ANTI-MICROBIAL ACTIVITY OF LEAF EXTRACT Avicennia marina TO TOTAL MICROBIAL AND Staphylococcus epidermidis FROM FISH PINEKUHE

Jaka Frianto Putra Palawe, Eko Cahyono, Ely John Karimela Study Program Marine Products Processing Technology, Department of Fisheries and maritime Politeknik Negeri Nusa Utara North

Abstract

The purpose of this research is to know the anti-microbial activity of ethanol extract from leaves of mangrove api-api (Avicennia marina) to total microbe and staphylococcus epidermidis isolated from pinekuhe fish roomates. This research was divided into two stages: extraction by maceration method using ethanol solvent distillation 57% result from capticus beverage and anti- microbial activity of Kirby Bauer test paper disc method. The research method used is an experimental research method with complete randomized design with the parameters of the extract concentration that is 0%, 20%, 40%, 60%, 80% and positive control of chloroamphenicol. The analysis of data used Chi Square method continued with normality test and Kruskal-Wallis nonparametric test. The results of anti-microbial activity on the total microbial testing did not show any real effect, whereas on staphylococcus epidermidis Showed a real influence with 95% confidence level. The highest influence is the concentration of 80% with the average diameter of the clear zone is 11.67 mm and the cancel is at 20% concentration with the mean diameter of the clear zone is 8:33 mm. This study Proves that the secondary metabolites of mangrove leaves has an active antibacterial compound on staphylococcus epidermidis.

Keywords: Mangrove, Anti bacteria, Pinekuhe Fish, Staphylococcus epidermidis.

INTRODUCTION some phytochemical analysis on various body Mangrove is a vegetation typical of coastal tissues Avicennia spp plant is known that the ecosystems. Mangrove functions such as leaves of the plant contains alkaloids, saponins, retaining waves, wind storms, protection from glycosides, tannins, flavonoids in the leaves. abrasion and mudguards (Badruddin 1993). Triterpenoids found on all parts, especially the Mangrove trees live in a community in an area leaves and roots. Steroids are not found in all that often people refer to the mangrove forest. parts of the plant (Floreal, 2009). Mangroves can produce secondary metabolites Pinekuhe is typical smoked fish products are active as an anti-microbial (Naiborhu, made from round 2002), results of previous studies that reported scad (Decapterus sp). Pinekuhe smoked fish that the active compounds of anti- has a unique shape and has its own inflammatory, anti-oxidant, anti-bacterial and characteristics and not found in other regions anti-virus from extracts of various species of of Indonesia. Smoke Fish Pinekuhe only in mangrove (Withanawasam, 2002). Several Sangihe and generally processed traditionally, previous studies have been conducted to popular with consumers both in and outside determine the bioactive compounds produced Sangihe. (Karimela, 2013). Palawe (2014) by the leaves of Avicennia marina by doing showed that the smoked fish pinekuhe of Sangihe Islands Regency is not appropriate incubator (YCO-N01), waterbath (Nesco), national quality standard, with a value above magnetic stirrer (Vienna Type 206), laminary the maximum ALT smoked fish SNI 1.0 x 105 flow (Panasonic), microscope (Motic Type colonies / g. Staphylococcus epidermidis is a DMB01) and autoclave (Midnif). species of bacteria most commonly found Research methods living or as normal flora in human skin (Grice et al., 2009). The number of colonies of This research was conducted in two Staphylococcus epidermidis can achieve 1 x phases, 1) Extraction by maceration method 106 CFU per cm2 of human skin (Fey, 2014), includes the manufacture of ethanol solvent therefore, staphylococcus epidermidis has the and maceration. 2) Anti-Microbial Activity greatest potential to contaminate pinekuhe fish, Test (Kirby Bauer) include inoculation total because fish pinekuhe still processed microbial and Staphylococcus epidermidis and traditionally vulnerable cross-contamination. standardized test microbes. Treatment of mangrove leaf extract were expected to reduce the rate of growth of Production of Ethanol microbes on pinekuhe smoked fish, smoked Solvents used in this study is ethyl alcohol fish pinekuhe remember is the hallmark of /ethanol obtained by distillation at a Sangihe Islands Regency and very popular temperature of liquid to gas phase change of with the local community. The purpose of this ethanol that is 78 ° C. Raw material for making study was to determine the antimicrobial ethanol, which is used in this study is derived activity extract of leaves of Avicennia marina from a typical beverage distillation Minahasa on the total microbial and staphylococcus derived from palm juice is traditionally known epidermidis isolated from fish pinekuhe. as the rat cap/cap tikus. Distillation is done using a water bath to control the temperature MATERIALS AND METHODS and Soxhlet tube as distillation media. Soxhlet Materials and tools tube used in this study is a modified soxhlet The materials used in the study of tube by closing the exhaust hole feedback from mangrove Avicennia marina leaf samples the flask into solvent extractor locations, After taken from the Talengeng bay, Central distillation, ethanol is distilled further testing Tabukan Subdistrict, Sangihe Regency, North pure levels Sulawesi Province, Pinekuhe smoked fish, solvent is ethanol, distilled water, alcohol, The extraction of mangrove Avicennia Nutrient Broth, Nutrient Agar, 0.9% NaCl, marina leaves by maceration method spritus, paper disc, cotton, aluminum foil, The extraction of mangrove Avicennia tissue, and paper labels. The equipment used is marina leaves done by maceration method spectrophotometer (Mecasis optizen 2120 uv), using ethanol solvent distillation. Mangrove blender (Philips), micropippet (Dummo), leaves obtained washed with fresh water to remove salt, dirt or epiphytes on leaves, rinsed Standardization of Microbial Test with running water and distilled water to Determination of bacterial growth curve ensure clean leaves. Dried in an oven to dry was performed using adsorbtion test using which will be chopped finely and then blended spectrophotometric method with a wavelength until smooth and sifted. Samples were finely of 600 nm (Fey, 2014) for Staphylococcus macerated. Maceration carried out for 2 x 24 epidermidis and a wavelength of 500 nm for hours at room temperature with ethanol solvent mixed cultures / Total Plate Count (Sutton, with the sample and solvent ratio is 3: 7, 2011). Done by inoculating bacterial culture soaked in Erlenmeyer flask. Maceration perfomed by one loop into 10 ml nutrient process conducted over 2 days with repetition broth, then incubated for 24 hours and the as much as 2 times, the samples will be filtered measurement of adsorbtion every two hours. using Whatman filter paper. The results of the filtration (filtrate) is then inserted in the cup Antimicrobial Activity Test (Kirby Bauer) which had previously been weighed weight. In Testing the effectiveness of extracts of the cup evaporated solvent is by using an oven Avicennia marina as an anti-microbial use to accelerate the evaporation process. After a diffusion test according to Kirby-Bauer dry solvent extract obtained is then weighed method topical (Lay, 1994). On media smeared and then stored in a freezer that will be used the microbial loop of the test results in order for further testing, from the Nutrient media pinekuhe fish samples, then paper disc that contains extracts Inoculation Total Microbial and of Avicennia marina has been placed on the Staphylococcus epidermidis media and pressurized to extract permeate the The microbes in the inoculation in this media properly. The reading of the results is study is derived from microbes derived from done after incubation at 350C for 18-24 hours pinekuhe smoked fish that was purchased from by measuring the diameter of the area barrier the traditional market in the old port Tahuna. (clear zone) around the paper disc using graph Smoked fish samples are then wrapped with paper or ruler. aluminum voil sterilized and filled in a plastic Experimental design box. Pinekuhe smoked fish samples was then diluted with a dilution of 1 x 10-1. Samples The experimental design used in this study were diluted with 09.9% NaCl physiological is completely randomized design (CRD) with solution. After reconstitution dilution grown to treatments used in this test is extract ethanol media Nutrient agar slant culture order is then mangrove leaves with a concentration of 0%, stored as perfomed for further testing. 20%, 40%, 60% and 80% (w / v), while the Staphylococcus epidermidis cultures were control Kloroamfenikol positive use. taken from research Karimela (2017). Deuteronomy That done 3 times.

