Halophyte Plants as Potential Sources of Anticancer Agents: A Comprehensive Review

Salt-tolerant plants (halophytes) are widely distributed worldwide in several environments such as coastal salt marshes, sand dunes, and inland deserts. To cope with the harsh conditions that characterize those habitats, which include high salinity and radiation levels, such plants have developed morphological and physiological traits, the latter including the synthesis and accumulation of important secondary metabolites such as alkaloids and polyphenols. While essential in maintaining plant homeostasis, these compounds are highly valued in the medical field for the treatment of several human diseases, including cancer. Cancer is one of the most life-threatening disorders worldwide, which accentuates the need to improve current cancer therapies and minimize potential adverse secondary side-effects. In this context, the pharmacological evaluation of natural compounds has attracted growing interest since nature has already provided some important anti-cancer drugs. This review compiles, for the first time, research regarding the anticancer activity of halophytes from different families, including, whenever possible, the bioactive molecules involved in such therapeutical properties along with possible mechanisms of action. The introduction section provides some pertinent information regarding cancer and a summary of the most important characteristics of halophytes. The next section gives information regarding the in vitro and in vivo cytotoxic properties of several halophyte species, grouped by families, including contents in bioactive metabolites and proposed modes of action, if possible. Lastly, the conclusion presents the most relevant metabolites and/or promising species and extracts that could be further explored in anticancer drug research.


Introduction
Cancer is a group of diseases characterized by malignant neoplasms arising from the abnormal and uncontrolled cell proliferation that invades and destroys the surrounding tissue and, if not controlled, can result in death [1,2]. With an established complex link between cancer and aging and the increased risk factors of an unhealthy lifestyle, cancer is undoubtfully a major health problem in developed and developing countries [3][4][5][6][7]. It is estimated that, by 2040, the number of new cancer cases diagnosed per year will be higher than 27.5 million worldwide, with 16.3 million cancer deaths due to population growth and aging [8]. Globally, lung and breast cancers are the most frequently diagnosed and are the leading causes of cancer-related death in men and women, respectively [9]. Significant advances have been made in cancer research in the last 20 years concerning its general biology, prevention, and treatment [10]. Currently, chemotherapy is one of the most common cancer therapeutic approaches, although secondary effects are known and include a negative impact on the immune system, and the effectiveness of this therapeutic

Aizoaceae Family
Aizoaceae contains mostly succulent plants with 1910 species currently recognized, distributed across 125 genera [41,42]. The Mesembryanthemum genus, for example, is used in traditional medicine to treat several diseases such as hepatic conditions and diabetes, and it contains several bioactive compounds, including flavonoids and catechins [43]. An ethanol crude extract from leaves of Mesembryanthemum crystallinum L. (ice plant), and obtained hexane, ethyl acetate, butanol, and water fractions were tested for their effect on the growth of HCT116 cells [44] (Table 1). The application of the ethyl acetate and butanol fractions resulted in a dose-dependent inhibition of cell growth (at 125, 250, and 500 µg/mL, applied for 24 and 48 h), as observed in the MTT assay, a reduction in the levels of intracellular ROS (at 250 and 500 µg/mL, using 2 ,7 -dichlorofluorescin-diacetate), and apoptosis induction (at 250 and 500 µg/mL, during 48 h). Treatment with the butanol fraction also resulted in cell-cycle arrest in the G2/M phase [44]. Samples were evaluated for total phenolic content (TPC), using the Folin-Ciocalteau (F-C) assay, and it was observed that the butanol fraction had the highest TPC (5.4 mg gallic acid equivalent/g) [44], suggesting that phenolics may be related to the detected cytotoxicity. However, no attempt was made by the authors to identify the active molecules present in this sample.
The application of ethanol, methanol, acetone, hexane, and diethyl-ether extracts from whole plants of Sesuvium portulacastrum L. (sea purslane) decreased the viability of MDA-MB-231, IMR32, and HCT116 cell lines, as assessed by MTT after 24 h of incubation, using doxorubicin as the standard [45]. The hexane extract had the highest cytotoxic activity with IC 50 values of 942.07, 703.40, and 407.87 µg/mL for MDA-MDB-231, IMR32, and HCT116 cell lines (Table 1). Despite the considerably high IC 50 values obtained, the possible mechanisms of action of the extracts were evaluated using phase contrast-inverted microscope observation and propidium iodide staining, which revealed the presence of typical features of apoptotic cells, including nuclear condensation, cell shrinkage, and the presence of apoptotic bodies [45] (Table 1). No attempt was made by the authors to unravel the chemical composition of the extracts.

