Disclosing the bioactive metabolites involved in the in vitro anthelmintic effects of salt-tolerant plants through a combined approach using PVPP and HPLC-ESI-MSn

Total phenolics, total flavonoids and condensed tannins contents

The phenolic contents of the extracts are presented in Table 2. The phenolic content of the extracts is presented in Table 2. The total phenolic content of all species ranged between 14.2 and 226.3 mg GAE eq. g⁻¹ DW extract while the total flavonoid content ranged between 13.3 and 45.4 mg QE g⁻¹ DW. Lopes and colleagues (2016) reported higher TPC values for C. mariscus (254 mg GAE g⁻¹ DW), C. soldanela (144 mg GAE g⁻¹ DW), I. crithmoides (141 mg GAE g⁻¹ DW), L. monopetalum (248 mg GAE g⁻¹ DW) 80% acetone water extracts, except for P. lentiscus (130 mg g⁻¹ DW), but lower flavonoid contents in comparison to our work (1.26–13.8 mg rutin g⁻¹ DW)¹⁵. In another work, H. italicum picardi infusions and decoctions of aerial organs have been previously described as rich sources of flavonoids (91.8–119 mg rutin 200 mL⁻¹)²⁷. Moreover, a lower combined TPC value was detected in P. coronopus leaves and flowers extracts of different polarities (72.1 mg GAE g⁻¹ DW) but increased TFC levels (282.8 mg rutin g⁻¹ DW)²⁸. In this study, total condensed tannins were detected only in three species, in the following concentration order: P. lentiscus > L. monopetalum > C. mariscus. In agreement, tannins were formerly detected in the same three formerly mentioned species, although at lower concentrations (6.63–38.7 mg CE g⁻¹ DW, extract)¹⁵. It is worth to mention that dissimilarities between our results and those of other authors may be the reflection of different extraction methodologies and standards employed as well as environmental and plant-related factors.

In vitro anthelmintic properties

Table 3 summarizes the results of the in vitro activity of salt-tolerant plant extracts against H. contortus L3 larvae and eggs and T. colubriformis L3 larvae and eggs obtained in LEIA and EHIA assays. Lentisk (P. lentiscus) exhibited the highest activity on LEIA (IC₅₀ = 27.8–29.7 µg mL⁻¹) and egg hatching processes (IC₅₀ = 197.7 and 223.9 µg mL⁻¹), without significant differences between GIN species. Lentisk is an evergreen shrub with high polyphenol content and previous results have shown both in vitro and in vivo anthelmintic properties^29,30,31,32. In previous studies, P. lentiscus extracts (acetone, ethanol and/or water) exhibited less than 20% larvae exsheathment and migration at 1200 µg mL^−129,31. Nevertheless, the results for the in vitro egg hatching assay are herein, to the best of our knowledge, described for the first time.

Following P. lentiscus, L. monopetalum, C. mariscus and H. italicum. picardi extracts exhibited the most promising results towards both GIN species and life stages (Table 3). Limoniastrum monopetalum is a highly salt-tolerant shrub, widely distributed in the Mediterranean area, and was as effective as P. lentiscus in LEIA (p < 0.05), with IC₅₀ values lower than 50 µg mL⁻¹ (no significant difference between the two tested parasites; p > 0.05). In EHIA, L. monopetalum was also the most active species, besides P. lentiscus, with similar activity towards both parasites (IC₅₀ = 1999.9 and 2102.5 µg mL⁻¹, respectively). Cladium mariscus, or sawgrass, is an evergreen grass-like plant occurring in coastal saltmarshes in the Mediterranean region. C. mariscus extract inhibited L3 larvae exsheathment (IC₅₀ = 77.8–88.9 µg mL⁻¹), without significant differences between both parasite species (p > 0.05). In contrast, in the EHIA, C. mariscus was more effective towards H. contortus (IC₅₀ = 1496.6 µg mL⁻¹) than T. colubriformis (IC₅₀ = 2575.5 µg mL⁻¹; p < 0.05). Helichrysum italicum subsp. picardi (everlasting) is an aromatic salt tolerant plant commonly found in sandy soils, such as sand dunes, along the Southern European coast. Everlasting extract exhibited IC₅₀ values ranging between 92.8–132.5 µg mL⁻¹ on LEIA, and 2947.7–3707.5 µg mL⁻¹ on EHIA. Interestingly, H. contortus larvae and eggs were more susceptible to the H. italicum picardi extract than those of T. colubriformis (p < 0.05).

