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Jurnal Ilmu dan Teknologi Kelautan Tropis, Vol. 7, No. 2, Hlm. 603-612, Desember 2015 ANTIBACTERIAL ACTIVITY ASSAY OF MANGROVE EXTRACTS AGAINST
SALMONELLA TYPHI AND LISTERIA MONOCYTOGENES
UJI AKTIVITAS ANTIBAKTERI EKSTRAK MANGROVE TERHADAP SALMONELLA
TYPHI DAN LISTERIA MONOCYTOGENES
Apon Zaenal Mustopa1*, Rifqiyah Nur Umami1, dan Melki2
1Research Center for Biotechnology, Indonesian Institute of Sciences, Bogor, Indonesia 2Marine Science Department, Faculty of Natural Science, Sriwijaya University, Palembang *Email: [email protected] Aktivitas antibakteri ekstrak tanaman mangrove, Avicennia marina, Sonneratia caseolaris (berasal
dari Teluk Payo, Banyuasin, Sumatera Selatan), Ceriops tagal, Rizhopora apiculata, dan Sonneratia
alba (berasal dari Sadai, Bangka Selatan) terhadap Salmonella typhi dan Listeria monocytogenes
diteliti menggunakan uji disk difusi agar. Ekstraksi dilakukan menggunakan solven organik (metanol,
etil asetat dan aseton secara berurutan). Sebagian besar dari ekstrak yang diuji menunjukkan potensi
aktivitas antibakteri terhadap kedua patogen. Ekstrak metanol kulit batang S. alba dan buah A.
marina menunjukkan zona hambat yang cukup besar (15 mm) terhadap S. typhi. Ekstrak aseton daun
S. alba menunjukkan zona hambat terbesar (14 mm) ketika diuji terhadap L. monocytogenes.
Purifikasi parsial lebih lanjut dari ekstrak terpilih yang menunjukkan daya hambat besar dilakukan
menggunakan kromatografi kolom gel silika dengan berbagai komposisi eluen yang memiliki
perbedaan polaritas. Fraksi ketiga dari ekstrak metanol daun S. alba yang dielusi dengan kloroform:
metanol (1:5) menghasilkan zona hambat yang besar (23 mm) terhadap S. typhi. Fraksi ketiga dan
ketujuh dari ekstrak aseton daun S. alba yang dielusi dengan etil asetat:metanol (7:3) menghasilkan
zona hambat yang besar (15 mm) terhadap L. monocytogenes. Sedangkan fraksi keenam dari ekstrak
metanol buah A. marina yang dielusi dengan kloroform:metanol (6:4) menghasilkan zona hambat
terbesar (17 mm) terhadap L. monocytogenes. Hasil ini mengindikasikan bahwa ekstrak mangrove
dapat dikembangkan menjadi biomaterial untuk industri biofarmasi maupun biopreservasi.
Kata kunci: aktivitas antibakteri, mangrove, kromatografi kolom
ABSTRACT
The antibacterial activities of mangrove species, Avicennia marina, Sonneratia caseolaris (collected
from Teluk Payo, Banyuasin, South Sumatera), Ceriops tagal, Rizhopora apiculata, and Sonneratia
alba (collected from Sadai, South Bangka) were screened against Salmonella typhi and Listeria
monocytogenes by agar disk diffusion assays. Extractions were conducted using organic solvents
(methanol, ethyl acetate, and acetone, subsequently). Most of the extracts tested showed potential
antibacterial activity against both pathogens. The methanol extracts of the bark from S. alba and the
fruit from A. marina showed particularly large inhibition zones (15 mm) against S. typhi. The acetone
extract of S. alba leaves showed the largest inhibition zone (14 mm) when tested against L. Monocy-
togenes. Further partial purifications of selected extracts which showed strong inhibition were
performed by silica gel column chromatography using various eluent compositions with different
polarities. The third fraction of methanol extract from S.alba leaves eluted with chloroform:methanol
(1:5) resulted in a remarkably large inhibition zone (23 mm) against S. typhi. The third and seventh
fractions of acetone extract from S. alba leaves eluted with ethyl acetate:methanol (7:3) resulted in a
large inhibition zones (15 mm) against L. monocytogenes. In addition, the sixth fraction of methanol
extract from A. marina fruit eluted with chloroform : methanol (6:4) resulted in the largest inhibition
zone (17 mm) against L. monocytogenes. These results indicated that mangrove extracts could be
developed as potential biomaterials for biopharmaceutical as well as biopreservation industries.
