IN VITRO ANTIBACTERIAL EFFICACY OF LEAVES EXTRACT OF Centela asiatica AGAINST Vibrio harveyi AND Aeromonas hydrophila

Disease infection is one of the limiting factors that affect productivity in aquaculture and has caused economic losses. Luminescent vibrios and motile aeromonas septicemia (MAS) are diseases caused by Vibrio harveyi and Aeromonas hydrophila bacteria, respectively. Certain plants have antimicrobial compounds and can potentially be used to treat the diseases, such as Centella asiatica. In the present study, the crude leaves extracts of C. asiatica were examined for its antibacterial potential using methanol solvents against V. harveyi and A. hydrophila bacteria. Different concentrations of 50 mg/mL and 100 mg/mL were checked for its antibacterial activity. The crude extract was also tested for phytochemistry content and LC50 using Brine Shrimp Lethal Assay. The crude extracts of C. asiatica showed a remarkable antibacterial activity with inhibition zone of 10.57 mm against A. hydrophila and 21.14 mm against V. harveyi. The phytochemistry test result showed that C. asiatica leaves contain alkaloid, phenol, and tannin compounds. The acute lethal concentration (LC50) of C. asiatica after 24 hours exposure to the extract mixture was 254 mg/L. The results confirmed the potential use of C. asiatica extracts as a source of antibacterial compounds.


INTRODUCTION
Aquaculture is the fastest-growing food-producing sector in the world with annual growth reaching 9%. This growth is driven by the increased of seafood demand along with global population growth (Allan & Burnell, 2013). Aquaculture activities are not only required to be able to provide food source but also to sustain aquaculture production and product trade. The growth of aquatic biota was influenced by infection of diseases. Disease infection is one of limiting factors that affect productivity in aquaculture and has caused economic losses due to various diseases caused by bacteria, fungi, parasites, and viruses (Flegel et al., 2008).
Here, we focus on disease caused by luminescent vibrios (i.e., Vibrio harveyi) and motile aeromonas septicemia (MAS) diseases caused by Aeromonas hydrophila. Vibrio harveyi is a bacterium that causes shrimp mass mortality. These bacteria attack shrimp larvae in hatcheries and shrimp cultivated in on growing ponds and known as luminescent shrimp disease. The shrimp infected by this bacteria will glow in the dark and usually attack the larvae at zoea, mysis and post larvae stages. V. harveyi are gram-negative, usually motile rods, facultative anaerobes and types of bacteria that live in the sea and have high salinity resistance (Thompson et al., 2004). While A. hydrophila attacked all types of freshwater fish in the tropics and were often isolated from diseased catfish. This disease usually occurs when the fish condition decreases due to stress and poor water quality (Sarono et al., 1993).
There are many approaches and mitigation efforts to reduce the negative impacts caused by pathogenic agents, including the use of high quality seed, best culture management, and application of biotechnology. In addition, many farmers, especially in developed countries, have improved their capacity to respond quickly and effectively to emergent disease situations. In most developing countries, the use of antibiotics is common to control fish disease distribution. However, antibiotics to treat bacterial diseases has been prohibited because uncontrolled antibiotics use may lead to resistant bacteria. This is In vitro antibacterial efficacy of leaves extract of Centela asiatica ..... (Rukisah) viewed as potential health issue related to the transmission of resistant bacteria from aquaculture environment to humans (Cabello, 2006). Other related risk is the introduction of non-pathogenic bacteria in the environment which contain antimicrobial resistance genes causing genetic contamination on the aquatic environment (Serrano, 2005). Because of their toxic and carcinogenic side effects, the use of modern therapeutic drugs is being restricted. Thereby, interest in finding alternative medicinal ingredient for fish diseases from natural source without undesirable effects has increased greatly (Bothon et al., 2013).
Centella asiatica (L.) is a tropical medicinal plant from Apiaceae family native to Asian countries such as India, Sri Lanka, China, Indonesia, and Malaysia as well as South Africa and Madagascar (Jamil et al., 2007). The leaves of C. asiatica had traditional uses as treatment of asthma, ulcers, leprosy, lupus, vein diseases (Brinkhaus et al., 2000;Kartnig, 1988), memory improvement (Gupta et al., 2003;Rao et al., 2005), antidepressant (Chen et al., 2003), antibacterial, antifungal (Ullah et al., 2009), psoriasis (Sampson et al., 2001), and anti-cancer agent (Babu et al., 1995). C. asiatica contains a variety of compounds including asiaticoside, centeloside, madecassoside, brahmoside, glycosides, saponine, asiatic centoic acid, centelic acid, centoic acid, madecassic acid, and oxyacathicoside. Some of these compounds are known to have the most active antimicrobial capability which could even inhibit tuberculosis bacteria (Kurniawati et al., 2005). Methanol extract of C. asatica has been investigated for antimicrobial activity against A. hydrophila and Edwardsiella tarda (Purkait et al., 2018). However, there are currently no reports on the ability of C. asiatica extracts to inhibit the growth of V. harveyi. This study was aimed to evaluate the antibacterial activity of C. asiatica against V. harveyi and A. hydrophila.

