Antimalarial Activity of Sea Sponge Extract of Stylissa massa originating from waters of Rote Island

Received: 18th September 2020 Revised: 14th March 2021 Accepted: 23rd March 2021 Online: 30th April 2021 Research on the isolation, toxicity test, antimalarial test, and identification of the active compound from the ethyl acetate fraction of Stylissa massa sponge from Oenggae waters, Rote Island, has been conducted. This study aimed to investigate the antimalarial activity of the ethyl acetate fraction of the Stylissa massa sponge. Isolation was carried out by the extraction method using a mixed solvent of methanol: dichloromethane of 3: 2 (v/v), then the extract was partitioned in a solvent mixture of ethyl acetate: water of 1: 2 (v/v). The ethyl acetate extract obtained was separated by column chromatography using the gradient polarity system method. The toxicity test of each fraction was carried out by the Brine Shrimp Lethality Test (BSLT) method, and the antimalarial test was carried out by the haematin polymerization inhibition method. Identification of compounds from the active fraction in the antimalarial test was carried out using Liquid Chromatography-Mass Spectrometry (LC-MS). The extraction yield was 1.14 g (0.23%) of the ethyl acetate extract in the form of a dark brownish-yellow oily solid. Separation by column chromatography resulted in 15 fractions. Toxicity test results showed the four most active fractions with LC5O values, which are very promising for new drug discovery. The IC50 value in the antimalarial activity test of the four fractions indicated that the Stylissa massa sponge ethyl acetate extract was more active than the standard chloroquine compound (115 pg/mL). The LC-MS analysis indicates that fraction 11 contains two compounds that have been reported, and 1 compound is unknown. In contrast, fraction 14 indicates that it contains three compounds that have been reported and one unknown compound.


Introduction
Malaria is still a problem that needs attention. The resistance of Plasmodium falciparum to antimalarial drugs is the cause of the spread of malaria to new areas. It causes the re-emergence of malaria in areas that have been eradicated. Based on a World Malaria Report 2019 report, there are 228 million malaria cases globally, and 405 ,000 have died [1]. Thus, the discovery of new compounds as candidates for antimalarial drugs is essential.
Exploration of antimalarial compounds from marine materials, especially sponges, has been relatively developed in this decade. Manzamine A from the Haliclona oculata sponge, Diacarnuperoxide M from the Diacarnus megaspinorhabdosa sponge, Psammaplysin H, and Tsitsikammamine C from the Zyzzya sp. sponge are some of the compounds reported to have antimalarial activity [2, 3 , 4 , 5l -Stylissa massa sponges are widespread in tropical seas such as the Banda Sea, Sulawesi Sea, Mergui Islands, and Papua New Guinea [6]. Several compounds that have potential as medicinal substances have been isolated from sponges of this species, such as Stylissatin A (cyclic peptide) as an anti-inflammatory agent, Stylissatin B as an antitumor agent, Spongiacidin C as a USP 7 inhibitor, and aldisine alkaloid as a MEK-i inhibitor [ 7 , 8, 9 , 10]. Currently, the exploration of bioactive compounds and antimalarial activity tests of the Stylissa massa sponge has not been widely carried out. This sponge is commonly found in the waters of the island of Rote (the southern border island of Indonesia) but has not been widely studied.
Recent studies have shown that ethyl acetate extract from Stylissa carteri sponge has antimalarial activity with an IC 50 of 20.56 pg / mL [11]. The Stylissa carteri sponge under the same genus as Stylissa massa allows a similar compound in the Stylissa massa sponge. Other studies have shown that some of the antimalarial compounds that have been reported are less polar isolated from less polar solvents, such as dichloromethane and ethyl acetate. So, this research was conducted to provide information about the antimalarial activity of the Stylissa massa sponge extract.

Methodology
This research was conducted in several stages, starting with sampling. Furthermore, activities are carried out in the laboratory, such as extraction, phytochemical testing, component separation, toxicity testing, and antimalarial testing. LCMS data were used to predict the compound content in each fraction.

Sponge Sampling
The Stylissa massa sponge was taken from Oenggae Waters, Rote Island, East Nusa Tenggara, at a depth of 7 meters, in the distance of 600 meters from the coastline. The dive was carried out at 10.00-12.00 Central Indonesian Time on June 27 , 2020, then cleaned of foreign material and other organisms and then put into a temporary storage box containing an ice pack. The samples were then freeze-dried and stored.

