Antimalarial Potential of a Marine Sponge Tedania Ignis Against Plasmodium Falciparum

Antimalarial Potential of a Marine Sponge Tedania Ignis Against Plasmodium Falciparum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antimalarial Potential of a Marine Sponge Tedania Ignis Against Plasmodium Falciparum Caio S. Moura, Yasmin Annunciato, Thais B. V. Dantas, Wéldion G. M. Júnior, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4320042/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction 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. General Experimental Procedures 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 o C 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 MS 2 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 MS 2 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 MS 2 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% CO 2 and 5% O 2 . 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% (IC 50 ) 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 IC 50 values for each plate were assessed using the SYBR Green I assay. The IC 50 values from the three incubation times were compared to ascertain any significant differences in inhibitory potency (IC 50 ) resulting from each duration ( Fig. 1 A ) . 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 IC 50 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 3x10 6 cells/100 µL/well and transferred in a CO 2 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% (CC 50 ), 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 IC 50 and CC 50 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 IC 50 and CC 50 were compared using the program GraphPadPrism 8.0.1. 3. Results 3.1 Biological Results The SYBR green experiments conducted to determine the extract's IC 50 exhibited promising results against various laboratory strains of P. falciparum ( Fig. 2 A and 2 B ) . 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. 2 C). 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 IC 50 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 IC 50 ratio 24/72 h and 48/72 h for CQ was 1.5 ± 0.7. In the case of PYR, the IC 50 s 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 IC 50 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 IC 50 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. 3 A and 3 C ) . By contrast, viable parasites were observed after incubation with PYR after 24 h ( Fig. 3 B ) . 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 CC 50 > 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] + , C 18 H 35 NO) and octadecanamide ( 1b , m/z 284.2949 [M + H] + , C 18 H 37 NO) 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] + , C 27 H 44 O 2 ) [29, 30] and austinoeol ( 2b , m/z 415.2116 [M + H] + , C 24 H 30 O 6 ) [31] MS/MS data yielded the ions fragments matching to literature, 2a ( m/z 383.3523 [M + H - H 2 O] + ; m/z 365.3351 [M + H − 2H 2 O] + ; 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 (C 11 H 16 N 5 O 5 ) and 127.0502 (C 5 H 6 N 2 O 2 ), 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 (C 7 H 8 NO 2 + ) (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 CO 2 ); 79.0214 and 65.0384. Dibutyl phthalate ( 5a ) was identified based on the protonated molecular ion at m/z 279.1592 (C 16 H 22 O 4 ) 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 ([C 8 H 7 O 4 ] + ) with m/z 167.0335 loses H 2 through rearrangement. In addition, the ion at m/z 149.0234 can be formed through phthalic acid loss of H 2 O (− 18.0101 Da) ( Figure S1 -S20, supplementary material, additional data are given in Online Resource 1 ). 4. Discussion 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 IC 50 value 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 CC 50 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 IC 50 3D7 value of 4.87 ± 1.26 µg/mL (17.50 µM) and low toxicity when evaluated on Chang liver cells (IC 50 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. Declarations 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 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. 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. References Aguiar ACC, Parisi JR, Granito RN, Ramos Freitas De Sousa L, Cláudia A, Renno M, Gazarini ML, Lorenzo-Morales J (2021) marine drugs Metabolites from Marine Sponges and Their Potential to Treat Malarial Protozoan Parasites Infection: A Systematic Review. doi: 10.3390/md19030134 Akihisa T, Taguchi Y, Yasukawa K, et al (2006) Acerogenin M, a Cyclic Diarylheptanoid, and Other Phenolic Compounds from Acer nikoense and Their Anti-inflammatory and Anti-tumor-Promoting Effects. Chem Pharm Bull (Tokyo) 54:735–739. https://doi.org/10.1248/CPB.54.735 Allard PM, Genta-Jouve G, Wolfender JL (2017) Deep metabolome annotation in natural products research: towards a virtuous cycle in metabolite identification. Curr Opin Chem Biol 36:40–49. https://doi.org/10.1016/J.CBPA.2016.12.022 Alves C, Silva J, Pinteus S, et al (2018) From marine origin to therapeutics: The antitumor potential of marine algae-derived compounds. Front Pharmacol 9:353805. https://doi.org/10.3389/FPHAR.2018.00777/BIBTEX Alves J, Gaspar H, Silva J, Alves C, Martins A, Teodoro F, Susano P, Pinteus S, Pedrosa R (2021) Unravelling the anti-inflammatory and antioxidant potential of the marine sponge cliona celata from the portuguese coastline. Mar Drugs 19. doi: 10.3390/MD1911063 Amelo W, Makonnen E (2021) Efforts Made to Eliminate Drug-Resistant Malaria and Its Challenges. Biomed Res Int 2021:. https://doi.org/10.1155/2021/5539544 Arriel-Elias MT, Pereira AK, Arriel GCTF, et al (2023) Molecular networking as a tool to annotate the metabolites of Bacillus sp. and Serratia marcescens isolates and evaluate their fungicidal effects against Magnapothe oryzae and Bipolaris oryzae. 3 Biotech 13:1–11. https://doi.org/10.1007/S13205-023-03547-6/FIGURES/5 Blaženović I, Kind T, Ji J, Fiehn O (2018) Software Tools and Approaches for Compound Identification of LC-MS/MS Data in Metabolomics. Metabolites 2018, Vol 8, Page 31 8:31. https://doi.org/10.3390/metabo8020031 Costantino V, Fattorusso E, Mangoni A, et al (2009) Tedanol: A potent anti-inflammatory ent-pimarane diterpene from the Caribbean Sponge Tedania ignis. Bioorg Med Chem 17:7542–7547. https://doi.org/10.1016/J.BMC.2009.09.010 Costantino V, Fattorusso E, Mangoni A, et al (2012) Tedarenes A and B: Structural and Stereochemical Analysis of Two New Strained Cyclic Diarylheptanoids from the Marine Sponge Tedania ignis. J Org Chem 77:6383. https://doi.org/10.1021/jo300295j Dahari DE, Mohamad Salleh R, Mahmud F, et al (2016) Anti-malarial Activities of Two Soil Actinomycete Isolates from Sabah via Inhibition of Glycogen Synthase Kinase 3β. Trop Life Sci Res 27:53. https://doi.org/10.21315/TLSR2016.27.2.5 Davies LP, Cook AF, Poonian M, Taylor KM (1980) Displacement of [3H] diazepam binding in rat brain by dipyridamole and by 1-methylisoguanosine, a marine natural product with muscle relaxant activity. Life Sci 26:1089–1095. https://doi.org/10.1016/0024-3205(80)90255-6 Dery V, Duah NO, Ayanful-Torgby R, et al (2015) An improved SYBR Green-1-based fluorescence method for the routine monitoring of Plasmodium falciparum resistance to anti-malarial drugs. Malar J 14:. https://doi.org/10.1186/S12936-015-1011-X Dillman RL, Cardellina JH (1991) Aromatic secondary metabolites from the sponge tedania ignis. J Nat Prod 54:1056–1061. https://doi.org/10.1021/NP50076A021 Do Céu De Madureira M, Paula Martins A, Gomes M, et al (2002) Antimalarial activity of medicinal plants used in traditional medicine in S. Tomé and Prı́ncipe islands. J Ethnopharmacol 81:23–29. https://doi.org/10.1016/S0378-8741(02)00005-3 Engel S, Pawlik JR (2000) Allelopathic activities of sponge extracts. Mar Ecol Prog Ser 207:273–281. https://doi.org/10.3354/MEPS207273 Fagundes T da SF, da Silva LRG, Brito M de F, et al (2021) Metabolomic fingerprinting of Brazilian marine sponges: a case study of Plakinidae species from