Seaweeds to the rescue of forgotten diseases: a review

Seaweed metabolites against trypanosomiasis

Trypanosomiasis is a vector-borne NTD caused by the kinetoplastid parasite genus Trypanosoma and transmitted to humans by tsetse fly (Glossina genus) or triatomine bug bites (Table 1). The human African trypanosomiasis (HAT), also known as sleeping sickness, is caused by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense and is common in Africa, while Trypanosoma cruzi is responsible for Chagas’ disease, which is common in Latin America. More than 10,000 people die annually due to Chagas’ disease with 25 million people at risk of acquiring the disease. Thanks to the efforts of WHO, national control programmes, bilateral cooperation and NGOs, the number of new human African trypanosomiasis cases reported between 2000 and 2012 decreased. Only 2804 new HAT cases have been reported in 2015 (WHO 2018).

The promastigotes and epimastigotes (forms of the parasite in insect hosts), as well as the bloodstream trypomastigotes (human infective forms) have been used in phenotypic screenings, while the latter is the clinically relevant form of the parasite. In Trypanosoma cruzi infection, both the (intracellular) amastigote and the trypomastigote life cycle stages take place in the human host (Jones et al. 2013). As discussed below, the only metabolite class reported from seaweeds with antitrypanosomal activity are terpenes, belonging to sesqui- (C15) and diterpenes (C20) subclasses which are shown in Figure 1:

1. Sesquiterpenes: Several curcuphenol type monocyclic aromatic sesquiterpenes have been obtained from organic extracts of the green alga Udotea orientalis growing on the Papua New Guinean gorgonian coral Pseutopterogorgia rigida (Sabry et al. 2017). The known compound (+)-curcudiol (Figure 1-1) showed the best antitrypanosomal activity against Trypanosoma cruzi amastigotes (the intracellular form of the parasite) with an IC₅₀ value of 10 μm, although the authors did not report the in vitro efficacy of other isolated compounds. Notably, curcuphenol type sesquiterpenes have been mostly reported from sponges and corals including the soft coral P. rigida.

   Several studies have investigated the inhibitory activity and mechanisms of the chlorinated bicyclic sesquiterpene elatol (Figure 1-2), the major component of the Brazilian red seaweed Laurencia dendroidea. Elatol is a small molecule with multiple chiral centers. In the initial work by Veiga-Santos et al. (2010), however, its specific rotation was not measured or mentioned, instead the authors have used only the name elatol, while in follow up studies the name (−)-elatol was used. Herein we use the same trivial names and structures as reported by the authors. This compound inhibited the epimastigote, trypanomastigote and intracellular amastigote forms of Trypanosoma cruzi with IC₅₀ values of 45.4, 1.38 and 1.01 μm, respectively. For comparison, the reference drugs used in testing different life forms of the parasite exert IC₅₀ values of 7.8 μm (benznidazole), 12.8 μm (crystal violet) and 24.3 μm (benznidazole). Hence elatol is more potent in vitro than the anti-Chagas drug benznidazole against the trypanomastigote and intracellular amastigote forms, that are present in the human host of T. cruzi. Furthermore, elatol shows little toxicity against the mammalian LLCMK₂ cells (selectivity index 20), and cause three times lower hemolysis (21%) than amphotericin B at the highest test concentrations (Veiga-Santos et al. 2010). In two follow-up studies, Desoti et al. (2012, 2014) performed mechanistic studies on the trypanocidal activity of (−)-elatol on both life stage forms. On trypomastigotes, (−)-elatol induced polarization of the mitochondrial membrane, mitochondrial superoxide anion formation, loss of cell membrane and DNA integrity. Hence its effect on trypanosomes has been associated with oxidative stress triggered by mitochondrial dysfunction (Desoti et al. 2012). When tested on amastigotes, (−)-elatol again induced the formation of superoxide anions (O₂˙⁻), caused DNA fragmentation and the formation of autophagic compartments. It has been hence concluded that the trypanocidal action of (−)-elatol involves the induction of the autophagic and apoptotic death pathways triggered by an imbalance of the redox metabolism of T. cruzi (Desoti et al. 2014).