Keywords: antibacterial activity, mangrove, column chromatography
@Ikatan Sarjana Oseanologi Indonesia dan Departemen Ilmu dan Teknologi Kelautan, FPIK-IPB Antibacterial Activity Assay . . I. INTRODUCTION
derived from mangrove extracts. The current study aims to explore the potentials of some Health problems caused by bacterial selected mangrove species collected from pathogens derived from food spoilage re- Indonesian mangrove forests, with emphasiz- mains to be a risk factor particularly in ed to screen their antibacterial activities aga- developing countries which have sanitation inst two representative foodborne pathogens- issues. Antibiotics and chemicals are com- Salmonella typhi and Listeria monocy- monly used as therapeutic medicines against bacterial infections. However, maltreatments S. typhi is a Gram-negative bacterium of antibiotics and chemotherapeutic agents that belongs to the family Enterobacteri- are heading to the new problem of drugs aceae. S. typhi infection could lead to the resistance. Thus, the discovery of new and development of typhoid or enteric fever and safe antibacterial compounds is continuously remains as a threat for public health. Some needed to work against resistant pathogens. risk factors such as the oversuse, misuse and The widespread use of plants for inappropriate antibiotics prescribing could be medication is already known for a long responsible for the increasing number of period of time. Plant extracts and phyto- multi-drug resistance problem among S. typhi chemicals have the potentials as alternative strains, including ineffective or sub-optimal treatments against bacterial infections. Indo- therapy using ampicillin, chloramphenicol, nesia has been blessed with enormous tropi- trimethoprim-sulphamethoxazole, and fluoro- al plant species that may represent a source quinolones such as ciprofloxacin, and oflo- of natural medicines. An interesting group of xacin (Zaki and Karande, 2011; Butler, plants in this regard are mangroves. According to Kusmana (2014), there are L. monocytogenes is a Gram-positive approximately 3.2 million hectares of mang- bacterium. L. monocytogenes is a causative rove area in Indonesia with over 202 mang- agent of listeriosis, however, unlike any other rove species. However, natural medicines foodborne pathogens, it has the ability to derived from mangroves are still inadequa- survive food-processing technologies, and tely studied due to their limited distribution. could grow even in properly refrigerated Mangroves are typical plants that live food. People usually get infected through the in littoral zone with harsh environmental consumption of contaminated food. Although conditions such as anaerobic soils, wide it is a relatively rare infection, L. Monocyto- range of saline concentrations, tidal waves, genes is one of the most virulent foodborne winds and extreme temperatures. Mangrove pathogens since it could lead to severe infec- plants often produce diverse groups of tion with up to 30% fatality rates even with unique chemical components as secondary early and adequate antibiotics treatment. The metabolites for protection and adaptation in most common clinical manifestation of such challenging conditions. Those com- listeriosis is meningitis, and usually being pounds often have interesting antibacterial, treated with ampicillin, penicillin or a combi- antiviral and antifungal activities. Neverthe- nation of ampicillin and gentamycin. Other less, there are only limited investigations that options are trimethoprim-sulfamethoxazole, have been conducted to identify the metabo- erythromycin, vancomycin, fluoroquinolones lite agents, which might be responsible for (ciprofloxacin) and quinolones (levofloxacin their bioactivities (Bandaranayake, 2002; and moxifloxacin). Some L. monocytogenes Eldeen and Effendy, 2013). isolates were reported to have low resistance In vitro screening for antibacterial to these antibiotics (Allerberger and Wagner, activity could be one of the initial steps for 2010; Hernandez-Milian and Payeras-Cifre, the discovery of new antimicrobial agents Mustopa et al. II. METHODS
longer the contact between solvents and materials the more the bioactive compounds 2.1. Mangrove Plants Collection
will be extracted. Furthermore, the selected The mangrove plants, Avicennia solvents should be less toxic and should not marina (Forsk.) Vierh., and Sonneratia influence the antibacterial assay results caseolaris (L.) Engl., were collected from (Ncube et al., 2008). Based on the fact that Teluk Payo, Banyuasin, South Sumatera, most of the identified bioactive compounds Indonesia. The locations extend from which possess antibacterial activity are com- 02°26'08.6" to 02°26'12.45"S and 104°45' monly less polar or not water soluble, org-52" to 104°45'59.9"E. While Ceriops tagal anic solvents (methanol, ethyl acetate and (Perr.) C. B. Rob., Rizhopora apiculata Bl., aceton) were used in this study. and Sonneratia alba J.E. Smith, were The powdered samples were extracted collected from Sadai, South Bangka, Indo- with a