The Evaluation of Phenolic Acids and Flavonoids Content and Antiprotozoal Activity of Eryngium Species Biomass Produced by Biotechnological Methods

Three species from the Eryngium L. genus—E. campestre, E. maritimum, and E. planum, plants with a rich chemical composition, were selected for phytochemical and biological studies. The applied biotechnological methods allowed to obtain the biomass of these rare or protected species in the form of multiplied shoots (stationary system) and roots cultured in a liquid medium (agitated system). In the extracts from the raw material obtained under in vitro conditions, the content of selected phenolic acids and flavonoids (HPLC-DAD method) as well as the total of polyphenols (Folin–Ciocalteu assay) were quantified. The highest amount of all phenolic compounds was found in extracts from E. planum roots (950.90 ± 33.52 mg/100 g d.w.), and the lowest from E. campestre roots (285.00 ± 10.07 mg/100 g d.w.). The quantitatively dominant compound proved to be rosmarinic acid. The highest amounts were confirmed for E. planum root extract (694.58 mg/100 g d.w.), followed by E. planum (388.95 mg/100 g d.w.) and E. campestre (325.85 mg/100 g d.w.) shoot extracts. The total content of polyphenols was always increased in the biomass from in vitro cultures in comparison to the analogous organs of intact plants of each species. The obtained extracts were assessed for antiprotozoal activity against Acanthamoeba sp. The strength of biological activity of the extracts correlated with the content of phenolic compounds. To our knowledge, this is the first report on the amoebicidal activity of E. campestre, E. maritimum, and E. planum extracts from biomass produced by biotechnological methods.


Introduction
Eryngium L. (Sea Holly, Eryngo) comprises about 230-250 species and is, therefore, the largest genus of the Saniculoideae subfamily from the Apiaceae family. This taxon is widespread in Central Asia, America, Central, and Southeast Europe, North Africa, and Australia [1,2]. Some of them, including E. campestre, E. maritimum, and E. planum, are described in Flora Europaea [3]. Among Polish taxa of the genus, E. maritimum is endangered and protected, E. planum and E. campestre are rare and can be found only in restricted regions [4].
Based on the review of the scientific literature up to now, the phytochemical investigations of those three Eryngium species revealed the presence of triterpenoid saponins,

