Skip to main content
NCBI home page
Molecules logoLink to Molecules
. 2020 Mar 20;25(6):1416. doi: 10.3390/molecules25061416

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

Małgorzata Kikowska 1,*, Dariusz Kruszka 2, Monika Derda 3, Edward Hadaś 3, Barbara Thiem 1
PMCID: PMC7144402  PMID: 32244952

Abstract

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.

Keywords: alpine eryngo, in vitro shoot culture, phenolic compounds, triterpenoid saponins, phytochemical analysis, Acanthamoeba treatment

1. 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 in shoots of in vivo plants and in vitro shoot cultures [6,7]. Roots, phytochemically investigated in the study of Le Claire et al., are known to contain chlorogenic acid, R‒(+)‒rosmarinic acid and its derivative – R‒(+)‒3′‒O‒β‒d-glucopyranosyl rosmarinic acid [4]. Caftaric acid, chlorogenic acid, neochlorogenic acid, isochlorogenic acid, 3,4-dihydroxyphenylacetic acid, caffeic acid, and rosmarinic acid were detected in the leaves of in vivo plant and in vitro shoot cultures [6,7]. The dominant components identified in the essential oil of the aerial part of the plant were caryophyllene, bicyclogermacrene, germacrene, cariophyllene oxide, α‒bisabolol, and camphor. Furthermore, the essential oil showed a promising antiphytoviral effect, which is probably correlated with a high content of β-caryophyllene and caryophyllene oxide [5].

In vitro cultures of E. alpinum are a part of the collection of the protected species of the Department of Pharmaceutical Botany and Plant Biotechnology of Poznan University of Medical Sciences (PUMS). The use of in vitro cultures of alpine eryngo allows for conducting the phytochemical analyses and testing further biological activities of this species without depleting its natural sites to obtain the plant material. Since the seeds exhibit strong dormancy and a low germination rate, the generative propagation seems to be unsatisfactory to provide the raw material. In this regard, in vitro cultures of a number of the medicinal plant species offer an alternative source of uniform and renewable biomass, usually with high biosynthesis capacity for the desired compounds, with the same optical stereometry as in nature, and thus provide the valuable raw material. An important advantage of in vitro cultures is the possibility of intensifying biosynthesis and affecting the accumulation of desired metabolites in biomass, applying various biotechnological methods. It is possible to obtain biomass in a continuous large-scale production process [8].

Acanthamoeba is a single-celled eukaryote existing in two forms: dormant cysts and vegetative trophozoites. The infective free-living amoeba gains entry into body through eyes and ulcerated skin, which may lead to epithelial and ocular keratitis and granulomatous amoebic encephalitis. Treatment of acanthamoebosis is difficult and not always effective [9,10,11]. In humans, due to the problems in the treatment of opportunistic and pathogenic 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 [10,12,13].

The aim of the study was to obtain shoot biomass of E. alpinum under in vitro conditions and to conduct phytochemical analysis of the crude extracts as well as to evaluate the activity against Acanthamoeba sp. Shoots were developed from meristematic tissue of lateral buds. Then, they were multiplied in vitro on the artificial media by means of the axillary bud proliferation technique. The protocol of shoot multiplication allows for harvesting the high-quality and uniform raw material from alpine eryngo without decreasing the medicinal quality and quantity of bioactive compounds.

The novelty of this manuscript is the indication of the presence of coumarins, triterpenoid saponins, amino acids, dipeptides, and other compounds for the first time for this species. The manuscript aims to enrich the knowledge of phenolic compounds with unexplored phenolic acids, flavonoids, and their derivatives. The present report is the first to show complete phytochemical screening of this important taxon and discusses the antiamoebic activity of Eryngium alpinum L. shoots, both from in vitro cultures and in vivo plants.