Data analysis 1.4 1.233 1.315 1.307 1.298

1.2 1.003 Normality of data analysis using chi square 1 0.834 test and non-parametric test Kruskal Wallis test 0.8 0.611 with a level of 95%. Furthermore, the analysis 0.60.398 0.401 0.4 also descriptive methods of regression and

Adsorbtion Adsorbtion on OD 600 nm 0.2 correlation. 0 0 2 4 6 8 10 12 14 16 18 Hour RESULTS AND DISCUSSION Figure 1. Growth curve of Staphylococcus Extraction Results epidermidis Ethanol for extraction testing in Research 2 1.7541.7721.771.781.767 and Industry Standards of Indonesia located in 1.8 1.511 1.6 1.322 City , results obtained 1.4 1.2 1.01 1 0.817 from testing the levels of ethyl alcohol / 0.6180.62 0.80.5180.538 ethanol distillate result is 56.9% or 57%. The 0.6 0.4 extraction of mangrove leaf maceration adsorbtion on OD 500 nm 0.2 0 method obtained yield as much as ± 10%. 0 2 4 6 8 10 12 14 16 18 20 22 24 26

Hour The growth curve of Staphylococcus Figure 2. Growth Curve of Total Plate Count epidermidis and Total Plate Count Total Plate Count and Staphylococcus The growth curve of Staphylococcus epidermidis Counting. epidermidis (Figure 1) shows that there Table 1. Total Plate Count and Staphylococcus logartimic phase on two and twelve hours to epidermidis number. the value indicated by the surge adsorbtion. the Staphylococcus epidermidis lowest adsorbtion is 0.401 and highest is 1.315. TIME Total Colony Total Plate Count growth curve (Figure 2) 8 3:42 x 107 shows that there logartimic phase at the sixth 10 6.75 x 108 hour and fourteenth hour evidenced by the 12 1:12 x 109 surge in the value of adsorbtion. the lowest Total Plate Count adsorbtion is 0.62 and highest is 1.754. In the Time Total Colony logarithmic phase, the nutritional needs of 12 1:18 x 108 these bacteria can be fulfilled optimally so that 14 1:26 x 109 bacteria can make the growth process quickly 16 3:12 x 109 and can respond with a variety of external factors influence it takes. The results of calculations of total plate count (Table 1) of the past six hours shows that the number of colonies units that meet the standards for antibacterial testing are on 10 hour of Staphylococcus epidermidis, and 12 hour for Total Plate Count due to its value above 1 x 108 colonies / ml.

