Effect of extracts from several sponges of Yucatan Coast on Giardia lamblia and preliminary chemical investigation of the bioactive extract of Haliclona (Reinera) tubifera

Abstract In this study, twenty-four organic extracts from six marine sponge species, collected at shallows of Yucatan, Mexico, were evaluated against Giardia lamblia trophozoites and Vero cells. The dichloromethane and hexane extracts of Haliclona tubifera exhibited the highest antigiardiasic activity (IC50 = 1.00 and 2.11 µg/mL, respectively), as well as high selectivity (SI = 41.8 and > 47.4, respectively), while ethyl acetate and methanol extracts of Cinachyrella alloclada, and methanol extract of Suberites aurantiaca showed moderate activity. Contrastingly, the extracts of Halichondria magniculosa and Oceanapia nodosa were considered non actives. Consequently, the dichloromethane extract of H. tubifera were subject to an exploratory chemical study, isolating cholesterol, two benzaldehyde derivatives, three benzoic acid derivatives, cytosine, and thymine. Graphical Abstract


Introduction
Human giardiasis is an infectious disease caused by the flagellate protozoan Giardia lamblia, affecting close to 1 billion persons each year, of which around 200 million develop symptoms (Riches et al. 2020).
Nowadays, it is possible to find this parasitosis disease throughout either developed or developing countries, but its prevalence is 10-fold higher in the last ones.Unfortunately, the inhabitants of underprivileged socioeconomic areas of those countries bear most of the burden (Al-Jawabreh et al. 2019).
Non-bloody foul-smelling diarrhoea, burping, bloating, epigastric pain, cramps, nausea, vomiting, and weight loss are the typical symptoms of an infection by G. lamblia.Most acute giardiasis cases last 4-6 weeks, but others can become chronic (Escobedo et al. 2010).The chronic stage of giardiasis leads to malabsorption syndrome causing a malnutrition state and delaying the physical growth and cognitive-intellectual development of small children (Halliez and Buret 2013).
Currently, the first-line treatment of human giardiasis relies on 5-nitroimidazoles, as well as on benzimidazoles or nitazoxanide.Nevertheless, in not a few cases, it is necessary to use secondary agents such as furazolidone, chloroquine, quinacrine, or paromomycin (Riches et al. 2020).
Regrettably, all antigiardial drugs have important limitations.One of these limitations is the variety of adverse effects these drugs provoke in patients.Some of these effects can be transient, like gastrointestinal discomfort, headache, vertigo, irritability or nausea.In contrast, others, such as leukopenia, hepatitis, pancreatitis, neurotoxicity, and teratogenic and mutagenic effects, are severe (Riches et al. 2020).
Increasing drug resistance is another limitation of antigiardial drugs.Indeed, this is a critical issue for anti-Giardia treatment because, during the last decade, the rates of treatment failure associated with parasite resistance increased significantly (Mørch and Hanevik 2020).
This scenario makes it necessary to keep a continuous search for sources of new effective and safe antigiardial drugs.In this context, it is well-known that natural products are a promising source of compounds with potential application in pharmacy and medicine.
Consequently, extracts from different parts of many terrestrial plants have been screened for their activity against Giardia trophozoites leading to the isolation of a substantial number of compounds with antigiardial activity (Calzada and Bautista 2020).In contrast, very few marine organisms have been investigated regarding their content in antigiardial compounds.
The coastal and marine territory of the Yucatan Peninsula, in southeastern Mexico and surrounded by the Gulf of Mexico and the Caribbean Sea, presents high habitat diversity, where 50 sponge species grow.Shallows and coastal lagoons of Yucatan, a state located at the north portion of Yucat an peninsula, harbour nine of those 50 species (Torruco-G omez and Gonz alez-Sol ıs 2010).The potential of these nine species as a source of compounds with antigiardial activity, as in most marine sponge species, remains unexplored.Therefore, as a part of our bioprospecting studies of marine invertebrates of the Yucatan state, Mexico, the organic extracts from Cinachyrella alloclada, Clathria (Clathria) foliacea, Halichondria magniconulosa, Haliclona (Reinera) tubifera, Suberites aurantiaca, and Oceanapia nodosa were screened for their in vitro activity against Giardia lamblia trophozoites.Furthermore, the dichloromethane extract from H. tubifera, was subject to a preliminary chemical study.

