Phytochemical and antibacterial properties of sea cucumber (Muelleria lecanora) from Barrang Lompo Islands, Makassar South Sulawesi

Barrang Lompo Island Waters is home for different species of marine biota of sea cucumber (Muelleria lecanora). Many sea cucumber species have been used as health supplements because they contain bioactive compounds that are beneficial to people in Indonesia. Given this, our study was designed to investigate the phytochemical, and antibacterial properties of crude acetone, methanol and hexane extract of sea cucumber using maceration extraction methods. The sea cucumber extract was prepared and the phytochemical profile was studied by analysing Gas Chromatography-Mass Spectrometry (GC-MS). Results showed that the extracts were a complex mixture of numerous compounds; many of which were present in trace amounts antioxidants and antimicrobial; hexadecanoic acid, methyl ester, 9-octadecenoic acid (z) -, methyl ester (stearic acid methyl ester), octadecanoic acid, methyl ester, 2-[(hexadecyloxy)methyl]oxirane, cholest5-en-3-yl acetate, ergosta-14,22-dien-3-ol, acetate,(3.beta.,5.alpha.,22e), 5,8,11,14eicosatetraenoic acid, methyl ester, (all-z) epa/omega 3, pentacosane, hexatriacontane, and 9-hexadecenoic acid, methyl ester, (Z). The extract was also evaluated for activity against three pathogenic bacterial strains (Escherichia coli, Staphylococcus aureus and Salmonella) using the disc diffusion method. The extract exhibited clear zones of inhibition against the tested bacteria. Maximum inhibitory zone concentration values were demonstrated to be: Escherichia coli = 6.84 mm , Staphylococcus aureus = 7.22 mm, and Salmonella = 7.87 mm. These results revealed the significant potential of sea cucumber as a source of antioxidants and antimicrobial agents and also highlight the necessity of further purification and characterisation of solitary bioactive compounds for their prospective applications in pharmaceutical industries, food, and nutraceutical (food functional).