It is well recognized that the anthelmintic activity is affected by the class, structure and concentration of secondary metabolites⁷. Moreover, these metabolites have different effects, depending on the target parasite species and life development stages⁷. A higher susceptibility of H. contortus in comparison to T. colubriformis, as observed for C. mariscus and H. italicum picardi extracts, has been previously documented for other bioactive plants, such as sainfoin, and individual chemical structures, depending on the ratios of prodelphinidins/procyanidins^10,33,34. The authors suggest that such differences can reflect dissimilarities on the composition of specific parasite sheath proteins, that interact differently with the chemical groups^33,34. The same conclusion can be driven for differences among parasite stages, as the eggshell and larvae coat differ in their structural components, which has also been recorded with conventional anthelmintic drugs^7,35. This may explain the results obtained for P. coronopus, which was more active against larvae exsheathment (IC₅₀ = 94.0 and 212.4 µg mL⁻¹), and inactive towards eggs, of both parasite species, at the maximum concentration tested. Overall, IC₅₀ results obtained in LEIA are frequently reported as lower than EHA, suggesting that infective L3 larvae are more susceptible than eggs^36,37.

Calystegia soldanella, C. maritima and M. marina co-occur in sand dunes along the Algarve coastline while I. crithmoides can be found in highly saline environments, such as saltmarshes. These four species were mildly to poorly active on both assays (Table 3). Interestingly, while I. crithmoides was mostly ineffective in this study, its related species, I. viscosa 70% ethanolic extract exhibit anthelmintic properties against the larvae exsheathment of a mixture of Teladorsagia circumcincta and T. colubriformis parasites³², suggesting significant chemical diversity among the genus.

Overall, the nine plant extracts had comparable effects between the two GIN species (Spearmen correlation; R² = 0.96; p < 0.01). In addition, a negative correlation between the total phenolic content and the anthelmintic activity was noted, particularly with H. contortus parasites (Spearmen correlation; R² = 0.783; p < 0.05), suggesting that these metabolites may be involved in the anti-parasitic nematode’s effects.

Role of polyphenols in the anthelmintic activity: PVPP as a polyphenol binding agent

In order to ascertain the role of polyphenols in the anthelmintic properties, the four plant extracts presenting results for both LEIA and EHIA were selected for further studies using PVPP. PVPP is a polyphenol inhibitor, as it binds to tannins and flavonoids, removing these metabolites from the solution²⁶. Thus, if after PVPP exposure a loss of the anthelmintic activity is observed, it can be assumed that polyphenols are most probably responsible for the activity once they were formerly removed.

The effects of the addition of PVPP to extracts on EHIA and LEIA are illustrated in Figs. 2 and 3, respectively. The application of all the extracts with PVPP largely restored the egg hatching process (Fig. 2) to control values, suggesting that polyphenols are most probably involved in the inhibition of this life stage development. Vargas-Magaña and colleagues (2014), while exploring the role of polyphenols on the anthelmintic effects of several extracts of tannin-containing tropical plants on EHIA, concluded that the main mechanism of action was by impairing larvae eclosion from the eggs³⁸. Likewise, we noted a high number of larvae trapped inside the eggs after the application of these active extracts (data not shown).

In contrast to EHIA, results with PVPP varied on LEIA (Fig. 3): the application of the L. monopetalum extract, resulted in 60–70% of larvae exsheathment of both parasite species after PVPP addition for 60 min, in contrast to 0% in the non-treated sample; the extract from H. italicum picardi pre-incubated with PVPP remained mostly completely active. Subtle changes were observed for C. mariscus (approx. 20–40% of larvae exsheathment after 60 min of treatment) for both parasite species, while P. lentiscus had only around 20% of larvae exsheathment at 60 min, after PVPP treatment. These results suggest that other bioactive metabolites, alone or in synergy, can be present in all extracts tested, especially for H. italicum picardi, P. lentiscus, and C. mariscus. In agreement with our results, other authors already reported that P. lentiscus extracts remain active on GIN larvae migration after exposure to PVPP²⁹.