Results and Discussion
The introduction of selected Eryngium species (E. campestre, E. maritimum, and E. planum) into in vitro culture conditions was previously described [10][11][12]. Material for biotechnological, phytochemical, and biological research came from stabilized cultures of the described species. Briefly, initial shoot cultures originated from aseptic seedling explants (fruits obtained from plants from natural sites) of E. campestre and E. planum, or stem fragments with nodes (young plants collected from a botanical garden) of E. maritimum. Despite many attempts, it was not possible to obtain seedlings from axenic seeds of E. maritimum continuing their development in in vitro cultures. For the subsequent growth of shoot cultures and the regeneration of the largest number of shoots, the media were supplemented with plant growth regulators. Adventitious root cultures were initiated from explants derived from rooted plantlets, on a solid medium in the process of micropropagation ( Figure 1).
Shoot multiplication by the development of axillary buds of three Eryngium species is a fast and effective strategy for obtaining a relatively large number of new shoots ( Table 1). The studied species were characterized by a high shoot induction coefficient (96.3-100%). For these species, it was possible to obtain a high number of shoots (except for E. maritimum), which guarantees obtaining the necessary biomass for phytochemical and biological analyzes. Shoot multiplication by the development of axillary buds of three Eryngium species is a fast and effective strategy for obtaining a relatively large number of new shoots ( Table  1). The studied species were characterized by a high shoot induction coefficient (96. 3-100%). For these species, it was possible to obtain a high number of shoots (except for E. maritimum), which guarantees obtaining the necessary biomass for phytochemical and biological analyzes. The shoots were characterized by the correct morphology and physiology, did not show hyperhydricity, necrosis, or callus formation at the shoot base, therefore, these raw materials can be described as homogeneous and of high quality ( Figure 1).
The presence of cytokinin in the medium was an important factor in the process of shoot multiplication. According to the literature, 6-benzyladenine (whether used alone or in combination with auxin) influenced the number of micro-shoots formed in in vitro cultures. This relationship was also observed for other species belonging to the Apiaceae family: Thapsia garganica L. [13], Arracacia xanthorrhiza Bancr. [14], or Anethum graveolens L. [15].
Roots play an important role not only in plant growth and development but also constitute a place of accumulation of secondary metabolites, in large part also those useful from a pharmaceutical point of view. Moreover, the adventitious roots show significant productivity, which is why they are often an important source of these substances [16]. In this study, a method of obtaining and cultivating adventitious roots in liquid media was  The shoots were characterized by the correct morphology and physiology, did not show hyperhydricity, necrosis, or callus formation at the shoot base, therefore, these raw materials can be described as homogeneous and of high quality ( Figure 1).
The presence of cytokinin in the medium was an important factor in the process of shoot multiplication. According to the literature, 6-benzyladenine (whether used alone or in combination with auxin) influenced the number of micro-shoots formed in in vitro cultures. This relationship was also observed for other species belonging to the Apiaceae family: Thapsia garganica L. [13], Arracacia xanthorrhiza Bancr. [14], or Anethum graveolens L. [15].
Roots play an important role not only in plant growth and development but also constitute a place of accumulation of secondary metabolites, in large part also those useful from a pharmaceutical point of view. Moreover, the adventitious roots show significant productivity, which is why they are often an important source of these substances [16]. In this study, a method of obtaining and cultivating adventitious roots in liquid media was developed for the domestic species of Eryngium. The main pharmaceutical raw material Eryngium is the root of intact plants, rich in secondary metabolites, therefore, in vitro cultures of these organs were established.
Undoubtedly, rosmarinic acid (RA) was the phenolic acid found in the highest concentration in all of the tested shoot and root in vitro cultures of Eryngium spp. The richest source of RA, based on our estimations, were root (694.58 mg/100 g d.w.) and shoot (388.95 mg/100 g d.w.) cultures of E. planum (Table 3). Within all detected phenolic compounds, RA was quantitatively dominant also in E. maritimum (181.27 mg/100 g d.w.-root; 174.51 mg/100 g d.w.-shoot cultures) and in E. campestre (100.91 mg/100 g d.w.-root; 325.85 mg/100 g d.w.-shoot cultures) ( Table 3). RA was found most notably also before in Eryngium species, especially in shoots and roots of intact plants and micropropagated in vitro plantlets: E. campestre [12], E. maritimum [11], and E. planum [10]. The qualitative screening analysis of Eryngium genus performed by the team of Le Claire revealed the presence of rosmarinic acid in the root extracts of 11 Eryngium species [17].
