Antibacterial activity of Rhizophora apiculata extract originated from Inner Ambon Bay against selected pathogen bacteria

Inner Ambon bay, Ambon Island, Maluku Province, is a good place for mangrove to growth. Some mangroves have been known as a source of germplasm that has antimicrobial properties. This research goals to study the antibacterial activity of various extract of Rhizophora apiculata from the coast of the Inner Ambon Bay, against several pathogenic bacteria. The experimental design was complete random design with two factors, firstly, the type of extract solvent was extract namely methanol, ethyl acetate and n-hexane, and the second four strains of bacteria consist of Gram positive strains: Bacillus cereus and Gram negative strains: Escherichia coli, Salmonella typhi Staphylococcus aureus.The highest antibacterial activity was resulted by ethyl acetate extracts with an average value of inhibition zone was 18.64±3.88b mm, then followed by methanol extract 15.02±5.17b mm and n-hexane extract 8.48±1.25a mm. The strongest extract against the bacterial pathogen was ethyl acetate extract. The most susceptible bacteria to the three extracts tested with an inhibition zone was (18.02±7.49b) mm, followed by B. cereus, E. coli and S. aureus with the inhibition zones were (13.48±4.50a) mm, (12.94±4.69a) mm and (11.74±3.90a) mm, respectively.


Introduction
Inner Ambon bay, Ambon Island, Maluku Province, is a good place for mangrove to growth. Mangrove in Ambon Bay are generally dominated by 4 genera namely Avicennia officinalis, Bruguiera cylindric, Rhizophora apiculata, and Sonneratia alba. Rhizophora apiculata and Sonneratia alba were the most abundant species with a height of trees can reach 30 m with a diameter reaching > 41 cm [1].
The primary purpose of the mangroves is to prevent the coastal from abrasion so that it stays in balance and beautiful. Mangroves are also home to a variety of wildlife such as various of mollusks, echinoderms, fish, crustaceans, birds, epiphytic plants and other various of biota. Mangroves are also known as spawning areas, nursery ground, and feeding ground for several types of marine biota. Besides protecting the land, mangrove have functionate as a wildlife conservation area and sedimentation prevention. Mangroves can also be used for commercial purposes such as the source of wood for exported, leather for tannins, charcoal, paper materials, medicines and food [2]. Beside above mention, some species of mangrove have known potentially to use as medicinal purpose. Some mangroves have been known as a source of germplasm that has antimicrobial properties. Many mangrove plants especially Rhizophoraceae species show a strong antimicrobial properties [3]. It has been reported that some extracts of Rhizophora species have wide varieties of pharmacological properties like antifungal, antibacterial, antiseptic, anti-inflammatory, antiulcer and efficacy in wound healing [4]. [5] reported the extract of R. apiculata and Bruguiera gymnorrhiza from the coast of Burmanallah, South Andaman, India, has an antimicrobial and anti-fungal activity against Aspergillus niger, Klebsiella pneumoniae, Escherichia coli, Shigella flexneri, Salmonella typhi and it has been explored the active fraction named tannin. While [6] reported that antibacterial isolates from endophytic fungi from According to [7] revealed that n-hexane and chloroform extracts of the leaves of Rhizophora mucronata showed a great inhibition against Bacillus subtilis,Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Aspergillus niger and a moderate inhibition against Bacillus subtilis, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Aspergillus niger. [8] reported the results of chemical and biological characterization studies of leaves, roots, where the best extracts produced by leaves came from secondary metabolites of flavonoids and tannins. [9] reported the roots, stems, and leaves endophytic antibacterials of Rhizophora mucronata Lam come from the coastal area of Lombok Island (Gili Sulat) had an antibacterial activity from moderate (8-12mm) to strong (> 12mm) against B. cereus and P. aeruginosa, while [10] reported the presence of antibacterial activity of endophytic fungi of Rhizophora mucronata from Segara anakan has a moderate to low activity against Escherichia coli and Staphylococcus ureus. [11] reported Actinomycetes isolate originated from Rhizophora apiculata rhizosphere, were able to inhibit the growth of Escherichia coli,Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus sp. [12] reported there was an antibacterial activity of mangrove leaf extract, Rhizophora mucronata, Sonneratia alba and Exoecariaagallocha from Chorao Island, Goa against human pathogenic bacteria Staphylococcus aureus, Streptococcus sp, Salmonella typi, Proteus vulgaris and Proteus mirabilis, phytochemical components found in human pathogenic bacteria Staphylococcus aureus, Streptococcus sp, Salmonella typi, Proteus vulgaris and Proteus mirabilis. saponins, glycosides, tannins, flavonoids, phenols and essential oils in mangrove leaves.
Because of the great antimicrobial potential of mangroves in various places in Indonesia and even in the world, it has inspired writers to study the antibacterial properties of mangrove leaf extract (Rhizopora apiculata) originated from Inner Ambon Bay, Maluku Indonesia. This research goals to study the antibacterial activity of various extract of Rhizophora apiculata from the coast of the Inner Ambon Bay, against several pathogenic bacteria.

