Phytochemical Screening and Acanthamoebic Activity of Shoots from in Vitro Cultures and in Vivo Plants of Eryngium alpinum L.—The Endangered and Protected Species

Genetically uniform shoots of Eryngium alpinum L. cultured in vitro were subjected to the qualitative analysis applying the UPLC-HESI-HRMS technique. In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from the natural environment. The phytochemical screening of the crude methanolic extracts of shoots, both from in vitro cultures and in vivo plants, revealed the presence of phenolic acids, coumarins, flavonoids, triterpenoid saponins, amino acids, or dipeptides. Active compounds detected are known to have medicinal importance, and for this reason, the present study represents a preliminary investigation of the extracts against pathogenic and opportunistic amoeba. Among the extracts tested, the extract of shoots from in vitro cultures exhibited remarkable amoebicidal action against trophozoites. On the second day of treatment, the extract at the concentrations of 5 mg/mL, 2.5 mg/mL, and 0.5 mg/mL showed the highest antiamoebicidal effect: the inhibition of trophozoites reached 81.14%, 66.38%, and 54.99%, respectively. To our best knowledge, the present report is the first to show the phytochemical screening and to discuss the antiamoebic activity of Eryngium alpinum L. shoots, both from in vitro cultures and in vivo plants.


Introduction
Eryngium alpinum L. is a perennial herb in the Saniculoideae subfamily of the Apiaceae family [1]. It is native the European Alps. The population of the plant is in decline. The species is protected by law: the Habitats Directive; the Convention on the Conservation of European Wildlife and Natural Habitats, the European Habitat Directive of Natura 2000, and the national red lists/books of protected species [2].
Due to the unavailability of the plant material, little research on this taxon was carried out. Only a few papers indicated the presence of phenolic acids, flavonoids and the essential oil in the organs of in vivo plants [3][4][5][6][7]. The identification of flavonoids, namely quercetin and kaempferol, in leaves of alpine eryngo was described by Crowden et al. [3]. Moreover, isoquercetin and quercitrin were detected  Primary explants failed to respond to MS medium without plant growth regulators, that is why this variant was withdrawn from our investigation. The hormonal investigation, regardless of the combinations and the concentration used, resulted in the response of explants (100%) and gave the largest number of new cloned shoots, with the value between 5.50 ± 0.86 and 6.79 ± 0.48. The values of the mean number of shoots calculated per one explant were not significantly different regardless of the increase in concentration of BAP and GA3 in the culture medium, on which shoots grew ( Figure  1; Table 1).
It is worth noticing that shoots grew vigorously, did not develop roots spontaneously, and also did not show any signs of verification or callusing at base, which is important for obtaining uniform shoot biomass. This study indicated the alternative method for effective and rapid shoot multiplication of E. alpinum. However, the increase in the concentration of BAP did not provide the highest biotechnological parameters compared to our previous studies [6,7]. In the case of E. planum, the highest mean number of shoots developed via axillary buds was 15.58 ± 0.54-17.10 ± 0.60 shoots per explant, depending on the culture media: MS + BAP 1.0 mg/L + IAA 1.0 mg/L or MS + BAP 1.0 mg/L + IAA 0.1 mg/L [14]. More shoots (13.30 ± 3.73), comparing to the control, were obtained for E. campestre when cultured on the same media composition as for E. planum [15]. The efficiency of shoot multiplication for E. maritimum varied between 1.2 ± 0.20 and 4.4 ± 0.24 shoots per explant on the different media variants. The highest value was observed for shoots growing on MS media supplemented with BAP 1.0 mg/L and IAA 0.1 mg/L [16].