2. Diterpenes: Organic extracts of the brown alga Bifurcaria bifurcata collected from different habitats have shown strong antitrypanosomal activity against the bloodstage trypomastigotes of Trypanosoma brucei rhodesiense (Spavieri et al. 2010, Gallé et al. 2013). Bioactivity-guided fractionation of the ethyl acetate (EtOAc) extract of French B. bifurcata (IC₅₀ 1.7 μm=0.53 μg ml⁻¹) has yielded the acyclic diterpene eleganolone (Figure 1-3). Eleganolone is the major diterpenoid constituent of the alga and shows moderate growth inhibitory activity against T. brucei rhodesiense (IC₅₀ 45 μm=13.7 μg ml⁻¹) and Trypanosoma cruzi (IC₅₀ 58 μm=17.7 μg ml⁻¹), with low selectivity indices around or lower than 4. The highest antiprotozoal activity (IC₅₀ value 7.9 μm) and selectivity (SI 21.6) observed for eleganolone is towards the blood stage forms of the malaria parasite Plasmodium falciparum (Gallé et al. 2013). The higher activity observed for the crude extract may be due to a synergistic effect of the other components found in the extract.

Figure 1:

Seaweed terpenes with trypanocidal and leishmanicidal activity: 1. curcudiol; 2. elatol; 3. eleganolone; 4. bifurcatriol; 5. obtusol; 6. triquinane derivative; 7a. pachydictyol; 7b. isopachydictyol; 8. dolabelladienetriol; 9. 4-acetoxydolastane; 10a. 3R-tetraprenyltoluquinol; 10b. 3S-tetraprenyltoluquinol; 11a. 3R-tetraprenyltoluquinone; 11b. 3S-tetraprenyltoluquinone; 12. Atomaric acid; 13. Atomaric methyl ester derivative acid. 14. Quinone. 15. Fucosterol.

Another new, linear diterpene recently isolated from Irish B. bifurcata is bifurcatriol (Smyrniotopoulos et al. 2017). Bifurcatriol (Figure 1-4) is a minor compound and the first acyclic natural product with two stereogenic centers whose absolute stereochemical structure has been elucidated by Vibrational Circular Dichroism (VCD) spectrometry and DP4/NMR analyses. Both African and American trypanosomes were moderately inhibited in vitro by compound 4 with IC₅₀ values of 11.8 μg ml⁻¹ (Trypanosoma brucei rhodesiense) and 47.8 μg ml⁻¹ (T. cruzi). The cytotoxicity of the compound against mammalian L6 cells is 56.6 μg ml⁻¹. Similar to eleganolone, P. falciparum is the most susceptible protozoan parasite against bifurcatriol (IC₅₀ value 0.65 μg ml⁻¹).

Seaweed metabolites against leishmaniasis

Leishmaniasis is another NTD caused by the protozoan parasite genus of Leishmania, which is transmitted by the bite of over 90 species of infected female phlebotomin sandflies (Table 1). The three main forms of the disease threaten large populations in Latin America, Africa, Asia, Middle East and even Mediterranean countries. Visceral leishmaniasis (VL), also known as kala-azar caused by Leishmania donovani and Leishmania infantum is almost always fatal if left untreated. The causative agents of the most widespread form of the disease, cutaneous leishmaniasis (CL) include Leishmania braziliensis and Leishmania amazonensis. The etiological agent of the mucosal leishmaniasis (ML) can be several Leishmania sp. e.g. L. braziliensis and L. amazonensis. Approximately 700,000 to 1 million new cases of leishmaniasis and up to 30,000 deaths occur annually (WHO 2018). The insect (promastigote) and human (amastigote) life stage forms have been used for assessment of the determination of natural products with leishmanicidal activity.