Description of the study sites
Location of sampling encompass the coastal area of the Inner Ambon Bay waters, Ambon Island, Maluku Province (Figure 1), where there many species of manggrove growth, especially R. apiculata. In Ambon Bay, the forest of mangrove were an important ecosystem supporting the development and protection of Ambon City.

Sample extraction preparation
The leaf of R. apiculata were carried to the Laboratory of Fisheries Products Technology, Pattimura University Ambon. The mangrove leaves were shade dried and powdered, then as much as 50 g leaf powdered soaked with a solution of methanol, ethyl acetate and n-hexane as much as 200 ml each, then macerated for 48 hours. The extract was then filtered by using Whatman paper filter No. 41. The filtrate is concentrated by using a vacuum evaporator at 50ºC. The extract was weighed, stored in a bottle in the refrigerator at 5ºC and the yield percentage was calculated using the following formula: Yield extract (Yield)% = R / S 100, where R = extracted weight by leaf residue and S = sample weight.

Preparation of bacterial inoculums
All tested bacteria strains were subcultured overnight on nutrient agar Mueller-Hilton at 37⁰C. Using 5 ml of sterile salt water, each of the bacteria tested was harvested and measured by using spectrophotometer at 580 µm and diluted to reach a decent cell count of 10 7 CFU / ml.

Preparation of paper disk
Paper disk used for screening the antibacterial activity were Whatmann No.1 filter paper 6 mm diameter size. The extract of R. apiculata mixed with 1 ml of 5% Dimethyl sulfoxide (DMSO). The discs were impregnated by n-hexane, ethyl acetate and methanol extract then screen their antibacterial activity. The positive control used was amoxillin (100 mg/ml) and the blind control was 5% DMSO.

Antibacterial assay
Method proposed by [13] was used to determine the antibacterial activity of R. apiculata. Muller Hinton agar was poured to the dish as much as 20 ml and it allowed to jell, for the use in susceptibility test against pathogenic bacteria. Bacterial inoculums suspension as much as 0.1 ml was poured and spreaded uniformly. Extract impregnated paper dish were placed on the surface of the Muller Hinton Agar. Positive controlled using an amoxillin (100 mg/ml) and the 5% DMSO was used as a blind control. The Petri dish were incubated at 37C for 24 hours. The zone of inhibition was observed and measured in millimeters. Each test in these experiments was repeated three times for concordance.

Statistical analysis
The experimental design was complete random design with two factors, firstly, the type of extract solvent, namely: N-Hexane, Ethyl acetate and Methanol, and secondly, the types of pathogenic bacteria consist of: E. coli, B cereus, S. thipy and S. aureus the experimental replicated 3 times. Then the variance was analyzed to determine the influenced between treatments. If there is an influenced then continued by the Least Significant Difference test (LSD) to determine the differences between treatments.

