Antimalarial Potential of a Marine Sponge Tedania Ignis Against Plasmodium Falciparum

doi:10.21203/rs.3.rs-4320042/v1

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Antimalarial Potential of a Marine Sponge Tedania Ignis Against Plasmodium Falciparum

https://doi.org/10.21203/rs.3.rs-4320042/v1

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Across the decades, malaria disease has been a public health problem. The use of natural products to treat malaria saved many lives over the centuries, but the parasite resistance against all the medicines available on the market has made it difficult. In this context, exploring new natural treatments from biodiverse ecosystems, such as oceans, holds promise. Marine sponges have been used in different ways to treat diseases, their secondary metabolites and endoperoxides showed antiplasmodial activity in different assays. With that in mind, the species of marine sponge Tedania ignis, was evaluated against sensitive and resistant laboratory P. falciparum strains and presented potential antimalarial activity. Moreover, the speed of action of the extract of T. ignis was assessed demonstrating a fast-active profile. Regarding cellular cytotoxicity assays, the compound under investigation did not exhibit cytotoxic effects on two cell lines: HepG2, derived from human hepatoma, and HEK 293 (Human Embryonic Kidney). The active extract of T. ignis was investigated using UHPLC-HRMS and for data analyses was used Global Natural Products Social Molecular Networking (GNPS). Eight metabolites were dereplicated belonging to classes of alkamides, terpenes, nucleobases, alkaloid, and benzoic acid ester. Among the compounds assigned, dibutyl phthalate is highlighted for its known antimalarial potential, which may contribute to the bioactivity of T. ignis extract revealed in this work.

Bioproducts

Tedania ignis

Antiplasmodial activity

Plasmodium.

Malaria is a public health problem that mainly affects countries with a tropical climate and is economically underdeveloped. Caused by an obligate intracellular parasite of the phylum Apicomplexa, Plasmodium spp. completes its life cycle after passing through an intermediate host in which its asexual cycle takes place, and a definitive host (Anopheles sp.) where the sexual phase of its development occurs (Meibalan, et al. 2017). Among several species, six are of great epidemiological importance for humans: P. falciparum, P. vivax, P. ovale curtisi, P. ovale wallikeri, P. malariae, and P. knowlesi, (World malaria report 2023). The estimate is known that 3.2 billion people, which is equivalent to almost half of the world's population, are exposed to endemic areas of the disease. In 2022 there were 249 million cases of malaria in Brazil and 608 thousand deaths registered worldwide (World Malaria Report 2023).

The fight against malaria eradication remains a challenge, especially within an era characterized by successive generations of resistant strains in pharmacology (Amelo et tal. 2021). Artemisinin has been classified as a first-line of malaria treatment, however, like all the other drugs on the market, there are already cases of resistance to artemisinin combinations (Amelo et al. 2021). Artemisinin-resistant cases have been reported in Southeast Asia and certain regions of Africa. Given this situation, the urgency to discover new drugs to treat this disease makes the scientific community explore new candidate molecules so that soon it will be possible to reduce the number of deaths and cases of malaria (Su et al. 2019).

Marine sponges (phylum Porifera) are ancient sessile filter-feeding metazoans that are distributed worldwide in aquatic ecosystems (2018). They can produce high levels of cytotoxic compounds, the majority coming from the secondary metabolism protecting them against predation, overgrowth by fouling organisms, and/or competition for space (Perdicaris et al. 2013). Sponges contain several secondary metabolites that provide them with competitive advantages (Engel et al. 2000) and protect them from epibiont overgrowth (Thakur et al. 2003) and biofouling (Qian et al. 2015). A comparative analysis performed by Kong and coworkers (2010) showed that marine natural products are superior to terrestrial natural products in terms of chemical novelty and significant bioactivity.

Previous works investigating secondary metabolites from T. ignis, revealed the presence of indoles, carbazole, carboline, and benzene derivatives (Lhullier et al. 2019; Dillman et al. 1991), several diketopiperazines and atisane derivatives (Schmitz et al. 1983), macrocyclic polyketide tedanolide (Schmitz et al. 1983), tedanol (Costantino et al. 2009) and macrocyclic diaryl ether heptanoid tedarenes (Costantino et al. 2012). Among the identified classes of secondary metabolites, diarylheptanoids (tedarenes) are known for their antiprotozoal (Takahashi et al. 2004) and antimalarial potential (Saxena et al. 2016, together with, antitumor (Ishida et al. 2002) and anti-inflammatory activities (Akihisa et al. 2006). Also, tedanol, which is a brominated and sulfated ent-pimarane-type diterpene alcohol, turned out to be biologically attractive because it showed a potent anti-inflammatory activity (Costantino et al. 2009).