This technique aims to obtain a large number of homogeneous plants, using only a small fragment of the donor plant, in a relatively short time. Plant multiplication via axillary bud development, as used in our experiment, provides a renewable, inexhaustible amount of the raw material, allowing for the assessment of the phytochemical profile and testing the biological activity  Primary explants failed to respond to MS medium without plant growth regulators, that is why this variant was withdrawn from our investigation. The hormonal investigation, regardless of the combinations and the concentration used, resulted in the response of explants (100%) and gave the largest number of new cloned shoots, with the value between 5.50 ± 0.86 and 6.79 ± 0.48. The values of the mean number of shoots calculated per one explant were not significantly different regardless of the increase in concentration of BAP and GA 3 in the culture medium, on which shoots grew ( Figure 1; Table 1).
It is worth noticing that shoots grew vigorously, did not develop roots spontaneously, and also did not show any signs of verification or callusing at base, which is important for obtaining uniform shoot biomass. This study indicated the alternative method for effective and rapid shoot multiplication of E. alpinum. However, the increase in the concentration of BAP did not provide the highest biotechnological parameters compared to our previous studies [6,7]. In the case of E. planum, the highest mean number of shoots developed via axillary buds was 15.58 ± 0.54-17.10 ± 0.60 shoots per explant, depending on the culture media: MS + BAP 1.0 mg/L + IAA 1.0 mg/L or MS + BAP 1.0 mg/L + IAA 0.1 mg/L [14]. More shoots (13.30 ± 3.73), comparing to the control, were obtained for E. campestre when cultured on the same media composition as for E. planum [15]. The efficiency of shoot multiplication for E. maritimum varied between 1.2 ± 0.20 and 4.4 ± 0.24 shoots per explant on the different media variants. The highest value was observed for shoots growing on MS media supplemented with BAP 1.0 mg/L and IAA 0.1 mg/L [16].
This technique aims to obtain a large number of homogeneous plants, using only a small fragment of the donor plant, in a relatively short time. Plant multiplication via axillary bud development, as used in our experiment, provides a renewable, inexhaustible amount of the raw material, allowing for the assessment of the phytochemical profile and testing the biological activity of the extracts, which is particularly important in the case of a rare and endangered plant. In addition, it is the alternative method of clonal multiplication of a plant from a different climate zone and of a low germination rate [8].  of the extracts, which is particularly important in the case of a rare and endangered plant. In addition, it is the alternative method of clonal multiplication of a plant from a different climate zone and of a low germination rate [8].