1. Sesquiterpenes: dos Santos et al. (2011) have tested the leishmanicidal effect of the halogenated sesquiterpene elatol (no specific rotation mentioned), the major component of the Brazilian red seaweed Laurencia dendroida against Leishmania amazonensis. Against the promastigote forms, elatol showed IC₅₀ and IC₉₀ values of 4.0 and 7.5 μm, respectively. The activity against the intracellular amastigotes was 10-fold higher (IC₅₀ 0.45 μm), but the cytotoxicity towards macrophages was less selective (CC₅₀ 1.4 μm) affording a selectivity index (SI) value of 3. Electron microscopy studies showed that elatol caused ultrastructural changes on both promastigote and amastigote forms of the parasite L. amazonensis. This includes remarkable swelling of mitochondrion, destabilization of the plasma membrane and extension of the endoplasmic reticulum, indicating autophagy.

   Another Brazilian group (da Silva Machado et al. 2011) has obtained 5 sesquiterpenes by antileishmanial activity-guided isolation of two populations of Laurencia dendroida from the southeastern Brazilian coast. (−)-Elatol showed activity against both promastigote and amastigote forms of Leishmania amazonensis with IC₅₀ values of 9.7 and 4.5 μg ml⁻¹, respectively. The toxicity against macrophages was low (IC₅₀ 112.9 μg ml⁻¹). Obtusol (Figure 1-5), a similar sesquiterpene with an additional bromine atom had similar activity towards promastigotes (IC₅₀ 6.2 μg ml⁻¹), amastigotes (IC₅₀ 3.9 μg ml⁻¹) and macrophages (IC₅₀ 133.5 μg ml⁻¹). The triquinane derivative (Figure 1-6) had practically identical antileishmanial activity against both life stages (IC₅₀ values 43.8 and 48.7 μg ml⁻¹, respectively) and low toxicity (160.2 μg ml⁻¹). None of the compounds promoted the elevated nitric oxide production by macrophages (da Silva Machado et al. 2011).

2. Diterpenes: Bifurcatriol, a highly oxygenated acyclic diterpene from the brown alga Bifurcaria bifurcata inhibited the growth of amastigotes of Leishmania donovani with an IC₅₀ value of 18.8 μg ml⁻¹. As mentioned above its cytotoxicity against mammalian L6 cells is 56.6 μg ml⁻¹ (Smyrniotopoulos et al. 2017).

   A 3:1 mixture of hydroazulene (prenyl guaiane)-type bicyclic diterpenes, pachydictyol and isopachydictyol (Figure 1-7a/7b) has been isolated from the dichloromethane extract of the Brazilian brown alga Dictyota menstrualis that showed significant activity against Leishmania amazonensis promastigotes (IC₅₀ 0.75 μg ml⁻¹). However the antileishmanial activity of the isolated isomeric mixture is moderate (IC₅₀ 23.5 μg ml⁻¹) and equal to their toxicity against macrophages (CC₅₀ 30 μg ml⁻¹), indicating that the mixture non-selectively inhibits the growth of the parasite (Lira et al. 2016). In Brazil, the co-infection of HIV and mucosal, muco-cutaneous and cutaneous forms of leishmaniasis is very common. Soares et al. (2012) assessed the inhibitory activity of dolabelladienetriol (Figure 1-8, no optical sign stated), a dolabellene-type diterpene, against several life stages of L. amazonensis. The motivation for this study was that dolabelladienetriol is a potent inhibitor of HIV-1 reverse transcriptase enzyme (Cirne-Santos et al. 2008). Based on the activity against promastigotes (95.5% inhibition at 100 μm concentration), the efficacy of dolabelladienetriol was evaluated on intracellular amastigotes. The compound showed only moderate activity (IC₅₀ 43.9 μm). Dolabelladienetriol reduced the NO, transforming growth factors (TNF-α and TGF-β) production and nuclear factor NF-κB nuclear translocation. Importantly, it inhibited the growth of Leishmania parasites in HIV-1-co-infected human macrophages, making it a promising compound against this disease.