For the chemical investigation of natural products, a hyphenated technique of liquid chromatography combined with electrospray ionization high-resolution mass spectrometry (LC-HRMS) has been used for metabolite annotation. Tandem mass spectrometry (MS/MS) with quadrupole time-of-flight (QTOF) mass spectrometer together with global natural product social molecular networking (GNPS) is an effective tool for metabolites dereplication in complex samples such as marine organisms (Nothias et al. 2018; Fagundes et al. 2021). The chemical fingerprinting of extracts with biological potential is essential in the search for new bioactive metabolites (Allard et al. 2017; Mendes et al. 2023). Taking this information into account, this study aimed to evaluate the antiplasmodial activity of the T. ignis sponge ethanolic extract and to dereplicate it by LC-HRMS.

2.1 Materials

The solvents with HPLC grades were purchased from J.T. Baker (Philipsbur, USA). The LC-HRMS system consisted of an Ultra-High Performance Liquid Chromatography (UHPLC), model Infinity II 1290 (Agilent Technologies, Santa Clara, CA, USA), and a high-resolution mass spectrometer (HRMS) containing a quadrupole time-of-flight mass analyzer (QTOF, Impact HD) with an electrospray ionization (ESI) source (Bruker Daltonics, Bremen, Germany) (FAPESP 2014/50244-6).

2.2 Sponge material

The sample of sponges T. ignis (family Tedaniidae) was collected in high hydrodynamic coasts, in the intertidal zone, in São Sebastião, north coast of São Paulo state, Brazil, in the area of Praia Grande (23°49'23.76"S, 45°25'01.79" W) and Enseada do Araçá (23 No. 81'73.78 "S, 45 ° 40'66.39" W, São Sebastião, Brazil). Taxonomic identification was conducted using standard methods based on the morphological characteristics inherent to these sponges. Samples were immediately rinsed with saltwater, placed separately in containers with salt water, and transported to the laboratory in thermal boxes. In the laboratory, they were washed with Milli-Q water, weighed, stored at -20°C with proper identification, and then lyophilized.

It is important to notice that this study is registered in the National Management System of Genetic Patrimony (Sistema Nacional de Gestão do Patrimônio Genético, SisGen, registration number A4D38EE).

2.3 Extraction and sample preparation

The sample material of the sponge (3.96 g) was extracted with ethanol (EtOH) using Ika Ultra Turrax (T 25) for 5 min, 20.000 rpm at room temperature. The extraction process was performed in triplicate using 150 mL of EtOH as solvent. Thus, the extracted material was filtered and rotatory evaporation at 40 ^oC afforded crude extract dry materials (EETi, 56.7 mg).

The extract obtained was used to investigate antimalarial activity and for chemical fingerprinting.

2.4 LC-HRMS

The EETi sample (2.0 mg) was prepared using ethanol: methanol(20:80 v/v) in ultrasson for 10 min. Afterward, an aliquot of this extract mixture was diluted in ultrapure water at a final concentration of 500 µg/mL and resubmitted to ultrasson for 5 min. The mixture was centrifuged for 10 min at 12.000 rpm for analyses on HRMS (Gonçalves et al. 2020).

A XSelect® HSS T3 (2.5 µm particle size; 100 x 2.1 mm) (Waters, Milford, MA, USA) analytical column was used with a mobile phase composed of water (A) acetonitrile (B), with 0.1% v/v of formic acid which was added to both solvents. A linear gradient of 5 to 95% B in 20 min, keeping the 95% B composition for another 2 min, was used at a flow rate of 0.4 mL/min, 5 µL of injection volume, and a temperature of 40°C.