Amaranthaceae Family
Amaranthaceae currently comprises 2398 species grouped in 182 genera [46], which can be found in a wide range of habitats from arid and semiarid regions to saline environments and subtropical areas; it contains flavonoids, phenolic acids, terpenes, and triterpene saponins, and it displays important pharmacological properties such as anticarcinogenic [47,48].
Arthrocnemum sp. is a small genus of succulent halophytes present in coastal marshes of South Africa, Asia, Mediterranean, Europe, and Northern America, and it contains several compounds such as phenols, flavonoids, and tannins with described therapeutic properties, including antibiotic, hypoglycemic, and antitumoral [49,50]. The application of an 80% methanol extract from shoots of Arthrocnemum indicum (Willd.) Moq. significantly reduced the viability of Caco-2 cells, as observed by the MTT assay [50] (Table 1). Fluorescence microscopy observation through DAPI (4,6-diamidino-2-phenylindole) staining showed that cells exhibited a decline of DNA synthesis, while flow cytometry allowed for the observation of cell-cycle arrest at the G2/M phase after 72 h of exposure to the extract at the concentration of 100 µg/mL [51] ( Table 1). The extract was profiled by liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS), and high levels of phenolic compounds were detected such as gallic acid, cyanidin, chrysoeriol, quercetin, catechol, syringic acid, and luteolin (Table 2), although their cytotoxicity toward Caco-2 cells was not established [51]. However, it is known that some of the detected compounds exhibit antitumoral properties. For example, chrysoeriol was identified as a selective inhibitor of the PI3K-AKT-mTOR pathway and, therefore, linked with cell cycle regulatory effects; it also reduced the proliferation of human multiple myeloma cells (RPMI 8226 and KM3, with IC50 values of 26 and 35 µmol/L at 48 h) but not of peripheral blood mononuclear cells (PBMNCs) [52].
Several Atriplex species are halophytic [53]. An ethanol extract from leaves of Atriplex halimus L. (sea orache) significantly reduced the viability of HepG2 cells (IC 50 = 54.86 µg/mL after 24 h of incubation) ( Table 1) via apoptosis induction linked to the expression of TP53, BCL2, and BAX genes [54] (Table 1). No information was provided by the authors regarding the chemical components of such extract, but it is known that A. halimus is characterized by the presence of different bioactive compounds, including syringetin derivatives and the flavonol glycosides atriplexoside A , which may be related to the detected cytotoxic activity [55][56][57][58][59][60].
Chenopodium contains weedy herbs native to Asia, Europe, and America, rich in phenolics, saponins, and triterpenoids, conferring them therapeutic properties such as laxative, analgesic, and anticarcinogenic [61]. The edible species Chenopodium album L. (lamb's quarters) is valued in traditional medicine for its anticancer properties [62]. A petroleum ether extract of branches and leaves from C. album reduced the viability of A549 cells in a dose-dependent manner, showing an IC 50 of 33.31 µg/mL using gemcitabine as a positive control, which was linked to cell-cycle arrest at the G1 phase [63] (Table 1). Essential oil from whole plants of Chenopodium ambrosioides L. (Mexican tea) decreased the viability of MCF7 cells by inducing DNA fragmentation (IC 50 values of 18.75, 9.45, and 10.50 µg/mL at 6, 24, and 48 h, respectively) when compared to the control treatment [64] ( Table 1). In another study, an essential oil of the same species also displayed a significant cytotoxic activity toward RAJI cells, with an IC 50 value of 1.0 µg/mL, which was probably related to the high levels of ascaridole, detected using GC with flame ionization detection (GC-FID), GC-MS, and proton nuclear magnetic resonance (1H-NMR) [65] (Tables 1 and 2). Ascaridol is a monoterpene with a strong in vitro capacity to decrease the growth of Pharmaceutics 2022, 14, 2406 6 of 45 different tumor cell lines and is, therefore, considered as a strong candidate for the treatment of cancer [65]. Chenopodium quinoa Willd is a pseudocereal originated from the Andes of South America with recognized nutritional and functional properties that rendered the species the status of "functional food" [66]. Polysaccharides were extracted from quinoa seeds with petroleum ether and purified by column chromatography, revealing mainly galacturonic acid and glucose monosaccharides. This polysaccharide fraction was applied toward cancer (SMMC 7721 and MCF7) and "normal" (L02 and MCF 10A) cell lines, for 24 and 48 h, allowing for IC 50 values ranging from 53.4 to 121.4 µg/mL, without affecting "normal" cell viability [66] (Table 1).
Salicornia species (glasswort, sea asparagus, or samphire) are edible succulent halophytes highly appreciated in gourmet cuisine due to their organoleptic properties [67]. The ethyl acetate and methanol leaf extracts from Salicornia europaea L. reduced the viability of MCF7 cells with IC 50 values of 97.9 and 117.1 µg/mL for the ethyl acetate and methanol extracts, respectively, after 24 h of incubation [68]. A qualitative analysis of the extracts identified tannins and saponins, which may be associated with the in vitro antitumor capacity. Moreover, a GC-MS analysis showed the presence of 32 and 29 compounds in corresponding ethyl acetate and methanol extracts, respectively, which were not assessed individually against the cancer cells [68]. Tannins are phenolic compounds with a high molecular weight, while saponins contain a steroidal or triterpenoid aglycone and one or more sugar chains [69]. Both groups of compounds exhibit antitumoral properties; for example, saponins generally decrease cellular proliferation by binding to cholesterol structures on cell membranes, forming pores and holes by binding to specific receptors followed by the induction of apoptosis, while tannins can hamper cancer cell proliferation through antioxidant processes and apoptosis induction [70,71]. However, the presented results must be analyzed with caution, since the phytochemical analysis reported by the authors only targets lipophilic compounds, without unraveling the possible cytotoxic molecules present in the methanol (polar) extract.
Suaeda comprises around 80 to 100 succulent species distributed in semideserts, deserts, and seashores. These species are usually edible and contain several bioactive components, such as phenolics, flavonoids, and terpenoids, conferring them with bioactive properties, including anticarcinogenic [72,73]. A hexane extract from leaves of Suaeda ruticose (L.) Forssk. (shrubby seablight) reduced the viability of HCT116, HepG2, and MCF7 cell lines, being more effective toward HCT116 cells (IC 50 = 17.2 µg/mL) [74] (Tables 1 and 4). The cytotoxicity of the extract was tested using the sulforodamine B (SRB) assays for 72 h using doxorubicin as a positive control with IC 50 values of approximately 0.5 µg/mL. This extract caused cell-cycle arrest at the G0-G1 phase and induced apoptosis, especially in HCT116 cells that exhibited chromatin condensation and membrane blebbing ( Table 4). The extract was profiled using liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS) and several molecules were identified, including monoterpenes (dihydrojasmone, jasmolone, terpinene-4-ol), diterpenes (pimaric acid, steviol, and momilactone B), and phenolics (quercinol, zingerone, zingerol, and neovaflan) ( Table 4), but its contribution to the detected cytotoxicity was not directly established [74]. However, there are several reports on the antitumoral properties of phenolic compounds [56][57][58][59]. Specifically, zingerone reduced the viability of HCT116 cells through ROS-mediated apoptosis in colon cancer cells [75]. Monoterpenes and diterpenes have reported cytotoxic properties [76,77]. For instance, dihydrojasmone was identified as one of the active compounds toward HeLa and NIH/3T3 cell lines, in a methanol extract from Rumex hastatus D. Don [78], while steviol significantly reduced the viability of MCF7 cells via apoptosis induction [79]. A dichloromethane extract from the shoots of S. fruticosa significantly reduced the viability of A549 (IC 50 = 49 µg/mL), DLD1 (IC 50 = 10 µg/mL), Caco-2 (IC 50 = 140 µg/mL), and HT-29 cells (IC 50 = 12 µg/mL), which was assessed using the resazurin reduction test after 48 h of incubation (Tables 1 and 4). Nevertheless, the bioactive compounds present in the chemical profile of the extracts were not identified [80]. Additional studies on the anticarcinogenic activity of other halophytic species of this family are summarized in Table 1.