2. Results and Discussion

2.1. In Vitro Shoot Culture

Eryngium alpinum L. (Figure 1) was introduced into in vitro cultures and shoot cultures were established in our laboratory to study their capability of producing bioactive compounds under controlled conditions [6,7]. The biotechnological parameters of E. alpinum shoot multiplication under controlled conditions are presented in Table 1.

Figure 1.

Figure 1

Open in a new tab

Eryngium alpinum L.: (a) shoots of in vivo plants (b) shoots from in vitro cultures.

Table 1.

The effect of selected plant growth regulators—BAP (6-benzylaminopurine), IAA (indole-3-acetic acid) and GA3 (gibberellic acid) present in MS media on shoot multiplication ratio and length of multiplied shoots of Eryngium alpinum L. after 40 days of culture.

No. Cytokinin
[mg/L]		 Gibberellin
[mg/L]		 Auxin
[mg/L]		 Mean No. of Shoots ± SE Mean Length of Shoots [cm] ± SE
1. BAP 2.0 GA3 1.0 - 6.56 ± 0.35 ns 2.85 ± 0.06 a,b
2. BAP 2.0 GA3 1.0 IAA 1.0 6.79 ±0.48 2.57 ± 0.80 b
3. BAP 1.0 GA3 1.0 IAA 1.0 6.33 ± 1.21 2.08 ± 0.21 c
4. BAP 1.0 GA3 0.5 IAA 1.0 5.50 ± 1.04 3.03 ± 0.20 a
5. BAP 1.0 GA3 0.5 IAA 1.0 5.50 ± 0.86 3.01 ± 0.15 a,b
Open in a new tab

Mean values within a column with the same letter are not significantly different at p < 0.05. (Duncan’s Multiple Range Test).

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 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].

2.2. 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 Figure 2 and Figure 3.

Figure 2.

Figure 2

Open in a new tab

The qualitative analyses of the Eryngium alpinum L. samples from shoots of in vivo plants (black) and in vitro cultures (red). The base peak chromatograms in negative ion mode were obtained using the UPLC-HESI-II-HRMS system.

Figure 3.

Figure 3

Open in a new tab

The UPLC-UV chromatograms of shoots from in vivo plants (green) and in vitro cultures (blue) of Eryngium alpinum L. extracts recorded at 270 nm (1,2) and 330 nm (3,4).

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.

Table 2.

The annotated compounds in the shoot extracts of Eryngium alpinum L. from in vitro culture and in vivo plant. Metabolites detected by UPLC-HESI-II-HRMS. The compounds were characterized by the exact mass and the fragmentation pattern in negative and positive ion mode.

