Cuttlefish (Sepia Sp.) Ink Extract As Antibacterial Activity Against Aeromonas Hydrophila

INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 8, ISSUE 11, NOVEMBER 2019 ISSN 2277-8616

Faizal Zakaria, Mohamad Fadjar, Uun Yanuhar

Abstract: Aeromonas hydrophila is a gram negative opportunist bacterium associated with aquatic animal disease. Cephalopod ink has shown potential antiretroviral activity. The ink extracts of cuttlefish showed antibacterial effect. This study aims to investigate the antibacterial activity of the methanolic extract of the ink of cuttlefish (Sepia sp.) against Aeromonas hydrophilla. The shadedried ink sample from approximately 30g ink sacs obtained from 15 animals were immersed separately in methanol (1:3 w/v) solvents for overnight. Dried extract was used for the experiments. Isolate of Aeromonas hydrophila was originated from Jepara Brackishwater Aquaculture Center. The average yield percentage of cuttlefish tintan extract obtained was 4.86%. The results of the MIC test in table 5. show that the highest average absorbance value was obtained at a concentration of 50 ppm which was equal to 1,716 nm and the lowest absorbance was obtained at a treatment dose of 300 ppm at 0.841 nm while the Mc Farland tube was 0.933 nm. The results of antibacterial test on table 2 showed antibacterial activity of cuttlefish ink extract at concentration negative control showed diameter zone of 5 ± 1.2 mm, at positive control showed diameter zone of 31 ± 1.2 mm, at 250 ppm result 19 ± 0.9 mm, at 300 ppm result 22 ± 1.4 mm, at 350 ppm result 31 ± 1.2 mm.

Index Terms: Antibacterial; Cuttlefish Ink; Extract;Sepia sp.;Aeromonas hydrophila ——————————  ——————————

1. INTRODUCTION 2.2 Yield of Cuttlefish Ink Extract (Sepia sp.) Aeromonas hydrophila is a gram negative opportunist Rendemen analysis of ink extract based on comparison of bag bacterium associated with aquatic animal disease (1). A. weight (containing ink) samples to the weight of the extract of hydrophila causes mass mortalities in several species the ink contained. including Carps, Snake heads, Gouramies and Cat fishes and are considered as an etiological agent of several diseases 2.3 Bacterial preparation such as emaciation, haemorrhagic septicaemia, asymptomatic Isolate of A. hydrophila was originated from Jepara septicaemia, ulcerative infection, tail rot and fin rot (2). It also Brackishwater Aquaculture Center. These bacteria were kept o causes Motile Aeromonas Septicaemia (MAS) and Epizootic in Trypticase Soy Agar (TSA) media at 4 C and sub-cultured Ulcerative Syndrome (EUS) as a primary pathogen (3). Trypticase Soy Broth (TSB) in overnight before use. Indiscriminate use of antimicrobials for disease control in human beings and animals has increased the natural 2.4 Determination of MIC value emergence bacterial resistance (4) Emergence of multiple Minimum Inhibitory Concentration (MIC) is defined as the resistance has greatly limited the effectiveness of most of the lowest concentration of a compound /extract/drug that antibiotics. It is therefore necessary to search for novel completely inhibits the growth of the microorganism in 24 hrs. antibacterial compounds with therapeutic potential for which Determination of MIC value for the extract that showed high the pathogens may not have resistance (5). The class antibacterial activity was determined by Microbroth dilution Cephalopoda comes under the phylum Mollusca, which method. MIC tests were carried out at doses of 1 ppm, 50 involves squid, cuttlefish, octopuses and nautilus (6). ppm, 100 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, and Cephalopods occur in all marine habitats and are famous for mc farlanstandar .This test is carried out by inserting a their defenses, from their fast getting escape movements to bacterial inoculum of 1 ose into each tube, stored in an changes in colouration that can be cryptic, disruptive or incubator at a temperature of 30 ° C to 24 hours. Observe the startling, to arm autotomy, to toxin venom and to inking (7). turbidity in each tube with the degree of turbidity of the media. Cephalopod ink has shown potential antiretroviral activity (8). The spectrophotometer with the wavelength used in A Squid and cuttlefish eject ink from their ink sac to escape from Hydrophilla was 600nm to determine the absorbance value of their predators (9). The ink extracts of cuttlefish showed each turbidity at each dose, the smallest concentration which antibacterial effect (10). This study aimstoanalyse showed no bacterial growth (negative) in the MIC test. theantibacterialactivityofthecuttlefishink (Sepia sp.) extract against A. hydrophilla. 2.5 Antibacterial Test Using Disc Diffusion Assay The analysis of antimicrobial properties was started by dipping 2 MATERIAL AND METHOD a sterile cotton swab into adjusted suspension. The cotton swab was rotated in the suspension and pressed on the wall of 2.1 Extracts preparation the universal bottle to remove excess fluid. The cotton swab Cuttlefish (Sepia sp.) was washed with sterile water and then with suspension was spread on Mueller-Hinton agar surface dissections were made for removing the ink sacs. The ink was and repeated twice to ensure an even distribution of inoculum. gently squeezed out of the ink sac and air-dried. The The disc containing antimicrobial compound was pressed on shadedried ink sample from approximately 30g ink sacs the surface of agar to ensure a complete contact. Other discs obtained from 15 animals were immersed separately in were placed evenly on the agar surface giving enough methanol (1:3 w/v) solvents for overnight. The extracts then surrounding for microorganism inhibition. The petri dish with inoculum and the antimicrobial disc was inverted and filtered through Whatmann No.1 filter paper. The filtrate was o poured in previously weighed petridishes, evaporated to incubated at 35 ± 2 C for 24h. Inhibition zone formed after dryness and the dried extract was used for the experiments. incubation time was measured and recorded.

