Cytotoxic Test of Ethanol Extract of Bintaro Fruit and Peel ( Cerbera odollam Gaertn.) against Artemia salina Leach Larvae

: Bintaro ( Cerbera odollam Gaertn). Plants belonging to the Apocynaceae family have properties such as antifungal, insecticide, antioxidant and antitumor. Bintaro fruit contains alkaloids, flavonoids, terpenoids, tannins and saponins. Bintaro plants are poisonous because they contain alkaloid group compounds which are toxic. This study aims to obtain data and information regarding the toxicity ratio of bintaro rind and bintaro fruit ( Cerbera odollam Gaertn.) to Artemia salina Leach larvae by determining the value of Lethal Concentration 50 (LC 50 ). This type of research was experimental with quantitative methods, using Artemia salina Leach larvae. Each extract was divided into 4 concentration groups. In the ethanol extract of bintaro fruit peel, namely at concentrations of 500, 1000, 1500 and 2500 ppm. Meanwhile, bintaro fruit extract at concentrations of 1000, 1500, 2000 and 2500 ppm, each of which was carried out 3 replications with 10 larvae. Data analysis using probit analysis showed that the ethanol extract of bintaro fruit peel ( Cerbera odollam Gaertn.) had LC 50 = 701.455 μg/mL which was categorized as toxic. Meanwhile, the ethanol extract of bintaro fruit ( Cerbera odollam Gaertn.) has LC 50 = 1199.5 μg/mL which is categorized as nontoxic.


INTRODUCTION
Medicinal plants are all types of plants that can be used as medicinal ingredients, both singly and in mixtures. Medicinal plants are plants that can contain active substances that function to treat certain diseases or if they do not contain certain active substances but contain the resultant effect/synergy of various substances that play a role in the treatment of certain diseases (Supriat, H.S. et al., 2023). Cytotoxic test is an in vitro toxicity test using cell culture which is used to detect the presence of cytotoxic activity of a compound (Haryoto, et al., 2013). Toxicity tests on plant extracts are usually carried out to determine the level of safety of an extract (Vitalia, N., et al. 2016). Toxicity tests are widely used in tracing toxic compounds derived from natural products (Supriat, H.S. et al., 2023). One of the methods used for cytotoxic testing is the method Brine Shrimp Lethality Test (BSLT). BSLT is a test method using Artemia salina Leach larvae. as a test animal (Fatimah & Santoso, 2020). This test method is based on active plant compounds which are toxic and capable of killing Artemia salina Leach larvae (Vitalia, N., et al. 2016). The test results are expressed as LC50 (Kurniawan & Ropiqa, 2021). In addition, this method is also easy to do, cheap, fast, and accurate (Maukar, et al., 2013). One of the natural ingredients that has medicinal properties is the Bintaro plant (Cerbera odollam Gaertn). Plants from the Apocynaceae family have properties such as antifungal, insecticide, antioxidant and antitumor (Supriat, H.S. et al., 2023). Bintaro fruit has three layers, namely the outer skin (epikarp), the middle layer resembling fibers such as coconut fiber (mesokarp), and the inner layer in the form of fruit seeds (endocarp) (Ulung, 2014). Bintaro plant skin and leaves contain pseudondicaan. Bintaro seeds contain 0.6-1% serberin, serberoside, teviridosid, fatty oil, odoline (bitter substance), and papagan poison. The sap contains a laxative (Utami, 2008). Empirically, Bintaro fruit sap is used as fish, rat, pig and mosquito repellent.

A.
Extract Manufacturing Bintaro fruit simplicia and bintaro fruit rind (Cerbera odollam Gaertn.) obtained were weighed at 200 grams. The simplicia extraction process uses the maceration method with 96% ethanol solvent until it is submerged in a tightly closed container for 24 hours. The extraction process was carried out until the maceration solution was close to colorless. The extract is obtained by filtering the residue with the extract using filter paper. The extract solution was evaporated using a rotary vacuum evaporator at a temperature of 55-57 o C to obtain a thick extract (Syarif, et al., 2022).

B.
Cytotoxic Test a.
Hatching of Artemia salina Leach larvae Artemia salina Leach eggs were weighed as much as ±50 mg and then put into an incubator vessel which was provided with a partition so that it had two sides of the chamber, namely the open side and the closed (dark) side. The incubator vessel is filled with sea water which has been filtered with whatman paper number 42. Then the aerator is placed in the vessel and illuminated with an incandescent lamp. After 48 hours, the eggs that have hatched become nauplii. Then the nauplii are given yeast suspension as a food ingredient and can be used as test animals .

b. Preparation of Sample and Control Solutions
The stock solution was prepared by weighing 2,500 mg each of the ethanol extract of bintaro fruit rind and the ethanol extract of bintaro fruit dissolved in 2 mL of sea water. If the sample is insoluble or poorly soluble, 0.1-50 μg or 2 drops of 1% dimethyl sulfoxide (DMSO) is added and seawater is added until the volume reaches 250 mL to obtain a stock solution concentration of 10,000 ppm. Test solutions of fruit peel ethanol extract were made in concentrations of 500 ppm, 1000 ppm, 1500 ppm and 2500 ppm. For the control (0 ppm) was done without the addition of extracts. The fruit ethanol extract test solutions were made in concentrations of 1000 ppm, 1500 ppm, 2000 ppm and 2500 ppm. For the control (0 ppm) was done without the addition of extracts .

c. Cytotoxic Test Brine Shrimp Lethality Test (BSLT) Method
Each sample extract concentration was made in 3 replications. Then, at each solution concentration, 10 Artemia salina Leach larvae were put into the vial. The control was added with 10 mL of seawater without the test solution. Furthermore, observations were made after 24 hours and then counted the number of dead larvae from each vial then followed by probit analysis to determine the LC50 value. .
The toxicity test was carried out in each sample extract group which was divided into 5 test groups, namely 4 treatment groups and 1 control or comparison group (seawater).