Apiaceae Family
Apiaceae currently comprises 3916 species distributed across 457 genera dispersed worldwide [81], and it is characterized by the presence of polyacetylenes displaying important bioactivities, including anticarcinogenic [82]. In this family, there are two genera of halophytic species, Eryngium sp. and Crithmum sp., with described cytotoxic properties against human cancer cells.
Eryngium includes approximately 250 species present in Eurasia, North Africa, North and South America, and Australia. Eryngium species are frequently used as ornamentals or as a food source and are valued in traditional medicine as a diuretic, to treat diarrhea, headaches, and digestive problems. These properties are potentially associated with the presence of terpenoids, triterpenoids, polyacetylenes, flavonoids, and coumarins [83]. The cytotoxic effects of aqueous extracts from shoots and roots of Eryngium maritimum L. were assessed against human cancer lines (HepG2, HEP-2, and U138MG) and on a "normal" cell line (Vero), using the MTT assay after 24 h of incubation [70] ( Table 1). The root extracts were overall more cytotoxic, and the IC 50 values ranged from 30.3 to 50 µg/mL. However, a strong reduction in cellular viability was also observed in the "normal" cell line, which suggest no selectivity toward cancer cells [70] (Table 1). Although the phytochemical profile of the extracts was not established by the authors, other reports identified several cytotoxic molecules in E. maritimum, including saponins, which create pores and perforations on the cellular membranes by binding to specific receptors, followed by apoptosis induction by, for example, stimulation of the cytochrome c-caspase 9-caspase 3-pathways [70,71].
Crithmum maritimum L. (sea fennel) is traditionally used as a cooking ingredient and in folk medicine for its stimulating and diuretic effects [84]. An ethyl acetate extract from the whole sea fennel plant reduced the proliferation of Huh7 and HepG2 cell lines, by interfering with the cell cycle, specifically on the shift of phases with increasing number of cells in the G0/G1 phase after 24 h of incubation and in the G2/M phase after 48 h [85]. The extract induced an increase in necrotic and apoptotic cancer cells, assessed by cytofluorimetric analysis although the values of IC 50 , but the chemical profile of the extract was not assessed [85] (Table 1).

Asteraceae Family
Asteraceae currently contains 1733 genera and 35,988 species [86], including several used as ornamentals (e.g., Calendula and Chrysanthemum) or for food (e.g., Helianthus annuus) and medicinal (e.g., Artemisia sp. and Echinacea sp.) purposes [87]. Achillea millefolium L. (yarrow) is an aromatic perennial herb with traditional medicinal uses such as wound healing and anti-inflammatory activity, and it contains different classes of bioactive compounds, including flavonoids and terpenoids [88]. A methanol extract from yarrow shoots was applied toward prostate cancer (DU145) and "normal" skin (HFFF2) cells, at different concentrations (20, 100, 500, 1000, and 2000 µg/mL), alone or in combination with bleomycin, an anticancer agent. The yarrow extract was not toxic toward "normal" cells, but had significantly enhanced cytotoxicity induced by bleomycin showing 60% and 49% survival rate at doses of 1000 and 2000 µg/mL, respectively, which may indicate that this extract contains molecules able to improve the effectiveness of bleomycin, while minimizing negative side-effects caused by toxicity toward "normal" cells. [88] ( Table 1). The extract was not profiled for chemical components, but there are reports of the presence of several cytotoxic compounds in yarrow, such as achillinin A (guaianolide) and casticin (flavonoid) [88,89]. In another work, petroleum ether, ethyl acetate, methanol, and water extracts were prepared from aerial organs of yarrow and tested for toxicity, using the MTT assay, on human cancer cell lines [90] ( Table 1). The strongest cytotoxic effect was observed after application of the ethyl acetate extract on HeLa (IC 50 = 0.58 µg/mL) and K562 cells (IC 50 = 0.73 µg/mL), followed by the water (MCF-7, IC 50 = 0.87 µg/mL) and the petroleum  (Tables 1 and 4) [90]. The cytotoxic activity of these extracts may be associated with its contents in phenolic acids, such as apigenin and chlorogenic, p-coumaric, and rosmarinic acids (Table 4), which showed the capacity to block oncogenic pathways due to the activation of caspases [91]. A methanol extract from aerial parts of Limbarda crithmoides (L.) Dumort (commonly known as Inula crithmoides L.) was able to decrease the viability of acute myeloid leukemia cells (OCI-AML3) when applied at 100 and 200 µg/mL, for 24 h [92] (Table 1). This extract was submitted to a solventsolvent partitioning, affording n-hexane, dichloromethane, and aqueous methanol-soluble fractions. The hexane and dichloromethane fractions exhibited a strong cytotoxicity toward OCI-AML3 cells at concentrations of 15 or 10 µg/mL, which was ascribed to an increase in apoptotic cells, especially in the G0/G1 phase by the mitochondria-dependent pathway. The hexane extract was then further fractionated, leading to the isolation of two molecules, which were identified by NMR as the thymol derivatives 10-acetoxy-8,9-epoxythymol tiglate and 10-acetoxy-9Z-chloro-8,9-dehydrothymol, with the latter being the most active, causing a decrease in cell viability at 1.25 µg/mL associated with apoptosis induction [92].