Fernando de Noronha Archipelago. Anal Bioanal Chem 413:4301–4310. https://doi.org/10.1007/S00216-021-03385-6/FIGURES/5 Fattorusso E, Taglialatela-Scafati O (2009) Marine Antimalarials. Mar Drugs 7:130. doi: 10.3390/MD7020130 Feng YL, Singh R, Chao A, Li Y (2022) Diagnostic Fragmentation Pathways for Identification of Phthalate Metabolites in Nontargeted Analysis Studies. J Am Soc Mass Spectrom 33:981–995. https://doi.org/10.1021/JASMS.2C00052/ASSET/IMAGES/LARGE/JS2C00052_0007.JPEG Ferraz CG, Ribeiro PR, Verde B V., et al (2023) Metabolite Profiling of Pleurotus ostreatus Grown on Sisal Agro-Industrial Waste Supplemented with Cocoa Almond Tegument and Wheat Bran. Chem Biodivers 20:e202300346. https://doi.org/10.1002/CBDV.202300346 Gonçalves Neto GS, Da Costa RP, Zanini CL, et al (2020) Chemical Prospection of Qualea grandiflora Mart. Fruit and Stem Extracts and Their in vitro and in vivo Antiplasmodial Activity. Article J Braz Chem Soc 31:. https://doi.org/10.21577/0103-5053.20200034 Ishida J, Kozuka M, Tokuda H, et al (2002) Chemopreventive potential of cyclic diarylheptanoids. Bioorg Med Chem 10:3361–3365. https://doi.org/10.1016/S0968-0896(02)00164-5 Kong DX, Jiang YY, Zhang HY (2010) Marine natural products as sources of novel scaffolds: achievement and concern. Drug Discov Today 15:884–886. https://doi.org/10.1016/J.DRUDIS.2010.09.002 Kouchaksaraee RM, Farimani MM, Li F, et al (2020) Integrating Molecular Networking and 1H NMR Spectroscopy for Isolation of Bioactive Metabolites from the Persian Gulf Sponge Axinella sinoxea. Marine Drugs 2020, Vol 18, Page 366 18:366. https://doi.org/10.3390/MD18070366 Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. Journal of Parasitology 65:418–420. https://doi.org/10.2307/3280287 Le Manach C, Scheurer C, Sax S, et al (2013) Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum. Malar J 12:. https://doi.org/10.1186/1475-2875-12-424 Lhullier C, Moritz MIG, Tabalipa EO, et al (2019) Biological activities of marine invertebrates extracts from the northeast brazilian coast. Brazilian Journal of Biology 80:393–404. https://doi.org/10.1590/1519-6984.213678 Lhullier C, Moritz MIG, Tabalipa EO, Sardá FN, Schneider NFZ, Moraes MH, Constantino L, Reginatto FH, Steindel M, Pinheiro US, Simões CMO, Pérez CD, Schenkel EP, Agronômico E, Ferreira AC (2020) Brazilian Journal of Biology Biological activities of marine invertebrates extracts from the northeast brazilian coast. Braz J Biol 80:393. doi: 10.1590/1519-6984.213678 Li Z, Dong F, Sun Y, et al (2022) Qualitative and Quantitative Analysis of Six Fatty Acid Amides in 11 Edible Vegetable Oils Using Liquid Chromatography–Mass Spectrometry. Front Nutr 9:857858. https://doi.org/10.3389/FNUT.2022.857858/BIBTEX Lu Z, Wang Q, Wang M, et al (2017) Using UHPLC Q-Trap/MS as a complementary technique to in-depth mine UPLC Q-TOF/MS data for identifying modified nucleosides in urine. Journal of Chromatography B 1051:108–117. https://doi.org/10.1016/J.JCHROMB.2017.03.002 Meibalan E, Marti M (2017) Biology of malaria transmission. Cold Spring Harb Perspect Med 7:. https://doi.org/10.1101/cshperspect.a025452 Mendes L da C, Dantas TBV, Severino RP, et al (2023) Using molecular networking and docking to explore arginase inhibitors among Drimys brasiliensis chemical constituents. Medicinal Chemistry Research 32:2208–2218. https://doi.org/10.1007/S00044-023-03123-W/TABLES/2 Negm WA, Ezzat SM, Zayed A (2023) Marine organisms as potential sources of natural products for the prevention and treatment of malaria. RSC Adv 13:4436–4475. doi: 10.1039/D2RA07977A Nothias LF, Nothias-Esposito M, Da Silva R, et al (2018) Bioactivity-Based Molecular Networking for the Discovery of Drug Leads in Natural Product Bioassay-Guided Fractionation. J Nat Prod 81:758–767. https://doi.org/10.1021/ACS.JNATPROD.7B00737/SUPPL_FILE/NP7B00737_SI_001.PDF Parra LLL, Bertonha AF, Severo IRM, et al (2018) Isolation, Derivative Synthesis, and Structure-Activity Relationships of Anti-Parasitic Bromopyrrole Alkaloids from the Marine Sponge Tedania brasiliensis. J Nat Prod 81:188. https://doi.org/10.1021/ACS.JNATPROD.7B00876 Perdicaris S, Vlachogianni T, Valavanidis A (2013) Issue 3 • 1000114 Nat Prod Chem Res ISSN: 2329-6836 NPCR, an open access journal Perdicaris. Nat Prod Chem Res 1:3. https://doi.org/10.4172/2329-6836.1000114 Pita L, Rix L, Slaby BM, et al (2018) The sponge holobiont in a changing ocean: from microbes to ecosystems. Microbiome 2018 6:1 6:1–18. https://doi.org/10.1186/S40168-018-0428-1 Qian PY, Li Z, Xu Y, et al (2015) Mini-review: Marine natural products and their synthetic analogs as antifouling compounds: 2009–2014. http://dx.doi.org/101080/089270142014997226 31:101–122. https://doi.org/10.1080/08927014.2014.997226 Quiann RJ, Gregson RP, Cook AF, Bartlett RT (1980) Isolation and synthesis of 1-methylisoguanosine, a potent pharmacologically active constituent from the marine sponge tenadia digitata. Tetrahedron Lett 21:567–568. https://doi.org/10.1016/S0040-4039(01)85558-1 Razzazi-Fazeli E, Kleineisen S, Luf W (2000) Determination of cholesterol oxides in processed food using high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionisation. J Chromatogr A 896:321–334 Roy RN, Laskar S, Sen SK (2006) Dibutyl phthalate, the bioactive compound produced by Streptomyces albidoflavus 321.2. Microbiol Res 161:121–126. https://doi.org/10.1016/J.MICRES.2005.06.007 Saxena A, Yadav D, Mohanty S, et al (2016) Diarylheptanoids Rich Fraction of Alnus nepalensis Attenuates Malaria Pathogenesis: In-vitro and In-vivo Study. Phytotherapy Research 30:940–948. https://doi.org/10.1002/PTR.5596 Schmitz FJ, Vanderah DJ, Hollenbeak KH, et al (1983) Metabolites from the Marine Sponge Tedania ignis. A New Atisanediol and Several Known Diketopiperazines. Journal of Organic Chemistry 48:3941–3945. https://doi.org/10.1021/JO00170A011/SUPPL_FILE/JO00170A011_SI_001.PDF Shaaban M, Abd-Alla HI, Hassan AZ, et al (2012) Chemical characterization, antioxidant and inhibitory effects of some marine sponges against carbohydrate metabolizing enzymes. Org Med Chem Lett 2:30. https://doi.org/10.1186/2191-2858-2-30 Sittie AA, Lemmich E, Olsen CE, et al (1998) Alkamides from Phyllanthus fraternus. Planta Med 64:192–193. https://doi.org/10.1055/S-2006-957405/BIB Su XZ, Lane KD, Xia L, et al (2019) Plasmodium Genomics and Genetics: New Insights into Malaria Pathogenesis, Drug Resistance, Epidemiology, and Evolution. Clin Microbiol Rev 32:. https://doi.org/10.1128/CMR.00019-19 Sumner LW, Amberg A, Barrett D, et al (2007) Proposed minimum reporting standards for chemical analysis: Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics 3:211–221. https://doi.org/10.1007/S11306-007-0082-2/METRICS Takahashi M, Fuchino H, Satake M, Agatsuma Y, Sekita S. (2004) In Vitro Screening of Leishmanicidal Activity in Myanmar Timber Extracts, Biological and Pharmaceutical Bulletin, 2004, Volume 27, Issue 6, Pages 921-925, Released on J-STAGE June 01, 2004, Online ISSN 1347-5215, Print ISSN 0918-6158, https://doi.org/10.1248/bpb.27.921. Thakur NL, Hentschel U, Krasko A, et al (2003) Antibacterial activity of the sponge Suberites domuncula and its primmorphs: potential basis for epibacterial chemical defense. Aquatic Microbial Ecology 31:77–83. https://doi.org/10.3354/AME031077 Tian C, Ni J, Chang F, et al (2016) Bio-Source of di-n-butyl phthalate production by filamentous fungi. Sci Rep 6:19791. https://doi.org/10.1038/srep19791 Trager W, Jensen JB (1976) Human Malaria Parasites in Continuous Culture. Science (1979) 193:673–675. https://doi.org/10.1126/SCIENCE.781840 van den Wildenberg SAH, Streng AS, van den Broek R, et al (2022) Quantification of uracil, dihydrouracil, thymine and dihydrothymine for reliable dihydropyrimidine dehydrogenase (DPD) phenotyping critically depend on blood and plasma storage conditions. J Pharm Biomed Anal 221:115027. https://doi.org/10.1016/J.JPBA.2022.115027 Vossen MG, Pferschy S, Chiba P, Noedl H (2010) The SYBR Green I malaria drug sensitivity assay: performance in low parasitemia samples. Am J Trop Med Hyg 82:398–401. https://doi.org/10.4269/AJTMH.2010.09-0417 Wang M, Carver JJ, Phelan V V., et al (2016) Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nature Biotechnology 2016 34:8 34:828–837. https://doi.org/10.1038/nbt.3597 Wood K V., Bonham CC, Miles D, et al (2002) Characterization of betaines using electrospray MS/MS. Phytochemistry 59:759–765. https://doi.org/10.1016/S0031-9422(02)00049-3 World malaria report 2023. https://www.who.int/publications/i/item/9789240086173. Accessed 16 Jan 2024 Xie JW, Huang LF, Hu W, et al (2010) Analysis of the Main Nucleosides in Cordyceps Sinensis by LC/ESI-MS. Molecules 2010, Vol 15, Pages 305-314 15:305–314. https://doi.org/10.3390/MOLECULES15010305 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx GraphicalAbstract.