Extraction
solution into a vial containing ethyl acetate extract, then dropping Wagner ' s reagent. The ethyl acetate extract was positive for alkaloid compounds if a brown precipitate was formed [12].
The terpenoid test in this study used a vanillin-H2SO 4 reagent. This reagent was prepared by dissolving 0.1 g of vanillin in a mixture of 16 mL concentrated H2SO 4 and 4 mL of ethanol. The terpenoid test was carried out by dissolving the ethyl acetate extract in methanol, then dropping the vanillin-H2SO 4 reagent. Ethyl acetate extract was positive for terpenoids if there was a change in color to purple [12].
The saponin test was carried out by dissolving the ethyl acetate extract with distilled water in the vial, then shaking the vial. The saponin-containing compounds were characterized by a stable foam for approximately 15 minutes [12].
The steroid test in this study used the Liebermann-Burchard reagent. The steroid test was done by dissolving ethyl acetate extract with 2 mL of chloroform in a vial, adding ten drops of acetic anhydride and three drops of concentrated H2SO 4 . The steroid -containing ethyl acetate extract was characterized by a color change from red to green / blue [12].

Separation of Ethyl Acetate Extract Components by Column Chromatography
The separation of ethyl acetate extract components was carried out using column chromatography. The column was attached to the stative vertically, and cotton was added to the bottom of the column. Silica gel 60 (mobile phase) before use was activated by heating at 120°C for 2 hours. Before using, the column was filled with n-hexane to be rinsed. The activated Silica gel 60 was made a slurry with n-hexane. The slurry was then put into the column and made homogeneous. After filling finished, the top of the column was wrapped with aluminum foil to prevent drying on the silica gel surface. The column was then conditioned for one night.

. Brine Shrimp Lethality Test (BSLT) for ethyl acetate extract
The toxicity test was carried out using the brine shrimp lethally test (BSLT) method by Meyer et al. [ 13 ]. 500 g of the sponge was cut into small pieces and then macerated with the solvent mixture methanol: dichloromethane of 3 : 2 (v / v) for 2 x 1 hour. The maceration results were filtered and separated with a separating funnel to obtain dichloromethane extract. An evaporator evaporated the dichloromethane extract at a temperature below 35°C until a concentrated dichloromethane extract was obtained. The concentrated dichloromethane extract was then partitioned with ethyl acetate: water of 1: 2 (v / v). This process is carried out in a separating funnel with three repetitions. The ethyl acetate layer was separated and evaporated by an evaporator at 40°C until ethyl acetate extract (concentrated extract) was obtained.

. Phytochemical Test
The alkaloid test in this study used Wagner ' s reagent. This reagent was prepared by mixing 2.5 g of iodine with 2 g of KI in 200  The egg hatching started with preparing a container ( 30 x 10 x 15 cm) filled with seawater. A glass separator with a hole was inserted into the container to form a bulkhead. The container made consisted of two parts, which were dark and light. The hatchery was then equipped with a lamp and aerator, which was located on the bright side. Eggs were put in a dark container. The eggs were hatched for ± 24 hours with the help of heat. Shrimp larvae that were 48 hours old were used in the toxicity test.

.2. Test and control material preparation
A 250 pg / mL stock sample solution was prepared by dissolving 2 mg of the test material in 50 pL DMSO in an Eppendorf tube. The solution was then rotated with a vortex mixer to get the maximum solubility. The solution was then added by seawater to a total volume of 8 mL. The control stock solution was prepared by infusing 50 pL of DMSO into Eppendorf without any test material. The solution was then added by seawater to a total volume of 8 mL.

6.25
A total of 100 pL of each concentration was added to the 96 microculture wells. The absorbance value of each level was read using a spectrophotometer Elisa reader at = 405 nm. The standard curve for haematin y = a + bx was made by plotting the x-axis as concentration and the y-axis as absorbance.

3 . Preparation of stock solution for each fraction
The stock solution for each fraction with a 5.0 mg / mL concentration was prepared by weighing 2 mg of the sample in an Eppendorf tube. 40 pL of DMSO 100% was added, shaken until the sample dissolved and homogeneous. 360 pL of distilled water was added and homogenized.

4 . Preparation of concentration series for each fraction
Each fraction was prepared with a concentration of

. 3 . Toxicity test
Ten larvae of Artemia salina aged 48 hours were inserted into 1.5 mL Eppendorf tubes, then added with stock sample solutions with volume variations of 1200; 600; 300 ; 150 and 75 pL. The tube was then put in seawater until the limit mark so that the final concentration of the sample was 200; 100; 50 ; 25 , and 12.5 pg / mL. 1200 pL of control stock solution was put into a 1.5 mL Eppendorf tube, then 10 Artemia salina larvae were added, and seawater was added to the limit mark. Each treatment was repeated three times. The observation of the number of dead larvae was carried out after 24 hours using a magnifying glass.