Brassicaceae Family
Brassicaceae is one of the largest dicotyledon family of flowering plants, including model species and commercial crops, with 341 genera and 3921 species recognized at the moment [93,94], but few studies have described the potential anticarcinogenic properties of halophytes belonging to this family. One example is the annual halophyte Cakile maritima Scop. (sea rocket), which is confined to maritime strandlines of sand and has agronomic (oilseed and phytoremediation) and medicinal (diuretic, antiscorbutic, and purgative) properties [95]. Hexane, ethyl acetate, and methanol fractions were obtained from a methanol extract prepared from aerial organs of sea rocket and tested for antiproliferative properties on Caco-2 and HeLa carcinoma cells [96] ( Table 1). The hexane fraction significantly reduced the viability of Caco-2 and HeLa cells, with IC 50 values of 12 and 126 µg/mL after 24 h of incubation; respectively. Cisplatin was used as the positive control with values of 69 µg/mL (Caco-2) and 85 µg/mL (HeLa). The extract was profiled using GC-MS, and the major molecules identified were, by area, 2-hydroxy-1,8-cineole, decane, and limonene, which may contribute to the detected antiproliferative activity [96] (Table 4). For instance, limonene has been reported with anticarcinogenic activity in HepG2 cells due to apoptosis induction [97].

Convolvulaceae Family
Convolvulaceae currently contains 50 genera and 1952 species widely distributed in tropical and temperate regions of the world [98,99]. Calystegia soldanella (L.) R. Br. ex Roem. & Schult. (shore bindweed) is a perennial edible herb commonly found in coastal sand dunes and foredunes of South Korea, East Asia, Europe, and the Pacific. Shore bindweed is traditionally used for the treatment of, for example, rheumatic arthritis and scurvy, and it displays relevant biological properties, including anticancer [100]. An 85% aqueous fraction from combined methylene chloride and methanol crude extracts obtained from whole plants of shore bindweed significantly decreased HepG2 cellular viability after 24 h of incubation, in a concentration-dependent manner, via cell-cycle arrest at the G0-G1 and S phases and apoptosis induction [100]. In another study, a methanol crude extract from whole plants of the same species exhibited a potent cytotoxic activity toward A549 cells (human lung cancer) and Col2 cells (human colon cancer), with IC 50 values of 8.0 µg/mL and 27.4 µg/mL, respectively [101]. A hydroalcoholic extract from shoots of Cressa cretica L. decreased the viability of HepG2 cells (IC 50 value = 2300 µg/mL after 72 h of incubation), by increasing the expression of the proapoptotic protein BAX in detriment to antiapoptotic proteins (BCL2) [102].

Cymodoceaceae Family
This family presently contains six genera and 17 species of seagrasses [103,104]. A water leaf extract from Cymodocea rotundata Ehrenb Hempr. ex Aschers. was supplemented with silver nitrate (AgNO 3 , 1 M), to produce silver nanoparticles (AgNPs). AgNPs have several medical applications, including coating of medical devices and wound dressings, and they exhibit cytotoxic activity [105]. The obtained AgNPs exhibited high cytotoxicity toward MG63 cells with an IC 50 value of 25.31 µg/mL after 48 h of incubation [105]. AgNPs were also produced by combining a water leaf extract of Cymodocea serrulata (R. Br.) Aschers. & Magnus and AgNO 3 (1 M), and they reduced the viability of A549 cells in a direct doseresponse manner (IC 50 = 100 µg/mL after 24 h of incubation). The higher cytotoxicity of the AgNPs was ascribed to the easy permeability to the cellular barriers and their high affinity to biological macromolecules, as well as their capacity to release ROS [106]. No attempt was made to identify the active molecules present in those samples [105,106]. A hydroethanolic extract from shoots of the same species inhibited the proliferation of HepG2 cells (IC 50 value of 82.92 µg/mL after 24 h of incubation with camptothecin as a positive control with an IC 50 value of 8 µg/mL) [107]. The extract had a high concentration of tannins, flavonoids, and terpenoids, with described antitumoral properties [108], but no attempt was made by the authors to identify the possible bioactive molecules [107].