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4320042","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":309544862,"identity":"0ab059cd-e5da-48b5-9235-2390141235b8","order_by":0,"name":"Caio S. 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The IC\u003csub\u003e50 \u003c/sub\u003evalues were determined based on the antiplasmodial activity of marine sponge against (A) a sensitive laboratory strain of \u003cem\u003eP. falciparum\u003c/em\u003e (NF54) and (B) resistant strain (DD2), respectively. (C) IC\u003csub\u003e50\u003c/sub\u003e values obtained from duplicate assays conducted on both sensitive and resistant \u003cem\u003eP. falciparum\u003c/em\u003e strains.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4320042/v1/7e98169665ddaaa3ecd44d3f.png"},{"id":58030710,"identity":"8f25aa43-71b8-45b7-be43-7abf418df4ed","added_by":"auto","created_at":"2024-06-10 07:45:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":105720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of speed of action of antimalarials and marine sponge in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. falciparum \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edevelopment. (A) \u003c/strong\u003eIC\u003csub\u003e50\u003c/sub\u003e ratio values after 24, 48 and 72 hours.\u0026nbsp; \u003cstrong\u003e(B)\u003c/strong\u003e The parasitemia at 72 h was represented by boxplots and \u003cstrong\u003e(C)\u003c/strong\u003e images for \u003cem\u003eTedania ignis\u003c/em\u003e, Pyrimethamine, and Chloroquine.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4320042/v1/6c7a8f5b2f29a07eebf3d235.png"},{"id":58029267,"identity":"11630929-118d-44a0-875c-70f95cd569cf","added_by":"auto","created_at":"2024-06-10 07:29:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":183000,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity assay images of human cell lineages after incubation with marine sponge extract \u003cstrong\u003e(A)\u003c/strong\u003e HepG2 and \u003cstrong\u003e(B)\u003c/strong\u003e HEK293 cells before treatment and wells after treatment with 72 hours incubated in the presence of \u003cem\u003eT.ignis \u003c/em\u003eextract, showing cell viability.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4320042/v1/89894d452296a5a740652eb9.png"},{"id":58030144,"identity":"ebce1fa5-b33e-43d1-a209-243f73be2d18","added_by":"auto","created_at":"2024-06-10 07:37:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":175592,"visible":true,"origin":"","legend":"\u003cp\u003eNetworking (GNPS) clusters of \u003cem\u003eTedania ignis\u003c/em\u003e obtained with experimental data of MS/MS spectra in positive ion mode (+)-ESI. The Clusters highlighted are related to annotated classes of compounds known for antiprotozoal and/or antiplasmodial activities.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4320042/v1/6fa0f322c13b7ef4fc08196e.png"},{"id":62780530,"identity":"9bd50f03-8a37-48e2-aebf-be9eac8bde9e","added_by":"auto","created_at":"2024-08-19 11:44:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1266184,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4320042/v1/b9ca03f5-2e3e-42a9-9c63-bbbb546a2dc8.pdf"},{"id":58029269,"identity":"1649b2a6-d854-427f-9531-b15e7a93b38d","added_by":"auto","created_at":"2024-06-10 07:29:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2788531,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4320042/v1/8cd8cb1ab53a9a97f3240e65.docx"},{"id":58030142,"identity":"a6249ac4-2fdf-459d-a57a-36282d6a53ac","added_by":"auto","created_at":"2024-06-10 07:37:32","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":226267,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-4320042/v1/cf72dd1eb2e5d8243485f2a0.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAntimalarial Potential of a Marine Sponge Tedania Ignis Against Plasmodium Falciparum\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMalaria 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, \u003cem\u003ePlasmodium spp.\u003c/em\u003e completes its life cycle after passing through an intermediate host in which its asexual cycle takes place, and a definitive host (\u003cem\u003eAnopheles\u003c/em\u003e sp.) where the sexual phase of its development occurs (Meibalan, et al. 2017). Among several species, six are of great epidemiological importance for humans: \u003cem\u003eP. falciparum, P. vivax, P. ovale curtisi, P. ovale wallikeri, P. malariae, and P. knowlesi\u003c/em\u003e, (World malaria report \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The estimate is known that 3.2\u0026nbsp;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\u0026nbsp;million cases of malaria in Brazil and 608 thousand deaths registered worldwide (World Malaria Report \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 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. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMarine sponges (phylum \u003cem\u003ePorifera\u003c/em\u003e) 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. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Sponges contain several secondary metabolites that provide them with competitive advantages (Engel et al. 2000) and protect them from epibiont overgrowth (Thakur et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and biofouling (Qian et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). A comparative analysis performed by Kong and coworkers (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) showed that marine natural products are superior to terrestrial natural products in terms of chemical novelty and significant bioactivity.\u003c/p\u003e \u003cp\u003ePrevious works investigating secondary metabolites from \u003cem\u003eT. ignis\u003c/em\u003e, revealed the presence of indoles, carbazole, carboline, and benzene derivatives (Lhullier et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dillman et al. 1991), several diketopiperazines and atisane derivatives (Schmitz et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), macrocyclic polyketide tedanolide (Schmitz et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), tedanol (Costantino et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and macrocyclic diaryl ether heptanoid tedarenes (Costantino et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Among the identified classes of secondary metabolites, diarylheptanoids (tedarenes) are known for their antiprotozoal (Takahashi et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and antimalarial potential (Saxena et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, together with, antitumor (Ishida et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) and anti-inflammatory activities (Akihisa et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). 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. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor 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. