27 In Vitro Citotoxicity Assays of Seagrass (Enhalus acoroides) Methanol Extract from Soropia Coastal Waters in Southeast Sulawesi Province Theosobia Grace Orno1, Agnes Rantesalu2 1Department of Health Analyst, Poltekkes Kemenkes Kendari, Kendari, Indonesia 2Department of Health Analyst, Poltekkes Kemenkes Kupang, Kupang, Indonesia Correspondence: Theosobia Grace Orno, Poltekkes Kemenkes Kendari, Jl. Jend A.H. Nasution No. G14 Anduonohu, Kendari, Southeast Sulawesi, Indonesia Zip Code : 93231 Email: theosobiagraceorno@gmail.com Received: February 11, 2020 Revised: March 13, 2020 Accepted: April 13, 2020 Abstract The studies analysing the use of natural ingredients as an alternative treatment in the field of pharmacology are developing very rapidly. One of researches that is quite promising in the pharmaceutical industry is the application of marine materials. Marine materials that are frequently used consist of shellfish, algae, sponges and seagrass. Several studies on toxicity tests have shown that the methanol extract of seagrass (from species Enhalus acoroides) is more toxic than the other seagrass family. This study aims to test the toxicity level of Seagrass (E. acoroides) extract from Soropia coastal waters. The research method in this study was an experimental laboratory using E. acoroides Seagrass as a sample that was obtained from Soropia Coast, Konawe Regency, Southeast Sulawesi Province. The sample was extracted using methanol as a solvent by macerating it and was tested for its toxicity using the Brine Shrimp Lethality Test (BSLT) method. Toxicity test results showed that the samples with a concentration of 10 ppm, 100 ppm and 1000 ppm in leaves extracts produced an LC50 value of 404.88 ppm, while the stem and root extracts has a value of LC50 > 1000 ppm. The test was continued with higher concentration of leaves extracts consisted of 250 ppm, 500 ppm, and 1000 ppm. The toxicity test showed an LC50 value of 0.7309; which means that it was very toxic. The methanol extract of Seagrass (E. Acoroides) is potential to be used for further analysis and anticancer formulations. Keywords Cytotoxicity, Enhalus acoroides, soropia coastal waters, southeast regency INTRODUCTION The research and development on new compounds with unique structures and interesting pharmacological activities have been isolated from marine biota. In general, these compounds are beneficial for humans as it containts high-value bioactive compounds that can be used in both the research and industrial setting. A very complex and dynamic interaction of marine Ina J Med Lab Sci Tech 2020; 2(1): 27-33 Theosobia Grace Orno, et al. 2 8 ecosystems trigger marine biota to produce secondary metabolites that has a function to defend their lives against competitors, predators and parasite. Since the last ten years, the study of marine natural products has began to be carried out in Indonesia (1). Corals, sponges, algae and seagrasses are marine organisms that are often used as research materials to find new raw material for medicines. However, among these organisms, seagrass is still relatively new in the development of raw materials of new drugs. Seagrass is a group of closed seeded plants (angiosperms) and single piece plants (monocots) that are able to live permanently below the sea level. Because seagrasses live permanently below the sea level, seagrass is classified as benthic organisms. These organisms produce secondary metabolites to maintain their survival from external disturbances both physicochemically and biologically (2). Several studies have shown that seagrass contains compounds that are potential as an anticancer (3), antioxidants (4), and antibacterial agents (5). The research on the potential of some seagrasses as anticancer have been carried out in severeal palces in Indonesia including the Pramuka Island waters, Lampung Waters, Morotai and Spermonde waters. The results declared that it has acute cytotoxic potential which can be applied as a new raw material for anticancer drug (6). The toxicity tests intend either to evaluate the safety of a compound or to detect the anticancer activity of a compound. In the Soropia Waters of Konawe Regency, Southeast Sulawesi Province, the data on the distribution of seagrass has been extensively studied, but the research on the level of toxicity of active compounds contained in seagrass is still lacking. The Soropia sub-district is a coastal area where natural products in the form of marine materials can be studied and utilized for various sectors including the health sector. The reason of choosing Enhalus acoroides as a research sample is based on Aprilyani et al's (15) research which analysed the seagrass distribution in Southeast Sulawesi waters, where E. acoroides ranks first in the seagrass distribution in all stations (7). This study tests the cytotoxicity of seagrass in Soropia waters by using the Brine Shrimp Lethality Test (BSLT). This test is usually used in screening natural marine bioactive compounds because it shows a correlation within in vitro cytotoxic methods and a specific anticancer test (8). Some methods can also be employed in this test, such as Lemna Assay, Potato disc as well as sell culture