Salt-Tolerant Plants as Sources of Antiparasitic Agents for Human Use: A Comprehensive Review

Parasitic diseases, especially those caused by protozoans and helminths, such as malaria, trypanosomiasis, leishmaniasis, Chagas disease, schistosomiasis, onchocerciasis, and lymphatic filariasis, are the cause of millions of morbidities and deaths every year, mainly in tropical regions. Nature has always provided valuable antiparasitic agents, and efforts targeting the identification of antiparasitic drugs from plants have mainly focused on glycophytes. However, salt-tolerant plants (halophytes) have lately attracted the interest of the scientific community due to their medicinal assets, which include antiparasitic properties. This review paper gathers the most relevant information on antiparasitic properties of halophyte plants, targeting human uses. It includes an introduction section containing a summary of some of the most pertinent characteristics of halophytes, followed by information regarding the ethnomedicinal uses of several species towards human parasitic diseases. Then, information is provided related to the antiprotozoal and anthelmintic properties of halophytes, determined by in vitro and in vivo methods, and with the bioactive metabolites that may be related to such properties. Finally, a conclusion section is presented, addressing perspectives for the sustainable exploitation of selected species.


Introduction
Human parasitic diseases are predominantly linked to tropical or subtropical areas. However, climate change and the increased mobility of humans and animals trigger vector migration and the upsurge of parasitic infections in developed countries. Parasitic diseases continue to play a major role in human health, particularly infections caused by protozoans and helminths, such as malaria, trypanosomiasis, leishmaniasis, Chagas disease, schistosomiasis, onchocerciasis, lymphatic filariasis, and helminthiases, which are common in tropical regions and cause millions of morbidities and deaths every year [1,2].
For centuries, nature has been a source of medicines for the treatment of a vast array of diseases, with the first records of the ethnomedicinal uses of plants dating back to 2600 BC in Mesopotamia [3]. The easy access to terrestrial plants helps to explain their popularity as a source of bioactive products and innovative drug leads used in the pharmaceutical industry [4]. The importance of plant-based molecules as antiparasitic agents was reinforced by the Nobel Prize laureates in physiology or medicine in 2015, where Youyou Tu was honored for her discovery of artemisinin, a novel drug for the therapy against malaria, which was derived from Artemisia annua L., a plant used in the Chinese traditional medicine [5].
Most research targeting the identification of antiparasitic agents has focused on glycophytes, but salt-tolerant plants (halophytes) have lately aroused the interest of the scientific community due to their multiple medicinal properties, including antiparasitic activities.

Methodology
This review provides a focused overview of the potential use of halophytes as sources of bioactive molecules and/or natural products to tackle human parasitic diseases. A systematic search was performed to find all English articles with available full text related to the subject from 1993 until December 2022. Searches were performed by consulting several databases, including PubMed, Web of Science, Embase, and Google Scholar (as a search engine). The keywords "ethnomedicinal", "traditional use", "halophyte" and/or "salt tolerant plant" were used, alone or in combination with, for example, "antiparasitic", "antiprotozoal", and "anthelminthic" and its hyphenated variations. The classification of the plant species as halophytes was confirmed by a search in the eHALOPH database, and/or the description of their occurrence in coastal areas.
Herbal medicine has always had a fundamental role in human welfare, and it is still of utmost importance in many cultures today. In this sense, the ethnopharmacological knowledge of populations can serve as a base to search for novel bioactive compounds. Considering this, scientific research has explored and produced numerous studies about medicinal plants' biological properties and chemical constituents not only to advance plant drug discovery but also to validate the plant's traditional uses. However, for halophytes with folk uses, research on their bioactivities and subsequent evidence are still scarce for many of those plants. The next sections review some of the most relevant published work related to antiprotozoal (Section 4) and anthelmintic (Section 5) properties of halophyte species.

Halophyte Plants as Sources of Antiprotozoal Agents
Aligned to their traditional uses as antiparasitic agents, halophytes have proven by in vitro and in vivo research approaches their potential as sources of molecules with activity towards different protozoa species. Most antiprotozoal studies on natural products focus particularly on neglected tropical diseases (NTDs), a group of twenty infectious illnesses that include, for example, leishmaniasis, human African trypanosomiasis (HAT), Chagas disease, and schistosomiasis. NTDs affect more than 1 billion people worldwide, particularly very poor populations in tropical and subtropical areas in 149 countries [38][39][40]). Leishmaniasis is caused by more than 20 Leishmania species, while trypanosomiasis is ascribed to Trypanosoma, either the Trypanosoma brucei complex (sleeping sickness, human African trypanosomiasis) or T. cruzi (Chagas disease, American trypanosomiasis) [40]. Malaria, referred to as a "disease of poverty", is no longer recognized as an NTD [41] and is caused by protozoa of the genus Plasmodium, namely P. falciparum, P. vivax, P. malariae, and P. ovale, which are specific for humans [42].