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fagundes et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The chemical fingerprinting of extracts with biological potential is essential in the search for new bioactive metabolites (Allard et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mendes et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Taking this information into account, this study aimed to evaluate the antiplasmodial activity of the \u003cem\u003eT. ignis\u003c/em\u003e sponge ethanolic extract and to dereplicate it by LC-HRMS.\u003c/p\u003e"},{"header":"2. General Experimental Procedures","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eThe 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sponge material\u003c/h2\u003e \u003cp\u003eThe sample of sponges \u003cem\u003eT. ignis\u003c/em\u003e (family Tedaniidae) was collected in high hydrodynamic coasts, in the intertidal zone, in S\u0026atilde;o Sebasti\u0026atilde;o, north coast of S\u0026atilde;o Paulo state, Brazil, in the area of Praia Grande (23\u0026deg;49'23.76\"S, 45\u0026deg;25'01.79\" W) and ​​Enseada do Ara\u0026ccedil;\u0026aacute; (23 No. 81'73.78 \"S, 45 \u0026deg; 40'66.39\" W, S\u0026atilde;o Sebasti\u0026atilde;o, 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\u0026deg;C with proper identification, and then lyophilized.\u003c/p\u003e \u003cp\u003eIt is important to notice that this study is registered in the National Management System of Genetic Patrimony (Sistema Nacional de Gest\u0026atilde;o do Patrim\u0026ocirc;nio Gen\u0026eacute;tico, SisGen, registration number A4D38EE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Extraction and sample preparation\u003c/h2\u003e \u003cp\u003eThe 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 \u003csup\u003eo\u003c/sup\u003eC afforded crude extract dry materials (EETi, 56.7 mg).\u003c/p\u003e \u003cp\u003eThe extract obtained was used to investigate antimalarial activity and for chemical fingerprinting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 LC-HRMS\u003c/h2\u003e \u003cp\u003eThe 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 \u0026micro;g/mL and resubmitted to ultrasson for 5 min. The mixture was centrifuged for 10 min at 12.000 rpm for analyses on HRMS (Gon\u0026ccedil;alves et al. 2020).\u003c/p\u003e \u003cp\u003eA XSelect\u0026reg; HSS T3 (2.5 \u0026micro;m 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 \u0026micro;L of injection volume, and a temperature of 40\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe 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 \u0026ordm;C, capillary voltage 4500 V, end plate offset 500 V, collision cell energy 10 eV, ion energy 5 eV, transfer time 50 and 90 \u0026micro;s, pre pulse time 6 \u0026micro;s and full-MS scan range 50-1300 \u003cem\u003em/z\u003c/em\u003e. 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 \u003cem\u003em/z\u003c/em\u003e analyzed (Gon\u0026ccedil;alves 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Data Processing\u003c/h2\u003e \u003cp\u003eThe MS\u003csup\u003e2\u003c/sup\u003e 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 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gnps.ucsd.edu\u003c/span\u003e\u003cspan address=\"http://gnps.ucsd.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e The molecular networks were generated according to the standard protocol (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ccms-ucsd.github.io/GNPSDocumentation/\u003c/span\u003e\u003cspan address=\"https://ccms-ucsd.github.io/GNPSDocumentation/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e available on GNPS platform (Wang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter running a spectral clustering algorithm (MS-Cluster software), data sets were downloaded from GNPS pages into Cytoscape (version 3.8.2) together with MS\u003csup\u003e2\u003c/sup\u003e features allowing for analysis of the network. In the molecular network, clusters comprised nodes joined by edges for compounds with similar product ions.\u003c/p\u003e \u003cp\u003eTo generate molecular networking the mass tolerance was 0.02 Da for the precursor peaks, and MS\u003csup\u003e2\u003c/sup\u003e 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.\u003c/p\u003e \u003cp\u003eThe 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 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=7398f47d17ed430d84b3e842a0c7d34d\u003c/span\u003e\u003cspan address=\"https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=7398f47d17ed430d84b3e842a0c7d34d\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Maintenance of \u003cem\u003eP. falciparum in vitro\u003c/em\u003e and SYBR Green assay\u003c/h2\u003e \u003cp\u003eFor the culture of the \u003cem\u003eP. falciparum\u003c/em\u003e NF54 strain, the parasites were cultivated as described by Trager and Jensen (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). 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 37\u0026ordm;C 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\u003csup\u003e+\u003c/sup\u003e human erythrocytes. Twenty-microliter aliquots from serial dilutions, ranging between 100 \u0026micro;g/mL to 0.04 \u0026micro;g/mL, of the compounds under examination were prepared in a 96-well plate. Then, 180 \u0026micro;L of the culture with adjusted hematocrit and parasitemia were added per well. The plate was incubated at 37\u0026ordm;C in a humidified incubator with an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e and 5% O\u003csub\u003e2\u003c/sub\u003e. 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% (IC\u003csub\u003e50\u003c/sub\u003e) was obtained by analyzing dose-response curves plotted in GraphPad Prism 8.0 (Dery et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Vossen et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Speed of action assay\u003c/h2\u003e \u003cp\u003eTo categorize the compounds based on their fast or slow-acting profiles, two protocols were simultaneously conducted, adapted from ( Le Manach et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 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 \u003cem\u003eP. falciparum\u003c/em\u003e NF54 strain, wherein\u0026thinsp;\u0026gt;\u0026thinsp;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 IC\u003csub\u003e50\u003c/sub\u003e values for each plate were assessed using the SYBR Green I assay. The IC\u003csub\u003e50\u003c/sub\u003e values from the three incubation times were compared to ascertain any significant differences in inhibitory potency (IC\u003csub\u003e50\u003c/sub\u003e) resulting from each duration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Morphology assay\u003c/h2\u003e \u003cp\u003eAssociated with the speed of action assay, we conducted the morphology assay. In this analysis, parasites were incubated (2% hematocrit and 0.5% parasitemia, with \u0026gt;\u0026thinsp;90% in ring form) with 5 times the IC\u003csub\u003e50\u003c/sub\u003e 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 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Maintenance of cell culture\u003c/h2\u003e \u003cp\u003eTwo 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Cytotoxicity with resazurin assay\u003c/h2\u003e \u003cp\u003eCytotoxicity protocol, as detailed by C\u0026eacute;u 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\u0026deg;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 3x10\u003csup\u003e6\u003c/sup\u003e cells/100 \u0026micro;L/well and transferred in a CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C overnight to promote adherence. Next, 100 \u0026micro;L of complete medium with different concentrations of the compound (\u003cem\u003eT. ignis\u003c/em\u003e), ranging from 200 \u0026micro;g/mL to 0.195 \u0026micro;g/mL, was added to each well. After 72 hours, 40 \u0026micro;L 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% (CC\u003csub\u003e50\u003c/sub\u003e), 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe concentration and results of the IC\u003csub\u003e50\u003c/sub\u003e and CC\u003csub\u003e50\u003c/sub\u003e 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 IC\u003csub\u003e50\u003c/sub\u003e and CC\u003csub\u003e50\u003c/sub\u003e were compared using the program GraphPadPrism 8.0.1.