Results and discussion
Twenty-four extracts belonging to six sponge species collected in Yucatan coastal shallows (Supplementary material, Table S1) were screened in vitro against Giardia lamblia trophozoites (IC 50 ) and mammalian normal cells (CC 50 ) (Supplementary material).According to a previous report in this study, an extract was considered to have antigiardial activity if it showed an IC 50 10 mg/mL (Cantillo-Ciau et al. 2010).Also, it was considered that the antiprotozoal activity exhibited by an extract was not due to in vitro cytotoxicity if its selectivity index (SI ¼ CC 50 /IC 50 ) 10 (Vonthron-S en echeau et al. 2003).The results of the bioassays are summarized in Supplementary material, Table S2.
Seven extracts showed IC 50 's ranging from 1.0 to 9.74 mg/mL; consequently, they were considered to have antigiardial activity.The most active extracts were the dichloromethane and hexane extracts from H. tubifera, followed by the hexane extract of C. foliae, exhibiting IC 50 's ¼ 1.00, 2.11 and 3.44 mg/mL, respectively.Surprisingly, both extracts of H. tubifera were highly selective toward G. lamblia; indeed, the hexane extract resulted in the most selective one with a SI > 47.4 while the dichloromethanic extract showed a SI ¼ 41.8 being the second one.
Importantly, the H. tubifera dichloromethane extract meets both used criteria to define an antiprotozoal compound as a hit (Pink et al. 2005), which are a remarkable antiprotozoal activity (IC 50 1 lg/mL) as well as an excellent selectivity index (SI 10).Furthermore, the IC 50 of this extract was only 4.2-fold higher than that shown by positive control, metronidazole, the current drug used in the treatment of giardiasis.
In contrast to the low-polarity extracts, the ethyl acetate extract was three-fold less active against G. lamblia, besides exerting significant effects (Suffness and Pezzuto 1991) on normal mammalian cells (CC 50 ¼ 25.6 lg/mL).Conversely, the methanolic extract did not affect protozoan and Vero cells.
The ethyl acetate and methanol extracts of C. alloclada, and methanol extract of S. aurantiaca showed moderate antigiardial activity (CI 50 's 6.42-9.74mg/mL) and, curiously, their cytotoxic was low; while the extracts of H. magniconulosa and O. nodosa were non active (Supplementary Table S1).
Polyacetylenes have been the only class of compounds with antigiardiasic properties isolated from marine sponges (Quiñoa and Crews 1988;Sala et al. 2019).Interestingly, Haliclona genus has been recognized to be a source of compounds derived from the polyketide pathway, including polyacetylenes (Zhou et al. 2015) and sphingoids (Biegelmeyer et al. 2015).
Regrettably, it was not possible the isolation of polyacetylenes; nevertheless, the finding of aromatic compounds suggests that H. tubifera is a potential source of compounds derived from the polyketide pathway.

Experimental
See Supplementary material.

Conclusions
To the best of our knowledge, for the first time, extracts from marine sponge species growing in shallows of Yucatan, a state belonging to Yucatan Peninsula, Mexico, were screened for their in vitro activity against G. lamblia trophozoites.The dichloromethane extract of H. tubifera meets the criteria for an antiprotozoal compound to be considered a hit.The presence of benzaldehyde and benzoic acid derivatives in H. tubifera dichloromethane extract strongly suggests that this species is a potential source of polyketides.Finally, a further major study of this extract, and the hexane extracts of the same species and of Clathria foliacea, should be performed to isolate the compounds responsible for their antigiardial activity.