In Vitro Activities and Bioactive Constituents
Most of the reports on the antiparasitic activity of halophyte species include an in vitro screening, followed by the determination of the chemical composition of raw extracts, and less frequently of purified fractions or pure compounds. Sixteen species belonging to 14 different families have been described with in vitro antiprotozoal activity, and those reports are summarized in Table 2.
Essential oils are described as a composite mixture of volatile molecules obtained from aromatic plants, mostly by hydrodistillation, and display highly relevant biological properties, including antiparasitic activities. Essential oils were the main target to evaluate the potential antiprotozoal properties of halophyte species, mostly against Leishmania and Trypanosoma parasites. For example, the essential oil of flowering aerial parts of Crithmum maritimum was highly effective towards T. brucei parasites (EC 50 = 5.0 µg/mL), which was linked to its mono-terpene hydrocarbon content, such as in limonene (EC 50 = 5.6 µM; Figure 1) and sabinene (EC 50 = 6.0 µM; Figure 1) [43]. However, they were less effective against L. infantum promastigotes (IC 50 = 122 and 205 µg/mL, respectively) [44]. The antiparasitic features of major compounds, including monoterpene hydrocarbons, sesquiterpene hydrocarbons, oxygen-containing sesquiterpenoids, and diterpenoids, are well described [44]. In turn, the essential oil of leaves and fruits of Pistacia lentiscus exerted high inhibitory effects against promastigotes of Leishmania major, L. tropica, and L. infantum, with IC 50 values varying between 8 and 26.2 µg/mL [45]. The major volatile components were myrcene and α-pinene in leaves, and α-pinene and limonene, in fruits, all reported with antileishmanial activities [46,47]. In another work, essential oils from leaves of P. lentiscus collected from two areas in Tunisia were tested against L. major intramacrophage and axenic amastigote forms [48], displaying moderate activities against intracellular amastigote (IC 50 = 12.5-35.6 µg/mL), and high activity against L. major axenic amastigote forms (IC 50 = 0.5 µg/mL) [48]. The main compounds were identified as pinene, β-myrcene, D-limonene, O-cymene, terpinen-4-ol, β-pinene, and α-phellandrene, which may disrupt parasite intracellular metabolic pathways [48]. Mar. Drugs 2023, 21, x FOR PEER REVIEW 9 of 21 Some essential oils from other species have been described with antiparasitic activities, but with higher IC50 values, such as those from P. oleracea leaves and stems (IC50 = 360 and 680 µg/mL) on L. major promastigotes [67], or from F. vulgare seeds against T. vaginalis (MLC = 1600 µg/mL) [54]. The essential oil of D. ambrosioides aerial organs have been investigated for its in vitro activity against L. amazonensis and L. donovani, being highly active towards their epimastigotes (IC50 = 21.3 µg/mL) and trypomastigotes (IC50 = 28.1 µg/mL), as well as towards T. cruzi amastigotes (IC50 = 50.2 µg/mL) [49]. Terpinolene was the major active component [49]. In addition, ascaridole, identified as the main component of D. ambrosioides leaves' essential oil, also exhibited in vitro activity against E. histolytica parasites, which are responsible for amebiasis, a parasitic disease considered a public health problem in developing countries [52].
Extraction with organic solvents has less expression than the extraction of essential oils, but even so, it also proves to be very effective in the extraction of compounds with antiprotozoal activity from salt-tolerant species. In this context, Oliveira and colleagues [60] performed an in vitro screening of T. cruzi trypomastigotes on 94 samples belonging to 31 halophytes species from Southern Portugal. From those, the dichloromethane extract of Juncus acutus roots was the most active (IC50 < 20 µg/mL), which was further fractionated, affording one active fraction with an IC50 of 4.1 µg/mL and selectivity index (SI) of 1.5. The active constituents were identified as phenanthrenes, dihydrophenanthrenes, and benzocoumarins [60]. C. maritimum flower decoction also presented anti-T. cruzi activity with an EC50 value of 17.7 µg/mL and SI of 5.65, and a fraction rich in falcarindiol has an increased activity (EC50 = 0.47 µg/mL) and selectivity (SI = 59.6) [53].