   The 4-acetoxydolastane diterpene (4R,9S,14S)-4α-acetoxy-9β,14α-dihydroxydolast-1(15),7-diene (Figure 1-9, no optical sign stated) has been isolated by activity-guided extraction of another brown alga, Canistrocarpus cervicornis from Brazil. This tricyclic diterpene (dos Santos et al. 2011) exhibited promising antileishmanial effects with IC₅₀ values of 2.0 μg ml⁻¹, 12.0 μg ml⁻¹, and 4.0 μg ml⁻¹ against promastigote, axenic amastigote and intracellular amastigote forms, respectively, of Leishmania amazonensis. The compound had a very high selectivity window with a 93-fold higher toxicity to protozoans than the macrophages. Similar to other terpenes, it also caused ultrastructural changes and depolarization in mitochondria, and an increase of lipid peroxidation. This successful compound warrants further studies to identify its exact mechanisms of action.

3. Meroterpenes: Meroterpenes are terpenes of mixed biogenesis, and are known for their antiprotozoal activity. By applying antileishmanial activity as a guide, de Sousa and coworkers (2017) have isolated the epimeric mixtures of two meroterpenes (3R)- and (3S)-tetraprenyltoluquinol (Figure 1-10a/10b) and (3R)- and (3S)-tetraprenyltoluquinone (Figure 1-11a/11b), from the hexane extract of the brown alga Cystoseira baccata collected from Portuguese waters. The hexane extract decreased the viability of the promastigote forms of Leishmania infantum moderately (74% at 250 mg ml⁻¹ concentration), and demonstrated the efficacy of mixtures of these compounds (IC₅₀ 44.9 μm for (3R)- and (3S)-tetraprenyltoluquinol, and 94.4 μm for (3R)- and (3S)-tetraprenyltoluquinone). Their cytotoxicities against mouse peritoneal macrophages were close or similar to each other (IC₅₀ 126.6 and 84.5 μm) and to the reference drug miltefosine (130.3 μm); however the selectivity was still very low. These compounds also interfered with mitochondria, inducing cytoplasmic vacuolization, and disrupted the mitochondrial membrane (de Sousa et al. 2017).

   Atomaric acid (Figure 1-12) is a meroterpene and a major chemical component of the tropical brown alga Stypopodium zonale (Dictyotaceae). The lipophilic extract of this seaweed was selected for its high leishmanicidal activity (IC₅₀ 0.27 μg ml⁻¹) against the amastigotes of Leishmania amazonensis (Costa Soares et al. 2016). Atomaric acid and its semi-synthetically prepared methyl ester derivative (Figure 1-13) was active against L. amazonensis promastigotes (up to 100% inhibition at 100 μm) and inhibited the amastigote forms with IC₅₀ values of 20 μm (9 μg ml⁻¹, 12) and 23 μm (10 μg ml⁻¹, 13). When tested on murine peritoneal macrophages, they showed low toxicity with acceptable SI indices around 8 and 20. Nitric oxide and reactive oxygen species (ROS) are potent leishmanicidal mediators. Both compounds 12 and 13 were found to modulate macrophage activity by inhibiting NO production and stimulating the production of ROS (Soares et al. 2016). Another meroterpene possessing a quinone function (Figure 1-14) has been the subject of a Japanese patent by Kimura et al. (2011). This compound was isolated from the brown alga Sargassum angii (formerly Sargassum yamadae) and reported to exhibit both in vitro and in vivo anti-leishmanial effects in a mouse model (Kimura et al. 2011).