3 RESULTS AND DISCUSSION results of measurements using a spectrophotometer MIC Yield of Cuttlefish Ink Extract (Sepia sp.) Analysis of the yield results worth a concentration of 250 ppm. This is indicated by of ink extracts based on the comparison of bag weight the absorbance value of the concentration treatment which is (containing ink) samples to the weight of the extract of the ink 0.933 nm, which is smaller than the standard Mc Farland produced. The yield of ink can be seen in the table 1. absorbance value of 0.993 nm while the treatment concentration of 200 ppm with an absorbance value of 1.148 TABLE 1 nm has passed Mc Farland standard. These results indicate AVERAGE YIELD PERCENTAGE OF CUTTLEFISH INK EXTRACT that at a concentration of 250 ppm there has been a growth inhibition of Aeromonas hydrophilla. Weight of extract % yield Sampel Weight of ink bag (Gram) (Gram) Antibacterial test Antibacterial test concentration used based on the MIC test. In 1 5.4 ± 0,36 0.280 ± 0.22 5.20% this study using K + doses (positive control using 2 5.6 ± 1,62 0.263 ± 0.69 4.71% oxytetracyclin antibiotics), 250 ppm, 300ppm 350 ppm and K- 3 4.2 ± 1,05 0.195 ± 1.04 4.66% (negative controls without the administration of cuttlefish ink Average 4.86% extract (Sepia sp.) And antibiotics).

Description: Samples 1, 2, and 3 are replicates Based on the TABLE 3 table above, it is found that the yield of the bags respectively is THE RESULTS OF ANTIBACTERIAL TEST 5.4 ± 0.36 grams; 5.6 ± 1.62 grams; and 4.2 ± 1.05 grams. Extracts obtained respectively are 0.280 ± 0.22gram; 0.263 ± 0.69gram; and 0.195 ± 1.04. The average yield percentage of Doses (PPM) Inhibition Zone (mm) cuttlefish ink extract obtained was 4.86%. Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of 250 19 ± 0.9 a compound /extract/drug that completely inhibits the growth 300 22 ± 1.4 of the microorganism in 24 hrs. 350 27 ± 1.6 Positive 31 ± 1.2 Negative 5 ± 1.2 TABLE 2

MINIMUM INHIBITORY CONCENTRATION (MIC) TEST OF CUTTLEFISH The results of antibacterial test on table 2 showed antibacterial INK EXTRACT (SEPIA SP.) ON BACTERIAL GROWTH AND INHIBITION activity of cuttlefish ink extract at concentration negative ZONE OF AEROMONAS HYDROPHILLA control showed diameter zone of 5 ± 1.2 mm, at positive

