In Vitro Citotoxicity Assays of Seagrass (Enhalus acoroide.
Methanol Extract from Soropia Coastal Waters in Southeast Sulawesi Province Theosobia Grace Orno1.
Agnes Rantesalu2 Department of Health Analyst.
Poltekkes Kemenkes Kendari.
Kendari.
Indonesia Department of Health Analyst.
Poltekkes Kemenkes Kupang.
Kupang.
Indonesia Correspondence:
Theosobia Grace Orno.
Poltekkes Kemenkes Kendari.
Jl.
Jend A.
Nasution No.
G14
Anduonohu.
Kendari.
Southeast Sulawesi.
Indonesia Zip Code : 93231 Email:
theosobiagraceorno@gmail.
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 .
rom species Enhalus acoroide.
is more toxic than the other seagrass family.
This study aims to test the toxicity level of Seagrass (E.
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) 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.
which means that it was very toxic.
The methanol extract of Seagrass (E.
Acoroide.
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 these compounds are beneficial for humans high-value compounds with unique structures and compounds that can be used in both the interesting pharmacological activities have research and industrial setting.
A very been isolated from marine biota.
In general, complex and dynamic interaction of marine Theosobia Grace Orno, et al.
ecosystems trigger marine biota to produce evaluate the safety of a compound or to detect secondary metabolites that has a function to the anticancer activity of a compound.
defend their lives against competitors, predators and parasite.
Regency.
Southeast Sulawesi Province, the Since the last ten years, the study of data on the distribution of seagrass has been marine natural products has began to be extensively studied, but the research on the carried out in Indonesia .
Corals, sponges, level of toxicity of active compounds algae and seagrasses are marine organisms contained in seagrass is still lacking.
The that are often used as research materials to Soropia sub-district is a coastal area where find new raw material for medicines.
natural products in the form of marine However, among these organisms, seagrass is materials can be studied and utilized for still relatively new in the development of raw various sectors including the health sector.
materials of new drugs.
Seagrass is a group The reason of choosing Enhalus acoroides as of closed seeded plants .
and a research sample is based on Aprilyani et al's single piece plants .
that are able to .
research which analysed the seagrass live permanently below the sea level.
distribution in Southeast Sulawesi waters.
Because seagrasses live permanently below where E.
acoroides ranks first in the seagrass the sea level, seagrass is classified as benthic distribution in all stations .
In the Soropia Waters of Konawe These This study tests the cytotoxicity of secondary metabolites to maintain their seagrass in Soropia waters by using the Brine survival from external disturbances both Shrimp Lethality Test (BSLT).
This test is physicochemically and biologically .
usually used in screening natural marine Several studies have shown that seagrass bioactive compounds because it shows a contains compounds that are potential as an correlation within in vitro cytotoxic methods and a specific anticancer test .
Some antibacterial agents .
The research on the methods can also be employed in this test, potential of some seagrasses as anticancer such as Lemna Assay.
Potato disc as well as have been carried out in severeal palces in Indonesia including the Pramuka Island Tetrazolium Salt/MTT).
Among the four waters.
Lampung Waters.
Morotai and methods.
BSLT is highly recommended by Spermonde waters.
The results declared that Anderson in the toxicity test because it has a it has acute cytotoxic potential which can be correlation of up to 95% confidence level in applied as a new raw material for anticancer specific anticancer tests.
Although MTT also drug .
The toxicity tests intend either to gets the same results as BSLT.
BSLT is .
, .
, (Microculture Ina J Med Lab Sci Tech 2020.
: 27-33
Theosobia Grace Orno, et al.
easier, faster, cheaper and practical method samples were separated into the roots, stems .
This study used methanol as an extraction and leaves and each parts was arranged in solvent based on the results of previous aluminum trays.
The E.
acoroides samples studies by Aulia Fajarullah on the Extraction were dried using oven dryer with a Seagrass temperature of 50AC for 4 days.
Drying extraction process uses several types of process was conducted until the samples solvent, where methanol solvent yields the reached a moisture content below 10%.
highest extract .