4. Sterols (triterpene): Fucosterol is a common triterpene in some brown algae. In a recent study Becerra et al. (2015) isolated fucosterol (Figure 1-15) by using supercritical fluid (SFE) and pressurized solvent (PSE) extraction techniques from the Argentinian brown seaweed Lessonia vadosa. Fucosterol was active against the extracellular promastigotes and intracellular amastigotes of both Leishmania infantum (IC₅₀ values 45 and 10.3 μm, respectively) and Leishmania amazonensis (IC₅₀ 55 and 7.9 μm, respectively), and posed no toxicity against the host macrophagic cell line (IC₅₀ >100 μm). These results may suggest a selective activity towards amastigotes or the immunomodulatory effect of 15 by modifying the response of macrophages (Becerra et al. 2015).

5. Sulfated polysaccharides: In in vitro macrophage models, the commercially obtained fucoidan (size not mentioned) eliminated the intracellular amastigotes of both antimony-susceptible and -resistant Leishmania donovani (over 90% inhibition at 50 μm concentration, no IC₅₀ determination). Oral administration of fucoidan (200 mg kg⁻¹ day⁻¹, 3 times weekly) in BALB/c mice infected with in antimony-susceptible and -resistant L. donovani achieved full parasite clearance in mouse liver and spleen (Kar et al. 2011). When administered 15 days post-infection, fucoidan evoked resistance to reinfection. This effect has been linked to the direct induction of NO and ROS as well as the switch of CD4+ T cell mediated immune responses. Hence fucoidan appears to have both curative and immunomodulatory activity against L. donovani, the causative agent of visceral leishmaniasis (Kar et al. 2011).

Pires et al. (2013) have purified sulfated polysaccharides (with molecular size ranging from 25 kDa to 200 kDa) from four seaweeds including the red algae, Solieria filiformis, Botryocladia occidentalis and Gracilaria caudata and a green alga Caulerpa racemosa. These polymers were tested in vitro against the promastigotes of Leishmania amazonensis. The most active SP was that obtained from C. racemosa, which displayed an EC₅₀ value of 34.5 μg ml⁻¹, while B. occidentalis and S. filiformis-derived SPs were less active (EC₅₀ 63.7 and 137.4 μg ml⁻¹). However, all three SPs were found to be toxic against J774 macrophages, resulting in selectivity indices ranging from 0.42 to 1.42.

Seaweed against dengue and other mosquito-borne infections

Dengue is the most important mosquito-borne viral disease in the world (WHO 2018). Dengue virus (DENV) is an enveloped virus (genus Flavivirus) classified into four different serotypes (DENV-1, -2, -3 and -4), all of them capable of causing the disease. Dengue is transmitted by the bites of female mosquitoes Aedes aegypti and Aedes albopictus that cause a flu-like illness, and occasionally develops into a potentially lethal complication called severe dengue. This mosquito also transmits other closely related enveloped viruses such as Zika (ZIKV) and chikungunya (CHIKV) (Table 1). ZIKV infection is a tremendous risk for pregnant women because ZIKV can seriously damage the brain development of the fetus in utero (WHO 2018). CHIKV virus, an arbovirus of the genus Alphavirus, has significantly expanded its geographical distribution due to increased migration of infected people. Despite the importance of these mosquito-borne viral diseases, treatments are only preventative and/or palliative. Until the recent tetravalent dengue vaccine licensure (Table 1), the only approach to control the transmission of these virus diseases has been through the control of the Aedes mosquito. At present, there are no specific antiviral treatments for these mosquito-borne viral diseases. As is common to the series of enveloped RNA-composed viruses, the viral replicative cycle starts with the attachment of virus to the surface of the host cell; therefore, blocking virus binding is a valuable anti-viral strategy because it allows establishment of a first barrier to suppress infection. Because of their ability to change cell surface properties, the antiviral effects of sulfated polysaccharides from seaweeds have been investigated in order to prevent the virus attachment.