Inhibition Capacity Testing Results of Inhibition Capacity Testing conducted on Total Plate Count from smoked Figure 4. Inhibition Capacity Testing against fish pinekuhe showed that the extract ethanol Staphylococcus epidermidis mangrove leaves showed no inhibition is shown with no emergence of a clear zone Table 2. Clear Zone Diameter of Inhibition around the paper disc this applies also to the Capacity Testing against Staphylococcus positive control, kloroamfenikol (figure 3). epidermidis REPEATED NO TREATMENT AVERAGE 1 2 3 1 Kloroamfenikol 6 6 6 6 Ethanol / 2 6 6 6 6 concentration 0% Concentration of 3 9 8 8 8:33 20% Concentration of 4 10 9.5 9 9:50 40% The concentration of 5 11 9.5 10.5 10:33 60% Figure 3. Inhibition Capacity Testing against The concentration of 6 14 10.5 10.5 11.67 Total Plate Count 80% Results of Inhibition Capacity Testing 14 conducted on Staphylococcus epidermidis y = 0.066x + 6.498 12 inoculation from smoked fish pinekuhe 10 11.67 10.33 showed that the extract ethanol mangrove (mm) 8 9.5 8.33 R² = 0.964 leaves showed the presence of the inhibition is 6 Clear Zone Diameter Diameter Zone Clear 6 indicated by the appearance of a clear zone 4 0 50 100 around the paper disc, whereas for the positive Concentration (%) control and the concentration of 0% or ethanol Figure 5. Graph regression Inhibition of showed no inhibition (Figure 4). Inhibition Staphylococcus epidermidis Capacity Testing against Staphylococcus epidermidis are shown in Table 2. The results of the normality test data analysis results obtained chi squared value (X2) / X count = 10.1917 and X2 table = 9.21. Because the X count > X table, we conclude that the data are not normally distributed followed by methyl ester and cis-9- Hexadecenal. Besides non-parametric statistical test of Kruskal phenol content is a component that is toxic to Wallis test. The results of the analysis of non- the microbes that are pathogenic (Aboaba et parametric Kruskal Wallis test (Sheskin, 2000) al., 2006), showed the value of H (12.6)> X2 table value Kloroamfenikol is a compound used as (9.49) with a 95% confidence level. therefore it a positive control, kloroamfenikol used for can be concluded that the concentration of the reacting positively to the broad spectrum extract have significant effect on the inhibition because it is active on gram-positive and gram- of Staphylococcus epidermidis. The lowest negative manner disturbing work namely the scores were found in concentrations of 0% process of protein synthesis at the ribosome with a diameter of 6 mm and have no (site of protein synthesis) 50S subunit of the inhibition zone/clear zone, just a 6 mm 70S ribosome contained in bacterial cells diameter paper disc. The highest value (Brown and Smith, 2012). From the results of contained at a concentration of 80% with a this test showed that kloroamfenikol used do diameter of 11.67 mm. Descriptive analysis of not have antibacterial activity against linear regression (Figure 5) shows a positive Staphylococcus epidermidis and Total Plate trend with a formula is Y = 0,066x + 6.498 and Count isolated from fish pinekuhe. it is caused the coefficient of determination R2 = 0, 964 by bacteria that inoculated was resistance to which gives the sense that the concentration kloroamfenikol. affects the diameter of the inhibition of 96.4% Staphylococcus epidermidis a bacterial with the means to have influence very strong. species coagulation negative staphylococci According Poompozhil and (CNS) living naturally in human skin infection Kumarasamy (2014), phytochemical content most likely to cause problems (Vuong et al, contained in Avicennia marina namely 2002). Staphylococcus epidermidis most infect alkaloids, flavonoids, phenols, saponins, humans in a hospital environment passing the tannins and Terpenoid. And Rajakumar Dinesh tools that are used (Ziebuhr, 2006). S. Kumar (2016) also perform extraction with epidermidis can produce a toxin groupphenol- ethanol and obtain the contents chemicals soluble modulins (PSMS) with the potential of namely: triterpenes, polyol, palmitic acid, acid damaging the red blood cells and white blood heksadekanoat, Terpenes alcohol, diterpene, cells in humans (Cheung et al, 2010 and (E)-9-octadecenoic acid ethyl ester, Mehlin et al 1999). dodekanoat acid, acid Oktadecanoat 2-methyl- methyl ester, cis-9-Hexadecenal, sugar CONCLUSION aldohexoses and nonanoat acid, which is Results of testing the anti-microbial antibacterial six components there in, namely activity against Total Plate Count showed no triterpene, terpene alcohol, diterpene, real influence, while against the dodekanoat acid, octadecanoic acid 2-methyl- staphylococcus epidermidis showed their real influence with a confidence level of 95%. 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