series of organic solvents at room tem- nesia, with sampling locations extend from perature according to the standard methods 03°00'02.1" to 03°00'15.2"S and 106°43'45. (Joel and Bhimba, 2010; Sahoo et al., 2012) 2" to 106°43'43.2"E. Information about the with some modifications. Briefly, to prepare environmental conditions including salinity, methanol extracts, 100 g of powdered temperature, pH and dissolve oxygen (DO) samples were soaked into 250 mL 80% me- of the locations were recorded. The mang- thanol and stirred. The solvent was subtituted rove samples were taxonomically identified in every 24 hours for 3 days. The total based on their morphological features at extracts were then filtered through Whatman Marine Science Department, Faculty of Na- No.1 filter paper. The filtrate was concentrat- tural Science, Sriwijaya University, South ed using a rotary evaporator at 50°C to get Sumatera, Indonesia. crude methanol extracts. To prepare ethyl acetate extracts, methanol extract residues 2.2. Mangrove Samples Extraction
were soaked into 250 mL 80% ethyl acetate The fresh samples were washed with and stirred. The solvent was subtituted in water to remove dirt and then dried in the every 24 hours for 3 days. The total extracts oven at 60°C for 3 days for leaf samples and were then filtered through Whatman No.1 up to 7 days for root, bark and fruit samples, filter paper. The filtrate was concentrated respectively. The dried samples were then using a rotary evaporator at 40°C to get crude crushed and powdered into fine particles. ethyl acetate extracts. To prepare acetone Instead of the fresh samples, the dried mang- extracts, ethyl acetate extract residues were rove plant materials were used since the soaked into 250 mL 80% acetone and stirred. differences in water content may affect the The solvent was subtituted in every 24 hours solubility. The dried samples were ground for 3 days. The total extracts were then into fine particles to increase the surface filtered through Whatman No.1 filter paper. areas, thus will increasing the extraction The filtrate was concentrated using a rotary evaporator at 40°C to get crude acetone The extraction method aims to sepa- extracts. The extracts were stored at 4°C in rate the bioactive compounds of the plant air-tight glass vials prior to in vitro antibac- parts from the inactive components using terial screening. selected solvents. The choice of the solvents for extraction had definite effects for the 2.3. Antibacterial Activity Assay
isolation of bioactive compounds since the Initial screening of potential anti- solvents will diffuse into the solid plant bacterial activity or commonly known as materials and solubilise the biocompounds antimicrobial susceptibility testing (AST) of with similar polarity during extractions. The the crude mangrove extracts was performed Jurnal Ilmu dan Teknologi Kelautan Tropis, Vol. 7, No. 2, Desember 2015 Antibacterial Activity Assay . . against Salmonella typhi (P2KIM collection) was eluted with various selected eluent and Listeria monocytogenes (BTCCB693) by compotitions initially tested on thin layer Kirby-Bauer agar disk diffusion test method chromatography (TLC) according to the (Bauer et al., 1966; CLSI, 2012). The AST method described by Harborne (1984), for method is formerly used as an essential optimization of the gradient polarities i.e., method to determine the microbial resistance chloroform: methanol (1:5); ethyl acetate : to antimicrobials. In this study, the AST methanol (7:3); and chloroform : methanol method was used as preliminary screening to (6:4). Individual fractions were then collect- elucidate the efficacy of antimicrobial ed and tested for antibacterial activity scree- candidates derived from mangrove extracts ning by the same method as described above. against representative microbial pathogens. Further TLC analysis using silica gel GF254 The agar diffusion based is the conventional (Merck) and chloroform: methanol (8:3) as reference method, however, the bactericidal eluent was carried out for the fraction with and bacteriostatic effects could not be the strongest antibacterial activity. The TLC differentiated using this method. results were visualized after heat treatment Briefly, the obtained crude extracts and under UV light 254 nm. dissolved in the respective solvents (20 μl) and positive controls (penicillin or chloram- III. RESULTS AND DISCUSSIONS
phenicol) were individually applied to the sterilized filter paper disks (Filtres Fioroni®, 3.1. Mangrove Samples Collection and
France; 6 mm in diameter) and then placed Extraction
