In vitro antiplasmodial activities of the fractions of Hyrtios reticulatus sponge extract

The discovery of new compounds sourced from nature, which are active against malaria, is very important. Sponge Hyrtios reticulatus from Bali, Indonesia, is one of the examples that can be investigated. The sponge samples were extracted using ethanol, followed by trituration fractionation and vacuum liquid chromatography to get the samples. The nine samples obtained, their main extracts, chloroform fraction, residue, and SF 1–9 were tested for activities against Plasmodium falciparum variants 3D7 and FCR3. The results showed that all samples had a moderate antiplasmodial activity, where the most active sample was SF 3 with an IC 50 of 12.98 ± 1.88 µg/ml on 3D7 and 19.81 ± 0.75 µg/ml on FCR3. This study discovered that H. reticulatus sponges had antiplasmodial activities and could be further used as a guide to finding a new antiplasmodial compound.


INTRODUCTION
Globally, there are an estimated 229 million cases of malaria, and 409,000 of them died in 2019 in 87 malariaendemic countries, making it the leading cause of morbidity and mortality in tropical countries. This is further exacerbated by the increase in parasite resistance to various drugs accompanied by the slow development of vaccines and drug discovery (World Health Organization, 2021). Malaria is caused by five species of Plasmodium spp., which include Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi. Plasmodium falciparum is the variant that is the most common cause of morbidity and mortality in malaria (Hyde, 2007;Kenangalem et al., 2019;Kotepui et al., 2020), and the variants that are often found in endemic areas are P. falciparum 3D7 and FCR3 (Molina-Cruz et al., 2012). Therefore, it is necessary to search for new compounds that are active against malaria (Capela et al., 2019;Fattorusso and Taglialatela-Scafati, 2009).
Marine resources are great sources for the synthesis of new molecules, and they need to be studied. According to evolutionary history, marine microorganisms are more diverse than land microorganisms (Anjum et al., 2016;Setyowati et al., 2009). During 2014-2015, numerous articles described several marine extracts as in vitro natural product discoveries to predict the activity in extracts of bioactive components (Mayer et al., 2020). A marine sponge is one of the invertebrate creatures which is intriguing to be investigated from the standpoint of drug development due to its potency in producing novel chemicals (Hikmawan et al., 2018;Kirsch et al., 2000;Setyowati et al., 2008). It is a significant source of new marine natural products, as this group of species has acquired most of the biomedically or ecologically relevant chemicals (Tajuddeen and Van Heerden, 2019).
In our research for new antiplasmodials of marine invertebrates, sponge Hyrtios reticulata was investigated. This sponge was used because, based on the results of previous studies, several species of the genus Hyrtios have antiplasmodium activity (Ju et al., 2018;Shady et al., 2017). Previous studies on Hyrtios reticulatus sponge have identified it as an abundant source of uncommon secondary metabolites, such as alkaloid, sesterterpene, and macrolides, and it has an activity as an inhibitor of the ubiquitin-activating enzyme, and anticancer and antimicrobial activities (Inman et al., 2010;Mahfur et al., 2022;Shady et al., 2017;Yamanokuchi et al., 2012). In this research, the sponge samples H. reticulatus (family of Thorectidae) were collected from Bali, Indonesia. This study aims to determine the antiplasmodial activity of crude ethanol extract and its fraction from the sample. Antiplasmodial activity will be tested against P. falciparum 3D7 and FCR3 variants.

Material and extraction process
Sponge H. reticulatus was collected by scuba diving in Bali, Indonesia. The permit for sampling was given by the Head of the West Bali National Park No. S.931/BTNBB/Kons/7/2017. The identity of the material was confirmed at the Marine Natural Product Laboratory at the University of Diponegoro, Indonesia. The sample obtained was washed to remove impurities and then reduced in size to make it easier to be extracted. The result (100 g) was extracted first with ethanol. The crude extract (6.2 g) obtained was then trituration-fractionated with chloroform to produce the chloroform fraction and residue.