The ionization experiments were carried out in positive mode. The parameters used for mass spectrometry ionization source were as follows: nebulizer 4.0 bar, dry gas flow 9.0 L/min, dry heater temperature of 180 ºC, capillary voltage 4500 V, end plate offset 500 V, collision cell energy 10 eV, ion energy 5 eV, transfer time 50 and 90 µs, pre pulse time 6 µs and full-MS scan range 50-1300 m/z. The acquisition was obtained in auto MS/MS mode (cycle time 3 sec) in experiments with different collision energies of 20, 25, 30, 35, and 40 eV for all m/z analyzed (Gonçalves et al. 2020). Data acquisitions were carried out using the Data Analysis 4.0 software (Bruker Daltonics GmbH, Bremen, Germany), Compound Crawler Smartformula 3D, and GNPS. For compounds identification manual data curation was also necessary comparing results with literature.

2.5 Data Processing

The MS² data acquired by LC-HRMS were converted into mzML (data files) using Data Analysis 4.0 (Bruker Daltonics, Bremen, Germany). Afterward, data was compressed using the program WinSCP client FTP and uploaded to the workflow into GNPS (http://gnps.ucsd.edu). The molecular networks were generated according to the standard protocol (https://ccms-ucsd.github.io/GNPSDocumentation/) available on GNPS platform (Wang et al. 2016).

After running a spectral clustering algorithm (MS-Cluster software), data sets were downloaded from GNPS pages into Cytoscape (version 3.8.2) together with MS² features allowing for analysis of the network. In the molecular network, clusters comprised nodes joined by edges for compounds with similar product ions.

To generate molecular networking the mass tolerance was 0.02 Da for the precursor peaks, and MS² fragment ion tolerance of 0.02 Da. A network was created where edges were filtered to have a cosine score above 0.65 and more than four matched peaks. Further, edges between two nodes were kept in the network if and only if each of the nodes appeared in each other's respective top 10 most similar nodes. The bordering of the molecular family was defined as 100. Finally, the spectra in the network were searched against GNPS' spectral libraries. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least six matched peaks.

The spectral libraries used for compound annotation were Mass Bank, NIST (National Institute of Standards and Technology), MoNa (MassBank of North America), and ReSpect. The data files deposited in GNPS can be accessed at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=7398f47d17ed430d84b3e842a0c7d34d.

2.6 Maintenance of P. falciparum in vitro and SYBR Green assay

For the culture of the P. falciparum NF54 strain, the parasites were cultivated as described by Trager and Jensen (1976). They were maintained in RPMI culture medium supplemented with 0.5% albumax (GIBCO) at a hematocrit of 2% and parasitemia of 5%. For the experiments, the culture was synchronized with a 5% sorbitol solution at 37ºC for 10 minutes (Lambros et al. 1979). For the SYBR Green assay and evaluation of antiplasmodial activity, the hematocrit of the culture was adjusted to 2%, and parasitemia to 0.5% by adding O⁺ human erythrocytes. Twenty-microliter aliquots from serial dilutions, ranging between 100 µg/mL to 0.04 µg/mL, of the compounds under examination were prepared in a 96-well plate. Then, 180 µL of the culture with adjusted hematocrit and parasitemia were added per well. The plate was incubated at 37ºC in a humidified incubator with an atmosphere of 5% CO₂ and 5% O₂. In each plate, negative and positive inhibition control wells were added, containing parasitized RBCs without added compounds and non-parasitized RBCs, respectively. After incubation, the SYBR Green protocol [53] was applied to evaluate the inhibition of parasite growth. The intensity values obtained post-SYBR staining were normalized as percentage viability relative to both the positive and negative controls. The minimum inhibitory concentration of 50% (IC₅₀) was obtained by analyzing dose-response curves plotted in GraphPad Prism 8.0 (Dery et al. 2015; Vossen et al. 2010).

2.7 Speed of action assay

To categorize the compounds based on their fast or slow-acting profiles, two protocols were simultaneously conducted, adapted from ( Le Manach et al. 2013).

The action duration of the compounds was assessed by preparing three identical plates (A, B, and C) with equivalent compound dilutions. Each plate was incubated with P. falciparum NF54 strain, wherein > 90% of the stages were in the ring form (synchronized), adjusted to 2% hematocrit and 0.5% parasitemia. The three plates were incubated with the inhibitor under growth conditions for durations of 24 hours (plate A), 48 hours (plate B), or 72 hours (plate C). Following the respective incubation periods, plates A and B underwent three washes with RPMI medium to eliminate the inhibitors and were subsequently incubated for an additional 48 and 24 hours, respectively. Plate C remained continuously incubated in the presence of the inhibitor throughout the entire period. Post-incubation, viabilities, and IC₅₀ values for each plate were assessed using the SYBR Green I assay. The IC₅₀ values from the three incubation times were compared to ascertain any significant differences in inhibitory potency (IC₅₀) resulting from each duration (Fig. 1A).