The remaining activity on LEIA for the majority of the extracts tested should be carefully analyzed, and two scientific questions arise. First, was the ratio of PVPP used insufficient to cope with the high phenolic content of the extracts? Despite being commonly used, Manoloraki et al. (2010) questioned this hypothesis when testing P. lentiscus for larvae migration after PVPP addition, since this species has a high polyphenol content, comparable to our results²⁹. On the other hand, are other bioactive metabolites present in the extracts that are also effective in inhibiting larvae exsheathment? For instance, different authors suggest that terpenes may be responsible for the remaining in vitro and in vivo anthelmintic properties of P. lentiscus after the addition of PVPP or polyethylene glycol (PEG), a similar inhibitor of polyphenols^29,30. Additionally, Botura and colleagues (2013) described that the flavonoid fraction of Agave sisalana Perrine (sisal) had higher activity on egg hatching, while the saponin fraction had mostly larvicidal effects³⁹. In an attempt to address these scientific questions, and elucidate the possible metabolites involved, we have conducted an HPLC-ESI-MSⁿ comparative analysis on the active samples, before and after PVPP treatment.

HPLC-ESI-MSⁿ comparative analysis of the chemical profile of non-treated and treated-PVPP samples

The HPLC-ESI-MSⁿ analysis was performed in the most active extracts, with and without PVPP. Obtained chromatograms are represented in Fig. 4 while the chemical profile of each species is depicted in Tables 4, 5, 6 and 7. The characterization of the compounds is detailed in Supplementary Material files.

The main constituents of P. lentiscus extract were flavonoid glycosides (mainly from myricetin and quercetin; approx. 53 mg g⁻¹ DW), and galloylquinic acid and di-O-galloylquinic acid isomers (60 mg g⁻¹ DW; Table 4; Suppl. files, Table I). In agreement to our findings, Romani et al. (2002) detected a high concentration of galloyl derivatives (5.3% DW), and a substantial amount of myricetin and quercetin glycosides (1.5% DW), extracted from a 70% ethanol solution of leaves⁴⁰. Hydrolysable tannins are a group of gallic acid esters associated with polyols (e.g., glucose, glucitol, quinic acid), and the etherification or oxidation of the galloyl groups leads to the formation of complex structures (gallotannins and ellagitannins)⁴¹. Plant extracts containing hydrolysable tannins with gallic acid units were more effective as anthelmintics than those containing condensed tannins⁴². Nevertheless, the oligomerization and molecular weight of tannins may affect the anthelmintic activity, as is the case, for example, of elagitannins and condensed tannins^34,43. Other metabolites present in lower concentrations in P. lentiscus extract with reported anthelmintic effects include flavan-3-ols and its galloyl derivatives, namely epigallocatechin (6.4 mg g⁻¹ DW), gallocatechin gallate (6.8 mg g⁻¹ DW) and catechin (5.0 mg g⁻¹ DW). Molan et al. (2003) found that the presence of the galloyl group on flavan-3-ols was crucial for the activity on T. colubrifomis egg hatching (20% vs. 100% inhibition at 1 mg mL⁻¹), and also more effective on immobilizing infective larvae (100% inhibition at 100–150 µg mL⁻¹)⁴⁴.

In P. lentiscus PVPP-treated samples, the concentration of flavonoid glycosides (0.17 mg g⁻¹ DW) and galloylquinic acid (2.2 mg g⁻¹ DW) drastically dropped (Suppl. files, Table I), which may justify the restoration of the egg hatching. On the other hand, the presence of these compounds in lower concentrations may explain the remaining activity on larvae. Nevertheless, compounds 2, 58, and 78 remained in this sample, and may also account for the activity.