Introduction
The increasing number of scientific research and journals published in recent decades has to do with functional materials derived from vegetable and animal natural resources, potentially as food functional, nutraceuticals and health supplement products that support nutritional value needs and improve body health. Among other marine animals, sea cucumber is a source of natural ingredients that contain functional ingredients and bioactive compound that can be used in biomedicine and food processing industries. Sea cucumber is a marine invertebrate of the Holothuroidea class, has rough skin on its outer and elongated body and contains a single branched gonad. This marine animal has a wide number of species, ranging from 1716 species, with the largest potential of biological diversity in the Asia-Pacific region. In Indonesia, it is known as "Teripang or Trepang", in France under the name "Beche-De-Mer", which means seafood products, and "Balate" in the islands of Guam or Chomorro. Sea cucumber includes living marine animals with complex environments, making it difficult to live in extreme environments, therefore the sea cucumber can produce biologically active secondary metabolites that can not be found and obtained from other marine animals (Pangestuti and Arifin, 2018).
The sea cucumber has a complete nutritional content, low-fat content, high protein, and rich essential amino acids, such as tryptophan, arginine, and lysine, as well as having a body wall consisting of non-soluble collagen which can be used as a nutritional supplement (Chen, 2004). Sea cucumber has been recognized as a traditional remedy for treating asthma, rheumatism, hypertension, impotence, constipation and burns (Wen et al., 2010). Other functions, among others, anti-coagulant (Nagase et al., 1995;Chen et al., 2011), anticancer (Janakiram et al., 2015), anti-inflammatory (Olivera-Castillo et al., 2018), antithrombotic (Mourão et al., 1998;Pacheco et al., 2000), antioxidant (Althunibat et al., 2009), antimicrobial (Beauregard et al., 2001;Hing et al., 2007), antihypertensive (Zhao et al., 2007), antiangiogenic (Roginsky et al., 2004;Tian et al., 2005), antitumor (Zou et al., 2003;Tong et al., 2005), and healing wound (San Miguel-Ruiz and García-Arrarás, 2007), due to the role of bioactive compounds present in the sea cucumber mainly the triterpene glycosides (saponin) (Miyamoto et al., 1990;Kerr and Chen, 1995;Aminin et al., 2010), phenolics (Mamelona et al., 2007), lectins Merca, 2004, 2005), sterols (glycosides and sulfate) (Han et al., 2009), peptides (Zhou et al., 2012), glycosaminoglycan (Kariya et al., 1990;Borsig et al., 2007), chondroitin sulfate (Ustyuzhanina et al., 2016), cerebrosides (Sugawara et al., 2006), and sulfate polysaccharides (Luo et al., 2013). This bioactive compound can be used as a potential antibacterial. Antibacterial is a compound that can suppress the growth and development of bacteria, the ability of bioactive compounds in the sea, which makes researchers interested in researching a sea cucumber. The need to find new antimicrobial material is increasing, because the growth and development of bacteria are currently able to be resistant to antibiotics, as well as the growing conventional antibiotics (Li et al., 2008). Nguyen et al. (2011) reported about potent α-Glucosidase inhibitory activity purified from the body wall of Sea Cucumber (Stichopus japonicus). The result shows that sea cucumber fatty acids can potentially be developed as a novel natural nutraceutical for the management of type -2 diabetes. Other research, indicating that sea cucumber Apostichopus japonicus, analysis in vitro show inhibitory zones on microbes strains Vibrio splendidus, Vibrio harveyi and Staphylococcus aureus (Jiang et al., 2014). The antibacterial activity of sea cucumber Actinopyga lecanora against some common pathogenic Grampositive bacteria (Bacillus subtilis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas spp.), was evaluated. It was indicated that sea cucumber extract was able to inhibit Staphylococcus aureus (Ghanbari et al., 2012). Five curvularin macrolides in isolation from sea cucumber Holothuria moebii, results in 11-hydroxycurvularins ((11S,15R)-11-hydroxycurvularin) and ((11R,15R)-11-hydroxycurvularin) also showed antibacterial activity inhibiting the growth of Escherichia coli (Ye et al., 2016). Previous studies on antibacterial activity of sea cucumber extracts have been reported against several pathogenic bacteria such as Escherichia coli, Salmonella, Listeria, and Staphylococcus aureus. Previous literature showed that sea cucumber is extensively studied for antioxidant and antimicrobial activities.
In this study, sea cucumber is used for investigating antioxidant and antimicrobial potential using maceration extraction with three solvents (acetone, hexane and methanol). The present work aimed to investigate the efficiency of different solvents for the phytochemical extraction from sea cucumber and to identify and analyze the bioactive compounds by GC-MS (Gas Chromatography-Mass Spectrometry). The antibacterial efficacy against pathogenic bacteria Escherichia coli, Salmonella and Staphylococcus aureus using disc diffusion methods was also investigated.

Materials
Sea cucumber phylum Echinodermata, family Holothuriidae and genus Muelleria lecanora (Figure 1) collected from the coast Barrang Lompo Island in Makassar, South Sulawesi, Indonesia. During the trip, sea cucumber is stored in a cooling box that contains an ice pack and is moved into the freezer after arriving in the laboratory and processed in Food Science and Instrumental Analysis Laboratory, Chemical Engineering Department, Politeknik Negeri Ujung Pandang, Indonesia. Tetracycline hydrochloride (CAS: 64-75-5) supplied by Sigma Aldrich (St. Louis, Missouri, United States), culture strains Staphylococcus aureus, Salmonella and Escherichia coli.

Preparation of extracts
The sea cucumbers were cleaned and dried in an oven at 70°C, then cropped small-minced. About 100 g of the sea cucumbers were homogenized and extracted using the maceration method with a ratio of volume 1:2 (V/V) methanol, acetone or n-hexane. Samples were allowed for 72 hrs with solvent replacement every 24 hrs and were stirring with orbital shakers. The resulting extraction (macerate) is then filtered and concentrated with a rotary evaporator at 40°C until the extract is obtained. The Supernatant is produced for each sample, then in centrifugation for 10 mins and stored at a temperature of 10°C, for use in analysis bioactive compound in GC-MS and antibacterial disc diffusion method.