The same research group also screened 25 salt-tolerant plant species from southern Portugal for promastigotes and intracellular amastigotes of L. infantum, and the highest activity was obtained with the dichloromethane extract of S. rubra and I. crithmoides aerial organs [56]. The active extracts from I. crithmoides were rich in phenolic acids (gallic, syringic, salicylic caffeic, coumaric, and rosmarinic acids) and flavonoids (epicatechin, epigallocatechin gallate, catechin hydrate, quercetin, and apigenin), while catechin hydrate was detected in S. rubra [56].
In addition, hexane and methanol extracts of F. vulgare seeds were highly active against T. vaginalis (MLC = 360 µg/mL) [54], whereas aqueous extracts exhibit anti-Blastocystis activity with IC50 values ranging between 223.8 and 174.9 µg/mL [55]. The predominant compounds in F. vulgare were hesperidin, ferulic, and chlorogenic acid [55].     Some essential oils from other species have been described with antiparasitic activities, but with higher IC 50 values, such as those from P. oleracea leaves and stems (IC 50 = 360 and 680 µg/mL) on L. major promastigotes [67], or from F. vulgare seeds against T. vaginalis (MLC = 1600 µg/mL) [54]. The essential oil of D. ambrosioides aerial organs have been investigated for its in vitro activity against L. amazonensis and L. donovani, being highly active towards their epimastigotes (IC 50 = 21.3 µg/mL) and trypomastigotes (IC 50 = 28.1 µg/mL), as well as towards T. cruzi amastigotes (IC 50 = 50.2 µg/mL) [49]. Terpinolene was the major active component [49]. In addition, ascaridole, identified as the main component of D. ambrosioides leaves' essential oil, also exhibited in vitro activity against E. histolytica parasites, which are responsible for amebiasis, a parasitic disease considered a public health problem in developing countries [52].
Extraction with organic solvents has less expression than the extraction of essential oils, but even so, it also proves to be very effective in the extraction of compounds with antiprotozoal activity from salt-tolerant species. In this context, Oliveira and colleagues [60] performed an in vitro screening of T. cruzi trypomastigotes on 94 samples belonging to 31 halophytes species from Southern Portugal. From those, the dichloromethane extract of Juncus acutus roots was the most active (IC 50 < 20 µg/mL), which was further fractionated, affording one active fraction with an IC 50 of 4.1 µg/mL and selectivity index (SI) of 1.5. The active constituents were identified as phenanthrenes, dihydrophenanthrenes, and benzocoumarins [60]. C. maritimum flower decoction also presented anti-T. cruzi activity with an EC 50 value of 17.7 µg/mL and SI of 5.65, and a fraction rich in falcarindiol has an increased activity (EC 50 = 0.47 µg/mL) and selectivity (SI = 59.6) [53].
The same research group also screened 25 salt-tolerant plant species from southern Portugal for promastigotes and intracellular amastigotes of L. infantum, and the highest activity was obtained with the dichloromethane extract of S. rubra and I. crithmoides aerial organs [56]. The active extracts from I. crithmoides were rich in phenolic acids (gallic, syringic, salicylic caffeic, coumaric, and rosmarinic acids) and flavonoids (epicatechin, epigallocatechin gallate, catechin hydrate, quercetin, and apigenin), while catechin hydrate was detected in S. rubra [56].
Overall, some authors defined that extracts with IC 50 values below 15 µg/mL and selectivity above 3 can be considered promising for further development as drug leads [69]. Following this guideline, the fraction of flower decoction of C. maritimum can be considered the most promising sample with anti-T. cruzi activity by coupling both criteria (EC 50 = 0.47 µg/mL; SI = 59.6) [53].

