Journal of BIOLOGICAL RESEARCHES ISSN: 08526834 | E-ISSN:2337-389X Volume 21| No. 1| December | 2015 Original Article ANTIBACTERIAL ACTIVITY OF BELILIK (Brucea javanica (L). MERR) AND BENTA (Wikstroemia androsaemofolia DECNE) TO INHIBIT THE GROWTH OF ENTEROPATHOGENIC BACTERIA Henny Helmi*, Idha Susanti, Noptian Asmara Agung, Sadam Kusen Biology Department, Bangka Belitung University ABSTRACT Several native Indonesia plants have been used to prepare traditional medicine since long time ago. One of common diseases in tropical country is diarrhea, it caused by the infection of enteropathogenic bacteria such as Enteropathogenic Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Shigella sp. Belilik (Brucea javanica (L.) Merr) and Benta (Wikstroemia androsaemofolia Decne) are herbals that utilized as medicine for diarrhea in Bangka Belitung, Indonesia. Parts of these plants are mostly can be utilized as medicine, such as leaf, root, and fruit. The aims of this study were to investigate the antibacterial activity of ethanol crude extract of B. javanica (root and fruit) and W. androsaemofolia (leaf and fruit) against enteropathogenic bacteria (EPEC, P. aeruginoa, S. aureus, Shigella sp.). Meth- od that used was papper disc diffusion. The results showed that at concentration 10 mg/mL, 20 mg/mL, and 30 mg/mL of B. javanica ethanol extract of both root and fruit could not inhibit the enteropathogenic bacteria, while the ethanol extract of leaf and fruit of W. androsaemofolia were shown inhibition activity on the growth of enteropathogenic bacte- ria. W. androsaemofolia leaf extract performed the best inhibition activity to the growth of EPEC (20.55±1.5mm) and S. aureus (22.14±4.5mm), it was better than kanamycin performance at the same concentration (30 mg/mL). In addition, ethanol extract of W. androsaemofolia fruit showed the best inhibition activity against Shigella sp. (19.64±1.8mm). Keywords: antibacterial, Brucea javanica (L.) Merr, enteropathogenic bacteria, Wikstroemia androsaemofolia Decne INTRODUCTION medicinal properties, such as antibacterial properties. Several medicinal plants that have been used today were Antibiotic resistance has become a global problem in known by the ancient cultures throughout the world (Zal- the world recently. One of the cases is the resistance of ika 1975 in Dipankar et al, 2011). Traditional usages of in some infectious pathogenic bacteria that caused entero- indigenous society can provide information as the founda- pathogenic disease, such as EPEC (Enteropathogenic E. tion of modern pharmaceutical development (Philip et al, coli), Staphylococcus aureus, Pseudomonas aeruginosa, 2009). and Shigella sp. Kusmala (2012). Noveria (2012) stated Indonesia is known as country with the second high- that those bacteria are resist to kanamycin, amoxillin, est plant diversity in the world after Brazil (Sampurno chloramphenicol, and tetracycline. The cases of resistance 2007). Supported by indigenous cultures that have wide case encourage researchers to find alternative drugs to knowledge of traditional medicine, Indonesia posseses treat various diseases caused by microbial infection such high diversity of medicinal plants. Bangka Belitung Is- as medicinal plants exploration. On the contrary to syn- lands is one of provinces in Indonesia which rich of folk thetic drugs, natural antimicrobials derived from plant are medicines. Bangka Belitung community commonly uti- not associated with negative effects. Furthermore, it has lize local plants such as belilik (B. javanica (L) Merr) and therapeutic potential to treat other diseases (Anand et al, benta (W. androsaemofolia Decne) to overcome diarrhea. 2011). B. javanica is commonly used to treat malaria, amoebic The number of researchers that explore natural dysentery, vaginal candidiasis, hemorrhoids, intestinal products in order to develop better drugs against cancer as worms, papilloma, and nasopharyngeal cancer. In addi- well as viral and microbial infections is increase dramati- tion, its root has benefit in malaria treatment, fever, and cally (Srinivasan et al, 2001). Total 80% of world’s popu- food poisonous (Adelia, 2010; Dalimartha, 2001). Root lation relies on traditional medicines for primary and stem mixture is effective for fever and indigestion healthcare and most of it involves the utilization of plant treatment (Hendrian & Haidah 1999). B. javanica fruit extracts (Sandhya et al, 2006). Various plants possess contains saponins and tannins that potential as antibacte- rial agents, it can be assumed that these compounds also Corresponding Author: can be found in root (Rahayu et al, 2009). Another plant Henny Helmi that Bangka Belitung community use for medicine is W. Biology Department, Bangka Belitung University phone : (0717) 