1. Sulfated polysaccharides: Rodrigues et al. (2017) reported a polysulfated fraction called ulvan (containing 11% sulfate and 6% uronic acids) from the green alga Caulerpa cupressoides collected in Northeastern Brazil, which showed an effective in vitro activity against DENV-1 in Vero (African green monkey kidney) cell line (96% percentage inhibition, EC₅₀ 0.35 μg ml⁻¹) with a high selectivity index (>714) and no cytotoxicity (CC₅₀ >1000 μg ml⁻¹). Infrared analysis revealed four SP fractions of distinct molecular weights ranging from 8 to >100 kDa (Rodrigues et al. 2017). The authors concluded that the strong antiviral activity displayed supported the hypothesis that the sulfation pattern on C-6 galactose residues was important in the inhibition of infection. They argued that the amount of sulfation (1.8-fold greater than the uronic acid content) and the effect of the chain length (of less than 100 kDa) could contribute in parallel to the in vitro antiviral effect of ulvan, suggesting that this SP interfered with virion envelope structures or masked viral structures, once they are necessary for adsorption or entry into cells.

In relation to brown seaweeds, the fucoidan of Cladosiphon okamuranus significantly inhibited DENV-2 infection (IC₅₀ 4.7 μg ml⁻¹) in baby hamster kidney (BHK-21) cells in a dose-dependent manner (Hidari et al. 2008). This fucoidan consists of a repeating unit of sulfated fucose and glucuronic acid residues in a molar ratio of 6.1:1.0:2.9 fucose, glucuronic acid and sulfate, respectively (Nagaoka et al. 1999). Treatment of the virus with 10 μg ml⁻¹ fucoidan reduced the infectivity by 20% compared with that in untreated cells, and two derivatives of this fucoidan generated by chemical modifications, such as elimination of the sulfated group or reduction of carboxylic acid, were also tested (Hidari et al. 2008). A fucose polymer was used for the control experiment. Desulfation from fucoidan showed marked suppression of inhibitory activity to 1% of that of fucoidan and a carboxy-reduced fucoidan derivative in which glucuronic acid was converted to glucose knocked out the effect of fucoidan against DENV2 infection. DENV2 particles bound exclusively to fucoidan, indicating that both glucuronic acid and sulfated fucose residues were involved in the interaction with envelope glycoprotein on DENV2. Of dengue virus serotypes, the DENV2 strain was highly susceptible to the Cladosiphon fucoidan. DENV3 and DENV4 were moderately susceptible and DENV1 showed no susceptibility. To elucidate the molecular basis of susceptibility to the Cladosiphon fucoidan, the nucleotide sequence of DENV1 was examined and multiple alignment analyses of the amino acid sequences of the proteins of the four serotype strains were performed. Results showed that amino acid residues at positions 295 and 310 (Lys295 and Lys or Arg310) are critically involved in the interaction with sulfated glycosaminoglycans, and contribute to the susceptibility of dengue virus to fucoidan (Hidari et al. 2008).

From Rhodophyta, a novel series of DL-galactan hybrids of the carrageenan- and agaran-types (molecule size ranging from 18 kDa to 77 kDa and sulfate content ranging from 8.6 to 29.2%) were isolated from the red seaweed Gymnogongrus torulosus (Estevez et al. 2001). All DL-galactan hybrids showed strong in vitro inhibitory effects against DENV-2 in Vero cells, with IC₅₀ ranging from 0.19 to 1.7 μg ml⁻¹ (Pujol et al. 2002). No cytotoxicity was observed with any of the galactans (CC₅₀ >1000 μg ml⁻¹) with the exception of only one fraction, which exhibited a CC₅₀ value of 822 μg ml⁻¹. Full inhibitory activity was achieved when the galactans were present during the virus adsorption period, confirming that the mode of action of these sulfated compounds is to interfere in the binding of the surface envelope glycoprotein with the cell receptor. The effects of DL-galactans were not reversible, since the withdrawal of the compounds after adsorption did not affect the antiviral activity.