No RT [min] Ion mode Observed m/z Reference m/z Delta (ppm) Molecular Formula Fragmentation Name MSI Level a Class b CIDc Ref.
1 2.58 [M + H]+ 175.11955 175.11896 3.4 C6H14N4O2 60.0564, 70.0660, 116.0717, 160.0981, 158.0928, 158.0921, Arginine 3 AA 6322
2 2.7 [M − H]− 191.0191 191.01973 −3.3 C6H8O7 57.0329, 111.0069, 129.0175, 191.0193, Citric acid 1 s CA 311
3 2.7 [M − H]− 243.06236 243.06226 0.4 C9H12N2O6 82.0280, 110.0228, 122.0231, 140.0340, 152.0331, 200.0553. Uridine 2 N 6029
4 2.7 [M − H]− 180.0657 180.06662 −5.1 C9H11NO3 72.0073, 93.0335, 119.0484, 163.0383, 180.0649, Tyrosine 1 s AA 6057 3
2.7 [M + H]+ 182.0817 182.08118 2.9 C9H11NO3 119.0493, 123.0442, 136.0758, 147.0441, 165.0553, 182.0824,
5 2.7 [M + H]+ 268.10495 268.10403 3.4 C10H13N5O4 57.0346, 136.0624, 268.1039, Adenosine 2 N 60961
6 2.73 [M − H]− 282.08435 282.08438 −0.1 C10H13N5O5 133.0141, 150.0403, Guanosine 2 N 135398635
7 2.93 [M + H]+ 276.14529 276.1445 2.9 C12H23NO7 86.0970, 132.1028, 212.1273, 230.1401, 258.1352, 276.1455 N-Fructosyl isoleucine 3 AA 137530247
8 3 [M − H]− 182.0449 182.04588 −5.4 C8H9NO4 108.0445, 120.0439, 138.0539, 182.0458 4-Pyridoxic acid 2 O 6723
9 3.65 [M + H]+ 192.06627 192.06552 3.9 C10H9NO3 146.0598, 192.1035 5-Hydroxy-3-indoleacetic acid 3 PH 1826
10 3.76 [M + H]+ 175.11923 175.11896 1.5 C6H14N4O2 60.0564, 116.0717, 160.0981, 158.0928, 158.0921 Arginine 3 AA 6322
11 3.77 [M + H]+ 209.09282 209.09207 3.6 C10H12N2O3 74.0249, 120.0449, 136.0759, 146.0599, 163.0859, 174.0547, 192.0658, Kynurenine 2 AA 846
12 3.92 [M − H]− 359.0995 359.0977 5 C15H20O10 - Glucosyringic acid 3 BA - 4
13 4.01 [M − H]− 338.08862 338.08789 2.2 C15H17NO8 132.0443, 176.0334 Indole + 1O, 1carboxy, O-Hex; 3 AA -
14 4.17 [M + H]+ 213.07613 213.07574 1.8 C10H12O5 149.0606, 195.0643 Trimethoxybenzoic acid 3 BA -
15 4.21 [M − H]− 315.0725 315.071 4.8 C13H16O9 108.0199, 152.0102, 315.0696 Dihydroxybenzoic acid hexoside 3 BA - 4
16 4.26 [M − H]− 167.035 167.03499 0.1 C8H8O4 108.0200, 123.0434, 152.0102, 167.0343 Vanillic acid 2 BA 8468
17 4.26 [M + H]+ 179.03462 179.03389 4.1 C9H6O4 105.6870, 107.0502, 123.0445, 133.0281, 151.0394, 179.1061 Dihydroxycoumarin 2 C -
18 4.26 [M − H]− 329.08838 329.08841 −0.1 C14H18O9 108.0200, 123.0434, 152.0102, 167.0343 Vanillic acid hexoside 3 BA -
19 4.39 [M − H]− 218.1031 218.10339 −1.3 C9H17NO5 71.0121, 88.0389, 99.0432, 116.0707, 140.2067 Pantothenic acid 3 O 6613
20 4.95 [M]+ 266.1389 266.13812 2.9 C14H20NO4 95.0861, 163.0382, 207.0648 Caffeoylcholine 2 HC 6440794
21 5.16 [M − H]− 203.08162 203.0826 −4.8 C11H12N2O2 116.0494, 142.0644, 159.0909, 186.0549, Tryptophan 1 s AA 6305 3