Materials
The chemicals used in this study were methanol, sulfuric acid (H 2 SO 4 ), hydrochloric acid (HCl), potassium iodide, iodine, glacial acetic acid, ferric chloride, chloroform, and media cultures (nutrient agar, nutrient broth, tryptic soy agar, and thiosulfate citrate bile salts sucrose) obtained from Water Quality Laboratory, Faculty of Fisheries and Marine Science, Borneo Tarakan University. The bacterial strains were V. harveyii and A. hydrophila obtained from the collection of the laboratory of Nutrition and Fish Feed, Faculty of Fisheries and Marine Science, Borneo Tarakan University, Indonesia.

Collection of Plant Material
C. asiatica leaves were collected in the early morning hours in Tarakan rural areas. The plants were washed with cold distilled water and then dried at room temperature for seven days. The samples were crushed properly by a metal mortar until a fine homogeneous dried powder was obtained and then placed in paper bags under free humidity conditions (Cipriano, 2001).

Preparing Plant Extract
Dried powder of C. asiatica leaves (40 g) was placed in a 500 mL flask, mixed with 160 mL of methanol, and tightly wrapped with aluminum foil. Extraction was carried out using an orbital shaker at 180 rpm for 24 hours, after which the suspensions were filtered using Whatman No. 1 filter paper. The filtrate was evaporated using rotatory evaporator maintained at 65°C to get dry extracts. Solvent-free extracts were transferred to the extract vials and stored at 4°C for further used.

Phytochemical Screening of C. asiatica Leaves
Phytochemical screening of C. asiatica to determine the presence of alkaloids, saponins, tannins, flavonoids, and phenol hydroquinone was done by using a standard procedure of Samejo et al. (2013).

Test for Tannins
About 0.5 g of a dried powdered sample of plant was boiled in 10 mL distilled water in test tube and then filtered. A few drops 0.1% of FeCl 3 solution were added to the filtrate. Blue-black precipitate indicated the presence of tannins.

Test for Alkaloids
About 2 mL of 2N HCL was added to 5 mL extracts and then heated with stirring in a water bath for 10 minutes. The cooled solution was filtered and added with few drops of Wagner's reagent. Reddish-brown precipitate indicated the presence of alkaloids.

Test for Saponins
About 1 g of dried powdered sample was boiled with 10 mL distilled water. Frothing precipitate indicated the presence of saponins.

Test for Flavonoids
About 0.5 g dried powdered plant sample was boiled in 10 mL ethanol and filtered. Few pieces of magnesium ribbon and few drops of concentrated HCl were carefully added to the filtrate. The red color indicated the presence of flavonoids.

Test for Phenol Hydroquinone
One mL of extracted sample (ethanol, n-hexaneethanol) was added with FeCl 3 1%. Terpenoids compound was indicated by the appearance of blue to black or purple color.

Brine Shrimp Lethality Test
The extracts of C. asiatica leaves were routinely evaluated in a test for lethality to brine shrimp larvae, with minor modifications. Toxicities of the compounds were tested at 10, 50, 100, and 300 ppm in 10 mL sea-water. Ten of the one-day age nauplii were used in each test and survivors were counted after 24 hours. Three replications were used for each concentration. The blank control was always present. The lethal concentration for 50% mortality after 24 hours of exposure, the chronic LC 50 , and 95% confidence intervals were determined using the probit method (Finney, 1971), as the measure of toxicity of the extract.

Antibacterial Activity
The antibacterial screening was carried out by agar disk diffusion method with the determination of diameters of inhibition zones made by C. asiatica extract against two bacterial strains (V. harveyi and A. hydrophila). Sterilized tryptic soy agar (TSA) and thiosulfate citrate bile salts sucrose (TCBS) were poured into plates individually and then inoculated with 100 µL suspension of tested bacteria (TSA for. A. hydrophila; TCBS for V. harveyi). One of five-millimeter discs of Whatman No. 1 filter paper was prepared and immersed in each of 1 mL extracts solution (50 mg/mL, and 100 mg/mL) in Aquadest. Chlorampenicol was used as positive control. The plates were incubated at 37°C for 18 hours. Antibacterial activity was evaluated by measuring the diameter (mm) of inhibition zones (Alabri et al., 2014).

Statistical Analysis
The antibacterial screening experimental results were run with four treatments in triplicate and expressed as mean ± SD and results were statistically evaluated using SPSS software version 16 at 5% significant level. Means were compared using Tukey's simultaneous test set at P<0.05 (Dhiman et al., 2016).