The Phytochemical Analysis of Shoots from In Vitro Cultures and In Vivo Plants
Shoots harvested from in vitro cultures as well as shoots from in vivo plants were subjected to the phytochemical analysis. The LC-MS base peak and the UV (270 and 330 nm) chromatograms of the Eryngium alpinum L. are presented in Figures 2 and 3.  The retention times (RT), the observed and reference exact ion mass, the fragmentation spectra and the details are presented for the annotated compounds in Table 2. The annotation of compounds was carried out by comparing the observed and calculated exact mass for ions and the fragmentation pattern in positive and negative ion modes. Identification was complemented by applying the commercially available standards. During the analysis, 98 compounds were annotated and nine compounds were confirmed using the external standards. The main annotated compounds were phenylpropanoids, such as flavonoids (F), hydroxycinnamic acid derivates (HCA), and coumarins (C). Benzoic acid derivates (BA) and triterpenoid saponins (TT) were recognized in the samples. Other annotated groups of compounds were amino acids (AA), nucleotides (NA), carboxylic acids, some vitamins, and phytohormones.       The retention times (RT), the observed and reference exact ion mass, the fragmentation spectra and the details are presented for the annotated compounds in Table 2. The annotation of compounds was carried out by comparing the observed and calculated exact mass for ions and the fragmentation pattern in positive and negative ion modes. Identification was complemented by applying the commercially available standards. During the analysis, 98 compounds were annotated and nine compounds were confirmed using the external standards. The main annotated compounds were phenylpropanoids, such as flavonoids (F), hydroxycinnamic acid derivates (HCA), and coumarins (C). Benzoic acid derivates (BA) and triterpenoid saponins (TT) were recognized in the samples. Other annotated groups of compounds were amino acids (AA), nucleotides (NA), carboxylic acids, some vitamins, and phytohormones. One of the major groups of compounds were hydroxycinnamic acid derivates, which include conjugates of coumaric, caffeic and ferulic acid with the hexose (neutral losses −162.0834, C 6 H 10 O 5 ) and the quinic acid (characteristic fragment m/z 191.0195, C 7 H 11 O 6 − ). Three conjugates of caffeic acid and quinic acid were annotated in the sample namely neochlorogenic acid (5-caffeoylquinic acid, RT = 5.57 min), chlorogenic acid (3-caffeoylquinic acid, RT = 6.06), and isochlorogenic acid (5Z-caffeoylquinic acid, RT = 7.02 min); they were previously described by Kikowska et al. [6,7]. The pseudo-molecular ion m/z 353.08743 corresponded with the formula C 16  Coumarins such as umbeliferone, scopoletin, 7-methoxycoumarin, and dihydroxycoumarin were recognized in our sample basing on the exact mass and the fragmentation pattern and were previously described for the different Eryngium species and the Apiaceae family [18], however, for the first time they were recognized in E. alpinum. Moreover, the conjugates with glucose were tentatively identified  tentatively recognized in the sample. Also, citric acid was identified as a major carboxylic acid. Twelve amino acids and dipeptides were observed in positive and negative ion mode. Three nucleotides were recognized as uridine, adenosine and guanosine. The pseudo-molecular ions at m/z 182.04490 (C 8 H 8 NO 4 − ) and 218.10310 (C 9 H 16 NO 5 − ) were annotated as 4-pyridoxic acid and pantothenic acid, the major vitamins in the extracts. Some of phytohormones such as 5-hydroxy-3-indoleacetic acid, gibberellic acid, 12-hydroxyjasmonic acid, jasmonic acid, and OPDA were putatively identified by means of the exact mass and the fragmentation pattern. The results of the study indicated that the extracts obtained from E. alpinum shoots, both from in vitro cultures in vivo plantlets, inhibited growth of Acanthamoeba sp. trophozoites to varying degrees (Tables 3-5; Figures 4 and 5).        The dependence of the effect on the extract concentration and treatment time was noted. The strongest effect was observed for leaves from in vitro shoot culture. The extract showed the highest antiamoebicidal effect already on the second day of treatment: indicated inhibition of trophozoites was 81.14%, 66.38%, and 54.99% at the concentrations of 5 mg/mL, 2.5 mg/mL, and 0.5 mg/mL, respectively (Table 4, Figure 4). The extract from shoots of in vivo plants at a dose of 0.5 and 2.5 mg/mL weakly inhibited the development of trophozoites (Table 3, Figure 3). The best IC50 index was calculated for leaves from the shoot culture extract. On the second day of treatment, the IC50 value was 0.35 mg/mL (Table 5). Due to the problems in the treatment of opportunistic Acanthamoeba spp. and the lack of effective but safe drugs, the search continues for substances of plant origin that, applied as combined therapy, could contribute to decreasing the effective doses of antibiotics used [10,12].