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Biological Results\u003c/h2\u003e \u003cp\u003eThe SYBR green experiments conducted to determine the extract's IC\u003csub\u003e50\u003c/sub\u003e exhibited promising results against various laboratory strains of \u003cem\u003eP. falciparum\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Notably, it recorded an average of 21.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5 \u0026micro;g/mL against the sensitive strain NF54 and an average of 30 \u0026micro;g/mL against the resistant strain DD2, in duplicate experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The extract's effectiveness against the resistant laboratory strain proves its antiplasmodial efficacy without cross-resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe action assay of the compounds was analyzed by SYBR Green and optical microscopy analyses. The IC\u003csub\u003e50\u003c/sub\u003e values relative to 72 h for the antimalarials chloroquine (CQ), and pyrimethamine (PYR) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The results after 24 h and 48 h incubation with chloroquine compared to the standard 72-hour assay, the IC\u003csub\u003e50\u003c/sub\u003e ratio 24/72 h and 48/72 h for CQ was 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7. In the case of PYR, the IC\u003csub\u003e50\u003c/sub\u003es for 24/72 h and 48/72 h were 1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 and 1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19. Regarding the sponge extract \u003cem\u003eT. ignis\u003c/em\u003e, it was possible to classify the action stage as fast from the SYBR Green assay, obtaining an IC\u003csub\u003e50\u003c/sub\u003e ratio of 24/72 h and 48/72 h for \u003cem\u003eT.ignis\u003c/em\u003e was 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 and 0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eIn parallel to the IC\u003csub\u003e50\u003c/sub\u003e 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 \u003cem\u003eT.ignis\u003c/em\u003e that didn\u0026rsquo;t show morphological development of the parasite, and no parasites were observed in the blood smears after 24 h \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. By contrast, viable parasites were observed after incubation with PYR after 24 h \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Moreover, a parasitemia level of 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87% and 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20% was determined in the blood smear of PYR collected at 72 h. The microscopy investigation confirmed that \u003cem\u003eT. ignis\u003c/em\u003e and CQ eliminated the parasite within the first 24 h of compound incubation. Whereas for PYR the parasites remained viable in microscopy smears.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe cytotoxicity assays result with \u003cem\u003eT. ignis\u003c/em\u003e revealed that both HepG2 and Hek 293 cells maintained viability even at the highest tested concentration of 200 \u0026micro;g/mL of the extract, indicating no toxicity and a CC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;200 \u0026micro;g/mL. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. illustrates the cells' condition before and after treatment with \u003cem\u003eT. ignis.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Dereplication of the active extract\u003c/h2\u003e \u003cp\u003eLC-HRMS has increasingly been used for non-target analysis of natural product extracts (Nothias et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The annotation of the chemical compounds of the ethanolic extract from \u003cem\u003eT. ignis\u003c/em\u003e 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) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gnps.ucsd.edu\u003c/span\u003e\u003cspan address=\"http://gnps.ucsd.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e (Allard et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mendes et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and with the software Compound Crawler (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://massbank.eu/\u003c/span\u003e\u003cspan address=\"https://massbank.eu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mona.fiehnlab.ucdavis.edu/\u003c/span\u003e\u003cspan address=\"http://mona.fiehnlab.ucdavis.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metlin.scripps.edu\u003c/span\u003e\u003cspan address=\"https://metlin.scripps.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e (Gon\u0026ccedil;alves et al. 2020).\u003c/p\u003e \u003cp\u003eThe 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 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. 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. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Blaženović et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn 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 (\u003cb\u003e1a\u003c/b\u003e, \u003cb\u003e1b\u003c/b\u003e), two terpenes (\u003cb\u003e2a\u003c/b\u003e and \u003cb\u003e2b\u003c/b\u003e), two nucleobases (\u003cb\u003e3a\u003c/b\u003e, \u003cb\u003e3b\u003c/b\u003e), an alkaloid (\u003cb\u003e4a\u003c/b\u003e), and a benzoic acid ester (\u003cb\u003e5a\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTwo nodes were annotated as alkamides, octadecenamide (\u003cb\u003e1a\u003c/b\u003e, \u003cem\u003em/z\u003c/em\u003e 282.2780 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e35\u003c/sub\u003eNO) and octadecanamide (\u003cb\u003e1b\u003c/b\u003e, \u003cem\u003em/z\u003c/em\u003e 284.2949 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e37\u003c/sub\u003eNO) confirmed by exact mass measurement and similar fragmentation patterns of \u003cb\u003e1a\u003c/b\u003e (m/z 97.1009; 95.0852; 83,0853; 69.0696) and \u003cb\u003e1b\u003c/b\u003e (m/z 102.0912) compared to literature data [27, 28]. The supplementary \u003cb\u003eTable S1\u003c/b\u003e shows detailed MS data information of annotated compounds.\u003c/p\u003e \u003cp\u003eThe ketocholesterol (\u003cb\u003e2a\u003c/b\u003e, \u003cem\u003em/z\u003c/em\u003e 401.3431 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, C\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e44\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) [29, 30] and austinoeol (\u003cb\u003e2b\u003c/b\u003e, \u003cem\u003em/z\u003c/em\u003e 415.2116 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, C\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e) [31] MS/MS data yielded the ions fragments matching to literature, \u003cb\u003e2a\u003c/b\u003e (\u003cem\u003em/z\u003c/em\u003e 383.3523 [M\u0026thinsp;+\u0026thinsp;H - H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e+\u003c/sup\u003e; m/z 365.3351 [M\u0026thinsp;+\u0026thinsp;H \u0026minus;\u0026thinsp;2H\u003csub\u003e2\u003c/sub\u003eO]\u003csup\u003e+\u003c/sup\u003e; m/z 175.1140) and \u003cb\u003e2b\u003c/b\u003e (\u003cem\u003em/z\u003c/em\u003e 147.0650, 135.0799, 107.0858, with the main fragment at \u003cem\u003em/z\u003c/em\u003e 119.0852).\u003c/p\u003e \u003cp\u003eCompounds from nucleoside molecular family (\u003cb\u003e3a\u003c/b\u003e and \u003cb\u003e3b\u003c/b\u003e) (Fagundes et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; van den Wildenberg et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) exhibited [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e ions at m/z 298.1139 (C\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e) and 127.0502 (C\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), respectively. Methylguanosine (\u003cb\u003e3a\u003c/b\u003e) fragments ions at \u003cem\u003em/z\u003c/em\u003e 166.0722 and 149.0461 are related to the loss of ammonia and cyanamide (Lu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTrigonelline (\u003cb\u003e4a\u003c/b\u003e) was identified by protonated molecular ion at \u003cem\u003em/z\u003c/em\u003e 138.0551 (C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) (Data Analysis 4 data) (Wood et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and fragments at \u003cem\u003em/z\u003c/em\u003e 120.0445; 110.0600; 94.0649 (43.9902 Da, loss of CO\u003csub\u003e2\u003c/sub\u003e); 79.0214 and 65.0384.