on nutrient agar (NA-Oxoid®, England) test Mangrove plants are generally under- plates inoculated with the overnight culture valued and poorly managed. However, nume- of pathogens which had been prepared from rous mangrove plants had been used in the suspension equivalent to 0.5 McFarland folklore medicine as alternative treatment turbidity standard (108 CFU/ml). Sterile for- againts human and animal diseases. In this ceps were used to assure complete contact regard, scientific evidence of their bioacti- between the paper disks and the surface of vities are considerably needed. Mangroves agar medium. The disks were arranged in a are usually need warm condition for survival, proper space to prevent the overlapping of thus most of their habitats are in tropical the inhibition zones. The whole set up was areas. In this study, five species of mangrove first incubated at 4°C for 2 hours to give plants (i.e., A. marina, C. tagal, R. apiculata, sufficient time for the samples to diffuse into S. alba and S. caseolaris) were collected the medium. Then the test plates were kept at from Indonesian mangrove habitats. Diffe- 37°C for 16, 20, or 24 hours to allow the rent plant parts of the mangrove species bacterial growth. The antibacterial activity (roots, barks, leaves and fruits) were selected assays were determined by measuring the as the samples for the screening of anti- diameter of inhibition zones formed around bacterial activity (Figure 1). In addition, the the paper disks. environmental factors such as salinity, tem- perature, pH, and dissolve oxygen that could 2.4. Partial Purification of Bioactive
affect the bioactive constituents of the sam- Compound
ples and leading to the differences in their Selected crude mangrove extracts bioactivities, were recorded (Table 1). which showed strong inhibition zone were applied into a column chromatography pack- 3.2. Antibacterial Activity Assay
ed with silica gel (60-120 Mesh, Merck) Food borne pathogens and drug based on the method of Mishra and Sree resistance microbes such as S. typhi and L. (2007) with some modifications. Each extract monocytogenes remain to be problems 606 http://itk.fpik.ipb.ac.id/ej_itkt72

Mustopa et al. A. marina S. caseolaris R. apiculata A. marina Figure 1. Photographs of mangrove plants collected in this study (personal collection, 1:10 in scale). Barks and leaves (a), Fruits (b). Table 1. Environtmental characteristics of mangrove habitat at Teluk Payo, Banyuasin, South Sumatera and Sadai, South Bangka. A. marina S. caseolaris R. apiculata Temperature (°C) Jurnal Ilmu dan Teknologi Kelautan Tropis, Vol. 7, No. 2, Desember 2015 Antibacterial Activity Assay . . among food industries. In the meantime, growth of S. aureus. The silvernanoparticles people are questioning the safety of anti- derived from water extract of the leaves from biotics and chemical preservatives. In this C. tagal exhibited antimicrobial activity study, natural inhibitors for food borne pa- against pathogenic bacteria and fungi (Dhas thogens derived from the extracts of selected et al., 2013). According to Pimpliskar et al. mangrove plants were evaluated. This study (2012), ethanol extract of the stem from R. revealed that the mangrove crude extracts apiculata showed antibacterial as well as derived from different plant parts and eluted antifungal activities. It is reported that etha- with different solvents show positive anti- nol extract of the leaves from S. alba could bacterial activities against both of S. typhi inhibit the growth of S. typhi (Sahoo et al., and L. monocytogenes. 2012), while methanol extract of the seeds Strong inhibition activities against S. from S. caseolaris exhibited growth inhibi- typhi were shown by methanol extracts of the tory effect against S. aureus (Mahadlek et al., bark from S. alba and the fruit from A. 2012). The methanol extract of S. caseolaris marina (15 mm, respectively), compared to also reported to have antibacterial activities the positive control (10 mm), after 16 hours against multi-drug resistants pathogens by observations (Table 2). While acetone extract Yompakdee et al. (2012). of the leaves from S. alba exhibited parti- cularly large inhibition zone against L. mono- 3.3. Partial Purification of Bioactive
cytogenes (14 mm), although it was smaller Compound
compared to the positive control (18 mm), Mangrove species are known as a rich after 24 hours observations (Table 3). This source of bioactive compounds including ste- variable results of inhibition zones could be roids, triterpenes, phenolics, saponins, flavo- due to the differences of bioactive com- noids, alkaloids and tannins (Bandara- pounds solubility among the crude extracts. nayake, 2002). These phytochemicals have Previous research articles also pro- toxicological, pharmacological and ecologi- vide informations about the biological activi- cal importance. A knowledge of the chemical ties of mangrove extracts. Abeysinghe (2010) constituents of the bioactive compounds de- reported that some mangroves species had rived from natural products is required since antibacterial activities against antibiotic re- it can be great value in discovering new sistant Staphylococcus aureus. Dhayanithi et sources of economic phytocompounds parti- al. (2012) reported that methanol extract of cularly in regard of mangrove plants. The the leaves from A. marina could inhibit the phytocompounds could be responsible for the
Table 2. Antibacterial activity of crude mangrove extracts against S. typhi after 16 hours
observation.