It was shown in our studies that the extract of leaves from in vitro shoot culture of E. alpinum at a dose of 0.5 mg/mL was effective in inhibiting trophozoites, which can be interpreted as favourable compared to the amoebicidal effect of the plant extracts such as Allium sativum at 3.9 mg/mL [28], Salvia staminea at 16 mg/mL [29], Peucedanum caucasicum, P. palimbioides, P. chryseum, P. The dependence of the effect on the extract concentration and treatment time was noted. The strongest effect was observed for leaves from in vitro shoot culture. The extract showed the highest antiamoebicidal effect already on the second day of treatment: indicated inhibition of trophozoites was 81.14%, 66.38%, and 54.99% at the concentrations of 5 mg/mL, 2.5 mg/mL, and 0.5 mg/mL, respectively (Table 4, Figure 4). The extract from shoots of in vivo plants at a dose of 0.5 and 2.5 mg/mL weakly inhibited the development of trophozoites (Table 3, Figure 3). The best IC 50 index was calculated for leaves from the shoot culture extract. On the second day of treatment, the IC 50 value was 0.35 mg/mL (Table 5).
Due to the problems in the treatment of opportunistic Acanthamoeba spp. and the lack of effective but safe drugs, the search continues for substances of plant origin that, applied as combined therapy, could contribute to decreasing the effective doses of antibiotics used [10,12].
The flavonoid-saponin fraction of the ethanolic extract from leaves of Eryngium planum L., at the concentration of 1 mg/mL, with the similar phytochemical pattern to E. alpinum, showed the amoebistatic effect-76% inhibition of amoebae growth on the third day of treatment. However, the flavonoid fraction from leaves at the concentration of 5 mg/mL revealed the 56.1% inhibitory effect and the phenolic acid fraction at the concentration of 2 mg/mL showed 36.8% inhibition. The authors concluded that the activity may be correlated with the saponin actions, which may be associated with the integration between those compounds and the cell wall of Acanthamoeba [27]. As stated by Mahboob et al. [34], the acanthamoebicidal effect of Lonicera japonica flower, which evoked a significant reduction of trophozoite viability, depends mostly on the major compound form the extract, that is chlorogenic acid. According to Bittner Fialová, rosmarinic acid and its derivates appeared to be promising anti-Acanthamoeba agents with the EC 50 values between 5.6 ± 0.3 mM and 19.1 ± 0.4 mM [35]. The biological study of the Passiflora spp. extracts from leaves and callus biomass revealed that all the samples showed amoebostatic and amoebicidal properties at the concentrations from 4 to 12 mg/mL. The authors tried to find a correlation between the studied activity and the presence of phenolic compounds, with particular emphasis on flavonoids [26]. Moreover, it is noteworthy that quercetin exhibited potent antiamoebic activities against Acanthamoeba [36]. These findings were accordingly similar to the results of the study performed on fractions of the ethanol extracts prepared from Frankenia thymifolia. The fractions showed moderate activity against Acanthamoeba castellanii, which may be associated with the presence of quercetin and its derivatives [37]. As it was shown in our study, E. alpinum shoots, in addition to the presence of phenolic acids and flavonoids, are characterized by a broad spectrum of coumarins. And as it results from numerous studies, phenolic compounds in the extracts of the species such as Allium sativum, Solidago virgaurea, Teucrium chamaedrys or Peucedanum spp. are responsible for the amoebicidal effect [11].
To our best knowledge, the present report is the first one that discusses the phytochemical screening and discusses the antiamoebic activity of Eryngium alpinum L. shoots from in vitro cultures and in vivo plants of this endangered species.