methos (Microculture Tetrazolium Salt/MTT). Among the four methods, BSLT is highly recommended by Anderson in the toxicity test because it has a correlation of up to 95% confidence level in specific anticancer tests. Although MTT also gets the same results as BSLT, BSLT is Ina J Med Lab Sci Tech 2020; 2(1): 27-33 Theosobia Grace Orno, et al. 2 9 easier, faster, cheaper and practical method (9). This study used methanol as an extraction solvent based on the results of previous studies by Aulia Fajarullah on the Extraction of Secondary Metabolites. Seagrass extraction process uses several types of solvent, where methanol solvent yields the highest extract (10.09%) and secondary metabolite compounds namely tannins, saponins, triterpenoids and steroids (10). MATERIALS AND METHODS This research was conducted at the Biochemistry Laboratory of Hasanuddin University for approximately one month. This research was an analytical study with a laboratory observation approach. Chemical and Reagents Methanol, E. acoroides, Artemia salina larvae were purchased in Laboratory of Poltekkes Kemenkes Kendari, Indonesia. Fresh leaves, root and rhizome of E. acoroides were used for experiments. It was collected from Soropia District, South Sulawesi Province, Indonesia. Sample Preparations The wet weight of seagrass samples was collected and was immediately placed in plastic bags containing sea water in order to prevent evaporation. Then, it was transported to the laboratory under cool condition. The seagrass samples of E. acoroides was washed thoroughly in deionized water to removed the biota that attached to it. In addition, the samples were separated into the roots, stems and leaves and each parts was arranged in aluminum trays. The E. acoroides samples were dried using oven dryer with a temperature of 50°C for 4 days. Drying process was conducted until the samples reached a moisture content below 10%. Furthermore, the samples were removed from the oven and were stored in a jar then was put in a dry place (Total Solid). The dried samples were milled by using grinder machines and the powder was then sieved manually by using mesh filter with size 30 mm. The fresh samples used in this experiment were 2 kg and it yielded 800 g powder after the drying and grinding process. Weight shrinkage in the seagrass samples were 60%. The powdered samples were stored in a refrigerator for further use. E. acoroides Extract Extraction were conducted by maceration method, namely by immersing the sample in a solvent within a few days. The maceration method was chosen for extraction because it is easy to do and only uses tools that are simple and easy to obtain (10,11). The dried seagrass sample was weighed as much as 50 g and was soaked in a 250 mL methanol solvent in a glass bottle and was macerated for 24 hours. The sample solutions were then filtered using filter paper through glass funnel and Whatman No. 42. The filtrate was concentrated to dryness by Ina J Med Lab Sci Tech 2020; 2(1): 27-33 Theosobia Grace Orno, et al. 3 0 rotation using evaporator under reduced pressure at temperature of 4°C. Finally, the crude extract from the seagrass sample was obtained in the form of a paste. Then, it was weighed to get the percentage of yield, and it was kept at -20°C prior to further analysis. Preparation of The Artemia salina Larvae A. salina eggs was prepared by soaking it in sea water for 10-15 minutes. Good egges would be settle while bad eggs would be float. As much as 50 mg of A. salina eggs were hatching in a container filled with sea water for 10-15 minutes. Then, the eggs at the bottom of the container were taken and were hatched in a container that also contained sea water under a 25-watt lamp and was equipped with an aerator. A. salina eggs hatched and became larvae after 24 hours. A. salina larvae that were good for the BSLT test were those that have a lifetime of 48 hours. If it died more than 48 hours, it was feared that A. salina's death was not caused by extract toxicity but rather by limited food supply (12). Toxicity Analysis The toxicity of E. acoroides crude extract was analyzed by using the Brine shrimp Letality Test (BSLT). This test were applied on A. salina L. This test aims to determine the level of toxicity of a natural material (13). BSLT test was carried out by describing a total of 10-15 shrimp larvae in each test bottle which was then added with a crude extract of seagrass from each tests. Test solutions with concentration of 250 ppm, 500 ppm and 1000 ppm were inserted into the bottle. Incubation was carried out for 48 hours and it was aerated. This test was carried out 3 times in each concentration series. The observation was run for 24 hours. The number of shrimp larvae death to determine the Lethal Concentration 50 (LC50) value was counted. LC50 is an assessment of the level of toxicity of a substance against 50% of larval deaths. Shrimp larvae defined have died while they were motionless for 10 seconds. The data were analyzed using Probit analysis to obtain the value LC50 by Microsoft Excel (2009) for Windows. Analysis were conducted by comparing the LC50, if the value of LC50 was higher than 1000 ppm, it would be categorized as toxic (13). RESULTS