2.8 Morphology assay

Associated with the speed of action assay, we conducted the morphology assay. In this analysis, parasites were incubated (2% hematocrit and 0.5% parasitemia, with > 90% in ring form) with 5 times the IC₅₀ value of the inhibitor, under growth conditions for 24 hours. Afterward, the parasites were washed to eliminate the inhibitor and further incubated until reaching 72 hours. Blood smears were subsequently taken at 0, 24, 48, and 72 hours after incubation and stained with Giemsa to track parasite development. Parasitemia growth was quantified via optical microscopy at 0, 24, 48, and 72 hours after incubation and compared with the untreated control. This assay aimed to mitigate potential SYBr labeling errors that might occur in the speed-of-action assay model, as deceased parasites could be falsely identified by SYBr green at the 24-hour mark (Fig. 1).

2.9 Maintenance of cell culture

Two distinct cell lines were utilized: HepG2, derived from human hepatoma, and HEK 293, derived from human embryos. These cell lines were acquired from the Cell Bank of Rio de Janeiro. They were cultured in RPMI medium supplemented with 10% fetal bovine serum, sodium bicarbonate, gentamicin, 2g/L glucose, and HEPES at pH 7.4. Specifically, the HEK-293 cells were maintained in RPMI culture medium with similar supplementation except for 1g/L glucose. The culture medium was refreshed every 2 days, and their morphology was observed using an inverted microscope until reaching the desired confluence for conducting the experiments.

2.10 Cytotoxicity with resazurin assay

Cytotoxicity protocol, as detailed by Céu de Madureira (2002), was conducted in duplicate using cells maintained in confluent cultures and subsequently washed with serum-free medium. Following the addition of 5 mL of 1x trypsin and incubated for 5 minutes at 37°C. The harvested cells were then suspended in a complete medium, and centrifuged, resulting pellet that was resuspended in 10% SBF. After cell counting, the cell suspension were distributed in 96-well microplates at a concentration of 3x10⁶ cells/100 µL/well and transferred in a CO₂ incubator at 37°C overnight to promote adherence. Next, 100 µL of complete medium with different concentrations of the compound (T. ignis), ranging from 200 µg/mL to 0.195 µg/mL, was added to each well. After 72 hours, 40 µL of Resazurin solution was introduced into each well followed by 4-hour incubation, with the reduction of Resazurin resulted in a color change from dark blue to pink. The microplates were then analyzed using a spectrophotometer with 570 nm and 630 nm filters. The cytotoxic concentration at 50% (CC₅₀), indicating the concentration where 50% of viable cells were observed in the presence of the test molecules and the control antimalarial, was determined by comparing it with cell cultures grown without the molecules, considered as exhibiting 100% growth.

2.11 Statistical analysis

The concentration and results of the IC₅₀ and CC₅₀ were evaluated based on the equation of the curve obtained by plotting the % of parasitemia regression vs the log of the concentration of extract. The average IC₅₀ and CC₅₀ were compared using the program GraphPadPrism 8.0.1.

3.1 Biological Results

The SYBR green experiments conducted to determine the extract's IC₅₀ exhibited promising results against various laboratory strains of P. falciparum (Fig. 2A and 2B). Notably, it recorded an average of 21.5 ± 6.5 µg/mL against the sensitive strain NF54 and an average of 30 µg/mL against the resistant strain DD2, in duplicate experiments (Fig. 2C). The extract's effectiveness against the resistant laboratory strain proves its antiplasmodial efficacy without cross-resistance.

The action assay of the compounds was analyzed by SYBR Green and optical microscopy analyses. The IC₅₀ values relative to 72 h for the antimalarials chloroquine (CQ), and pyrimethamine (PYR) are shown in Fig. 3. The results after 24 h and 48 h incubation with chloroquine compared to the standard 72-hour assay, the IC₅₀ ratio 24/72 h and 48/72 h for CQ was 1.5 ± 0.7. In the case of PYR, the IC₅₀s for 24/72 h and 48/72 h were 1.8 ± 0.3 and 1.16 ± 0.19. Regarding the sponge extract T. ignis, it was possible to classify the action stage as fast from the SYBR Green assay, obtaining an IC₅₀ ratio of 24/72 h and 48/72 h for T.ignis was 1.0 ± 0.2 and 0.93 ± 0.05.