Later, two homogeneous and chemically well characterized SPs obtained from Gymnogongrus griffithsiae and Cryptonemia crenulata, the κ/ι/ν carrageenan, and a DL-galactan hybrid (3-linked β-D-galactose 2- and 2,6-disulfated and 4-linked α-D- and α-L-galactose 6- and 2,6-disulfated besides 3,6.anhydro-α-D and α-L-galactose, with molecular size 236 kDa) were potent and selective inhibitors in vitro of DENV-2 in Vero cells with IC₅₀ around 1 μg ml⁻¹, CC₅₀ >1000 μg ml⁻¹, and selectivity indices >1000, values comparable to the reference polysaccharides heparin and DS8000 with IC₅₀ 1.9 and 0.9 μg ml⁻¹, respectively (Talarico et al. 2004, 2005). Nevertheless, these SPs were inactive in mosquito cells, and this differential susceptibility was related to the mode of entry of DENV into diverse cells. The authors concluded that the compounds blocked the initial steps of virus adsorption and internalization into the host cell (Talarico et al. 2007). Afterwards, the authors also assayed commercial iota-, lambda- and kappa-carrageenans for anti-DENV-2 activity in Vero cells and in C6/36 HT cells from Aedes albopictus, as model systems of mammalian and mosquito cells, respectively (Talarico et al. 2011). All commercial polysaccharides blocked DENV-2 infection in Vero cells, but only iota-carrageenans were virus inhibitors in mosquito cells. However, iota-carrageenan was less effective in mosquito cells in comparison with mammalian cells with EC₅₀ values in C6/36 HT cells 4.9–17.5-fold higher than in Vero cells, as determined by virus yield reduction assay. The authors reported the mode of action of iota-carrageenan to be strikingly different in both cell types: in Vero cells the inhibitory activity was exerted only at the initiation of the cycle, affecting virion binding. In mosquito cells, iota-carrageenans induced a subtle alteration in cells, detected by cell proliferation and protein synthesis analyses.

In relation to the inhibitory activity of seaweed compounds against ZIKV, very few studies have been completed. A recent study of Cirne-Santos et al. (2017) reported that organic extracts of Kappaphycus alvarezii, Caulerpa racemosa and Osmundaria obtusiloba collected from the Brazilian coast were able to inhibit ZIKV replication above 90% in Vero cell culture at low concentrations in a dose-dependent manner, with EC₅₀ values of 1.38, 1.98 and 1.82 μg ml⁻¹ and selective indices of 306, 369 and 288, respectively. Caulerpa racemosa extract presented the best CC₅₀ with a value of 732 μg ml⁻¹, followed by O. obtusiloba (525 μg ml⁻¹). To identify the step at which viral replication was inhibited, the authors performed a time-course experiment with the compounds administered at 3.2 μg ml⁻¹ and 1 h before infection, time 0 (immediately when virus added) and 1, 2 and 3 h after infection. Ribavirin at 5 μm was used as a control. Results showed that at time 0, all extracts were able to inhibit over 80% of viral replication and O. obtusiloba and Ribavirin maintained the inhibitory effect at the other times. The authors pointed out the significant viricidal effect of O. obtusiloba, inhibiting viral replication more than 80% at a concentration of 10 μm, showing it to be an excellent preventative agent, especially considering the effect directly on the virus particle. In a previous study, the same group reported the sulfolipid SQDG (1,2-di-O-acyl-3-O-(6-deoxy-6-sulfo-α-D-glucopyranosyl)-sn-glycerol) for this species, which also showed a potent antiviral activity against HSV-1 (EC₅₀ 42 μg ml⁻¹) and HSV-2 (EC₅₀ 12 μg ml⁻¹) (de Souza et al. 2012).