Extract of E. acoroides were found to inhibit the growth of A. salina on BLST test (Table 1). Based on the results of the toxicity test of seagrass extract (E. acoroides) which was divided into leaf extract, stem extract and root extract, leaf extract gave an LC50 value of 404.88 ppm (low toxic) while stem and root extract gave an LC50 value of >1000 which means it's not toxic. Ina J Med Lab Sci Tech 2020; 2(1): 27-33 Theosobia Grace Orno, et al. 3 1 Table 1. Toxicity Test Results of Seagrass (E. acoroides) Extract with BSLT Method Sample Conc (μg/mL) Log Conc Replication 1 (larvae) Replication 2 (larvae) Replication 3 (larvae) % Dead % Corrected Probit LC50 (ppm) Dead Life Dead Life Dead Life E. acoroides (leaf) 100 3 5 5 7 3 5 5 56.67 56.67 5.17 404.88 10 2 4 6 4 6 4 6 40 40.00 4.75 1 1 2 8 2 8 2 8 20 20.00 4.16 E. acoroides (stem) 100 3 2 8 2 8 3 7 23.33 23.33 4.27 >1000 10 2 2 8 1 9 3 7 20 20.00 4.16 1 1 0 10 0 10 3 7 10 10.00 3.72 E. acoroides (root) 100 3 2 8 2 8 4 6 26.67 26.67 4.38 >1000 10 2 2 8 1 9 0 10 10 10.00 3.72 1 1 1 9 0 10 1 9 6.67 6.67 3.50 Note: LC50 <1 (very toxic), >1 and <100 (moderate toxic), >100 (low toxic) (16). The toxicity test on leaf extracts with several levels of concentration including 250 ppm, 500 ppm and 1000 ppm represents in Table 2. Table 2. Toxicity Test Results of Seagrass (E. acoroides) Extract with BSLT Method No. Concentration (µg/ mL) Total Larvae Dead Precentation of Larvae Dead (%) LC50 (µg/ mL) 1 250 11 37 0,7309 2 500 21 70 3 1000 20 100 Note: LC50 <1 (very toxic), >1 and <100 (moderate toxic), >100 (low toxic) (16). DISCUSSION In this study, researchers chose seagrass species E. acoroides as research samples. The selection of this sample type was guided by Aprilyani et al's (15) research on mapping of seagrass distribution in Southeast Sulawesi waters, where E. acoroides ranks first in seagrass distribution in all stations (7). E. acoroides seagrass was then extracted using methanol as a solvent. Among the various types of solvents used in screening the secondary metabolites of the seagrass E. acoroides, methanol has been shown to have very high rendering activity and has the highest secondary metabolite content of four compounds namely tannins, saponins, triterpenoids and steroids (10). Methanol is a universal solvent that has polar (-OH) and nonpolar deciduous (-CH3) groups so that it can attract polar and nonpolar compounds (14). Bioactive compounds are usually having toxic at high doses. Therefore, in this study, the acute toxicity test used BSLT. The mechanism of death of A. salina was thought to be related to the function of compounds dissolved in seagrass extracts which can inhibit the feeding power of larvae (antifeedants/food dodgers). Therefore, toxicity analysis was performed to know the toxicity level of the E. acoroides extracts in order to determine their toxicity level. The way these compounds work is by acting as stomach poisoning (stomach poisoning). Ina J Med Lab Sci Tech 2020; 2(1): 27-33 Theosobia Grace Orno, et al. 3 2 Thus, if these compounds enter the larva body, the digestive apparatus will be disrupted. In addition, these compounds can inhibit the taste receptors in the mouth area of the larvae. This results in larvae failing to get a taste stimulus so they are unable to recognize their food until finally the larvae starve and die (13). The results of BSLT on leaf extract samples (E. acoroides) with dilution of 1 ppm, 10 ppm and 100 ppm gave LC50 values of 404.88 ppm with low toxicity interpretation, while stem extracts and root extracts gave LC50 values of >1000 ppm with non-toxic interpretation. Because only leaf extract that gave an LC50 value of 404.88 ppm, we conducted further tests on leaf extract with dilution variations of 250 ppm, 500 ppm and 1000 ppm. The calculation results show LC50 of 0.7309 ppm with a very toxic interpretation. Tannin is one of the secondary metabolites contained in the methanol extract of E. acoroides that can be used as an anticancer compound. Anti-cancer tannin activity occurs through the mechanism of inhibiting the work of enzymes, prevention of mutagenesis of cells that can cause cancer, and activation of cancer macrophage cells. The mechanism of action of this tannin uses histone deacetylase inhibitors (HDAC) (15). The role of Saponin as an anticancer has been known to inhibit the formation of Bcl-2 which is expressed too high, induce caspase-3 protein that is expressed too low, and can trigger G1 cell cycle arrest (16). Whereas, triterpenoids and steroids act as anti-cancer by activating apoptosis as well as making anti-proliferation (17). CONCLUSIONS The finding of this study revealed that E. acoroides methanol extract had bioactivity potentials. The extracts with a concentration of 250 ppm, 500 ppm and 1000 ppm has an acute toxic potential againts A. salina larvae, which is indicated by LC50 values. LC values of E. acoroides methanol extract is 0.7309 ppm, thus proving the existence of BSLT method. It is necessary to do further study to identify and isolate the active compounds that serve as anti-cancer agents to maximize the benefits of E. acoroides to human medicine. 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