Comparing the total phenolic acids contents estimated with the HPLC method, E. planum root cultures proved to be the richest source of these compounds (924.21 mg/ 100 g d.w.), mainly due to the high content of RA (Table 3). Moreover, high phenolic acid total contents were detected for E. campestre (739.26 mg/100 g d.w.) and E. planum (729.10 mg/100 g d.w.) shoot cultures (Table 3). The flavonoid estimations with HPLC showed also quantitative differences within individual compounds depending on studied species as well as Eryngium spp. (Table 3). Quantitative analyzes generally show that shoots from in vitro cultures have a higher content of flavonoids than roots grown under the same conditions. The main flavonoid for E. campestre was astragalin (57.05 mg/100 g d.w.-shoots, 6.21 mg/100 g d.w.-roots), for E. maritimum-rutoside (14.32 mg/100 g d.w.-shoots, 12.16 mg/100 g d.w.-roots) and for E. planum was quercitrin (11.25 mg/100 g d.w.-shoots, 14.41 mg/100 g d.w.-roots) ( Table 3). Comparing the total contents of flavonoids in in vitro cultures of analogous organs of Eryngium spp., a trend according to the formula can be noticed: E. campestre > E. maritimum > E. planum. Summing up, the most valuable source of flavonoids is the in vitro shoots of E. campestre, mainly due to the very high content of astragalin (Table 3).
Based on the comparison of total phenolic compounds contents estimated with the HPLC method, the richest source of these compounds was root cultures of E. planum (950.90 mg/100 g d.w.) and shoot cultures of E. campestre (846.27 mg/100 g d.w.) ( Table 3).
Quantitative studies of the content of polyphenols using spectrophotometric methods according to the modified method of Meng et al. [25] with the use of the Folin-Ciocalteu reagent were carried out comparatively for the raw materials of intact plants and organs (shoots and roots) from in vitro cultures of three Eryngium species (Table 4). The results are presented as the mean ± SD of six independent repetitions. Mean values within a column with the same letter are not significantly different at p < 0.05 using Duncan's multiple range test. The first letter of the alphabet for the highest values, the next for statistically significant decreasing values.
The content of the sum of polyphenols in the tested raw materials was always higher in the biomass from in vitro cultures than in the analogous organs from intact plants for selected species (Table 4). In particular, the root biomass from in vitro cultures was characterized by a much higher polyphenol content than the roots of intact plants. The highest content of total polyphenols (2254 of gallic acid equivalent (mg/100 g)) was found in the roots of in vitro cultures of E. campestre, which is consistent with the observation of their morphology-these were the youngest roots, characterized by a large number of lateral roots.
The antiprotozoal action of the extracts obtained from Eryngium, both from in vitro shoot and root cultures, was evaluated in vitro against Acanthamoeba sp. strain Ac55. The results of the study indicated that the extracts inhibited the growth of trophozoites to varying degrees. The dependence of the effect on the extract concentration and treatment time was noted (Tables 5-8).
In the case of E. campestre, the strongest effect was observed for in vitro shoot culture ( Table 5). The higher sample concentration was applied, the more effective activity was observed in comparison with the control during the same time interval. It can be noticed that the shoot extract activity was potent from the beginning of the treatment and remained at a similar level (5.0 mg/mL) or a significantly lower level (0.5 mg/L and 2.5 mg/L) during the next days, while the activity of the root extract increased with the time of treatment (0.5 mg/L and 2.5 mg/L).  Mean values within a raw (capital letters) and a column (small letters) with the same letter are not significantly different at p < 0.05 using Duncan's multiple range test. The first letter of the alphabet for the highest values, the next for statistically significant decreasing values. n = 18. MN-mean number of trophozoites; GI-growth inhibition.  In the case of E. maritimum, a stronger effect was observed for in vitro root culture (Table 6). Generally, the higher sample concentration was applied, the more effective activity was observed in comparison with the control during the same time interval. It can be noticed that the activity of both shoot and root extracts (0.5 mg/L and 2.5 mg/L) increased with the time of treatment, while the activity of the extract of a higher concentration (5.0 mg/L) remained at a statistically similar level.