Determination of The Phytochemicals
The qualitative phytochemical analysis data of extracts of C. asiatica leaves showed that the phytochemicals were present in the methanolic extracts (Tabel 1). However, flavonoid and saponin were absent. Phytochemical screening in the plant samples determined the biologically active compounds, which have different benefits such as antioxidant, antimicrobial, antifungal, and anticancer (Alabri et al., 2014;Suresh & Nagarajan, 2009). All secondary metabolite components displayed antioxidant and antimicrobial properties through different biological mechanisms. Most of the secondary metabolite components were isolated and identified in the polar plant extracts (Alabri et al., 2014;Gonzales-Guevarra et al., 2004).
The phytochemical screening of methanol extracts of C. asatica leaves showed the major presence of active chemical constituents such as alkaloids, phenol hydroquinone, and tannins. Alkaloids have been reported as powerful poison, and many alkaloids derived from medicinal plants showed biological activities like antimicrobial (Iqbal et al., 2015;Benbott et al., 2012) that had bioactivity against gram-positive bacteria (Iqbal et al., 2015;Omar et al., 1992). Tannins and its derivatives are phenolic compounds which are also bioactive constituents and has a role in plant defense system due to their antimicrobial activity (Sudira et al., 2011).

Brine Shrimp Lethality Test (BSLT)
The mortality percentages of brine shrimp and the LC 50 values after 24 hours (chronic) exposure to various extracts of C. asiatica are shown in Table 2. After 24 hours of exposure, increased mortalities of brine shrimps were recorded in the treatment using all prepared extracts. Maximum mortalities (50%) were observed in the 300 mg/L concentrations of the mixture extract tested. According to Meyer et al. (1982), LC 50 lower than 1,000 ppm is considered cytotoxic (active) on the evaluation of plant extracts by BSLT, while nontoxic (inactive) if it is greater than 1,000 ppm. Meanwhile, Ramos et al. (2009) stated that a compound categorized highly toxic if the LC 50 value is less than 1 mg/L, toxic if the LC 50 value range is 1-250 mg/L, weak toxicity if the LC 50 value is more than 250 mg/L. According to these results, C. asiatica extract as antimicrobial agent poses a low risk of harm against the brine shrimps. Phytochemical screening of the extract of C. asiatica showed that the extract contained secondary metabolites such as alkaloid, tannins and phenol hydroquinone. These phytochemicals have been implicated in the lower cytotoxicity activity demonstrated by the extracts of C. asiatica.

Antimicrobial Activity
C. asiatica extracts were investigated for antimicrobial activity assay against both the gram-negative (i.e., A. hydrophila and V. harveyi) bacteria by agar dif-In vitro antibacterial efficacy of leaves extract of Centela asiatica ..... (Rukisah) fusion method. Different concentrations of the sample extracts (i.e. 50 mg/mL and 100 mg/mL) were used for the antimicrobial tests. The extracts showed antimicrobial activity against all the microorganisms (Table 3) in both concentrations. The methanol extracts of C. asiatica leaves concentration of 50 mg/mL showed the maximum antibacterial activity against A. hydrophila and the diameter of the inhibition zone was 10.57 mm. V. harveyi was found to be more sensitive to C. asiatica with 14.16 and 21.14 mm inhibition zone concentration of 50 and 100 mg/mL, respectively. Most of the studies revealed the in vitro antimicrobial activities of plant extracts against different pathogens are difficult to compare due to the use of different extraction, solvents, microbial strains, and antimicrobial test methods (Dhiman et al., 2016).
The antibacterial activity of the leaves extracts of C. asiatica is caused by the presence of alkaloid, tannin, and phenol hydroquinone, which are known as potential antimicrobial agents. The alkaloid mechanism as an antibacterial is by intruding the peptidoglycan in bacterial cells causing unforming the cell walls layer, and subsequently the death of cells. Another mechanism for alkaloid is it inhibits topoisomerase enzyme of bacterial cell (Karou et al., 2005). The mechanism of phenolic compounds such as phenol hydroquinone is by attacking cell walls and cell membranes, and thus affecting their permeability and releasing intracellular constituents. Phenolic compounds also interfere with membrane functions such as electron transport, enzyme effect, and nutrient uptake. Thus, active phenolic compounds might have several targets, which could lead to the inhibition of bacterial growth (Amensour et al., 2010). Tannins are organic compounds that inhibit microbial growth by damaging cell walls of microbial and forming bonds  Table 3. Antimicrobial activity of C. asiatica leaves extracts against gram-negative bacteria Note: Each value is the mean standard deviation of minimum three observations. Different letters at the top of each bar showed significant differences among treatments (P<0.05) Indonesian Aquaculture Journal, 14 (2), 2019, 69-74 with functional proteins of microbial cell (Sudira et al., 2011). The mechanism of tannins is also through producing hydrogen bonds with proteins, which convert the structure and block the protein synthesis. Tannins were considered as a phenolic compound of plants with anti-oxidative effects (Khder, 2008).

CONCLUSION
The phytochemical qualitative analysis of C. asiatica by the methanol extract revealed the presence of three major bioactive compounds such as alkaloids, phenol hydroquinone, and tannins. This study suggests that those compounds are responsible for its antibacterial effect which lead to the conclusion that C. asiatica leaves extracts could be used antibacterial ingredient in treating V. harveyi and A. hydrophila infections.