422145 androsaemofolia. It commonly used as anti-malaria, e-mail : [email protected] moreover people also used this plant as cure for diarrhea. W. indica shares the same genus with W. androsaemo- Open Access www.berkalahayati.org 35 Antibacterial Activity of Belilik (Brucea javanica (L). Merr) and Benta (Wikstroemia androsaemofolia Decne) to Inhibit The Enteropathogenic Bacteria folia, because of that they contain similar active com- formation of red, yellow, or orange colour in the (Har- pound such as flavonoid, biflavonoid, coumarins, lignans, borne 1996). essential oils, and polysaccharides (Li et al, 2009). Like- wise, this plant allegedly also has antibacterial properties. Phenols As the matter of facts, those medicinal plants are 10 drops of 1% FeCl3 were added into 30 mg of ex- undoubtedly have important value to treatment of several tract. Phenols were represented by the production of diseases. The antibacterial activity of these plants against green, red, purple or dark black colour (Harborne 1996). enteropathogenic bacteria through in vitro have not clear- ly revealed yet. Therefore, this study investigated the an- Saponin tibacterial activity of B. javanica and W. androsaemofolia 30 mg of extract was re-extracted with 5 mL of di- against enteropathogenic bacteria. The aims of this study ethyl ether to divide it into two fractions (soluble and in- were to investigate the antibacterial activity of these soluble). Insoluble fraction of diethyl ether then added plants and compare their effectivity to commercial antibi- into 5 mL of water in a test tube then they were shaken. otic. This study will contribute in new approach of diar- Positive test result indicated by for about 1-3 cm foam rhea treatment. production in 15 minutes (Harborne, 1996). Tannins METHODS 30 mg of the extract was added into hot water and Plant material boiled for 5 minutes. The mixture then filtered and the Herbals that used in this study were belilik (B. ja- filtrate divided into 2 parts and each of it was added 1% vanica (L). Merr) and Benta (W. androsaemofolia of FeCl3. The presence of tannins and polyphenol were Decne). B. javanica were obtained from Selindung, characterized by the formation of blue-green colour. Be- Pangkalpinang, Bangka Island, while W. androsaemofolia sides of that, the presence of tannins will characterized by were from Tanjungpandan, Belitung Island Indonesia. the formation of a white precipitate after gelation addition (Harborne 1996). Extraction Extraction was done by soxhlet method. Each 100 g Triterpenoids and Steroids sample of B. javanica fruit, B. javanica root, W. an- Saponin soluble fraction in diethyl ether separated drosaemofolia leaves, and W. androsaemofolia fruit were and added with 10 drops of glacial CH3COOH and 2 crushed separately then soaked in 96% of ethanol and drops of concentrated H2SO4. The solution was shaken heated to get the perfect extract, at the end of the process, slowly and was left for few minutes. Steroids gave blue or solvent became clear. Solvent then were dried using vac- green colour, while triterpenoids gave red or purple col- cum rotary evaporator. Total yields of extraction were our (Harborne 1996). then measured. Tested Microorganism Phytochemical Analysis Four enterophatogenic bacterial strains were used in Phytochemical components were analyzed qualita- this study: EPEC, P. aerugionosa, S. aureus, and Shigella tively. Purpose of this analysis was use to investigate ac- sp. These bacteria were obtained from Microbiology La- tive compound contains in fruit and root of Belilik boratory, Bangka Belitung University, Indonesia. Bacteri- (Brucea javanica (L.) Merr) and W. androsaemofolia al strains were cultivated at 37°C and maintained on Nu- leaves and fruit. There are some active compound that trient Agar (NA) slant (Oxoid, USA) at 4°C. have been analyzed in both plants such as alkaloids, fla- vonoids, phenols, saponins, steroids, tannins, and triterpe- Antimicrobial activity assay noids. Antimicrobial activity of extracts was determined by observation of their ability against four pathogenic bacte- Alkaloids ria using disc diffusion method. Crude ethanol extract and 30 mg of extract were diluted in 10 mL of CHCl3 – antibiotic (kanamycin as control) were dissolved in NH3 and filtered, filtrate was accumulated into test tube. aquadest. Extract and antibiotic were tested in three dif- 3-5 drops of 2M sulfuric acid was added into the filtrate ferent concentrations (10mg/mL, 20 mg/mL, and 30 then shook until formed two layers. Acid layer (top layer) mg/mL). 20 ml of Nutrient Agar and 100 µL of bacteria was pipetted into another test tube, then Dragendorff rea- suspension at log phase (106-108 cell/mL) were poured gent was added. Alkaloids will be detected by