Caffeoylquinic and dicaffeoylquinic acids were the most abundant compounds in H. italicum picardi extract (150 mg g⁻¹ DW), followed by quercetin-O-glucosides (approx. 31 mg g⁻¹ DW; Table 5; Suppl. files, Table II). These findings were expected, since previous works identified high contents of these metabolites in aerial organs of the same species^27,45. Borges and colleagues (2019) found a significant correlation between the phenylpropanoid content (particularly chlorogenic acid, 1,3-dicaffeoylquinic, and 3,5-dicaffeoylquinic acids), and the ovicidal activity of 17 plant extracts from Pantanal wetlands against Haemonchus placei⁴⁶. Additionally, chlorogenic acid exhibited an IC₅₀ value of 92.4 μg mL⁻¹ against L3 larvae exsheathment of H. contortus, and was also effective on preventing larvae hatching from eggs (IC₅₀ = 520.8 μg mL⁻¹)⁴⁷. These results point out the potential of caffeoylquinic and dicaffeoylquinic acids to be the active metabolites of H. italicum picardi extracts. However, some O-glycosides are also present that may contribute to the detected activity. For example, Barrau and colleagues (2005) tested the activity of 3 flavonol glycosides (quercetin-3-O-rutinoside or rutin, kaempferol-3-rutinoside or nicotiflorin, and isorhamnetin-3-rutinoside or narcissin), and all reduced the migration of H. contortus L3 larvae in 25–35% when applied at 1200 µg mL⁻¹⁴⁸.

In H. italicum picardi PVPP-treated sample, although in lower concentrations, caffeoylquinic and dicaffeoylquinic acids remained in solution (8.3 mg g⁻¹ DW), from which chlorogenic acid was the main compound (6.3 mg g⁻¹ DW; Suppl. files, Table II). The high activity observed for the extract from H. italicum picardi treated with PVPP on larvae exsheathment is most likely due to the high content of chlorogenic acid remaining in the sample⁴⁷. Still, other caffeoylquinic and dicaffeoylquinic acids are present (2 mg g⁻¹ DW) that might also add to its effects. On the other hand, in EHIA the lower amount of these compounds in the PVPP-treated sample may have not be sufficient to inhibit egg hatching, since this process was completely restored. In fact, Borges and colleagues (2019) suggest that the concentration of monomeric and dimeric chlorogenic acid derivatives that enter in contact with eggs seems to be determinant for the activity, as observed for Melanthera latifolia ethanolic extract that had low concentrations of these compounds. and was considered inactive (up to 80% egg hatching at 50 mg mL⁻¹)⁴⁶.

Cladium mariscus acetone water extracts were previously reported as a rich source of polyphenols, particularly tannins by spectrophotometric methods, and chlorogenic, ferulic, and syringic acids were detected in higher amounts, through HPLC–DAD analysis^15,49. In agreement, in this study, C. mariscus extract was mainly composed of flavan-3-ols (epigallocatechin, catechin), proanthocyanidins (5.1 mg g⁻¹ DW), luteolin, C-glycosyl luteolin, a kaempferol glucoside, and an apigenin flavone (9.5 mg g⁻¹ DW; Table 6; Suppl. files, Table III). Flavan-3-ols and proanthocyanidins have well recognized anthelmintic effects^44,50, and therefore, they are most likely involved in the activity of C. mariscus extract. Also, the activity of the flavonoid luteolin on H. contortus larvae exsheathment has been previously established (IC₅₀ = 17.1 and < 71.5 µM)⁵¹. Interestingly, Klongsiriwet and colleagues (2015) found that luteolin, even at low concentrations (30 μM), display synergistic effects with procyanidins, leading to a fivefold lower IC₅₀ of the mixture in comparison to the procyanidin fraction alone (75.9 vs. 356 μg mL⁻¹)⁵¹. Having this in mind, the combination of proanthocyanidins and luteolin in C. mariscus extract could act synergistically in the inhibition of the egg hatching. Nevertheless, the activity on LEIA was only partially restored after PVPP addition (approx. 20–40% larvae exsheathment), i.e., the remaining metabolites are still exhibiting anthelmintic properties. In PVPP-treated samples, mainly C-glycosyl flavones (1.07 mg g⁻¹ DW) and, to a less extent chlorogenic acid, remained in solution while the catechin derivatives and luteolin were removed (Table 6; Suppl. files, Table III). As previously addressed, chlorogenic acid exhibits significant anthelmintic activity in vitro against H. contortus larvae exsheathment and egg hatching⁴⁷. Despite the activity described for luteolin, the investigation of the anthelmintic properties of its glycosides is lacking. In general, C-glycosyl flavones exhibit antioxidant and anti-inflammatory properties⁵², and two flavone-C-glycosides namely isoschaftoside and schaftoside shown strong toxicity (LC₅₀ = 114.66 μg mL⁻¹ and 323.09 μg mL⁻¹) against the plant-parasitic nematode Meloidogyne incognita⁵³. Moreover, it is worth noticing that compounds 20 and 39 are still unidentified, although present in PVPP-treated samples.