control showed diameter zone of 31 ± 1.2 mm, at 250 ppm result 19 ± 0.9 mm, at 300 ppm result 22 ± 1.4 mm, at 350 Treatment Dose (ppm) Absorbance (nm) ppm result 31 ± 1.2 mm. The inhibiton zone of bacteria increased simultantly increasing concentrations of extract of 50 1.716 ± 0.12 cuttlefish ink provide valuable information and highlight the 100 1.592 ± 0.02 potential of this extract in drug development as a candidate source of antibacterial agents being safe and edible. The 150 1.406 ± 0.01 results showed that administration of cuttlefish ink extract 200 1.148 ± 0.3 (Sepia sp.) can increase inhibition zone of Aeromonas hydrophilla bacteria. Treatment with cuttlefish ink extract with a 250 0.933 ± 0.22 dose of 350 ppm is the best dose. This is indicated by the 300 0.841 ± 0.14 inhibition zone value that approachesthe positive control of treatment. A study states that cuttlefish ink has antibacterial mcF 0.993 ± 0.02 activity (13). Several other researchers have also tested antibacterial activity on extracts from cuttlefish ink, the results The results of the MIC test in table 5. show that the highest of which say that the extract has inhibitory activity against average absorbance value was obtained at a concentration of several bacteria such as Pseudomonas aeruginosa, 50 ppm which was equal to 1,716 nm and the lowest Staphylococcus epidermidis, Klebsiella pneumoniae and E. absorbance was obtained at a treatment dose of 300 ppm at coli (14). This is presumably because cuttlefish ink has 0.841 nm while the Mc Farland tube was 0.933 nm. The melanin content where melanin itself has antibacterial activity number of bacterial cells can be measured by knowing although it has not been widely revealed. however, the turbidity or turbidity of the culture, if the culture media melanin concentration of cuttlefish ink extract (0.010 g / mL) becomes more turbid, the number of cells will increase (11). was able to kill bacteria so that after incubation 24 there was Nillai OD can show the amount of light that can be absorbed only 1 living cell (inhibition reached 99.99%) (15). in addition. by the cell expressed proportional to the number of cells that (16) reported that methanol extracts of Sepiellainermis ink exist, so that it can be interpreted that the higher the using GC-MS showed a mixture of oligomeric structures which concentration of the extract given, the greater the inhibitory was a combination of dihydroxy indol-2-carboxylic acid and activity. That the higher the concentration of an antibacterial dihydroxyindole. This methanol extract has inhibitory activity substance, the higher the antibacterial power (12). In Table 5 especially against Gram negative bacteria such as Proteus shows that with the provision of cuttlefish ink extract (Sepia vulgaris, Pseudomonas aeruginosa and E. coli. (17) reported sp.). The growth of the Aeromonas hydrophilladecreased as that dihydroxyindol and dicarboxylic acid from Sepia had concentration increases. The table above also shows the microbial inhibitory activity. Antimicrobial activity of 1176 IJSTR©2019 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 8, ISSUE 11, NOVEMBER 2019 ISSN 2277-8616

polysaccharide isolated from the cuttlebone of Sepia aculeate Edward J.K., 2000, Study oncephalopod’s ink for and Sepia brevimana and methanolic extract of body tissue of antiretroviral activity. Indian Journal of Experimental Sepia parshadi have been reported (18). Studies have shown Biology. 2000. 36: pp. 583-587. that methanolic extract of cephalopods showed maximum [10] Ortonne J.P., Voulot C., Khatchadourian C., Palumbo A., activity against human pathogens (18). The results obtained and Prota G. 1981). A reexamination of melanogenesis in that Aeromonas hydriphilla were effectively inhibited by the ink the ink gland of cephalopods. In: Pigment cell. Phenotypic extract of cuttlefish (Sepia sp.). The effect of the extract differs Expression in Pigment cells M.Seiji. Ed.) University Tokyo from strain to strain with respective to their concentration.. The Press, Tokyo. 1981. pp. 49-57. ink of cephalopods contains a rich array of chemical secretions [11] Nithya M, Ambikapathy V., and Panneerselvam A., (2011). to escape from predators (19) and contains many constituents Effect of pharaoh’s cuttlefish ink against bacterial of aversive compounds (20). There are previous reports on the pathogens. Asian Journal of Plant Science Research. 49: antibiotic effects of the fluid from the ink sac of cephalopods pp. 49-55. (21) and the antibacterial activity in the extracts of gill and ink [12] Nkere, C.K., and Iroegbu, C. (2005)., Antibacterial sac of cephalopods (22). The present study reported that the screening of the root, stem and stembark extracts of ink of cephalopods exhibit antimicrobial activity (23). 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