Cyperaceae Family
Cyperaceae currently comprises 92 genera and 5888 species with a cosmopolitan distribution [109,110]. Cyperus rotundus L. (nut grass) is a perennial halophyte species traditionally used in the treatment of several pathologies, such as stomach disorders [111]. Methanol, ethanol, and water extracts from nut grass rhizomes were tested toward MDA-MB-231 cells, for 24 h [112] ( Table 1). The highest reduction in cell viability was obtained with the ethanol extract, with an IC 50 value of 225 µg/mL, through apoptosis induction via upregulation of the death receptor 4 (DR4), DR5, and proapoptotic BAX, and downregulation of antiapoptotic survivin and BCL2 [112]. The active extract was not chemically profiled, but it is known that the rhizomes of nut grass are rich in several bioactive molecules, including flavonoids, tannins, and sesquiterpenes [113], with reported cytotoxic activity against cancer cell lines.
The genus Alhagi is distributed throughout Asia, Australia, and Europe and used traditionally for the treatment of, for example, gastroenteritis, ulcers, and rheumatoid arthritis [116]. Alhagi maurorum Medik (camelthorn) is used for its anti-inflammatory properties, which are ascribed to the presence of the triterpenoid lupeol [117]. In fact, lupeol was isolated from a methanol extract of camelthorn aerial parts, and it decreased the cellular viability of MCF7 and MDA-MB-231 cell lines, with one-fourth of IC 50 values >100 µg/mL (Table 1) [118]. The cytotoxic activity of lupeol was lower than that observed with its epoxide form, and it was related to the increase in mRNA expression levels of apoptosisrelated genes (TP53, caspase-3 and BAX) and decrease in BCL2 gene expression [118].
Glycyrrhiza (liquorice) contains legumes endogenous to Asia and southern Europe with reported anti-inflammatory and antiviral properties [119]. There are two Glycyrrhiza halophytic species with anticarcinogenic activity: Glycyrrhiza glabra L. and G. uralensis Fisch. Glycyrrhiza glabra is rich in phenolics, tannins, and especially glycyrrhizin, a triterpenoid saponin [120]. Glycyrrhizin reduced the proliferation of HeLa cells at 320 µM after 24 and 48 h of incubation, via apoptosis, through mitochondrial depolarization. Moreover, nuclear condensation, cell membrane lysis, and disintegration of organelles were observed in treated cells through phase-contrast microscopy [121]. The cytotoxic potential of the roots' methanol extracts from G. glabra, collected in nine different areas from Italy, Turkey, Syria, Russia, Afghanistan, and Uzbekistan, was assessed against "normal" (HaCaT) and cancer cell lines (A549 and HepG2) using the MTT assay, after 24 h of incubation [122]. Results disclosed variable cytotoxicity levels depending on the samples' collection location and season, potentially related to the influence of climatic conditions on the chemical composition of the plants. Only one sample from Afghanistan was active toward HepG2 cells (IC 50 of 248.5 µg/mL), while four extracts (from Italy, Afghanistan, and Syria) were cytotoxic toward A549 cells (IC 50 between 189.1 and 238.9 µg/mL) [122] (Table 1). However, samples were also cytotoxic toward "normal" cells (HaCat) ( Table 1). An ethanol root extract of G. glabra significantly reduced the proliferation of HT29 cells, at 200 µg/mL after 24, 48, and 72 h of incubation as detected by the MTT assay [123]. Polymerase chain reaction (PCR) studies revealed a downregulation of heat-shock protein 90 (HS90) gene expression that can be related to the reduction in cellular viability, since the HSP90 prevents tumor cells from undergoing apoptotic death; therefore, its blocking could assist active antitumor effects [124]. A reduction in HeLa cell viability above 80% was reported after application of an aqueous ethanol extract of G. uralensis rhizomes at the concentration of 1.84 mg/mL, but the determination of the IC 50 value was not reported [125]. Several cytotoxic compounds were identified in that extract using UPLC-ESI-Q-TOF, including isoquercitrin, 4 demethylpodophyllotoxin glucoside, and podophyllotoxin, all with described cytotoxic properties [125] (Table 2).
Melilotus contains 19 species of annual herbs widely distributed in North Africa and Eurasia [126]. Melilotus indicus L. All. (sweet clover), the only described halophyte within this genus, is used in traditional medicine as an analgesic, and it has reported cytotoxic activity [127,128]. A methanol extract from the aerial parts of sweet clover significantly decreased cell proliferation of HepG2 (IC 50 = 16.60 µg/mL) and SNU-182 cells (IC 50 = 13.21 µg/mL) after 24 h of incubation, using staurosporine as a positive control (Table 1) [127,128]. The extract was less cytotoxic toward L-02 cells (human "normal" hepatic) (IC 50 = 90.9 µg/mL) (Table 1) [127,128]. The application of the extract resulted in an increase in the number of apoptotic cells and loss of mitochondrial membrane potential (∆ψm) [128] ( Table 1). The chemical composition of the extract was not established by the authors.
Prosopis comprises 44 species, mainly small trees distributed in dry lands of America, Africa, and Asia. Several Prosopis species contain anti-inflammatory, antidiabetic, and anticancer compounds, namely, flavonoids, tannins, phenolics, and alkaloids [129]. Prosopis juliflora Sw. DC. (mesquite) is an invasive species in India with reported ethnomedicinal uses, for the treatment of eye and digestive disorders, to name a few [130]. Mesquite extracts display relevant bioactivities, including anti-inflammatory, ascribed mainly to its content in alkaloids [130]. A leaf methanol extract from Prosopis juliflora Sw. DC. (mesquite) had cytotoxic effects on Molt-4 cells, with IC 50 values of 90.5, 42.5, and 20.0 µg/mL after 24 h, 48 h, and 72 h of incubation, respectively, using mitomycin-C (6 µg/mL) as a positive control ( Table 1). The extract was less toxic toward "normal" cells (mitogen stimulated T-lymphocyte cultures from peripheral human blood) [130]. A genotoxic assessment using a cytokinesis-block micronucleus assay reported that the number of micronuclei showed an increasing pattern with the application of increasing concentration of the extract [130].
The genus Sesbania comprises 60 to 85 species of herbs, shrubs, and trees distributed mostly in the tropical and subtropical regions of Africa, Asia, Australia, and America. Sesbania species have relevant pharmacological properties such as anti-inflammatory and antidiabetic [131][132][133]. Sesbania grandiflora (Akatti) (sesbania or agathi), is a small perennial tree with high levels of vitamins and minerals associated with anti-inflammatory, analgesic, and antipyretic properties [134,135], and the anticancer properties of its fruit are mentioned in the Ayurvedic literature [136]. Water, ethanol, and acetone leaf extracts of S. grandiflora reduced the viability of IMR32 and HT29 cells with IC 50 values of 200 µg/mL after 24 h of incubation using doxorubicin as a control [137]. However, the chemical composition of the extract was not unraveled by the authors. Additional studies regarding the anticarcinogenic activity of other halophytes of this family are depicted in Table 1.