\u003c/p\u003e \u003cp\u003eDibutyl phthalate (\u003cb\u003e5a\u003c/b\u003e) was identified based on the protonated molecular ion at \u003cem\u003em/z\u003c/em\u003e 279.1592 (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) and the MS/MS spectra (Data Analysis 4 data) (Roy et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), observing the product ions at \u003cem\u003em/z\u003c/em\u003e 167.0335, 150,0266, 149.0234 and 121.0285 as expected. The benzoate ion at \u003cem\u003em/z\u003c/em\u003e 121.0285, can be produced when the ion phthalic acid ([C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e) with \u003cem\u003em/z\u003c/em\u003e 167.0335 loses H\u003csub\u003e2\u003c/sub\u003e through rearrangement. In addition, the ion at \u003cem\u003em/z\u003c/em\u003e 149.0234 can be formed through phthalic acid loss of H\u003csub\u003e2\u003c/sub\u003eO (\u0026minus;\u0026thinsp;18.0101 Da) (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S20, supplementary material, additional data are given in Online Resource 1\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSeveral 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.\u003c/p\u003e\n\u003cp\u003eThe 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;\u0026nbsp;Aguiar et al. 2021;\u0026nbsp;Fattorusso et al. 2009;\u0026nbsp;Lhullier et al. 2020). Studies have proven the use of \u003cem\u003eT. ignis\u003c/em\u003e as a solution for several aspects, they were screened for their potential antiproliferative, antibacterial, antiprotozoal and anti-herpes activities (Negm et al. 2023).\u003c/p\u003e\n\u003cp\u003eIn 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 \u003cem\u003eAplysina fulva\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Cladocroce caelum\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Cinachyrella apion\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Callyspongia sp.\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Desmapsamma anchorata\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Dysidea janiae\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Dragmacidon reticulatum\u0026nbsp;\u003c/em\u003ee \u003cem\u003eIrcinia strobilina\u003c/em\u003e,where the IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003evalue remained between 0.28 to 22.34µg/mL in \u003cem\u003eP. falciparum\u003c/em\u003e resistant strain (W2), accompanied by low cytotoxicity against the human cell line WI-26-VA4 which presented CC\u003csub\u003e50\u003c/sub\u003e values \u0026gt; 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.\u003c/p\u003e\n\u003cp\u003eThe necessity for new drugs is evident due to parasite strain resistance. The use of \u003cem\u003eT. ignis\u003c/em\u003e as a potential malaria treatment is a novel approach, supported by existing studies demonstrating antimalarial activity within the \u003cem\u003eTedania\u003c/em\u003e \u003cem\u003egenus\u003c/em\u003e. For instance, pseudoceratidine derivatives isolated from the marine sponge \u003cem\u003eTedania brasiliensis\u003c/em\u003e have shown antiparasitic activity against \u003cem\u003ePlasmodium falciparum\u0026nbsp;\u003c/em\u003e(Parra et al. 2018). As far as we know, this is the first study that reports the activity of \u003cem\u003eT. iginis\u003c/em\u003e against \u003cem\u003eP. falciparum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eRegarding the time-of-action assay, promising results were observed with \u003cem\u003eT. ignis\u003c/em\u003e. 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 \u003cem\u003eT. ignis\u003c/em\u003e, 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.\u003c/p\u003e\n\u003cp\u003eIn this view, chemical investigation of the active extract of \u003cem\u003eT. ignis\u0026nbsp;\u003c/em\u003ewas performed using LC-HRMS, allowing to dereplicate of eight metabolites. Among them, 1-methylguanosine (\u003cstrong\u003e3a\u003c/strong\u003e) was described in the Australian sponge \u003cem\u003eTedania digitata\u003c/em\u003e (Quiann et al. 1980; Davies et al. 1980), but the other compounds are revealed for the first time\u0026nbsp;in the \u003cem\u003eTedania\u003c/em\u003e genus. The annotated dibutyl phthalate (DBP) (\u003cstrong\u003e5a\u003c/strong\u003e) has been reported (Sittie et al. 1998) to be isolated from the marine sponge of \u003cem\u003eSmenospongia\u003c/em\u003e genus. Moreover,\u0026nbsp;it has been reported that naturally\u0026nbsp;occurring\u0026nbsp;filamentous fungi\u0026nbsp;produce\u0026nbsp;DBP by\u0026nbsp;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 \u003cem\u003eT. ignis\u003c/em\u003e 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).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePreviously, a study revealed that DBP (\u003cstrong\u003e5a\u003c/strong\u003e) was able to reduce \u003cem\u003eP. falciparum\u0026nbsp;\u003c/em\u003einfectivity with IC\u003csub\u003e50\u003c/sub\u003e\u003csup\u003e3D7\u003c/sup\u003e value of 4.87 ± 1.26 µg/mL (17.50 µM) and low toxicity when evaluated on Chang liver cells (IC\u003csub\u003e50\u003c/sub\u003e of 902.90 ± 2.96 µg/mL, SI\u0026gt;10) [49]. In addition, DBP showed 60.80% ± 1.29 of inhibition of \u003cem\u003eP. berghei\u003c/em\u003e NK65 \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003einfected 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 \u003cem\u003eT. ignis\u0026nbsp;\u003c/em\u003eextract in this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe current study demonstrates the potential pharmacological profile of the marine sponge extract from \u003cem\u003eT. ignis\u003c/em\u003e for treating the asexual forms of \u003cem\u003eP. falciparum\u003c/em\u003e. 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 \u003cem\u003eT. ignis\u003c/em\u003e 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 \u003cem\u003eT. ignis\u003c/em\u003e, contributing to a deeper understanding of its chemical composition.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP) for the financial support granted (Proc. no 2019/19708-0) and the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES, Finance Code 001). This research also received financial support from FAPEG (Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Goi\u0026aacute;s) Proc. no 202110267000075, (chamada p\u0026uacute;blica 004/2019) Brazil.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP) for the financial support granted (Proc. no 2019/19708-0) and the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES, Finance Code 001). This research also received financial support from FAPEG (Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Goi\u0026aacute;s) Proc. no 202110267000075, (chamada p\u0026uacute;blica 004/2019) Brazil.