. 4 . Toxicity Analysis
Toxicity represents %mortality (percentage of deaths) which is then analyzed to obtain LC 50 . The percentage of death (%mortality) at each concentration variant was calculated using equation 1 [ 14 ]. The %mortality data from each concentration variant were analyzed using the probit analysis method at SPSS 23 .
The LC 5o was determined from the analysis results with a probit value of 0.500 ( 50 %). (1)

5 . Haematin polymerization inhibition test
This test is based on the Basilico et al. [ 15 ] method with related modifications to the concentration of the λ solution and the test material [16]. 100 pL of 1 mM haematin solution in 0.1 M NaOH was put into the Eppendorf tube, then added 50 pL of test material with various levels of levels, namely 1.25 ; 0.625 ; 0.312 ; 0.156 ; and 0.078 mg / mL. To start the haematin polymerization reaction, 50 pL of the glacial acetic acid solution was added to the Eppendorf tube, which already contained the haematin solution and samples, then incubated at 37°C for 24 hours. The positive control was chloroquine, while the negative control was distilled water and 10% DMSO solution. After the incubation ended, the Eppendorf tube was rotated using a centrifuge at a speed of 8000 rpm for 10 minutes. The supernatant obtained was discarded, and the precipitate was washed three times with 200 pL DMSO. Each washing was carried out using centrifugation at a speed of 8000 rpm for 10 minutes. Then, the precipitate obtained was added with 200 pL 0.1 M NaOH. Every 50 pL of each solution was put into a 96 well microplate, and the absorbance was read with an Elisa reader at = 4 0 5 nm. The results of the absorbance value were used to calculate the hemozoin level. The value of the inhibitory activity of haematin polymerization was expressed in IC 50 , which is a number that indicates the concentration of the test material capable of inhibiting 50 % of haematin polymerization. The IC 50 value of haematin polymerization inhibitory activity was calculated using the probit analysis method at SPSS 23 .

Statistical analysis
Determination of the LC 50 and IC 50 values was carried out by entering the percentage of inhibition data as observed responses, 100 as the number of subjects, and the concentration as the concentration on SPSS 23 using the probit analysis method.

. Results and Discussion
The results of this study are presented based on the treatment of the method used in this study.

. Extraction
500 g of a wet sponge was dried with a freeze dryer to obtain a dry sponge weighing 71 g. The result of sponge maceration with solvent of methanol: dichloromethane was a very sticky yellow-brown extract. The yellow extract was partitioned with a mixture of ethyl acetate and water. This partitioning process aims to separate polar compounds and salts remaining in viscous extracts using a water solvent, while less polar compounds can be partitioned into ethyl acetate solvent. The results of the partitioning process showed three layers inside the separating funnel. The top layer is a layer of ethyl acetate which is dark yellow-brown. The middle layer is a light-yellow emulsion, while the bottom layer is a yellow water layer. The overall extraction process resulted in the form of ethyl acetate extract weighing 1.14 g ( 0.23 %).

. Phytochemical Profile
Phytochemical tests were carried out to obtain information about the class of compounds in the Stylissa massa sponge. A brown precipitate was seen in the alkaloid test, indicating that the ethyl acetate extract of Stylissa massa sponge was positive for alkaloid class compounds. Alkaloid compounds such as spongicidin c have been reported from Stylissa massa sponge [8]. The results of the terpenoid test also showed that the ethyl acetate extract of Stylissa massa was positive for the terpenoid class compounds. This is confirmed by a change in the color of the test solution to purple color. Terpenoids formamidoamphilect-11 isothiocyanato-15 -formamidoamphilect-n (20) -ene have been reported from this sponge [ 17 ]. Saponin test results on ethyl acetate extract of Stylissa massa did not show any foam, so it could be said that the Stylissa massa ethyl acetate extract did not contain saponin class compounds. In the steroid test, the ethyl acetate extract of Stylissa massa was indicated to contain steroid class compounds in which there was a change in the color of the test solution to green.

. Toxicity Test Results
In this study, BSLT was used as a preliminary test to determine the bioactivity of the ethyl acetate extract of Stylissa massa sponge. The DMSO used in this study was only 0.63 %, still below the allowable limit, so that it did not poison Artemia salina larvae [ 14 ]. The results of the SPSS 23 calculation show that the LC 50 value of ethyl acetate extract is 934.395 Pg/mL. According to Meyer et al. [ 13 ], toxic (active) compounds have an LC 50 value <1000 pg / mL. Thus, ethyl acetate extract can be classified as toxic because it has an LC 50 value <1000 pg / mL. The toxicity LC 50 value of each fraction is presented in Table 4 . Table 4 shows that the toxic compounds are in the middle fraction. As in general isolation, compounds that have the potential to have high toxicity are less polar compounds. This is consistent with the principle of medicinal chemistry that a drug must not be too polar or too nonpolar to enter the target organ [18]. Calculations using SPSS 23 show that fractions 8, 11, 12, and 14 are a series of fractions classified as very toxic. These fractions were then tested for antimalarial.