Result and Discussion
The yield of n-hexane, ethyl acetate and methanol extract of R. apiculatais is shown in Table 1. The yield was range of 5.98 -29.86%. The highest yield was found in methanol extract with an amount of 29.86%, followed by ethyl acetate extract and n hexane extract 6.36 and 5.98% respectively. The results of antibacterial test in Table 2 showed that the inhibitory zones of extracts (n-hexane, ethyl acetate and methanol) of R. apiculata leaf was in the range of 7.77 ± 0.23 to 23.72 ± 2.94 mm. [14] had classified the zone of inhibition based on their antibacterial strength as follows: <5 mm is called a weak inhibition power, 5-10 mm a moderate inhibition power and 10-20 mm strong inhibition power. According to that classification, the extract of R. apiculata was classified from moderate to strong power.
Analysis of variance resulted that the treatment of extract types (n-hexane, ethyl acetate and methanol) and types of bacteria E. coli (B1), B. cereus (B2), S. thypi (B3) and S. aureus (B4) have very significantly influenced the zone of inhibition (at p> 0.05). This means that there is at least one type of extract or bacterial treatment that has a zone of inhibition different from the others. The result of LSD test of extracts treated (n-Hexane, Ethyl acetate and Methanol) against the 4 species bacteria tested showed that there was a difference between n-hexane extract with methanol extract and ethyl acetate extract. The highest antibacterial activity was resulted by ethyl acetate extracts with an average value of inhibition zone was 18.64±3.88b mm, then followed by methanol extract 15.02±5.17b mm and n-hexane extract 8.48±1.25a mm. The strongest extract against the bacterial pathogen was ethyl acetate extract, statistically it was not significantly different from methanol extract but with n-hexane extract. [15] reported that extracts of n-hexane, ethyl acetate and methanol of R. apiculata against S. aureus produced inhibition zones 8 ± 0.5, 10 ± 0.3 and 11 ± 03 respectively. There was not a significant difference between the zone of inhibitory of n-hexane extract and the zone inhibitory of methanol, but the inhibition zone of ethyl acetate was very different, between 10 ± 0.3 with 16.50 ± 2.08 mm from the results of this study. [16] reported that the inhibition zone of methanol extract of R. apiculata leaves came from the waters of Tanjung Api-Api, South Sumatra against S. aureus and E.coli bacteria were 14 mm and 21.9 mm, respectively. [17] reported methanol extracts from R. stylosa zone of inhibition against E. coli and S. aureus bacteria 9.7 ± 0.6 mm and 8.7 ± 0.3 mm respectively. Secondary metabolites such as alkaloids, phenolics, steroids, terpenoids, tannins, flavonoids, saponins and quinons have been characterized from mangrove plants for toxicology and pharmacological purposes [18][19] [20].
The result of LSD test on the bacterial species treated showed that the zone of inhibition of bacteria S. thypi was significantly different from the others, where the S. thypi bacteria was the most susceptible bacteria to the three extracts tested with an inhibition zone was (18.02±7.49b) mm, followed by B. cereus, E. coli and S. aureus with the inhibition zones were (13.48±4.50a) mm, (12.94±4.69a) mm and (11.74±3.90a) mm, respectively (Table 2.). The results of this study indicated that extractof R. apiculata leaf is very effective to inhibit the growth of bacteria S. thypi. In contrast to the results of [21] S. typhi was the most resistant strain to plant extracts followed by E. coli and S. aureus.
The results of the positive control amoxillin against 4 species bacteria tested showed that when compared to the extract of R. apiculata leafs, an amoxillin only effective in killing S. thypi, while compared to other bacteria there is seems not significantly differences, especially for E. coli extract, ethyl acetate it could be seen that R. apiculata ekstract more effective than amoxillin ( Table 2, Figures  2 and 3). This means that because of E. coli has a resistant possibility to amoxillin, so that, an optimization and more in-depth studies of R. apiculata extract against various types of bacteria need to be done.  In addition to research on mangroves, many marine plants and animals that have a potential antibacterial. [22] reported traditional plant extracts have an antibacterial activity against pathogenic bacteria of Tilapia sp. [23] reported the sponge associated fungi showed inhibition zone against various types of vibriosis bacterial of shrimp growth disturbing.
According to [24], the seaweed extract of Phorphyra sp shows a good inhibition against 3 kinds of bacteria namely E. coli, Staphylococcus aureus and S. thypi, they mentioned the inhibition zones of methanol, ethyl acetate and n-hexane extracts from Phorphyra sp ranged from 6.21 -15.41 mm, the largest inhibition zone was 15.41 mm resulted by ethyl acetate extract against E. coli. [25] reported the active components of methanol extracts of R. mucronata leaf were tannins, saponins, flavonoids, phenol hydroquinone, triterpenoids, and alkaloids. Methanol extract has the greatest antibacterial activity with inhibition zones ranging from 3-12 mm.
The inhibitory zone of ethyl acetate extract from the seaweed species Sargassum sp was the most active extract against P. aeruginosa and M. luteus bacteria [26], while the methanol extract Sargassum sp was the most active extract against S. epidermidis bacteria. The most dominant bioactive compound from seaweed extract is steroid.

Conclusion
The results of this study indicated that extractof R. apiculata leaf is very effective to inhibit the growth of bacteria S. thypi, and the highest antibacterial activity was resulted by ethyl acetate extracts followed by methanol extract and n-hexane extract. An amoxillin only effective in killing S. thypi, while compared to other bacteria there is not significantly differences, especially for E. coli extract. Ethyl acetate ekstract of R. apiculata more effective than amoxillin. It needs more effort and in-depth studies of R. apiculata extract against various types of bacteria, in order to find the new and best antibacterial as a substitute for resistant antibacterial.