The Plant Material Origin
The fragments of the cuttings of Eryngium alpinum L.

Establishment of In Vitro Cultures
Young shoots with lateral buds were harvested. The collected explants were disinfected and transferred into basal MS medium [38] with plant growth regulators (PGRs), namely cytokinin BAP (6-benzylaminopurine), auxin IAA (indole-3-acetic acid), and gibberellin GA 3 (gibberellic acid) at the concentration of 1.0 mg/l (Table 1), 0.76% agar and pH set to 5.8 before autoclaving at 121 • C, 105 kPa for 20 min. All PGRs and agar originated from Sigma-Aldrich (Saint Louis, MO, USA). The cultures were placed in a growth chamber under controlled conditions, i.e., 21 • C with a 16 h light/8 h dark photoperiod, 55 µmol/m 2 s light, and subcultured every five weeks. Multiplication of shoots was repeated three times for each hormonal treatment using at least 10 explants (2-3 per flask).

Detection of Metabolites in the Extracts Using UPLC-HESI-II-HRMS
In order to conduct the phytochemical analysis, the exact amounts of fresh biomass from basal leaves of the intact plants as well as shoots from the in vitro cultures were dried at 40 • C for 24 h to a constant weight. The dried samples were extracted with 70% (v/v) EtOH (25 mg DW to 2.0 mL) in safe-lock tubes (Eppendorf, Hamburg, Germany). The samples were shaken at 3000 rpm for 20 min (IKA MS 3 Basic Vortex Mixer, Staufen, Germany) and centrifuged at 12,000 rpm, at 4 • C for 15 min (Allegra 21 centrifuge, Beckman Coulter, Brea, CA, USA). Supernatants were filtered through a 0.22 µm PTFE syringe filter (Φ 13 mm, Kinesis Ltd, St. Neots, U.K.). Aquity UPLC (Waters, Milford, MA, USA) with a high resolution Orbitrap mass spectrometer (Thermo Fischer, Bremen, Germany) were applied to the phytochemical analysis of the ethanolic extracts. BEH C13 column (1.7 µm, 2.1 × 150 mm, Waters) was used for separation of the samples (3 µL, partial loop mode) at 45 • C column temperature and 300 µL/min flow rate. 0.1% of formic acid in water (solvent A, MiliQ system, Merck, Darmstadt, Germany) and acetonitrile (solvent B, LC/MS grade, Merck) were used in gradient: initial-5% B, 20 min-75% B, 22 min-98% B, and isocratic 98% B for 24 min. The PDA detector scanned in the range 220-400 nm at frequency 20 spectra/s. The Orbitrap mass spectrometer equipped with the heated electrospray ion source II (HESI-II) operated in negative and positive ion mode. HESI II settings were: capillary voltage-2.5 kV (negative) and 3.5 kV (positive), sheath gas flow-35, auxiliary gas flow-10, sweep gas flow-3 arbitrary units, ion transfer tube temperature-400 • C, auxiliary gas heater temperature-350 • C, and S-lens RF level 50. The full-MS spectra were recorded at mass resolution of 70,000 in the range 150-2000 m/z and 200 ms maximum inject time. The data dependent MS2 spectra were recorded at resolution of 17,500. The data files were processed using Xcalibur Qual Browser (Thermo Fischer) and MSDIAL 3.9 software [39].

Acanthamoebic Activity Examination
In order to conduct the biological analysis, the exact amounts of fresh biomass from basal leaves of the intact plants as well as shoots from the in vitro cultures were dried at 40 • C for 24 h to a constant weight. Dried shoots from in vivo plants and in vitro cultures were extracted three times with EtOH 70% (v/v) at 95 • C. The extracts were concentrated under reduced pressure and used for the evaluation of the antiamoebic studies. The extract samples were weighed and then dissolved in 40 mL of DMSO (dimethyl sulfoxide). Distilled water was added to the solution to obtain the appropriate concentration. Then 200 mL of the appropriately diluted solution was added to 2 mL of trophozoites cultures to obtain the expected final concentrations (0.5, 2.5 and 5 mg/mL). In this study the strain of Acanthamoeba, isolated from the environmental sample, was used. This Acanthamoeba sp. strain was deposited in GenBank (NCBI) under the accession number KY203908. The pathogenicity of this strain was tested on laboratory animals. The research showed that this strain of amoebae is pathogenic for mice. The amoebae were axenically cultured on the liquid medium containing 2% Bacto-Casitone. Parasitological examination of the extracts was performed according to Derda et al. [9]. The study investigated the activity of the ethanol extracts from in vitro shoot cultures and in vivo plants. The increase in the number of parasites in culture was studied. Thoma hemocytometry chamber was used for cell counting. The amoebae were counted three times at 24 h intervals. The control consisted of cultured trophozoites without any extracts. The relationship between the extract concentration and the time of treatment for amoebae cultures was investigated.

The Statistical Analysis
The mean number of E. alpinum shoots and their length as well as the standard error were calculated in each hormonal variant of the culture medium. The data from biotechnological experiments were analyzed using a one-way analysis of variance (ANOVA) and the statistical significance was determined using Duncan's POST-HOC test (p-value < 0.05). All the analyses were conducted employing STATISTICA v. 13 (StatSoft, Inc. 2015). The mean number of amoebae and standard deviation were calculated in each measurement group. The statistical analysis was determined employing the Mann-Whitney and ANOVA tests. Statistical significance was defined as p < 0.05.

Conclusions
In vitro shoot culture of Eryngium alpinum L. can be considered a valuable alternative source of biomass that is rich in desired secondary metabolites such as phenolic acids, coumarins, flavonoids, and triterpenoid saponins. This is especially important for protected species, the collection of which from the natural environment is impossible. The results suggest that the extracts from E. alpinum may be promising natural products for Acanthamoeba treatment. Further studies are necessary to clarify which bioactive compounds are responsible for the observed activity.