Gas Chromatography-Mass Spectrometry(GC-MS)
Sea cucumber was analyzed using GC-MS using capillary column DB-5 (30 μm, 0.25 mm, 0.25 μm film) and Flame Ionization Detector (FID) operated in EI mode at 70 eV. 1 mL of the sea cucumber was added with 3 mL of methanol 96% in the reaction tube and vortex. The injectors and detector temperature are set at 220°C and 250°C. A sample was dissolved with 1 μl methanol, then injected and analyzed at 60°C for 2 minutes and then increased 3°C/min to 300°C, with Helium (He) is used as carrier gas (1 mL/min). This analysis will generate two GC data in the form of chromatogram which displays the peaks of the compound contained in the methanol, acetone or nhexane extract and the current MS (Mass Spectroscopy) data shows the molecular weight at each peak. Any peaks appearing on the GC chromatogram indicate a single molecule and have a fragmentation pattern displayed in the MS spectra. Based on the fragmentation pattern can be identified what compounds are contained in the sea cucumber sample.

Test microorganisms and culture media
Test microorganisms Salmonella, Staphylococcus aureus and Escherichia coli used in this study were obtained from the Microbiology Laboratory, Department of Biology, State University of Makassar. The bacteria was grown at 32 o C in nutrient broth (DIFCO Laboratories, Detroit, USA) following standard procedures (Keagle and Gersen, 2005).

Antimicrobial assay
Disc antibiotic blank (Whatman No. 1) was cut to size and sterilized with other equipment using autoclaved at 121 o C for 15 mins. Growth media microorganisms are 5 g nutrient agar (NA), dissolved 250 mL of aquadest in the Erlenmeyer 500 mL, then heated to homogeneous. The sodium chloride solvent was obtained by 0.9 g NaCl, then dissolved in a 100 mL volumetric flask, and inserted into the reaction tube 9 mL. Mc Farland solvent obtained, by mixing a solution of barium chloride (BaCl 2 ) 1.175% and a solution of sulphuric acid (H 2 SO 4 ) 1%, so that the solvent obtained Mc Farland 0.5% to be used as standard turbidity (absorbance 600 nm). Media nutrient agar, and sodium chloride solvent was sterilized using autoclave at temperature 121 o C for 15 mins.
Sterile nutrient agar 20 mL was poured in Petri dishes, allowed to set at 37°C and then inoculate uniformly with 0.1 mL of a 24 hr broth culture of test bacteria (Abubakar et al., 2012). Sea cucumber extract (0.25 g) were dissolved in 1 mL aqueous dimethylsulfoxide (DMSO) with tween 80 (0.5% v/v for easy diffusion) and sterilized by filtration through a 0.45 μm membrane filter. Under aseptic condition, each sterile disc (Whatman no. 5, 6 mm dia) was then dipped in 20 μL of the extracts and carefully placed on the agar plate using flame sterilized forceps, ensuring the discs were at least 2 cm separate from one another. After 30 mins, plates were inverted and incubated at 37°C for 48 hr, followed by measuring the inhibitory zone for each sample and the type of bacteria in mm. An experiment was carried out in duplicate and the averages diameter of zone of inhibition was recorded. Negative controls use a 10% DMSO solvent, and one paper disc is given a tetracycline of HCl as a positive control, antibacterial activity was classified as highly active (>10 mm), mild active (7-10 mm) and slightly active (6-7 mm) and less than 6 mm was taken as inactive (Chandra et al., 2011).