In Vivo Studies
Only a few authors evaluated the in vivo antiprotozoal potential of halophytes, including only three species and using mainly rodents as models. These reports are summarized in Table 3.
The most studied species was D. ambrosioides, investigated by five different authors. For instance, essential oil from its aerial parts was more effective against experimental cutaneous leishmaniasis by L. amazonensis in BALB/c mice than its pure main components, namely ascaridole, carvacol, and caryophyllene oxide [72]. In turn, hydroalcoholic extracts of its leaves displayed in vivo antimalarial properties against BALB/c mice infected with P. berghei intraperitoneally [51], and in vivo effects on C3H/HePas mice infected with L. amazonensis promastigotes [73]. Moreover, ascaridole was the main component in D. ambrosioides leaves' essential oil and exhibited in vitro and in vivo activity against E. histolytica parasites [52]. Besides, other authors reported the anti-Plasmodium efficacy of ethanolic extracts of A. officinalis flowers (P. falciparum IC 50 = 62.7 µg/mL; P. berghei suppression of parasitemia in vivo [400 mg/kg] = 62.86 %), and of P. major seeds (P. falciparum: IC 50 = 40.0 µg/mL; P. berghei suppression of parasitemia in vivo [400 mg/kg] = 22.5%) [65].

Halophyte Plants as Sources of Anthelmintic Agents
Supported by their traditional uses, some halophyte species have also demonstrated in vitro and in vivo potential as sources of molecules with anthelmintic activity. Most anthelmintic studies on natural products focus mainly on the model nematode Caenorhabditis elegansi, or gastrointestinal nematodes (GINs), namely Haemonchus contortus and Trichostrongylus colubriformis, a leading cause of production loss in agricultural animal systems worldwide [74,75]. Since different Trichostrongylus species, as for example, T. colubriformis, can infect humans in different areas of the world (e.g., Iran, Laos, Australia) [76], that species was included in this review.

In Vitro Activities and Bioactive Constituents
Most of the reports on the anthelmintic activity of halophytes were performed only in vitro, and only less than half included a chemical characterization of its major constituents. Twelve species from 9 families have been described with in vitro anthelmintic properties, and those reports are included in Table 4.
Oliveira and colleagues [77] have screened 80% acetone extracts from 8 halophyte species for their anthelmintic capacity using the Larval Ensheathment Inhibition Assay (LEIA) and Egg Hatching Inhibition Assay (EHIA) for T. colubriformis. P. lentiscus, L. monopetalum, C. mariscus, and H. italicum picardi were the most active in both GINs and life stages of the nematodes [77]. In particular, C. mariscus aerial parts collected during summer were more active against T. colubriformis (EC 50 = 77.8 µg/mL) [78]. Moreover, inflorescences presented the highest anthelmintic activity against T. colubriformis [EC 50 (LEIA) = 78.6 µg/mL; IC 50 (EHIA) = 848.2 µg/mL]. Flavan-3-ols, proanthocyanidins, luteolin, and glycosylated flavonoids are the main active components [78]. Some authors have also tested some halophyte extracts against S. mansoni, which is a helminth species and the causal agent of schistosomiasis in humans. Methanol extracts of C. ambrosioides reduced S. mansoni cercariae infectivity [79], as well as essential oils from F. vulgare that showed moderate in vitro activity against S. mansoni worms, but with more remarkable effects in egg development, possibly attributable to the two main constituents detected in the essential oil, I-anethole and limonene [80]. A compound isolated from G. inflata, licochalcone A (Figure 2), presented an LC 50 of 9 µM towards S. mansoni female and male adult worms [81].

Halophyte Plants as Sources of Anthelmintic Agents
Supported by their traditional uses, some halophyte species have also demonstrated in vitro and in vivo potential as sources of molecules with anthelmintic activity. Most anthelmintic studies on natural products focus mainly on the model nematode Caenorhabditis elegansi, or gastrointestinal nematodes (GINs), namely Haemonchus contortus and Trichostrongylus colubriformis, a leading cause of production loss in agricultural animal systems worldwide [74,75]. Since different Trichostrongylus species, as for example, T. colubriformis, can infect humans in different areas of the world (e.g., Iran, Laos, Australia) [76], that species was included in this review.