09%) and secondary Furthermore, the samples were removed from the oven and were stored in a jar then Secondary Metabolites.
namely tannins, saponins, triterpenoids and steroids .
was put in a dry place (Total Soli.
The dried samples were milled by using grinder
MATERIALS AND METHODS
machines and the powder was then sieved This research was conducted at the manually by using mesh filter with size 30 Biochemistry Laboratory of Hasanuddin The fresh samples used in this University for approximately one month.
experiment were 2 kg and it yielded 800 g This research was an analytical study with a powder after the drying and grinding process.
laboratory observation approach.
Weight shrinkage in the seagrass samples Chemical and Reagents were 60%.
The powdered samples were Methanol.
Artemia salina stored in a refrigerator for further use.
larvae were purchased in Laboratory of Poltekkes Kemenkes Kendari.
Indonesia.
acoroides Extract Fresh leaves, root and rhizome of E.
Extraction acoroides were used for experiments.
It was maceration method, namely by immersing collected from Soropia District.
South the sample in a solvent within a few days.
The Sulawesi Province.
Indonesia.
maceration method was chosen for extraction Sample Preparations because it is easy to do and only uses tools that are simple and easy to obtain .
collected and was immediately placed in The dried seagrass sample was weighed as plastic bags containing sea water in order to much as 50 g and was soaked in a 250 mL prevent evaporation.
Then, it was transported methanol solvent in a glass bottle and was to the laboratory under cool condition.
The macerated for 24 hours.
The sample solutions seagrass samples of E.
acoroides was washed were then filtered using filter paper through thoroughly in deionized water to removed the glass funnel and Whatman No.
The biota that attached to it.
In addition, the filtrate was concentrated to dryness by Ina J Med Lab Sci Tech 2020.
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The wet weight of seagrass samples was Theosobia Grace Orno, et al.
rotation using evaporator under reduced which was then added with a crude extract of pressure at temperature of 4AC.
Finally, the seagrass from each tests.
Test solutions with crude extract from the seagrass sample was concentration of 250 ppm, 500 ppm and 1000 obtained in the form of a paste.
Then, it was ppm were inserted into the bottle.
Incubation weighed to get the percentage of yield, and it was carried out for 48 hours and it was was kept at -20AC prior to further analysis.
This test was carried out 3 times in Preparation of The Artemia salina Larvae each concentration series.
The observation salina eggs was prepared by soaking it was run for 24 hours.
The number of shrimp in sea water for 10-15 minutes.
Good egges larvae death to determine the Lethal would be settle while bad eggs would be Concentration 50 (LC.
value was counted.
As much as 50 mg of A.
salina eggs LC50 is an assessment of the level of toxicity were hatching in a container filled with sea of a substance against 50% of larval deaths.
water for 10-15 minutes.
Then, the eggs at the Shrimp larvae defined have died while they bottom of the container were taken and were were motionless for 10 seconds.
The data hatched in a container that also contained sea were analyzed using Probit analysis to obtain water under a 25-watt lamp and was equipped the value LC50 by Microsoft Excel .
for with an aerator.
salina eggs hatched and Windows.
Analysis were conducted by became larvae after 24 hours.
salina larvae comparing the LC50, if the value of LC50 was that were good for the BSLT test were those higher than 1000 ppm, it would be that have a lifetime of 48 hours.
If it died categorized as toxic .
more than 48 hours, it was feared that A.
salina's death was not caused by extract toxicity but rather by limited food supply RESULTS Extract of E.
acoroides were found to .
inhibit the growth of A.
salina on BLST test Toxicity Analysis (Table .
Based on the results of the toxicity The toxicity of E.
acoroides crude extract test of seagrass extract (E.
which was analyzed by using the Brine shrimp was divided into leaf extract, stem extract and Letality Test (BSLT).
This test were applied root extract, leaf extract gave an LC50 value on A.
salina L.
This test aims to determine the 88 ppm .
ow toxi.
while stem and level of toxicity of a natural material .
root extract gave an LC50 value of >1000 BSLT test was carried out by describing a which means it's not toxic.
total of 10-15 shrimp larvae in each test bottle Ina J Med Lab Sci Tech 2020.