Previous works identified several phenolic compounds in L. monopetalum extracts including gallic, vanillic, ferulic, syringic, p-hydroxybenzoic, protocatechuic, chlorogenic, and trans-cinnamic acids, and also quercetin, apigenin, amentoflavone, flavones, methyl gallate, and myricetin^54,55. In the current work, the main metabolites identified in L. monopetalum extract were epigallocatechin, phenolic acids and derivatives, isorhamnetin sulfate, pinoresinol, methylated flavonoids sulfate and two oxylipins (Table 7). However, some of the major compounds, namely the methylated flavonoids sulfate 67, 71, 76, and 80 were not identified, as well as the minor metabolites 33, 46, and 55. The production of sulfated metabolites by plants is pointed out as an evolutionary trait to thrive in aquatic saline habitats, and part of the plant heavy metal detoxification mechanism^56,57. Indeed, L. monopetalum is a halophytic and metal accumulator shrub that thrives in saltmarshes under harsh biotic and abiotic stresses (e.g., tidal fluctuations, salinity, heavy metal soils, sunlight exposure, UV radiation). Sulfated phenolics were previously identified in other halophyte species, such as Limonium caspium (Willd.) Gams⁵⁸ and Halimione portucaloides (L.) Aellen⁵⁹. The pharmacological interest in sulphated flavonoids increased in the last decades, mainly driven by its hydrophobic nature, and many reported biological activities, like anti-coagulant, anti-viral, antioxidant, anti-inflammatory, antimicrobial⁶⁰.

Besides epigallocatechin (9.46 mg g⁻¹ DW), the concentration of isorhamnetin sulfate (65) was high in L. monopetalum (6.4 mg g⁻¹ DW) as well as phenolic acids and its derivatives (10.3 mg mL⁻¹ DW; 7, 16, 24, 32). Delgado-Nuñez and colleagues (2020) attributed the main anthelmintic effects of Prosopis laevigata Willd. M. Johnston to isorhamnetin, which caused 100% of mortality on H. contortus eggs at the lowest concentration tested (700 µg mL⁻¹), being also effective towards larvae (IC₅₀ = 2.07 mg mL⁻¹)⁶¹. The glycoside isorhamnetin-3-rutinoside decreased H. contortus L3 migration by 35% at 120 µg mL⁻¹⁴⁸. However, the activity of its sulfate structure is not reported. Among different classes of phenolic compounds, phenolic acids (i.e., caffeic acid, ferulic acid, and gallic acid) were the most potent anthelmintic metabolites against both H. contortus egg hatching (IC₅₀ values = 0.56–4.93 µg mL⁻¹) and larval development (IC₅₀ = 22–33 µg mL⁻¹)⁶². Nevertheless, one should keep in mind that structural modifications, such as glycosylation, methylation, and sulfation, may affect the bioactivity observed. For example, the substitution by a sugar unit in the quercetin structure showed a twofold increase in the larvicidal activity of rutin⁶². Still, studies concerning the anthelmintic effects of sulphated phenolics are missing. Since these metabolites are the main suspects as bioactive components of L. monopetalum extract, it would be interesting for further works to be conducted, not only confirming the anthelmintic effects of isolated compounds but also clarifying the role of sulfate in structure–activity relationship studies.

After PVPP treatment, the activity of L. monopetalum extract on larvae exsheathment was restored by approximately 60–70% to the control values. Although the remaining compounds may have contributed to the overall activity, the major anthelmintic effects were annulated. As some main metabolites of L. monopetalum (67, 71, 76, 80) remain to be identified and quantified, further studies on this species are required to completely understand its bioactive compound (s) and related anthelmintic properties.