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are included in this published article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCaio 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 Cl\u0026aacute;udia 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, W\u0026eacute;ldion Gon\u0026ccedil;alves Mesquita J\u0026uacute;nior, Larissa Ramos Guimar\u0026atilde;es da Silva, Quezia Bezerra Cass, Lorena Ramos Freitas de Souza conducted the chemical studies.All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAguiar ACC, Parisi JR, Granito RN, Ramos Freitas De Sousa L, Cl\u0026aacute;udia A, Renno M, Gazarini ML, Lorenzo-Morales J (2021) marine drugs Metabolites from Marine Sponges and Their Potential to Treat Malarial Protozoan Parasites Infection: A Systematic Review. doi: 10.3390/md19030134\u003c/li\u003e\n \u003cli\u003eAkihisa T, Taguchi Y, Yasukawa K, et al (2006) Acerogenin M, a Cyclic Diarylheptanoid, and Other Phenolic Compounds from Acer nikoense and Their Anti-inflammatory and Anti-tumor-Promoting Effects. Chem Pharm Bull (Tokyo) 54:735\u0026ndash;739. https://doi.org/10.1248/CPB.54.735\u003c/li\u003e\n \u003cli\u003eAllard PM, Genta-Jouve G, Wolfender JL (2017) Deep metabolome annotation in natural products research: towards a virtuous cycle in metabolite identification. Curr Opin Chem Biol 36:40\u0026ndash;49. https://doi.org/10.1016/J.CBPA.2016.12.022\u003c/li\u003e\n \u003cli\u003eAlves C, Silva J, Pinteus S, et al (2018) From marine origin to therapeutics: The antitumor potential of marine algae-derived compounds. Front Pharmacol 9:353805. https://doi.org/10.3389/FPHAR.2018.00777/BIBTEX\u003c/li\u003e\n \u003cli\u003eAlves J, Gaspar H, Silva J, Alves C, Martins A, Teodoro F, Susano P, Pinteus S, Pedrosa R (2021) Unravelling the anti-inflammatory and antioxidant potential of the marine sponge cliona celata from the portuguese coastline. Mar Drugs 19. doi: 10.3390/MD1911063\u003c/li\u003e\n \u003cli\u003eAmelo W, Makonnen E (2021) Efforts Made to Eliminate Drug-Resistant Malaria and Its Challenges. Biomed Res Int 2021:. https://doi.org/10.1155/2021/5539544\u003c/li\u003e\n \u003cli\u003eArriel-Elias MT, Pereira AK, Arriel GCTF, et al (2023) Molecular networking as a tool to annotate the metabolites of Bacillus sp. and Serratia marcescens isolates and evaluate their fungicidal effects against Magnapothe oryzae and Bipolaris oryzae. 3 Biotech 13:1\u0026ndash;11. https://doi.org/10.1007/S13205-023-03547-6/FIGURES/5\u003c/li\u003e\n \u003cli\u003eBlaženović I, Kind T, Ji J, Fiehn O (2018) Software Tools and Approaches for Compound Identification of LC-MS/MS Data in Metabolomics. Metabolites 2018, Vol 8, Page 31 8:31. https://doi.org/10.3390/metabo8020031\u003c/li\u003e\n \u003cli\u003eCostantino V, Fattorusso E, Mangoni A, et al (2009) Tedanol: A potent anti-inflammatory ent-pimarane diterpene from the Caribbean Sponge Tedania ignis. Bioorg Med Chem 17:7542\u0026ndash;7547. https://doi.org/10.1016/J.BMC.2009.09.010\u003c/li\u003e\n \u003cli\u003eCostantino V, Fattorusso E, Mangoni A, et al (2012) Tedarenes A and B: Structural and Stereochemical Analysis of Two New Strained Cyclic Diarylheptanoids from the Marine Sponge Tedania ignis. J Org Chem 77:6383. https://doi.org/10.1021/jo300295j\u003c/li\u003e\n \u003cli\u003eDahari DE, Mohamad Salleh R, Mahmud F, et al (2016) Anti-malarial Activities of Two Soil Actinomycete Isolates from Sabah via Inhibition of Glycogen Synthase Kinase 3\u0026beta;. Trop Life Sci Res 27:53. https://doi.org/10.21315/TLSR2016.27.2.5\u003c/li\u003e\n \u003cli\u003eDavies LP, Cook AF, Poonian M, Taylor KM (1980) Displacement of [3H] diazepam binding in rat brain by dipyridamole and by 1-methylisoguanosine, a marine natural product with muscle relaxant activity. Life Sci 26:1089\u0026ndash;1095. https://doi.org/10.1016/0024-3205(80)90255-6\u003c/li\u003e\n \u003cli\u003eDery V, Duah NO, Ayanful-Torgby R, et al (2015) An improved SYBR Green-1-based fluorescence method for the routine monitoring of Plasmodium falciparum resistance to anti-malarial drugs. Malar J 14:. https://doi.org/10.1186/S12936-015-1011-X\u003c/li\u003e\n \u003cli\u003eDillman RL, Cardellina JH (1991) Aromatic secondary metabolites from the sponge tedania ignis. J Nat Prod 54:1056\u0026ndash;1061. https://doi.org/10.1021/NP50076A021\u003c/li\u003e\n \u003cli\u003eDo C\u0026eacute;u De Madureira M, Paula Martins A, Gomes M, et al (2002) Antimalarial activity of medicinal plants used in traditional medicine in S. Tom\u0026eacute; and Prı́ncipe islands. J Ethnopharmacol 81:23\u0026ndash;29. https://doi.org/10.1016/S0378-8741(02)00005-3\u003c/li\u003e\n \u003cli\u003eEngel S, Pawlik JR (2000) Allelopathic activities of sponge extracts. Mar Ecol Prog Ser 207:273\u0026ndash;281. https://doi.org/10.3354/MEPS207273\u003c/li\u003e\n \u003cli\u003eFagundes T da SF, da Silva LRG, Brito M de F, et al (2021) Metabolomic fingerprinting of Brazilian marine sponges: a case study of Plakinidae species from Fernando de Noronha Archipelago. Anal Bioanal Chem 413:4301\u0026ndash;4310. https://doi.org/10.1007/S00216-021-03385-6/FIGURES/5\u003c/li\u003e\n \u003cli\u003eFattorusso E, Taglialatela-Scafati O (2009) Marine Antimalarials. Mar Drugs 7:130. doi: 10.3390/MD7020130\u003c/li\u003e\n \u003cli\u003eFeng YL, Singh R, Chao A, Li Y (2022) Diagnostic Fragmentation Pathways for Identification of Phthalate Metabolites in Nontargeted Analysis Studies. J Am Soc Mass Spectrom 33:981\u0026ndash;995. https://doi.org/10.1021/JASMS.2C00052/ASSET/IMAGES/LARGE/JS2C00052_0007.JPEG\u003c/li\u003e\n \u003cli\u003eFerraz CG, Ribeiro PR, Verde B V., et al (2023) Metabolite Profiling of Pleurotus ostreatus Grown on Sisal Agro-Industrial Waste Supplemented with Cocoa Almond Tegument and Wheat Bran. Chem Biodivers 20:e202300346. https://doi.org/10.1002/CBDV.202300346\u003c/li\u003e\n \u003cli\u003eGon\u0026ccedil;alves Neto GS, Da Costa RP, Zanini CL, et al (2020) Chemical Prospection of Qualea grandiflora Mart. Fruit and Stem Extracts and Their in vitro and in vivo Antiplasmodial Activity. Article J Braz Chem Soc 31:. https://doi.org/10.21577/0103-5053.20200034\u003c/li\u003e\n \u003cli\u003eIshida J, Kozuka M, Tokuda H, et al (2002) Chemopreventive potential of cyclic diarylheptanoids. Bioorg Med Chem 10:3361\u0026ndash;3365. https://doi.org/10.1016/S0968-0896(02)00164-5\u003c/li\u003e\n \u003cli\u003eKong DX, Jiang YY, Zhang HY (2010) Marine natural products as sources of novel scaffolds: achievement and concern. Drug Discov Today 15:884\u0026ndash;886. https://doi.org/10.1016/J.DRUDIS.2010.09.002\u003c/li\u003e\n \u003cli\u003eKouchaksaraee RM, Farimani MM, Li F, et al (2020) Integrating Molecular Networking and 1H NMR Spectroscopy for Isolation of Bioactive Metabolites from the Persian Gulf Sponge Axinella sinoxea. Marine Drugs 2020, Vol 18, Page 366 18:366. https://doi.org/10.3390/MD18070366\u003c/li\u003e\n \u003cli\u003eLambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. Journal of Parasitology 65:418\u0026ndash;420. https://doi.org/10.2307/3280287\u003c/li\u003e\n \u003cli\u003eLe Manach C, Scheurer C, Sax S, et al (2013) Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum. Malar J 12:. https://doi.org/10.1186/1475-2875-12-424\u003c/li\u003e\n \u003cli\u003eLhullier C, Moritz MIG, Tabalipa EO, et al (2019) Biological activities of marine invertebrates extracts from the northeast brazilian coast. Brazilian Journal of Biology 80:393\u0026ndash;404. https://doi.org/10.1590/1519-6984.213678\u003c/li\u003e\n \u003cli\u003eLhullier C, Moritz MIG, Tabalipa EO, Sard\u0026aacute; FN, Schneider NFZ, Moraes MH, Constantino L, Reginatto FH, Steindel M, Pinheiro US, Sim\u0026otilde;es CMO, P\u0026eacute;rez CD, Schenkel EP, Agron\u0026ocirc;mico E, Ferreira AC (2020) Brazilian Journal of Biology Biological activities of marine invertebrates extracts from the northeast brazilian coast. Braz J Biol 80:393. doi: 10.1590/1519-6984.213678\u003c/li\u003e\n \u003cli\u003eLi Z, Dong F, Sun Y, et al (2022) Qualitative and Quantitative Analysis of Six Fatty Acid Amides in 11 Edible Vegetable Oils Using Liquid Chromatography\u0026ndash;Mass Spectrometry. Front Nutr 9:857858. https://doi.org/10.3389/FNUT.2022.857858/BIBTEX\u003c/li\u003e\n \u003cli\u003eLu Z, Wang Q, Wang M, et al (2017) Using UHPLC Q-Trap/MS as a complementary technique to in-depth mine UPLC Q-TOF/MS data for identifying modified nucleosides in urine. Journal of Chromatography B 1051:108\u0026ndash;117. https://doi.org/10.1016/J.JCHROMB.2017.03.002\u003c/li\u003e\n \u003cli\u003eMeibalan E, Marti M (2017) Biology of malaria transmission. Cold Spring Harb Perspect Med 7:. https://doi.org/10.1101/cshperspect.a025452\u003c/li\u003e\n \u003cli\u003eMendes L da C, Dantas TBV, Severino RP, et al (2023) Using molecular networking and docking to explore arginase inhibitors among Drimys brasiliensis