RESULTS AND DISCUSSION
The cytotoxic test using the BSLT can be determined from the number of deaths of Artemia salina Leach due to the influence of extracts or natural compounds. The test results are expressed as LC50 (Kurniawan & Ropiqa, 2021). Good results and actively tested BSLT with the aim of knowing the LC50 (Wahyu Ningdyah, et al., 2015). Mortality values were determined using probit analysis to determine toxicity values using Lethal Concentration (LC05). The percentage of mortality of Artemia salina larvae can be calculated using the equation (Sumihe, et al. 2014). Using the linear regression equation is y = bx + a; y = 50% mortality in probit percentage, a = intercept or value obtained from the linear regression equation, x = log concentration, so that the LC50 value is obtained (Rahmi, et al., 2022). If LC50 < 30 ppm it is said to be very toxic, LC50 < 1000 ppm is said to be toxic, while LC50 > 1000 ppm then the test preparation is not toxic (Dona, et al., 2019).
The BSLT method cytotoxic test used Artemia salina larvae that were 48 hours old because at this time the larvae already had a high level of sensitivity due to their complete organs (Rahmi, et al., 2022). Based on its morphology, Artemia salina larvae that are 48 hours old have started to have mouths and digestive tracts but their food reserves have started to run out so they start looking for food (Fadhli & Hasanah, 2019). Artemia salina begins to eat in instar III, that is, after the digestive tract is formed. In instar III or 48 hours old, the larval body will metamorphose and increase its body resistance (Reskianingsih, A. 2014 The BSLT test was carried out by observing the mortality rate of the larvae after being incubated for 1x24 hours with the extract. The results obtained were then calculated as the LC50 (Lethal Concentration) value of the extract (Rani, Z., et al. 2022). The LC50 value was obtained from the percentage of death of Artemia salina larvae due to the extract (Alfath, T., 2018).
Based on the results of observations using the ethanol extract of bintaro fruit peels, it was shown that all concentration series groups caused death in larvae except for the control group which contained seawater. Concentrations of 500, 1000, 1500 and 2500 were used because these concentrations had a toxic effect on Artemia salina larvae. At a concentration of 2500 ppm it caused the highest average mortality of larvae, while at a concentration of 500 ppm it caused the lowest mortality of larvae. After probit analysis, it can be seen that the graph of the straight line equation Y = 5.5261X + 10.713. With the correlation coefficient obtained, namely R2 = 0.9973, which means that 99.7% change in the probit value is influenced by the concentration of the extract. The graph shows the log concentration on the probit value obtained from the percentage of larval mortality, then enter the Y value, namely the probit value of 50% of the test animals and obtain an X value = 5.52, so the LC50 antilog value is 5.52, which is 701.455 µg/mL. The LC50 results obtained were less than 1000 µg/mL. Where, if the LC50 ≤ 1000 ppm it can be concluded that the ethanol extract of bintaro fruit is toxic.
Based on the results of observations, it was shown that all concentration series groups caused death in the larvae except for the control group which contained seawater. Concentrations of 1000, 1500, 2000 and 2500 were used because these concentrations had a toxic effect on Artemia salina larvae. At a concentration of 2500 ppm it caused the highest average mortality of larvae, while at a concentration of 1000 ppm it caused the lowest mortality of larvae. Different concentrations in each test vial have different numbers of larval deaths, this shows that each concentration level has a different effect on larval mortality. The higher the concentration of the extract used, the higher the total number of larvae deaths. The graph shows the log concentration on the probit value obtained from the percentage of larval mortality, then enter the Y value, namely the probit value of 50% of the test animals and obtain an X value = 3.07, so the LC50 antilog value is 3.07, which is 1199.5 µg/mL. The LC50 results obtained were greater than 1000 µg/mL. Where, if the LC50 ≥ 1000 ppm it can be concluded that the ethanol extract of bintaro fruit is not toxic. The mechanism of larval death is thought to be related to the function of flavonoids and alkaloids. Flavonoids that inhibit the eating power of the larvae. The way toxic compounds work that cause the death of Artemia larvae is by acting as stomach poisoning (Rani, Z., et al. 2022). Alkaloid compounds have a toxin character, temporarily or permanently stopping appetite in insects so that it interferes with the growth and development of larvae and will only cause death in some time due to starvation. But in large quantities the alkaloids work as contact poisons and digestive poisons which will immediately kill the larvae and cause death because they attack vital organs such as the nervous system and affect heart activity (Kurniawan & Ropiqa, 2021). The data obtained was measured using the LC50 parameter (Lethal Concentration 50%) (Jannah, D. et.al., 2020). LC50 (Lethal Concentration 50) is the concentration of a substance that causes death in 50% of experimental animals, namely Artemia salina Leach larvae (Kurniawan & Ropiqa, 2021). Mortality in 50% of the experimental animals was calculated at a defined 24hour exposure period (Syahdana, et al., 2017). By entering the value (50% probit of death of the test animals) as y, the results of x are obtained as the log concentration value. The antilog of the x value is the LC50 value (Rani, Z., et al. 2022).

CONCLUSION
1. Bintaro fruit peel ethanol extract (Cerbera odollam Gaertn.) has an effect on Artemia salina Leach larvae. Where, the samples used have the potential to have toxic effects.