In the case of E. planum, a strong effect was observed both for in vitro shoot and root culture (Table 7). Generally, no statistical differences were observed in the activity of the extracts between the higher (2.5 mg/L) and the highest concentration (5.0 mg/L) during the same time interval. Moreover, no statistical differences were observed in the activity of the extracts between the 3rd and 4th day taking into consideration the highest concentrations of the extract. On the fourth day of exposure of trophozoites to the shoot culture extract, regardless of the concentration, cysts appeared.
The lowest IC 50 index was calculated for the E. planum shoot culture extract. On the second day of Acanthamoeba trophozoites treatment, the IC 50 value was 0.25 mg/mL. During the entire treatment period, the lowest IC 50 was obtained for the extract of E. campestre shoot culture and shoot and root cultures of E. planum ( Table 8).
The obtained extracts were assessed for antiprotozoal activity against Acanthamoeba sp. The strength of biological activity of the extracts correlated with the content of phenolic compounds. The highest amounts of phenolic acids and flavonoids were confirmed for E. planum root extract, followed by E. planum and E. campestre shoot extracts, which was correlated with the results of IC 50 for those extracts. The conducted studies showed a strong correlation between the activity of Eryngium species extracts on Acanthamoeba trophozoites proliferation with the sum of flavonoids and phenolic acids determined by using the HPLC-DAD method (R 2 in the range 0.7478-0.8424) (Figure 2). This effect of the synergistic action of phenolic compounds may be due to the presence in the plant materials of chlorogenic acid [26], rosmarinic acid [27], and quercetin [28], which are well known as amoebicidal agents.
Previously, the evaluation of the amoebicidal activity of E. planum was conducted for the extracts and fractions obtained from leaves and roots of intact plants. Among different fractions from leaf and root ethanolic extracts: flavonoid, flavonoid-saponin, and saponin assayed for the antiamoebic activity studies, the phenolic acid fraction from roots at the concentration of 5.0 mg/L showed the activity on the A. castellanii trophozoites. According to the authors, this activity is correlated with the antioxidant activity of phenolic acids. This class of phenolic compounds may cause damage to the plasma membrane, which results in a leakage of intracellular constituents from the cell [29]. For the alpine species (Eryngium alpinum), the activity of the extract from the leaves of intact plants and in vitro shoot cultures was compared. The extract of shoots multiplied in vitro showed the highest antiamoebicidal effect already on the second day of treatment: the percent of 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. The extract from shoots of intact plants at a dose of 0.5 and 2.5 mg/mL weakly inhibited the development of trophozoites [30]. related with the results of IC50 for those extracts. The conducted studies showed a strong correlation between the activity of Eryngium species extracts on Acanthamoeba trophozoites proliferation with the sum of flavonoids and phenolic acids determined by using the HPLC-DAD method (R 2 in the range 0.7478-0.8424) (Figure 2). This effect of the synergistic action of phenolic compounds may be due to the presence in the plant materials of chlorogenic acid [26], rosmarinic acid [27], and quercetin [28], which are well known as amoebicidal agents. In our experiment, on the fourth day of exposure of trophozoites to the E. planum shoot culture extract, regardless of the concentration, cysts appeared. The parasite has a strong ability to transform into a dormant cyst stage under stressful conditions [8]. Unfortunately, the cyst walls provide a physical barrier for drugs/extracts/fraction or isolated compounds to target amoeba residing within the shell [31]. In the authors opinion, the discovery of various antiacanthamoebic natural products tested in vitro has not been able to enter the drug development process due to their inefficacy against the cysts.
In the literature on the subject, more scientific information on the plant extracts with the amoebicidal or amoebistatic activity against pathogenic strains of Acanthamoeba spp. can be found regarding the extracts from aerial parts of Centaurea bella, C. daghestanica, Rhaponticum pulchrum, Tanacetum vulgare [32], roots and leaves of Rubus chamaemorus, Pueraria lobata, Solidago spp. [33], leaves and calluses of Passiflora spp. [34], calluses, leaves, and roots of micropropagated plantlets of Chaenomeles japonica [35]. Buddleia cordata is a plant of potential and practical use in the treatment of acanthamoebosis-the therapy uses extracts from the roots, bark, and fruits of this plant. It was noted that the chemically active substance in these extracts is linarin, which belongs to the flavones [36]. Flavonoids and phenolic acids are the phenolic compounds that may be found to exert a strong antiprotozoal effect [26][27][28]37]. The problem of treating acanthamoebiasis with the use of substances of plant origin has recently become very popular-the therapeutic properties of other plants are still being investigated.
To our knowledge, this is the first report on the amoebicidal activity of E. campestre, E. maritimum, and E. planum extracts from biomass produced by biotechnological methods.