Juncaceae Family
Juncaceae currently contains eight genera and 522 species [138] of herbs adapted to salty marshes or badly drained soils and accumulate different phytoconstituents such as flavonoids, triterpenes, steroids, and phenolic acid derivatives [139,140]. A phenanthrene, juncunol (1,7-dimethyl-5-vinyl-9,10-dihydrophenanthren-2-ol), was identified in a diethyl ether extract of leaves of Juncus acutus (L.) Torr. ex Retz. (spiny rush) and displayed selective in vitro cytotoxicity toward HepG2, HeLa, and MDA-MB-468 cell lines [141]. Juncunol had an IC 50 value of 18 µM in HepG2 cells after 72 h of incubation, determined using the MTT assay, and it induced an increase in the number of apoptotic cells in a concentration-dependent manner (IC 50 value ± 25%) accompanied by a decrease in the ∆ψm [141,142]. Juncunol induced cell-cycle arrest in the G0/G1 phase, while showing no hemolytic properties. In silico studies indicate that that compound seems to bind between GC base pairs and, thus, may act as a DNA intercalator [142].

Malvaceae Family
This family of flowering plants currently contains 243 genera and 5461 species [143]. A leaf decoction from Thespesia populnea L. Sol. ex Corrêa (portia tree) had a high content of total phenols and flavonoids and showed cytotoxic and antiproliferative properties toward HEP-2 cells, as observed in the MTT (IC 50 = 120.02 µg/mL after 24 h of incubation) and SRB (IC 50 = 77.06 µg/mL) assays [144]. Treated cells showed apoptotic characteristics including membrane blebbing, cell shrinkage, nuclear and cytoplasmic condensation, and formation of apoptotic bodies [144]. However, no attempt was made to identify the active molecules present in this sample. The chloroform-soluble fraction of a methanol bark extract of portia tree caused a reduction in cellular viability of MDA-MB-231 and MCF7 cells, with IC 50 values of 23.97 and 20.62 µg/mL respectively, after 24 h of incubation [145]. Chemical analysis of the extract using GC-MS revealed the presence of steroids such as cis-androsterone and fatty-acid derivatives, which display cytotoxic activity against cancer cell lines [108,145].

Myrtaceae Family
Myrtaceae presently includes 134 genera and 6614 species [146]. Focus is given to the genus Eucalyptus, especially Eucalyptus camaldulensis Dehnh, because is a salt-tolerant species with reported antitumoral properties. Eucalyptus spp. are mainly cultivated for timber and paper production, but the presence of bioactive compounds, such as triterpenoids, flavonoids, and tannins, confers E. camaldulensis with relevant bioactivities, including antitumoral [147]. Methanol, ethyl acetate, n-butanol, and water extracts from leaves of E. camaldulensis reduced the viability of MCF7 and MDA-MB-231 cell lines, according to the MTT and SRB assays [148]. The ethyl acetate extract had the highest cytotoxicity with IC 50 values of 26.7 and 7.9 µg/mL for MTT and SRB in MCF7 cells, and IC 50 values of 34.4 and 4.9 µg/mL for MTT and SRB in MDA-MB-231 cells after 24 h of incubation [148]. No attempt was reported by the authors to disclosure the molecules responsible for the detected cytotoxic activity.

Plantaginaceae Family
This family currently contains 101 genera and 2165 species [149], widely distributed worldwide but with preference for temperate zones [150]. Bacopa monnieri (L.) Wettst (water hyssop) is a wetland macrophyte and contains bacopaside (II) (saponine), with reported anticarcinogenic properties [151,152]. The anticarcinogenic properties of artificially digested (artificial saliva and artificial gastric juice) methanol extracts from B. monnieri reduced the motility (capacity of cell migration in a process of cancer development) of DU145 cells [153]. Nevertheless, the IC 50 value was not determined by the authors. The extracts, analyzed by HPLC, were rich in phenolic compounds such as chlorogenic, caffeic, and syringic acids and bacoside A, with reported anticarcinogenic properties [154][155][156][157].
Within the Plantaginaceae family, the Plantago genus is the largest with 275 annual and perennial species widespread around the world, some with traditional uses as antipyretic, anti-inflammatory, and antitumoral agents [158]. Plantago major L. (fleawort) is an important medicinal plant due to its richness in bioactive metabolites, such as alkaloids and flavonoids, especially luteolin-7-O-β-glucoside, with antitumoral properties [159]. Silver particles produced by combining a water extract from P. major seeds and silver nitrate (AgNO 3 , 0.1 M) significantly decreased the viability of MCF7 cells with an IC 50 value of approximately 12 µg/mL after 24 h of incubation [159]. However, no attempt was made by the authors to identify the bioactive compounds present in this extract. Information targeting other Plantaginaceae species is summarized in Table 1.

