activity and macrophage phagocytic activity [11].

4. Applications of Phyllanthus niruri L. as immunostimulant in culture fish

Several studies have been carried out to prove the effect of P. niruri as an immunostimulant that can increase the resistance of humans and animals including fish against disease. Several parameters can be used as indicators of an increase in the immune system or an increase in body resistance after being treated with compounds that act as immunostimulants, including antibody titres, white blood cells including their components, namely neutrophils, monocytes, lymphocytes and macrophages. Meanwhile, cytokines, antibodies, and complement proteins are included in the immune molecule. Synchronous interactions between the immune system and immune molecules can induce a good immune response, which can lead to resistance to pathogenic infections [32]. Antibody titer is one of the parameters to determine the effectiveness of an immunostimulant substance, by looking at the ability of serum proteins containing antibodies to collect and destroy antigens that enter the body [33].

Two sets of Oreochromis mossambicus fish groups, each consisting of 6 groups (n = 8 per group) were used as test fish to prove the ability of P. niruri as an immunostimulant by observing the levels of an body titers and neutrophil levels. The part of the P. niruri plant used was the leaf in the form of an aqueous extract. The extract was given to fish by intraperitoneal injection with various doses, namely 20, 2, 0.02 and 0.002 mg /kg fish body weight. To observe the activity of antibody titers, the first group of fish, after two days of administration of P. niruri leaf extract, were challenged with Sheep red blood cells (SRBC) which function as antigens. Sheep red blood cells (SRBC) are given by intraperitoneal injection with the first dose of 5% of 0.1 mL and a second dose of 25% (booster) of 0.1 mL given three days after the first dose, then observed for 60 days from the first injection. As for observing neutrophil activity, fish in the second set were challenged with 100 μL HA- BSA (heat aggregated-bovine serum albumin) with a concentration of 5 mg via intraperitoneal injection, then observations were made for 12 days. The results showed that administration of P. niruri aqueous extract resulted in a significant increase compared to controls in the activation of antibody and neutrophil responses. Of the various doses tested, the group of fish that were given P. niruri leaf aqueous extract at a dose of 20 mg /kg body weight gave the maximum increase in primary and secondary antibody responses on the 15th day of observation. Whereas the group of fish that were given P. niruri leaf aqueous extract at a dose of 0.02 mg /kg body weight resulted in a maximum increase in neutrophil levels on day 6 [34]. Tilapia fish (Oreochromis niloticus) that have been given P. niruri leaf extract through injection are able to survive against Aeromonas hydrophila bacteria. A concentration of 50 mg was significantly the best concentration with the highest survival of tilapia, which was 76.7% [35 2014]. carp (Cyprinus carpio) after being given feed mixed with P. niruri powder for 60 days was then challenged with A. hydrophila bacteria with a density of 103 CFU with the cohabitation method through water in a rearing aquarium, able to survive the attack of these bacteria. Giving P. niluri powder as much as 2% (10 g / 500 g feed) showed the highest fish resistance against attack by A. hydrophila bacteria, which was 100% [36] (Table 1).

The results of research conducted by Ma'at et al., (1996) [37] showed that P. niruri had an immunomodulating effect in vitro. P. niruri extract (50-200 mg /kg) can elicit both cellular and humoral immune responses. As the results of research by Nworu et al., (2010) [38] showed that P. niruri water extract (12.5-200 g /ml) could induce lymphocytes and macrophages in experimental animals.

In Table 1 it can be seen that the P. niruri plant used as an immunostimulant is the leaf part, in the form of extract or powder (flour) and the method of administration is also by injection or through feed. Testing the ability of these plants as immunostimulants in fish can be carried out using various challenge test methods and various observational indicators, among others by looking at antibody levels, neurophil levels and survival of fish after being challenged with pathogenic bacteria. The type of fish tested, the challenge test method and the dosage used influenced the results obtained.

Based on the results of several studies above, it proves that P. niruri can be used as an immunomodulator that can induce fish resistance to pathogenic bacteria. As according to the POM Agency (2006) [39] that P. niruri is an immunomodulator, which can improve or enhance the immune system. The main component and is an active compound as an immunostimulant which is contained in P niruri flavonoids. The mechanism of flavonoids as an immunostimulant is to activate the body's cells by sending intracellular signals to cell receptors, so that the body's cells work optimally. Apart from flavonoids, other compounds contained in P. niruri, including phyllanthin and hyphophyllanthin are two compounds that have antiinflammatory activity so that they can strengthen immunity. Another opinion states that P. niruri has an effect as an immunomodulator through activation and augmentation of the cellular immune system. Specifically P. niruri can activate neutrophils, macrophages, monocytes, T and B lymphocytes. The increase in phagocytosis by neutrophils indicates an accelerated process of eradicating pathogenic microbes, especially for extracellular pathogens, such as viruses, bacteria or fungi. P. niruri can induce an increase in monocytes and macrophages, which are able to lyse infecting intracellular pathogenic cells and expose these pathogenic cells to other immune components in the extracellular compartment. In addition, P niruri can modulate cytokine secretion, including stimulating IFN-γ, TNF- α, IL-4, IL-6, IL-12 and suppression of IL-10. This shows that P. niruri can induce the body's defense, by engaging the cellular immune system against foreign pathogens [40]. On the basis of the immunostimulatory properties possessed by P. niruri from a series of non-specific (neutrophil), specific (antibody) parameter tests and other tests, namely the survival rate of fish challenged with pathogenic bacteria can be used either as a routine feed supplement to activate the immune system of cultivated fish, other than that it can be used as an adjuvant in the use of vaccines.