5.16 [M + H]+ 205.09726 205.09715 0.5 C11H12N2O2 118.0661,132.0812, 146.0598, 159.0912, 170.0601, 188.0714,
22 5.18 [M]+ 250.1441 250.14322 3.5 C14H20NO3 - Coumaroylcholine 2 HC 6440550
23 5.3 [M − H]− 181.05003 181.05063 −3.3 C9H10O4 72.9914, 119.0483, 135.0436, 163.0382, 181.0498 Hydroxyphenyllactic acid 3 BA -
24 5.42 [M − H]− 339.07239 339.07214 0.7 C15H16O9 177.0192, Esculin 2 C 5281417
25 5.57 [M − H]− 353.08743 353.0878 −1 C16H18O9 161.0231, 173.0437, 179.0333, 191.0195, Neochlorogenic acid 1 s HC 5280633 1,3
26 5.58 [M − H]− 355.0672 355.06561 4.5 C15H16O10 135.0794, 147.0285, 163.0382, 179.0697, 191.0199, 209.0293 Coumaroyl + C6H9O8 3 HC -
27 5.7 [M + H]+ 167.07097 167.07027 4.2 C9H10O3 111.0448, 139.0757, 149.0232, 167.0708 Dimethoxybenzaldehyde 3 BA -
28 5.71 [M + H]+ 261.14514 261.1445 2.5 C11H20N2O5 84.0451, 86.0605, 132.1017, 198.1126, 244.1189, 261.1274 Glutamylleucine 2 AA 9856500
29 5.73 [M + H]+ 517.15778 517.15574 4 C22H28O14 193.0489, 178.0266, 165.0556, 133.0285 Scopoletin 7-O-dihexoside 2 C -
30 5.91 [M − H]− 341.08795 341.08726 2 C15H18O9 119.0336, 161.0236, 179.0339, Caffeic acid glucoside 2 HC 5281761 3
31 6.06 [M + H]+ 355.1022 355.10236 −0.5 C16H18O9 163.0385, Chlorogenic acid 1 s HC 1794427 3
6.06 [M − H]− 353.08755 353.0878 −0.7 C16H18O9 135.0437, 161.0226, 179.0334, 191.0546
32 6.17 [M + H]+ 295.12949 295.12949 0 C14H18N2O5 120.0811, 166.0859, 186.0907, 232.0963 Glutamylphenylalanine 2 AA 111299
33 6.3 [M − H]− ]− 399.09326 399.09299 0.7 C16H18O9 135.0437, 176.0116, 191.0346, 221.0073 Scopolin 2 C 439514
34 6.49 [M − H]− 367.1038 367.10199 4.9 C17H20O9 134.0357, 149.0238, 163.0483, 191.0562, 193.0493 Feruloylquinic acid 3 HC 10177048 3,4
35 6.5 [M − H]− 355.0665 355.06509 4 C15H16O10 147.0282, 191.0194, 209.0293 Coumaroyl + C6H9O8 3 HC -
36 6.77 [M + H]+ 223.06041 223.0601 1.4 C11H10O5 149.0244, 162.0302, 177.0907, 190.0266, 207.0280, Isofraxidin 2 C 5318565
37 7.00 [M + H]+ 773.21649 773.2135 3.9 C33H40O21 132.1144, 228.9691, 303.0504 Quercetin-3-O-dihexoside-7-O-rhamnoside 3 F 57393076
[M − H]− 771.20292 771.19839 5.9 151.0028, 178.9972, 300.0273, 446.0863, 625.1580
38 7.02 [M − H]− 353.08746 353.08621 3.5 C16H18O9 135.0437, 161.0226, 179.0334, 191.0546(100%) Caffeoylquinic acid
(Isochlorogenic acid)		 2 HC 5315832 1
[M + H]+ 355.10339 355.10236 2.9 137.0612, 163.0386, 201.0543
39 7.14 [M − H]− 337.09293 337.09219 2.2 C16H18O8 163.0483, 191.0562 Coumaroylquinic acid 2 HC 6441280 3
40 7.41 [M + H]+ 611.16309 611.16121 3.1 C27H30O16 303.0502 Quercetin-3-O-hexoside-7-O-rhamnoside 2 F 25080064 2
[M − H]− 609.15906 609.15612 4.8 299.0215, 301.0368, 447.0932, 463.0879