In parallel to the IC₅₀ shift assessment of the antimalarials, a parasite culture was maintained with drug pressure for 24 h, and blood smears were collected and analyzed at 0 h, 24 h, 48 h, and 72 h. This approach aimed to compare the effect of the standard antimalarials and marine sponge extracts by microscopy and determine both the parasitemia and the morphological evolution of parasites. Blood smears were collected after 24 h of incubation with the antimalarials CQ and T.ignis that didn’t show morphological development of the parasite, and no parasites were observed in the blood smears after 24 h (Fig. 3A and 3C). By contrast, viable parasites were observed after incubation with PYR after 24 h (Fig. 3B). Moreover, a parasitemia level of 2.0 ± 0.87% and 2.2 ± 0.20% was determined in the blood smear of PYR collected at 72 h. The microscopy investigation confirmed that T. ignis and CQ eliminated the parasite within the first 24 h of compound incubation. Whereas for PYR the parasites remained viable in microscopy smears.

The cytotoxicity assays result with T. ignis revealed that both HepG2 and Hek 293 cells maintained viability even at the highest tested concentration of 200 µg/mL of the extract, indicating no toxicity and a CC₅₀ > 200 µg/mL. Figure 4. illustrates the cells' condition before and after treatment with T. ignis.

3.2 Dereplication of the active extract

LC-HRMS has increasingly been used for non-target analysis of natural product extracts (Nothias et al. 2018). The annotation of the chemical compounds of the ethanolic extract from T. ignis was performed comparing experimental MS/MS data with Natural products screening libraries (Mass Bank, NIST, MoNa, and ReSpect) in Global Natural Product Social molecular networking (GNPS) (http://gnps.ucsd.edu) (Allard et al. 2017; Mendes et al. 2023) and with the software Compound Crawler (https://massbank.eu/; http://mona.fiehnlab.ucdavis.edu/; https://metlin.scripps.edu) (Gonçalves et al. 2020).

The analysis of MS/MS data in positive ion mode (+)-ESI, allowed us to identify eight compounds. The resulting molecular network included a total of 790 nodes with a minimum cosine score of 0.65, with 47 clusters revealing different metabolites with several clustering (Fig. 5). Of those, 80 spectra matched thereby the spectra from the libraries, and entries with higher errors were discarded from analysis, allowing to select 8 nodes related to compounds from marine organisms. Annotated compounds matched MS/MS product ions to literature data and databases by diagnostic evidence, using at least two orthogonal pieces of information, including evidence that excludes all other candidates, and level 2 of confidence identifications (Sumner et al. 2007; Blaženović et al. 2018).

In addition, manual data curation was performed to confirm annotated compounds via the GNPS platform by using Data Analysis 4 and analyzing experimental MS/MS data ion fragments. From the eight dereplicated compounds, two are alkamides (1a, 1b), two terpenes (2a and 2b), two nucleobases (3a, 3b), an alkaloid (4a), and a benzoic acid ester (5a).

Two nodes were annotated as alkamides, octadecenamide (1a, m/z 282.2780 [M + H]⁺, C₁₈H₃₅NO) and octadecanamide (1b, m/z 284.2949 [M + H]⁺, C₁₈H₃₇NO) confirmed by exact mass measurement and similar fragmentation patterns of 1a (m/z 97.1009; 95.0852; 83,0853; 69.0696) and 1b (m/z 102.0912) compared to literature data [27, 28]. The supplementary Table S1 shows detailed MS data information of annotated compounds.

The ketocholesterol (2a, m/z 401.3431 [M + H]⁺, C₂₇H₄₄O₂) [29, 30] and austinoeol (2b, m/z 415.2116 [M + H]⁺, C₂₄H₃₀O₆) [31] MS/MS data yielded the ions fragments matching to literature, 2a (m/z 383.3523 [M + H - H₂O]⁺; m/z 365.3351 [M + H − 2H₂O]⁺; m/z 175.1140) and 2b (m/z 147.0650, 135.0799, 107.0858, with the main fragment at m/z 119.0852).