. LC-MS Results of Fraction 11
LC-MS analysis of fraction 11 produces the chromatogram as shown in Figure 1. Figure 1 shows the rudimentary separation, especially after 5 minutes of separation. This may occur because the eluent system and the flow rate are not yet suitable, or in other words, the separation conditions are not optimal.

. Antimalarial test results
In the antimalarial test, the addition of acetic acid aims to initiate the polymerization process of haematin into hemozoin. The washing of hemozoin deposits is the most sensitive of the whole series of antimalarial tests. If the hemozoin deposits formed after 24 hours of incubation are lost during washing, the absorbance value on the Elisa reader is poor, and the hemozoin concentration data is inaccurate. This washing aims to remove the supernatant containing haematin which does not polymerize due to inhibition of the test material. The results of the hematin polymerization inhibition test for each test material are presented in Table 5 .  Palau 'amine is a pyrrole-imidazole hexacyclic alkaloid that contains a chain with eight chiral centers [20]. The compound, which was isolated from the Stylotella agminata sponge, has cytotoxic and immunosuppressive activities [21]. Palau 'amine was successfully synthesized for the first time in 2010. This synthesis was carried out again by another researcher in 2015 , where this synthesis route could explain the pharmacophores and the details of the mechanism of immunosuppressive activity [22,23 ]. The structure of the Palau'amine compound is presented in Figure 3 .  [ 24 ] discovered a compound from the Axinella verrucosa sponge from Mediterranean waters with a theoretical mass of 323.00 amu. The study revealed that a compound with a molecular mass of 323 amu was identified as hymenialdisine with the molecular formula CnHioBrNsCh . Based on the above literature, the compound with m / z of 323 in this study was thought to be a hymenialdisine compound with the molecular formula CnHioBrN502. The molecular formula is consistent with the nitrogen rule because it has an odd number of nitrogen atoms.  The hymenialdisine compound is a compound of the alkaloid group. This compound was first isolated in 1982 from the Axinella verrucosa sponge. This compound has also been isolated from the Stylissa massa sponge from the waters of Surigao, Philippines, and was reported to have MEK-i (a cancer target) inhibitor activity with an IC 50 value of 3 nM [10]. This compound has also been reported as an inhibitor of interleukin-2 production (IC 50 = 2.4 pM) and tumor necrosis factor (IC 50 of 1.4 pM) [ 25 ]. Several researchers have synthesized this β α compound, but a recent study succeeded in synthesizing hymenialdisine in 6 stages with a 44 % yield [26]. Feng et al. [ 27 ] stated that hymenialdisine has antifouling activity against P. viridis with EC 50 of 31.77 pg / mL, B. neritina with EC 50 of 3.43 pg / mL, and U. prolifera with EC 50 of 8.31 pg / mL. The structure of the hymenialdisine compound is presented in Figure 6. The ectyoplaside B compound was first isolated from the Ectyoplasia ferox sponge from the waters of the island of San Salvador, Bahamas. This white amorphous compound is a unique triterpene oligoglycoside with a sugar chain consisting of 2 -galactose units and 1arabinose unit. Ectyoplaside B was reported to have cytotoxic activity against cancer cells in the IC 50 range of 8.5 to 11.0 pg / mL [28]. The structure of the ectyoplaside B compound is presented in Figure 9 .

.2. Identification of the compound at a retention time of 4 094 minutes
The ESI-MS mass spectrum at a retention time of 4.094 minutes in Figure 10 shows positive ions at m / z      Figure 11. The hymenamide H compound is a cyclopeptide group compound previously isolated from the Hymeniacidon sp. sponge from the waters of Okinawa Island, Japan, with cytotoxic activity against L1210 cells with an IC 50 value of 6.3 pg / mL [ 30 ]. The structure of the hymenamide H compound can be seen in Figure 13 .  The ESI-MS mass spectrum at a retention time of 4.860 minutes in Figure 12 shows positive ions at the m / z peak of 942.39 [

. Conclusion
Based on this research, several things can be concluded. Isolation from Stylissa massa sponge resulted in 1.14 g ( 0.23 %) ethyl acetate extract with 15 fractions separated by column chromatography. The toxicity test results for fractions 8, 11, 12, and 14 showed that the LC 50 value was 13.080 , 1.014 ; 11.332 and 3.595 pg / mL, respectively; giving indications of toxicity. The results of the antimalarial activity test showed that the fractions 8, 11, 12, and 14 are the most active with IC 50 values of 82, 93 , 105 , and 96 pg / mL. Fractions 8,11,12, and 14 have potential as antimalarial agents with IC 50 values lower than chloroquine ( 115 pg / mL). The results of LC-MS analysis indicated that fraction 11 contained Palau'amine, hymenialdisine , and one unknown compound, while fraction 14 contained ectyoplaside B, hymenamide C, hymenamide H , and one unknown compound.