In relation to the mosquitocidal seaweed compounds, Yu et al. (2014) pointed out in a recent review that the halogenated sesquiterpene (−)-elatol from Laurencia dendroidea showed potent larvicidal effects (>91% mortality at 50 ppm, with lethal concentration 50 (LC₅₀) of 10.7 ppm) against Aedes aegypti larvae, while several fatty acids (capric, lauric, and myristic and palmitoleic acids) isolated from Cladophora glomerata showed activity against Aedes triseriatus, showing LC₅₀ values ranging from 3 to 14 ppm. More recently, Salvador-Neto et al. (2016) corroborated the larvicidal effect against A. aegypti of elatol and another halogenated sesquiterpene, (+)-obtusol, isolated from L. dendroidea. Elatol showed larvicidal activity with 10 ppm killing approximately 30% of the larvae within 24 h and at lower concentrations than elatol. However, using a concentration of 10 ppm, (+)-obtusol killed approximately 90% of A. aegypti larvae and, at lower concentrations, (+)-obtusol showed a reasonable larvicidal activity. It was noted that (+)-obtusol acted in a dose-dependent manner with LC₅₀ of 3.5 ppm. Moreover, histological analysis of the larvae exposed to (+)-obtusol revealed damage to the intestinal epithelium. Structurally, elatol and (+)-obtusol differ in only one double bond and the presence of an additional bromine atom (Salvador-Neto et al. 2016). The authors suggested that the presence of this additional bromine could be responsible for the increase in potency when compared to elatol.

Seaweed metabolites against schistosomiasis

Human schistosomiasis (also known as bilharzia, bilharziosis or snail fever) is a chronic parasitic disease caused by trematode flukes of the genus Schistosoma, which globally affects over 250 million people (WHO 2018). Among the Schistosoma species, Schistosoma mansoni is the most widely spread in Africa and Latin America. The adult male and female worms live within the veins of their human host, where they mate and produce fertilized eggs. The eggs are either shed into the environment through feces or urine or are retained in host tissues inducing a distinct immune-mediated granulomatous response that causes local and systemic pathological effects. These effects range from anemia, growth stunting, impaired cognition, and decreased physical fitness, to organ-specific effects such as severe hepatosplenism, periportal fibrosis with portal hypertension, and urogenital inflammation and scarring. In general, compounds with schistosomicidal activity lead to disruption of mating of males and females and alterations in the morphology or constitution of the protective tegument, or changes in the muscle activity of the parasite. There is currently only one method of treatment (monotherapy), the isoquinolinone drug praziquantel to suppress morbidity (Table 1). However, constant selection pressure through mass chemotherapy has yielded evidence of resistance to praziquantel. As discussed below, only one recent preliminary study on seaweeds with anti-schistosomal activity has been carried (Stein et al. 2015). In this study, the efficacies of 13 organic extracts of seaweeds from Brazil were tested in vitro against S. mansoni in a first trial, and the active extracts (at 500 μg ml⁻¹ exposure) were further evaluated at lower concentrations (100 μg ml⁻¹). Worm couples were incubated with different concentrations of seaweed extracts for 120 h and monitored after the first 2 h and then every 24 h to evaluate death, mobility reduction and couple detachment. Only extracts of Gracilaria ornata, as well as species belonging to the genera Dictyota (Dictyota dichotoma, Dictyota menstrualis and Dictyota mertensii and Laurencia (Laurencia catarinensis and Laurencia dendroidea) showed high activity at 100 μg ml⁻¹, with couple worm death time ranging from 24 to 120 h. The active extracts were analyzed chromatographically applying a UPLC–MS system in search of a common metabolite among all of the seaweeds. Stein et al. (2015) found an abundance (95%) of low weight molecules (<500 Da with a retention time of 76.1 min), indicating a similar secondary metabolite in the extracts (hexanoic and chloroformic extracts) with the molecular weight of a sesterterpene or a conjugated diterpene. From the three most active extracts, defined by the total mortality reached in 24 h, D. menstrualis and L. catarinensis yielded a common molecular weight of 181.123 Da, with a retention time of 36.1 min which coincided with the molecular weight of a sesquiterpene or a conjugated monoterpene.