Compounds from nucleoside molecular family (3a and 3b) (Fagundes et al. 2021; van den Wildenberg et al. 2022; Lu et al. 2017) exhibited [M + H]⁺ ions at m/z 298.1139 (C₁₁H₁₆N₅O₅) and 127.0502 (C₅H₆N₂O₂), respectively. Methylguanosine (3a) fragments ions at m/z 166.0722 and 149.0461 are related to the loss of ammonia and cyanamide (Lu et al. 2017).

Trigonelline (4a) was identified by protonated molecular ion at m/z 138.0551 (C₇H₈NO₂⁺) (Data Analysis 4 data) (Wood et al. 2002; Xie et al. 2010) and fragments at m/z 120.0445; 110.0600; 94.0649 (43.9902 Da, loss of CO₂); 79.0214 and 65.0384.

Dibutyl phthalate (5a) was identified based on the protonated molecular ion at m/z 279.1592 (C₁₆H₂₂O₄) and the MS/MS spectra (Data Analysis 4 data) (Roy et al. 2006; Feng et al. 2022), observing the product ions at m/z 167.0335, 150,0266, 149.0234 and 121.0285 as expected. The benzoate ion at m/z 121.0285, can be produced when the ion phthalic acid ([C₈H₇O₄]⁺) with m/z 167.0335 loses H₂ through rearrangement. In addition, the ion at m/z 149.0234 can be formed through phthalic acid loss of H₂O (− 18.0101 Da) (Figure S1-S20, supplementary material, additional data are given in Online Resource 1).

Several medicines derived from natural products have demonstrated their effectiveness over the years for the treatment of different diseases, and for malaria, the concept is no different. With the great biodiversity of existing ecosystems on the planet, the marine ecosystem presents infinite possibilities of molecules that can be experimented with, among them, there are already reports of antiplasmodial action derived from marine sponges, highlighting the action of secondary metabolites and endoperoxides of these animals.

The antimalarial activity of compounds from marine sponges has been verified in several studies. These compounds have shown promising structures as scaffolds for the development of drugs that can act on different cell processes during the parasite cell cycle (Alves et al. 2021; Aguiar et al. 2021; Fattorusso et al. 2009; Lhullier et al. 2020). Studies have proven the use of T. ignis as a solution for several aspects, they were screened for their potential antiproliferative, antibacterial, antiprotozoal and anti-herpes activities (Negm et al. 2023).

In the literature it is possible to analyze the antiplasmodial activity of extracts obtained from different marine sponge species as cited by Alves et al. and Aguiar et al.(Aguiar et al. 2021; Alves et al. 2018) who referenced in his work the antiplasmodial activity of 26 extracts obtained from Aplysina fulva, Cladocroce caelum, Cinachyrella apion, Callyspongia sp., Desmapsamma anchorata, Dysidea janiae, Dragmacidon reticulatum e Ircinia strobilina,where the IC₅₀value remained between 0.28 to 22.34µg/mL in P. falciparum resistant strain (W2), accompanied by low cytotoxicity against the human cell line WI-26-VA4 which presented CC₅₀ values > 89 µg/mL. The species tested by (Alves et al. 2018) was also collected on the coast of Brazil, showing the great potential of biodiversity and antiplasmodial activity that can be obtained in this region.

The necessity for new drugs is evident due to parasite strain resistance. The use of T. ignis as a potential malaria treatment is a novel approach, supported by existing studies demonstrating antimalarial activity within the Tedania genus. For instance, pseudoceratidine derivatives isolated from the marine sponge Tedania brasiliensis have shown antiparasitic activity against Plasmodium falciparum (Parra et al. 2018). As far as we know, this is the first study that reports the activity of T. iginis against P. falciparum.

Regarding the time-of-action assay, promising results were observed with T. ignis. The reduction in numbers was prominent shortly after the extract's application during the determined incubation periods (24, 48, and 72 hours). Light microscopy also revealed a decrease in parasitemia. Similarly, chloroquine exhibited swift action, effectively eradicating parasites within 24 hours of incubation, as indicated by SYBR Green and optical microscopy, contrasting the continuous growth observed with pyrimethamine. These findings highlight the efficacy and the fast action profile of T. ignis, among the tested compounds, this is of great importance in the rapid disappearance of disease symptoms after treatment and in the lower propensity to disseminate/generate resistant parasites.