Plumbaginaceae Family
Plumbaginaceae encompasses several species adapted to survive under saline conditions and currently includes 1138 species distributed across 26 genera [160]. The genus Plumbago contains 18 species (shrubs or perennial herbs) characterized by the presence of flavonoids, phenols and saponins, conferring several properties including anticarcinogenic [161,162]. The genus Limoniastrum Heist. ex Fabr. comprises the halophytic species Limoniastrum monopetalum (L.) Boiss. (≡ Statice monopetala L.) and Limoniastrum guyonianum Boiss. [163,164], thriving in coastal and saline dry areas of the Mediterranean and northern Saharan Africa [165][166][167][168]. Limoniastrum guyonianum is traditionally used in the treatment of gastric infections [169]. A gall aqueous extract from L. guyonianum reduced the viability of HeLa cells with IC 50 values of 170 and 140 µg/mL after 24 and 48 h of incubation, respectively [169]. This extract was rich in flavonoids and induced DNA hypomethylation and apoptosis due to its capacity to arrest cell-cycle progression in G2/M (Table 1). Due to the high levels of polyphenolic compounds detected in the extract, the antiproliferative and proapoptotic effects of such metabolites on HeLa cells were hypothesized by the researchers [169]. In another study, hexane, dichloromethane, ethanol, and methanol extracts from L. densiflorum (Guss.) Kuntze shoots were tested for cytotoxicity on cancer and "normal" cell lines using the resazurin reduction assay, after 48 h of treatment (Table 1) [170]. The dichloromethane extract was the most bioactive sample, with IC 50 values of 29 and 85 µg/mL toward A-549 and DLD-1, without significantly reducing the viability of a human skin fibroblast cell line ( Table 1). The major compounds present in that extracts were identified using RP-HPLC as trans 3-hydroxycinnamic acid, myricetin and isorhamnetin, which may be related to the observed cytotoxic activity [170].

Poaceae Family
This grass family currently includes 11,917 species divided across 796 genera [171,172]. In this family, only two genera of halophytes are reported with anticarcinogenic properties, namely, Cynodon and Echinochloa. The most representative Cynodon species is Cynodon dactylon L. Pers (Bermuda grass), a weed with several medicinal properties, such as antidiabetic, diuretic, and purifying, as well as in vitro cytotoxic properties [173]. The in vitro antitumoral properties of petroleum ether, dichloromethane, acetone, methanol/water (3/1), and water extracts from Bermuda grass were appraised on a breast cancer cell line, and cell viability was assessed by evaluating (3H)-hypoxanthine incorporation after 48 h of incubation [174] ( Table 1). The highest cytotoxicity values were observed after application of the water, acetone, and petroleum ether extract, with IC 50 values of 57.2, 38, and 39 µg/mL (Table 1) [174]. The acetone and ether petroleum extracts were profiled using LC-MS, allowing for the identification of several bioactive anthocyanins, namely, delphinidin, petunidin, malvidin, and cyanidin glucosides  Table 2). There is no information related to the specific anticancer properties of the detected anthocyanins; however, several others, such as delphinidin, cyanidin, malvidin, and corresponding glycosides, exhibit antitumoral properties in different cell lines [174]. An ethanol extract (70%) from grains of Echinochloa crus-galli (L.) P. Beauv. (cockspur grass) significantly reduced the viability of HCT-116 and HeLa cell lines, with IC 50 values of 11.2 and 12.0 µg/mL, respectively [175] (Table 1). The active ethanolic crude extract was fractionated using n-hexane, chloroform, ethyl acetate, and n-butanol, with the ethyl acetate fraction exhibiting the lowest IC 50 value (3.8 µg/mL). Fractionation of this active fraction allowed the isolation and identification of eight phenolic compounds, which exhibited significant cytotoxic activities toward the tested cells [175]. The most active molecules were 5,7-dihydroxy-3 ,4 ,5 -trimethoxy flavone and 5,7,4 -trihydroxy-3 ,5 -dimethoxy flavone (tricin), the latter with IC 50 values of 7.2, 8.6, 10.8, and 19.9 µM against HepG2, HeLa, HCT116, and MCF7 cell lines, respectively, with similar results to the commercial anticancer drug, doxorubicin [175]. Further data on the antitumoral properties of other species of the Poaceae family can be found in Table 1.

Portulacaceae Family
This family contains one genus with presently 150 species [176] distributed worldwide, including the edible Portulaca oleracea L. (purslane), a halophyte succulent annual plant with important nutraceutical and antioxidant properties [177,178]. The anticarcinogenic activity of this species can be ascribed to the presence of several compounds, such as portulacerebroside A, portulacanones B, and 2,2 -dihydroxy-4 ,6 -dimethoxychalcone [179] ( Table 2). Portulacerebroside A induced apoptosis in HCCLM3 cells via activation of the p38 MAPK and JNK-triggered mitochondrial death pathway [180]. An ethanol extract from purslane seeds reduced the viability (MTT assay) of HepG2 cells in a concentrationdependent manner with IC 50 values of approximately 75 µg/mL after 24 h of incubation via apoptosis induction according to cellular morphology modification, i.e., a reduction in cellular size and adhesion capacity (Table 1) [181]. Oil from seeds of the same species reduced the proliferation of HepG2 and A-549 cancer cell lines, at concentrations ranging from 250 to 1000 µg/mL as observed using the MTT and neutral red uptake assays, after 24 h of incubation in both cancer cell lines [182]. However, the high concentrations of the extract used (up to 1000 µg/mL) must be highlighted; even at 100 µg/mL, no reduction in cell viability was observed (Table 1) When treated with the highest concentration, cells exhibited morphological modifications typical of apoptotic cells, such as loss of cell adhesion capacity, shrinkage, and round shape [182]. However, the chemical profile of the extract was not determined by the authors.

Rhizophoraceae Family
This family includes mangroves and comprises currently 148 species and 15 genera [183,184]. It has two halophytic species with reported cytotoxic properties against human cancer cells, namely, Bruguiera gymnorhiza (L.) Lam (black mangrove) and Ceriops tagal (Pers.) C. B. Rob. (oriental mangrove) [185,186]. A methanol extract from black mangrove stem bark reduced the proliferation of HeLa, RAJI, and myeloma cell lines, with IC 50 values of 133, 504, and 384 µg/mL after 24 h of incubation using doxorubicin as a standard control, via apoptosis induction, since DNA fragmentation was detected by fluorescence with double staining (ethidium bromide-acrydine orange) [187].