In Vitro Activities and Bioactive Constituents
Most of the reports on the anthelmintic activity of halophytes were performed only in vitro, and only less than half included a chemical characterization of its major constituents. Twelve species from 9 families have been described with in vitro anthelmintic properties, and those reports are included in Table 4.
Oliveira and colleagues [77] have screened 80% acetone extracts from 8 halophyte species for their anthelmintic capacity using the Larval Ensheathment Inhibition Assay (LEIA) and Egg Hatching Inhibition Assay (EHIA) for T. colubriformis. P. lentiscus, L. monopetalum, C. mariscus, and H. italicum picardi were the most active in both GINs and life stages of the nematodes [77]. In particular, C. mariscus aerial parts collected during summer were more active against T. colubriformis (EC50 = 77.8 µg/mL) [78]. Moreover, inflorescences presented the highest anthelmintic activity against T. colubriformis [EC50 (LEIA) = 78.6 µg/mL; IC50 (EHIA) = 848.2 µg/mL]. Flavan-3-ols, proanthocyanidins, luteolin, and glycosylated flavonoids are the main active components [78]. Some authors have also tested some halophyte extracts against S. mansoni, which is a helminth species and the causal agent of schistosomiasis in humans. Methanol extracts of C. ambrosioides reduced S. mansoni cercariae infectivity [79], as well as essential oils from F. vulgare that showed moderate in vitro activity against S. mansoni worms, but with more remarkable effects in egg development, possibly attributable to the two main constituents detected in the essential oil, I-anethole and limonene [80]. A compound isolated from G. inflata, licochalcone A (Figure 2), presented an LC50 of 9 µM towards S. mansoni female and male adult worms [81].
Besides, other authors have focused on other less frequently tested parasite species. For instance, the roots of G. glabra contain glycyrrhizic acid ( Figure 2) that is very active in vitro against B. malayi microfilarae (IC50 = 1.20 µM), one of the most important causative agents of human lymphatic filariasis [82].  Besides, other authors have focused on other less frequently tested parasite species. For instance, the roots of G. glabra contain glycyrrhizic acid (Figure 2) that is very active in vitro against B. malayi microfilarae (IC 50 = 1.20 µM), one of the most important causative agents of human lymphatic filariasis [82].

In Vivo Studies
There is a reduced number of studies focusing on in vivo anthelmintic properties of halophytic species. In fact, to our best knowledge, only three species were tested, mostly using rodents and sheep as models (Table 5).
Pistacia lentiscus is the most studied halophyte species due to its high level of tannins, known to exhibit high anthelmintic activity, as confirmed by in vivo studies against T. colubriformis. Infected goats fed with aerials parts of P. lentiscus presented a 16% reduction in fecal oocyst counts, whereas, in lambs, the reduction ranged between 55.2 and 61.3% [84,85]. In turn, S. mansoni infected mice treated orally with a methanol extract of C. ambrosioides (1250 mg/kg/day) exhibited a 53.7% total worm burden decrease and a 60.3% ova/g tissue in liver reduction, 9 weeks post-infection [86]. Hymenolepis diminuta infected Wistar rats were treated with C. dactylon methanol extract (800 mg/kg) enabling a reduction of 77.6% in eggs and 79% in adult worms counts, respectively [83].

Conclusions
The importance of halophytes as sources of food and medicinal commodities is rising. Their role is of particular importance in the context of soil and water salinization since they can be commercially cultivated in underutilized saline areas and under sustainable saline agricultural systems. This review collected for the first time data on halophytes' ethnomedical properties towards human parasitic infections, as well as the existing scientific reports on its in vitro and in vivo experiments on antiprotozoal and anthelmintic properties. Sixteen species from 14 different families have reported antiprotozoal properties, with most studies focusing on different strains of Leishmania, Trypanosoma and Plasmodium parasites. In this context, eight pure compounds occurring in halophyte species were identified with antiprotozoal properties, namely limonene and sabinene against T. brucei, licochalcone A against L. major, oleanolic acid, kaempferol and quercetin anti-L. donovani, and betulinic acid and α-amyrin towards T. cruzi and P. falciparum, respectively.
In turn, 15 species from 12 families were studied for their anthelmintic capacity, mainly against Trichostrongylus gastrointestinal nematodes. Essential oils were the most represented type of extract, followed by extracts made with different combinations of water and organic solvents. From the in vivo studies, the most promising species is D. ambrosioides, including essential oil from aerial organs towards cutaneous leishmaniasis and hydroalcoholic extracts of its leaves, with antimalarial properties. Ascaridole, the major metabolite detected in D. ambrosioides leaves essential oil, exhibited in vitro and in vivo activity against E. histolytica parasites, responsible for amebiasis. Regarding anthelmintic properties, the most promising species is P. lentiscus, as confirmed by in vivo studies of T. circumcincta, and T. colubriformis nematodes. Furthermore, a few compounds were isolated from halophyte species with anthelmintic properties, such as licochalcone A from G. inflata with anti-S. mansoni activity, and glycyrrhizic acid obtained from G. glabra roots with in vitro activity against B. malayi microfilariae. Therefore, halophytes have shown to be promising sources of compounds with potential application as antiparasitic commodities for both humans and animals, including neglected tropical diseases related to protozoan, and gastrointestinal nematodes infections. The main gap in the search for halophytic products with antiparasitic activity is the isolation and identification of novel molecules. Thus, efforts must be made in this regard.

Conflicts of Interest:
The authors declare no conflict of interest.