In this view, chemical investigation of the active extract of T. ignis was performed using LC-HRMS, allowing to dereplicate of eight metabolites. Among them, 1-methylguanosine (3a) was described in the Australian sponge Tedania digitata (Quiann et al. 1980; Davies et al. 1980), but the other compounds are revealed for the first time in the Tedania genus. The annotated dibutyl phthalate (DBP) (5a) has been reported (Sittie et al. 1998) to be isolated from the marine sponge of Smenospongia genus. Moreover, it has been reported that naturally occurring filamentous fungi produce DBP by the shikimic acid metabolic pathway (Shaaban et al. 2012). Thus, it is unclear to us if this herein annotated metabolite originates from secondary metabolites of T. ignis or if it is produced by filamentous fungi associated with them. We can also not discard that it arises by accumulation from the environment. Alkamides are a class of metabolites identified in marine sponges showing antiparasitic activity, in which antiplasmodial potential has been related to the presence of an α,β,γ,δ-unsaturated conjugated amide (Tian et al. 2016).

Previously, a study revealed that DBP (5a) was able to reduce P. falciparum infectivity with IC₅₀^3D7 value of 4.87 ± 1.26 µg/mL (17.50 µM) and low toxicity when evaluated on Chang liver cells (IC₅₀ of 902.90 ± 2.96 µg/mL, SI>10) [49]. In addition, DBP showed 60.80% ± 1.29 of inhibition of P. berghei NK65 in vivo infected mice (at 300 mg/kg body weight dosage) (Tian et al. 2016; Dahari et al. 2016), which may be related to the antimalarial potential revealed in T. ignis extract in this work.

The current study demonstrates the potential pharmacological profile of the marine sponge extract from T. ignis for treating the asexual forms of P. falciparum. No cytotoxic effects were observed against HepG2 and Hek 293 cells. Furthermore, the extract exhibited rapid action against the asexual forms of the parasite. The chemical analysis of the active extract from T. ignis revealed the presence of a class of metabolites with potential antiprotozoal properties. Our investigation identified the bioactive compound dibutyl phthalate in the ethanolic extract, supporting its previously observed antimalarial potential. Additionally, LC-HRMS analysis facilitated the identification of various compounds within T. ignis, contributing to a deeper understanding of its chemical composition.

Acknowledgement

We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support granted (Proc. no 2019/19708-0) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001). This research also received financial support from FAPEG (Fundação de Amparo à Pesquisa do Estado de Goiás) Proc. no 202110267000075, (chamada pública 004/2019) Brazil.

Funding Declaration

Conflict of interest: The authors declare no conflict of interest.

Data Availability: All data generated or analyzed during this study are included in this published article

Author Contribution

Caio Silva Moura, Yasmin Annunciato, Erica Paloma Maso Lopes Peres, Lorena Ramos Freitas de Souza, Anna Caroline Campos Aguiar, Quezia Bezerra Cass, Renata Neves Granito, Ana Cláudia Muniz Renno, Marcos Leoni Gazarini Dutra wrote the main manuscript text.Caio Silva Moura, Yasmin Annunciato, Erica Paloma Maso Lopes Peres, Anna Caroline Campos Aguiar, Lorena Alessandra Bafoni, Marcos Leoni Gazarini Dutra performed the biological experiments.Thais Bertolino Vieira Dantas, Wéldion Gonçalves Mesquita Júnior, Larissa Ramos Guimarães da Silva, Quezia Bezerra Cass, Lorena Ramos Freitas de Souza conducted the chemical studies.All authors reviewed the manuscript.

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Antimalarial Potential of a Marine Sponge Tedania Ignis Against Plasmodium Falciparum

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Abstract

Figures

1. Introduction

2. General Experimental Procedures

2.1 Materials

2.2 Sponge material

2.3 Extraction and sample preparation

2.4 LC-HRMS

2.5 Data Processing

2.6 Maintenance of P. falciparum in vitro and SYBR Green assay

2.7 Speed of action assay

2.8 Morphology assay

2.9 Maintenance of cell culture

2.10 Cytotoxicity with resazurin assay

2.11 Statistical analysis

3. Results

3.1 Biological Results

3.2 Dereplication of the active extract

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

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