Plant Material
Fruits of E. campestre were collected from steppe reserve Owczary (Sękowa, Poland) and of E. planum from natural habitats in Poland (Lukaszewo, Kuyavian-Pomeranian province). Primary explants-shoot fragments with lateral buds of. E. maritimum-were shared by the Botanical Garden of Adam Mickiewicz University in Poznań (Poland). The appropriate explants were surface disinfected and introduced to the in vitro conditions according to the procedure adopted by Thiem et al. [10] and Kikowska et al. [11,12].

Shoot Cultures
Shoots were multiplied through an axillary branching method by repetitive transfer of suitable explant. Multishoots were divided into single shoots and transferred to new medium solidified with 7.6 g/L agar with the same supplementation every 5-6 weeks. E. campestre, E. planum, and E. maritimum were multiplied on MS medium [38] enriched with 6-benzyladenine (BA, 1.0 mg/L) and indole-3-acetic acid (IAA, 1.0 mg/L). The percentage of explants that proliferated buds, total number of shoots per explant, and length of shoots were recorded after 6 weeks of 18-19th subculture. Multiplication of shoots was replicated three times at least with 10 explants per treatment. The shoots were multiplied according to the procedure adopted by Thiem et al. [10] and Kikowska et al. [11,12].

Root Cultures
Root fragments with tips (1.0-2.0 cm long) obtained from axenic plantlets were used for adventitious root cultures initiation. The explants were transferred into MS liquid media with an auxin IAA (1.0 mg/L). The cultures were maintained on a rotary shaker at 100 rpm. Root cultures were inoculated into the same liquid media and the same culture conditions as the one employed for routine subculturing were applied. The root cultures were subcultured at 5-week intervals. The roots were maintained according to the procedure adopted by Thiem et al. [10] and Kikowska et al. [11,12].

Culture Conditions
The shoot cultures were grown under artificial light-55 µmol/m 2 s (16 h light/8 h dark photoperiod) and the root cultures were cultured in the darkness. Both cultures were maintained at a temperature of 21 • C ± 2 • C.

Total Phenolics Content
The content of the sum of polyphenols was determined with the Folin-Ciocalteu (Sigma-Aldrich, Saint Louis, MO, USA) reagent according to the modified method of Meng et al. [25]. The dried and weighed plant material (shoots and roots from in vitro cultures and intact plants) was extracted four times in 70% (v/v) MeOH in a water bath, under reflux, at a temperature of 95 • C, each time for 1 h. The obtained extracts are combined and concentrated to a volume of 25 mL on a vacuum evaporator at 40 • C. Briefly, 0.1 mL of each extract or gallic acid solution at different concentrations (0.02-0.08 mg/mL) was mixed with 0.1 mL of Folin-Ciocalteu reagent and allowed to react at room temperature for 3 min. Finally, 1.0 mL of aqueous solution of sodium carbonate (7.0%, w/v) was added, and the mixture was incubated in the dark at room temperature for 60 min. The blank sample contained water instead of the extract or gallic acid solution. From each extract, three samples were prepared, from each four analyzes were performed. The measurements were made at the wavelength λ = 760 nm, in the Perkin-Elmer Lambda (Norwalk, CT, USA) 35 UV/VIS apparatus. Gallic acid (GA) was used as an external standard to plot the calibration curve (y = 94.065x + 0.0331, R 2 = 0.9999) and the results were expressed as milligrams of GA equivalent per gram of dry weight of the plant material. The values were expressed as the mean of six replications ± SD.

Antiamoebic Activity
In this study, the Acanthamoeba sp. strain Ac55 (isolated from a patient with keratitis, T4 genotype) deposited in GenBank (NCBI) under accession number KP120880 was used. The amoebae were axenically cultured on a liquid medium containing 2% Bacto-Casitone. The methanolic extracts were dissolved in 50 µL of dimethylsulfoxide (DMSO) and then diluted with distilled water to obtain the appropriate concentrations. These dilutions were added to the axenic culture of amoebae containing 5 × 10 4 cells/mL at the concentrations of 0.5-5 mg/mL. The increase or decrease in the number of amoebae was checked at 24 h intervals for three days (2nd, 3rd, 4th) in a Thoma hemocytometer chamber. The control consisted of cultured trophozoites without extracts. The relationship between fraction concentration and the time of treatment of trophozoite cultures was investigated. The method was described by us previously [29][30][31][32][33][34].

Statistical Analysis
The data from biotechnological, phytochemical, and biological 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, Kraków, Poland).

Conclusions
Shoot and root in vitro cultures of Eryngium species may be considered a valuable alternative source of biomass that is rich in valuable bioactive compounds such as phenolic acids and flavonoids. The content of the sum of polyphenols in Eryngium species was always higher in the biomass from in vitro cultures than in the analogous organs of ground plants of each species. The strength of antiprotozoal activity of extracts correlated with the content of phenolic compounds determined in them. The results suggest that the extracts from Eryngium spp. may be promising candidates for Acanthamoeba treatment.

Data Availability Statement:
The data presented in this study are available from the authors.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the biomass/extracts are available from the authors.