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Theosobia Grace Orno, et al.
Table 1.
Toxicity Test Results of Seagrass (E.
Extract with BSLT Method Sample Conc .
/mL) Log Conc Replication 1 .
Dead Life Replication 2 .
Dead Life Replication 3 .
Dead Life Dead Corrected Probit .
Note: LC50 <1 .
ery toxi.
, >1 and <100 .
oderate toxi.
, >100 .
ow toxi.
The toxicity test on leaf extracts with several levels of concentration including 250
LC50
>1000 >1000 ppm, 500 ppm and 1000 ppm represents in Table 2.
Table 2.
Toxicity Test Results of Seagrass (E.
Extract with BSLT Method Concentration Precentation of LC50 No.
Total Larvae Dead (AAg/ mL) Larvae Dead (%) (AAg/ mL) 0,7309 Note: LC50 <1 .
ery toxi.
, >1 and <100 .
oderate toxi.
, >100 .
ow toxi.
universal solvent that has polar (-OH) and DISCUSSION In this study, researchers chose seagrass nonpolar deciduous (-CH.
groups so that it species E.
acoroides as research samples.
The can attract polar and nonpolar compounds selection of this sample type was guided by .
Aprilyani et al's .
research on mapping of Bioactive compounds are usually having seagrass distribution in Southeast Sulawesi toxic at high doses.
Therefore, in this study, waters, where E.
acoroides ranks first in the acute toxicity test used BSLT.
The seagrass distribution in all stations .
mechanism of death of A.
salina was thought acoroides seagrass was then extracted using to be related to the function of compounds methanol as a solvent.
Among the various dissolved in seagrass extracts which can types of solvents used in screening the secondary metabolites of the seagrass E.
ntifeedants/food acoroides, methanol has been shown to have toxicity analysis was performed to know the very high rendering activity and has the toxicity level of the E.
acoroides extracts in highest secondary metabolite content of four order to determine their toxicity level.
The way these compounds work is by acting as triterpenoids and steroids .
Methanol is a stomach poisoning .
tomach poisonin.
Ina J Med Lab Sci Tech 2020.
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Therefore.
Theosobia Grace Orno, et al.
Thus, if these compounds enter the larva action of this tannin uses histone deacetylase body, the digestive apparatus will be inhibitors (HDAC) .
The role of Saponin In addition, these compounds can as an anticancer has been known to inhibit the inhibit the taste receptors in the mouth area of formation of Bcl-2 which is expressed too the larvae.
This results in larvae failing to get high, induce caspase-3 protein that is a taste stimulus so they are unable to expressed too low, and can trigger G1 cell recognize their food until finally the larvae cycle arrest .
Whereas, triterpenoids and starve and die .
The results of BSLT on steroids act as anti-cancer by activating leaf extract samples (E.
with apoptosis as well as making anti-proliferation 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
CONCLUSIONS
extracts gave LC50 values of >1000 ppm with The finding of this study revealed that E.
non-toxic interpretation.
Because only leaf acoroides methanol extract had bioactivity extract that gave an LC50 value of 404.
The extracts with a concentration ppm, we conducted further tests on leaf of 250 ppm, 500 ppm and 1000 ppm has an extract with dilution variations of 250 ppm, acute toxic potential againts A.
salina larvae, 500 ppm and 1000 ppm.
The calculation which is indicated by LC50 values.
LC values results show LC50 of 0.
7309 ppm with a very of E.
acoroides methanol extract is 0.
toxic interpretation.
Tannin is one of the ppm, thus proving the existence of BSLT secondary metabolites contained in the It is necessary to do further study to methanol extract of E.
acoroides that can be identify and isolate the active compounds that used as an anticancer compound.
serve as anti-cancer agents to maximize the Anti-cancer benefits of E.
acoroides to human medicine.
through the mechanism of inhibiting the work of enzymes, prevention of mutagenesis of CONFLICT OF INTEREST cells that can cause cancer, and activation of There are no conflicts of interest.
cancer macrophage cells.
The mechanism of REFERENCES