Zone of inhibition (mm) methanol extracts ethyl acetate extracts acetone extracts root bark leaf fruit root bark leaf fruit root bark leaf fruit A. marina R. apiculata S. caseolaris Note: (-) : not determined. Positive control (penicillin) showed 10 mm of inhibition zone. 608 http://itk.fpik.ipb.ac.id/ej_itkt72 Mustopa et al. Table 3. Antibacterial activity of crude mangrove extracts against L. monocytogenes after 24 hours observation. Zone of inhibition (mm) methanol extracts ethyl acetate extracts acetone extracts root bark leaf fruit root bark leaf fruit root bark leaf fruit A. marina R. apiculata S. caseolaris Note: (-) : not determined. Positive control (chloramphenicol) showed 18 mm of inhibition zone. antibacterial activities as shown by the mang- (Table 5 and Figure 3). While chloram- rove extracts tested in present study. It might phenicol as positive control showed 20 mm be possible that each of the mangrove of inhibition zone. In addition, thin layer extracts contain multiple bioactive com- chromatography (TLC) analysis of the frac- pounds, and they inhibit bacterial growth in a tion of S. alba which exhibited the largest various ways. Bioactivity guided fractiona- inhibition zone was performed as initial tion using column chromatography was per- detection of bioactive compound (Figure 4). formed as an initial approach to separate the targeting compounds from the mixture within Table 4. Inhibition zone (mm) of column the crude extracts (Joel and Bhimba, 2010). fractions against S. typhi after 16 Some of the obtained fractions show- hours observation. ed stonger inhibition activities compared to the crude extracts. The third fraction of Methanol extract of methanol extract of the leaves from S. alba S. alba (leaves) eluted with chloroform: methanol (1:5) resulted in 23 mm of inhibition zone against S. typhi after 16 hours observation (Table 4 and Figure 2). While chloramphenicol as positive control also showed 23 mm of inhibition zone. The third and seventh frac- tions of acetone extract of the leaves from S. alba eluted with ethyl acetate: methanol (7:3) resulted in 15 mm of inhibition zones against L. monocytogenes. The sixth fraction of me- thanol extract of the fruit from A. Marina eluted with chloroform: methanol (6:4) resulted in 17 mm of inhibition zone against Positive control Chloramphenicol: L. monocytogenes after 20 hours observation Jurnal Ilmu dan Teknologi Kelautan Tropis, Vol. 7, No. 2, Desember 2015 Antibacterial Activity Assay . . Figure 2. The arrow indicates the inhibition zone of the third fraction of methanol extract of the leaves from S. alba eluted with chloroform : methanol (1:5) against S. typhi after 16 hours observation. Table 5. Inhibition zone (mm) of column fractions against L. monocytogenes after 20 hours Acetone extract of S. alba Methanol extract of A.marina ethyl acetate : methanol (7:3) chloroform : methanol (6:4) Positive control Chloramphenicol: 20 Chloramphenicol: 20 Note: (-) : negative. Figure 3. The arrows indicate the inhibition zone of : (a). The third and seventh fractions of acetone extract of the leaves from S. alba eluted with ethyl acetate : methanol (7:3) against L. monocytogenes after 20 hours observation. (b). The sixth fraction of methanol extract of the fruit from A. Marina eluted with chloroform : methanol (6:4) against L. monocytogenes after 20 hours observation. 610 http://itk.fpik.ipb.ac.id/ej_itkt72 Mustopa et al. Figure 4. Thin layer chromatography (TLC) analysis of the third fraction of methanol extract of the leaves from S. alba eluted with chloroform : methanol (8:3). Detection after heat treatment (a), detection under UV light 254 nm (b).
IV. CONCLUSION
thank Muhamad Ridwan, Ika Sari Kusuma-
wati and Dwi Setianingsih for technical The crude extracts derived from assistance. We also thank to reviewers who different parts of the mangrove plants with help to improve this paper. different solvents exhibited various level of antibacterial activities. The results suggest REFERENCES
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