Yield analysis extract of methanol, acetone and hexane
The effect of extraction time on the yield of sea cucumber obtained using various solvents (methanol, acetone and hexane) is shown in Figure 2. The solvent used is the polar methanol, acetone is semi-polar and the hexane is non-polar. The highest yield of 18.96% by using a methanol solvent, while the lowest yield of 0.14% using hexane solvent was obtained. Acetone solvent obtained a yield of 8.19%. This suggests that the ability of methanol solvent in extracting a compound is very good compared to other solvents.

GC-MS analysis
The presence of a bioactive compound in the methanol, acetone, and hexane extract of sea cucumber was characterized by GC-MS analysis ( Table 1).

Antibacterial activity
Antibacterial is a compound that can suppress growth and development of bacterial growth by using bioactive compounds found in sea cucumber, which are generally flavonoids and steroids. Disc diffusion method was followed to determine the antimicrobial activity. Antimicrobial activities of sea cucumber (Muellaria lecanora) extracts were evaluated against three bacterial (two gram-positive, one gram-negative) strains as shown in Table 2 and Figure 6.
The concentration of the acetone, hexane and methanol extract of sea cucumber leaves was calculated as 20 μL, were screened for sensitivity against the three solvents extract. Initial screening of sea cucumber for antibacterial activity on bacterial strains Escherichia coli Salmonella and Staphylococcus aureus was carried out  (Tilaoui et al., 2011) Table 1 (Cont.). GC-MS analysis for the methanol, acetone and hexane extract of sea cucumber FULL PAPER via Kirby-Bauer disc diffusion assay. The antibacterial activity was classified as highly active (>10 mm), mild active (7-10 mm) and slightly active (6-7 mm) and less than 6 mm was taken as inactive (Chandra et al., 2011).
An antibacterial activity can be known by the formation of a bright zone around the disc paper and the bright zone is the inhibitory zone. Methanol extraction exhibited a broad-spectrum antibacterial activity with a minimum zone diameter of 6.20 mm against bacteria Escherichia coli and hexane extraction a maximum zone diameter of 7.87 mm against bacteria Salmonella. Methanol extraction belongs to the antibacterial activity slightly active category with an average inhibitory zone of 6-7 mm bacteria Escherichia coli and mild active category with an average inhibitory zone of 7-10 mm against bacteria Salmonella and Staphylococcus aureus ( Figure 6). The activity of flavonoids inhibits the growth of bacteria by damaging the cell membrane, thereby inhibiting the synthesis of bacterial cell macromolecules (Dzoyem et al., 2013). The mechanism of the steroid works as an antibacterial by damaging the lipid membrane, so that liposomes leak. Steroids are also known to be interacting with membrane phospholipids, since their permeable nature to lipolytic compounds leads to decreased membrane integrity and morphology of impaired cells membranes, resulting in lysis and fragile cells (Madduluri et al., 2013). The preliminary antibacterial assay of the extracts showed different responses to the test strains with the best activity observed for acetone, methanol and n-hexane extracts of sea cucumber against bacteria gram-positive (Salmonella and Staphylococcus aureus), but not recommended for gram-negative bacteria such as Escherichia coli.

Conclusion
In summary, acetone and methanol were found to be the best solvent for phytochemicals extraction from Sea cucumber. The methanol and hexane extract showed a maximum number of bioactive compounds in preliminary phytochemical analysis and a good amount of antibacterial activity and total phenolics in the antioxidant activity. Special for methanol extract showed a bioactive compound as an anticancer and antiproliferative activity Lanost-8-en-3-ol, (3.beta.) and Farnesene epoxide. Both of these compounds work well in treating human gastric cancer which malignant cells and ability to stop the growth of cells and not allowing the cells to multiply rapidly. Antibacterial assay results from sea cucumber extract were very effective against bacteria gram-positive (Salmonella and Staphylococcus aureus). The bioautography analysis showed that the whole extract had free radical scavenging and antibacterial potential. GC-MS analysis revealed the presence of a good number of bioactive metabolites such as flavanoid and steroids in the extract. The results of this study implied that sea cucumber genus Muelleria lecanora have shown better antibacterial and antioxidant activities which could be used in the food and therapeutic applications.