Culture of P. falciparum
Parasites were cultured by using the Trager and Jensen method (Jensen and Trager, 1977). Plasmodium falciparum variants 3D7 and FCR3 were maintained at 2% hematocrit (human type O-positive red blood cells) in a complete culture medium at 37°C. The complete medium contained RPMI 1640 medium (Gibco-BRL; 24 mM NaHCO3) with the addition of 10% heat-inactivated O-positive human plasma, 20 mM N-2hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), and 2 mM glutamine (Biological Industries). All of the cultures were maintained in a standard gas mixture consisting of 1% O 2 , 5% CO 2 , and 94% N 2 . When parasitemia exceeded 2%, subcultures were taken; the culture medium was changed every second day. Erythrocytes infected with P. falciparum (ring stage, 1% parasitemia) were resuspended in a complete culture medium at a hematocrit of 1.5%. The suspension was distributed in 96-well microtiter plates (200 µl per well). Sample testing was carried out in triplicate. For each assay, parasite cultures were incubated with the samples for 48 hours in 5% CO 2 at 95% relative humidity and frozen until biochemical tests could be run.

Antiplasmodial activity assay
The antiplasmodial activity was studied in vitro against Plasmodium variants 3D7 and FCR3 by the micro method using the method described by Trager and Jensen. Compounds were dissolved in dimethyl sulfoxide (DMSO), and then they were diluted with a medium to achieve the required concentrations (in all cases, the final concentration contained 1% DMSO, which was found to be nontoxic to the parasite). Samples were placed in 96-well flat-bottomed microplates (Costar 3596) consisting of negative control (DMSO), samples, and positive control (chloroquine). Sample dilutions were made starting at 125 µg/ml for all of the compounds tested, except for chloroquine, the initial concentration of which was 500 ng/ml. Asynchronous cultures with parasitemia of 1%-1.5% and 1% final hematocrit were aliquoted into the plates and incubated for 72 hours at 37°C. Parasitemia was determined by observing the Giemsa-stained thin blood smears using a microscope. The culture of Plasmodium without sample was used as a negative control, which was considered a 100% growth of Plasmodium. Percentage of inhibition of parasitemia was calculated using the following formula: Inhibition% = parasetemia of negative control -parasitemia sample × 100 parasetemia of negative control

Statistical analysis
The potency of antiplasmodial activity was expressed as IC 50 which was calculated with the logarithm between % inhibition and the test concentration by statistical analysis regression probit using SPSS 16. The differences in IC 50 of the samples were analyzed using one-way analysis of variance (ANOVA) with a 5% level of significance, i.e., p ≤ 0.05.