Solanaceae Family
Solanaceae contains 100 genera and 2925 species currently recognized [188], many of them with a high worldwide economic importance as cultivated crops, such as Solanum lycopersicum L. (tomato) [189]. The number of halophytic species with anticarcinogenic activity in this family is scarce, and reports have mainly focused on Lycium barbarum L. (Chinese wolfberry). Chinese wolfberry's dried fruits are widely used in traditional Chinese medicine for its anticancer properties, potentially associated with the presence of different bioactive compounds, including polysaccharides (proteoglycans), scopoletin (phytoalexin), and 2-O-β-D -glucopyranosyl-L -ascorbic acid (vitamin C analog) [190]. A polysaccharide-rich aqueous extract from Chinese wolfberry fruits reduced the proliferation of SW480 and Caco-2 cell lines, as observed by the crystal violet and MTT assays, with IC 50 values of 600 and 650 µg/mL respectively, after 4 days of incubation, with a high capacity to arrest cellcycle progression in the G0/G1 phase [191]. An ethanol extract from the dried fruit residue of this species was cytotoxic toward A431 cells, with an IC 50 value of 873.7 µg/mL after 24 h of incubation, determined by water-soluble tetrazolium-8-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium] monosodium salt (CCK-8) assay [192]. The same authors conducted in vivo studies in mice submitted to an intraperitoneal injection of 50 mg/kg of the crude extract, reporting a lower volume and weight of the formed tumor and a higher survival rate of tumor-bearing nude mice [192]. Several phenolic amides were isolated from an ethanol extract from the stem of L. barbarum and tested toward glioma cell lines, using the MTS method [180]. 4-O-Methylgrossamide and grossamide exhibited the highest capacity to decrease the viability of GSC-3# and GSC-12# cell lines, especially the latter compound, with IC 50 values of 6.40 and 5.85 µg/mL respectively. [193]. The precise mechanism of action was not appraised for the isolated compounds; however, there are reports that phenolic amides can inhibit tumor necrosis factor-alpha (TNF-α) and nuclear factor-kappa B (NF-κB), which may be related to the detected cytotoxic effect [194].

Tamaricaceae Family
Tamaricaceae contains five genera with currently 110 species [195], widely distributed around the world, and several are halophytic [196,197]. Tamarix plants (salt cedar) contain several bioactive metabolites such as methylferulate and syringic acid with antineoplastic properties [198]. Syringic acid was isolated from a methanol extract of Tamarix aucheriana (Decne.) Baum aerial parts and was cytotoxic toward SW1116 and SW837 cells in a time-(24-72 h of incubation) and dose-dependent manner, with IC 50 values of 0.95 and 1.2 mg/mL, respectively [199]. Cytotoxicity was related to an increase in the percentage of apoptotic cells, attributed to the antimitogenic effect of syringic acid [199]. Methylferulate was isolated from the same type of extract and significantly reduced the viability of SW1116 and SW837 cells, with IC 50 values of 1.7 and 1.9 mM after 24 h of incubation, respectively, being responsible for cell-cycle arrest in the S and G2/M phases and apoptosis induction, together with a reduction in NF-κB DNA-binding activity [200].
Tamarix gallica L. is traditionally used as an expectorant, antidiarrheal, and laxative, potentially attributed to the presence of ellagic and gallic acids, anthocyanins, tannins, flavonones, isoflavonones, and resveratrol in its leaves [201]. A methanol extract from shoots, leaves, and flowers of T. gallica significantly reduced the viability of Caco-2 cells, related to a decrease in DNA biosynthesis, as observed using fluorescence microscopy after DAPI staining, while a reduction in the percentage of cells in the G0/G1 and S phases was detected using flow cytometry [202].

Conclusions and Future Perspectives
As stated in Section 1, we included in this review paper published work, which we considered relevant, related to the cytotoxic effects of halophytes. Such reports included results obtained with crude extracts, fractions, and pure compounds, using in vitro and in vivo assays, and they occasionally presented IC 50 values, phytochemical profile of the active samples, and identification of active molecules, along with proposed mechanisms of action. There are some drawbacks of the identified reports. For example, several authors tested considerably high concentrations of the target extracts, up to 1000 mg/mL, in some cases. Moreover, the IC 50 values were not reported in a high number of studies, which impairs a critical evaluation of the obtained results. Additionally, several reports failed to establish the chemical composition of the cytotoxic extracts and/or the identification of the molecules responsible for the detected activity. Lastly, information about the effect of the extracts on cell lines from nontumoral origin and, therefore, the selectivity of the extracts toward cancer cells was not always provided. According to the American National Cancer Institute NCI), only crude extracts/fractions displaying IC 50 values lower than 30 µg/mL in the preliminary assay are considered promising and should be further explored as sources of antitumoral compounds. Therefore, we consider that extracts from the species and extracts included in Table 3 could be explored in detail targeting the isolation of anticancer leads, namely, C. ambrosioides, S. fruticosa, A. millefolium, C. soldanella, C. rotundata, M. indicus, T. populnea, E. camaldulensis, P. major, P. lanceolata, E. crus-galli, and L. barbarum. Moreover, isolated compounds with IC 50 values ≤10 µM are considered promising anticancer leads. Therefore, we highlight the compounds in Table 3, namely, 6 -O-(n-butanol) ilekudinoside B ester (A. marina), 5,7-dihydroxy-3 ,4 ,5 -trimethoxy flavone and tricin (E. crus-galli), Portulacerebroside A (P. oleraceae), and grossamide (L. barbarum).  Cell-cycle arrest in the S and G2/M phases, apoptosis induction, reduction in NF-κB DNA-binding activity [200] Tamarix gallica L.
Author Contributions: P.G.-C., conceptualization, data curation, and writing-original draft preparation; P.C.-B., writing-review and editing; C.G.P., writing-review and editing; L.C., conceptualization, supervision, and writing-review and editing. All authors have read and agreed to the published version of the manuscript.