chemical constituents. Medicinal Chemistry Research 32:2208\u0026ndash;2218. https://doi.org/10.1007/S00044-023-03123-W/TABLES/2\u003c/li\u003e\n \u003cli\u003eNegm WA, Ezzat SM, Zayed A (2023) Marine organisms as potential sources of natural products for the prevention and treatment of malaria. RSC Adv 13:4436\u0026ndash;4475. doi: 10.1039/D2RA07977A\u003c/li\u003e\n \u003cli\u003eNothias LF, Nothias-Esposito M, Da Silva R, et al (2018) Bioactivity-Based Molecular Networking for the Discovery of Drug Leads in Natural Product Bioassay-Guided Fractionation. J Nat Prod 81:758\u0026ndash;767. https://doi.org/10.1021/ACS.JNATPROD.7B00737/SUPPL_FILE/NP7B00737_SI_001.PDF\u003c/li\u003e\n \u003cli\u003eParra LLL, Bertonha AF, Severo IRM, et al (2018) Isolation, Derivative Synthesis, and Structure-Activity Relationships of Anti-Parasitic Bromopyrrole Alkaloids from the Marine Sponge Tedania brasiliensis. J Nat Prod 81:188. https://doi.org/10.1021/ACS.JNATPROD.7B00876\u003c/li\u003e\n \u003cli\u003ePerdicaris S, Vlachogianni T, Valavanidis A (2013) Issue 3 \u0026bull; 1000114 Nat Prod Chem Res ISSN: 2329-6836 NPCR, an open access journal Perdicaris. Nat Prod Chem Res 1:3. https://doi.org/10.4172/2329-6836.1000114\u003c/li\u003e\n \u003cli\u003ePita L, Rix L, Slaby BM, et al (2018) The sponge holobiont in a changing ocean: from microbes to ecosystems. Microbiome 2018 6:1 6:1\u0026ndash;18. https://doi.org/10.1186/S40168-018-0428-1\u003c/li\u003e\n \u003cli\u003eQian PY, Li Z, Xu Y, et al (2015) Mini-review: Marine natural products and their synthetic analogs as antifouling compounds: 2009\u0026ndash;2014. http://dx.doi.org/101080/089270142014997226 31:101\u0026ndash;122. https://doi.org/10.1080/08927014.2014.997226\u003c/li\u003e\n \u003cli\u003eQuiann RJ, Gregson RP, Cook AF, Bartlett RT (1980) Isolation and synthesis of 1-methylisoguanosine, a potent pharmacologically active constituent from the marine sponge tenadia digitata. Tetrahedron Lett 21:567\u0026ndash;568. https://doi.org/10.1016/S0040-4039(01)85558-1\u003c/li\u003e\n \u003cli\u003eRazzazi-Fazeli E, Kleineisen S, Luf W (2000) Determination of cholesterol oxides in processed food using high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionisation. J Chromatogr A 896:321\u0026ndash;334\u003c/li\u003e\n \u003cli\u003eRoy RN, Laskar S, Sen SK (2006) Dibutyl phthalate, the bioactive compound produced by Streptomyces albidoflavus 321.2. Microbiol Res 161:121\u0026ndash;126. https://doi.org/10.1016/J.MICRES.2005.06.007\u003c/li\u003e\n \u003cli\u003eSaxena A, Yadav D, Mohanty S, et al (2016) Diarylheptanoids Rich Fraction of Alnus nepalensis Attenuates Malaria Pathogenesis: In-vitro and In-vivo Study. Phytotherapy Research 30:940\u0026ndash;948. https://doi.org/10.1002/PTR.5596\u003c/li\u003e\n \u003cli\u003eSchmitz FJ, Vanderah DJ, Hollenbeak KH, et al (1983) Metabolites from the Marine Sponge Tedania ignis. A New Atisanediol and Several Known Diketopiperazines. Journal of Organic Chemistry 48:3941\u0026ndash;3945. https://doi.org/10.1021/JO00170A011/SUPPL_FILE/JO00170A011_SI_001.PDF\u003c/li\u003e\n \u003cli\u003eShaaban M, Abd-Alla HI, Hassan AZ, et al (2012) Chemical characterization, antioxidant and inhibitory effects of some marine sponges against carbohydrate metabolizing enzymes. Org Med Chem Lett 2:30. https://doi.org/10.1186/2191-2858-2-30\u003c/li\u003e\n \u003cli\u003eSittie AA, Lemmich E, Olsen CE, et al (1998) Alkamides from Phyllanthus fraternus. Planta Med 64:192\u0026ndash;193. https://doi.org/10.1055/S-2006-957405/BIB\u003c/li\u003e\n \u003cli\u003eSu XZ, Lane KD, Xia L, et al (2019) Plasmodium Genomics and Genetics: New Insights into Malaria Pathogenesis, Drug Resistance, Epidemiology, and Evolution. Clin Microbiol Rev 32:. https://doi.org/10.1128/CMR.00019-19\u003c/li\u003e\n \u003cli\u003eSumner LW, Amberg A, Barrett D, et al (2007) Proposed minimum reporting standards for chemical analysis: Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics 3:211\u0026ndash;221. https://doi.org/10.1007/S11306-007-0082-2/METRICS\u003c/li\u003e\n \u003cli\u003eTakahashi M, Fuchino H, Satake M, Agatsuma Y, Sekita S. (2004) In Vitro Screening of Leishmanicidal Activity in Myanmar Timber Extracts, Biological and Pharmaceutical Bulletin, 2004, Volume 27, Issue 6, Pages 921-925, Released on J-STAGE June 01, 2004, Online ISSN 1347-5215, Print ISSN 0918-6158, https://doi.org/10.1248/bpb.27.921.\u003c/li\u003e\n \u003cli\u003eThakur NL, Hentschel U, Krasko A, et al (2003) Antibacterial activity of the sponge Suberites domuncula and its primmorphs: potential basis for epibacterial chemical defense. Aquatic Microbial Ecology 31:77\u0026ndash;83. https://doi.org/10.3354/AME031077\u003c/li\u003e\n \u003cli\u003eTian C, Ni J, Chang F, et al (2016) Bio-Source of di-n-butyl phthalate production by filamentous fungi. Sci Rep 6:19791. https://doi.org/10.1038/srep19791\u003c/li\u003e\n \u003cli\u003eTrager W, Jensen JB (1976) Human Malaria Parasites in Continuous Culture. Science (1979) 193:673\u0026ndash;675. https://doi.org/10.1126/SCIENCE.781840\u003c/li\u003e\n \u003cli\u003evan den Wildenberg SAH, Streng AS, van den Broek R, et al (2022) Quantification of uracil, dihydrouracil, thymine and dihydrothymine for reliable dihydropyrimidine dehydrogenase (DPD) phenotyping critically depend on blood and plasma storage conditions. J Pharm Biomed Anal 221:115027. https://doi.org/10.1016/J.JPBA.2022.115027\u003c/li\u003e\n \u003cli\u003eVossen MG, Pferschy S, Chiba P, Noedl H (2010) The SYBR Green I malaria drug sensitivity assay: performance in low parasitemia samples. Am J Trop Med Hyg 82:398\u0026ndash;401. https://doi.org/10.4269/AJTMH.2010.09-0417\u003c/li\u003e\n \u003cli\u003eWang M, Carver JJ, Phelan V V., et al (2016) Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nature Biotechnology 2016 34:8 34:828\u0026ndash;837. https://doi.org/10.1038/nbt.3597\u003c/li\u003e\n \u003cli\u003eWood K V., Bonham CC, Miles D, et al (2002) Characterization of betaines using electrospray MS/MS. Phytochemistry 59:759\u0026ndash;765. https://doi.org/10.1016/S0031-9422(02)00049-3\u003c/li\u003e\n \u003cli\u003eWorld malaria report 2023. https://www.who.int/publications/i/item/9789240086173. Accessed 16 Jan 2024\u003c/li\u003e\n \u003cli\u003eXie JW, Huang LF, Hu W, et al (2010) Analysis of the Main Nucleosides in Cordyceps Sinensis by LC/ESI-MS. Molecules 2010, Vol 15, Pages 305-314 15:305\u0026ndash;314. https://doi.org/10.3390/MOLECULES15010305\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bioproducts, Tedania ignis, Antiplasmodial activity, Plasmodium.","lastPublishedDoi":"10.21203/rs.3.rs-4320042/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4320042/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcross 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 \u003cem\u003eTedania ignis\u003c/em\u003e, was evaluated against sensitive and resistant laboratory \u003cem\u003eP. falciparum\u003c/em\u003e strains and presented potential antimalarial activity. Moreover, the speed of action of the extract of \u003cem\u003eT. ignis\u003c/em\u003e 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 \u003cem\u003eT. ignis\u003c/em\u003e 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 \u003cem\u003eT. ignis \u003c/em\u003eextract revealed in this work.\u003c/p\u003e","manuscriptTitle":"Antimalarial Potential of a Marine Sponge Tedania Ignis Against Plasmodium Falciparum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-10 07:29:28","doi":"10.21203/rs.3.rs-4320042/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1b504fe4-ae85-4896-b11b-322fa2e7e78d","owner":[],"postedDate":"June 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-19T11:36:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-10 07:29:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4320042","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4320042","identity":"rs-4320042","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}