RESULTS AND DISCUSSION
Hyrtios reticulatus is a marine sponge of the genus Hyrtios (family of Thorectidae) with specifications of dark brown branches, oscules with a diameter of up to 2 mm, 3-4 mm conules that are 1-2 mm high, and brown to orange tops of conules (De Forges, 2007;De Voogd, 2007). They are known to be a rich source of unusual secondary metabolites, such as alkaloids, sesterterpenes, and macrolides (Imada et al., 2013). Many of these metabolites have important biological activities (Shady et al., 2017).
In this research, we evaluated the in vitro antiplasmodial activity of H. reticulatus sponge. The evaluation began from the sponge H. reticulatus ethanol extract tested with P. falciparum 3D7 and FCR3 variants. The antiplasmodial activity of the sponge H. reticulatus extract showed that it had IC 50 42.01 ± 3.74 and 41.90 ± 2.32 µg/ml against 3D7 and FCR3, respectively. The activity of the extract sample included moderate activity criteria. The criteria for in vitro antiplasmodial activity of the extracts were divided into the following: IC 50 <1 µg/ml, potent; IC 50 <10 µg/ml, good; IC 50 10-50 µg/ml, moderate; IC 50 50-100 µg/ml, low; and IC 50 >100 µg/ml, inactive (Fattorusso and Taglialatela-Scafati, 2012). The fractionation step continued leading us to the fraction that was active against Plasmodium. The chloroform fraction showed that it had IC 50 34.15 ± 7.79 µg/ml on 3D7 and 21.23 ± 7.23 µg/ml on FCR3, while the residue had lower activity with IC 50 39.42 ± 7.57 and 39.05 ± 1.80 µg/ml against 3D7 and FCR3 Plasmodium variants, respectively.
The chloroform fraction had become a guide to reach the active compound, and from the fraction six subfractions were obtained. TLC was used to detect the characteristic type of the organic compounds in the fraction, whether polar or nonpolar, and groups of the compounds by separating them on a silica gel GF254 plate as the stationary phase using a specific solvent or eluent as the mobile phase. In this study, the eluent used for the six fractions was a mixture of N-hexane and ethyl acetate. The profiles of TLC of subfractions (Fig. 1) were different from each other. This visual detection method was chosen because it only requires simple equipment. Under 254 nm UV light, the substances in the fractions showed aromatic rings, conjugated double bonds, and unsaturated substances. Fluorescence under 366 nm showed that the fractions contained long-chain conjugated double bonds. Analysis continued to chemical detection by a spraying chromatogram with cerium sulfate, resulting in finding the fractions containing terpenes that were indicated by black color spots on the white background. Terpenes, also referred to as terpenoids, such as diterpenes and sesquiterpene, are involved in activities against microbial pathogens.
Based on the results of the one-way ANOVA statistical analysis of the IC 50 data on the 3D7 variant, it is divided into three groups: the first group is SF 3 and SF 2; the second group is SF 1 and SF 4; and the third group is SF 5 and SF 6, where the activities of group members do not have significant differences, i.e., p > 0.05, but have significant differences with other group samples, i.e., p < 0.05. The results of the one-way ANOVA statistical analysis of the IC 50 data on the FCR3 variant are divided into three groups: the first group is SF 3, SF 2, and SF 5; the second group is SF 1 and SF 4; and the third group is SF 6. The sample with the strongest activity is very likely to contain active compounds that are potent against P. falciparum 3D7 and FCR3 variants even though the subfraction activity is included in the moderate category (Kamaraj et al., 2014).
The activity of the samples is shown in Table 2 and specific SF 3 activity in Figure 2. The activity data in Table 2 show the response between concentration samples, %parasitemic growth, and %inhibition growth of parasitemia. The activity of the samples was concentration-dependent, which was seen at a high concentration of very little parasitemic growth and showed strong inhibitory activity. However, the smaller the concentration, the higher the parasitemia growth and the lower the inhibitory activity. The 3D7 and FCR3 parasitemia variants in the SF 3 sample did not grow at a concentration of 125 g/ml, but parasitemia growth increased when the concentration was reduced. The percentage of inhibitory growth of parasitemic 3D7 and FCR3 variants at 125 µg/ml is 100% and decreased with decreasing concentration.
Antiplasmodial activity in extracts, fractions, and subfractions is strongly influenced by the content of secondary metabolites that they have. The results of this antiplasmodial test can be used as a reference in an effort to find antiplasmodial active compounds. Previously, there were no reports on this sample related to antiplasmodial activity because the previous studies only reported that two types of sponges from the genus Hyrtios sponge had antiplasmodial activity. They were Hyrtios erectus and Hyrtios sp.
Hyrtios erectus has homofascaplysin A and fascaplysin against P. falciparum strain NF54 with IC 50 values of 24 and 34 ng/ ml (Shady et al., 2017) and smenotronic acid, ilimaquinone, and pelorol with 3.51 ± 0.63, 2.11 ± 0.23, and 0.80 ± 0.19 µM against the P. falciparum Dd2 strain, respectively (Ju et al., 2018). Hyrtios sp. has 15α-methoxypuupehenol against P. falciparum strain FcB1 with IC 50 1.4 µg/ml (Bourguet-Kondracki et al., 1999). Up until recently, there have been seven classes of antiplasmodials that have been shown to own antiplasmodial activity, such as endoperoxides, alkaloids, terpenes, polyphenols, quinones and polyketides, nonpeptide macrocyclic, and β-resorcylic lactone (Nogueira and Lopes, 2011). The mechanism of antiplasmodial activity from the sample above is unknown, but based on the previous research, the action mechanism of the antiplasmodial itself has several target mechanisms, such as the action mechanism in the cytosol, on  parasitic membranes, on food vacuoles, on mitochondria, and on apicoplasts (Rosenthal, 2003).

CONCLUSION
The fractions of H. reticulatus sponge extract have an antiplasmodial activity, especially SF 3 with IC 50 12.98 ± 1.88 µg/ml against P. falciparum 3D7 variant and 19.81 ± 0.75 µg/ml against P. falciparum FCR3, and have the potency to be researched about the new compound with potent antiplasmodial activity.