41 7.49 [M − H]− 345.13425 345.13437 −0.3 C19H22O6 143.0849, 221.1323, 239.1435, 273.1489 Gibberellic acid 3 O 6466
42 7.51 [M − H]− 755.2341 755.23986 −7.6 C33H40O20 176.8649, 227.0346, 255.0293, 284.0326, 285.0404, 609.1585 Kaempferol-O-rhamnodihexoside 3 F -
[M + H]+ 757.2229 757.21913 5.0 287.0552
43 7.71 [M − H]− 625.14482 625.14105 6 C27H30O17 151.0014, 178.9977, 255.0304, 271.0252, 300.0289, 301.0323 Quercetin-3-O-dihexoside 2 F 14185727 4
7.71 [M + H]+ 627.15741 627.15558 2.9 C27H30O17 159.8611, 281.4450, 303.0503
44 7.74 [M + H]+ 449.10812 449.10785 0.6 C21H20O11 130.1475, 287.0541, 299.0568, 329.0639, 353.0654 Luteolin-C-hexoside 3 F -
7.74 [M − H]− 447.09238 447.09329 −2 C21H20O11 285.0380, 297.0394, 327.0507, 357.0601
45 7.76 [M − H]− 367.1038 367.10291 2.4 C17H20O9 134.0357, 173.0386, 191.0546 Feruloylquinic acid 3 HC 10177048 3,4
[M + H]+ 369.11871 369.11801 1.9 117.0337, 145.0289, 149.0607, 163.0385, 177.0550, 195.0642
46 7.86 [M + H]+ 755.20820 755.20660 4.2 C33H38O20 127.0398, 145.0505, 303.0504 Unknown flavonoid 3 F -
[M − H]− ]− 753.18783 753.19064 −3.7 301.0349, 446.0853, 463.0864
47 8.03 [M + H]+ 227.12856 227.12779 3.4 C12H18O4 , 131.0861, 149.0969, 167.1077, 191.1070, 209.1177, 227.1267 12-Hydroxyjasmonic acid 2 O 5497122
48 8.17 [M − H]− 172.09709 172.09792 −4.8 C8H15NO3 130.0862, 172.0976 N-Acetylleucine 2 AA 70912
49 8.25 [M − H]− 609.14894 609.14612 4.6 C27H30O16 151.0018, 163.0026, 178.9970, 227.0341, 255.0299, 284.0318, 285.0379, 609.1639 Kaempferol-3-O-dihexoside 2 F 5282155 4
[M + H]+ 611.16367 611.16064 5 127.0398, 287.0541
50 8.4 [M − H]− 192.066 192.06662 −3.2 C10H11NO3 - Phenylacetylglycine 2 AA 68144
51 8.43 [M − H]− ]− 367.1038 367.10199 4.9 C17H20O9 191.0546 Feruloylquinic acid 3 HC 10177048
52 8.47 [M − H]− 609.14887 609.14612 4.5 C27H30O16 133.8210, 151.0018, 177.9567, 255.0308, 271.0236, 285.0425, 300.0290, 301.0323, 609.1639 Quercetin-3-O-rutinoside 1 s F 5280805 1
[M + H]+ 611.16321 611.16122 3.3 129.0572, 287.0541, 303.0504
53 8.67 [M − H]− 463.08749 463.0882 −1.5 C21H20O12 151.0036, 255.0304, 271.0255, 287.2002, 300.0289, 301.0315 Quercetin-3-O-galactoside 1 s F 5281643
[M + H]+ 465.10483 465.10275 4.5 142.7517, 257.0451, 285.0396, 303.0503
54 8.67 [M − H]− 593.15129 593.15118 0.2 C27H30O15 227.0337, 255.0302, 272.9905, 284.0320, 285.0383, 593.1517 Kaempferol-3-O-rutinoside 2 F 5318767 2
[M + H]+ 595.16882 595.16577 5.1 85.0296, 164.3077, 253.8810, 287.0542
55 8.67 [M − H]− 313.09305 313.09235 2.3 C14H18O8 121.0280 Glucovanillin 3 BA 6452133
56 8.68 [M + H]+ 609.14679 609.14557 2.0 C27H28O16 303.0501 Unknown 3 F -
57 8.7 [M − H]− 753.21992 753.22421 −5.7 C33H38O20 151.0018, 255.0304, 271.0245, 300.0290, 301.0324 609.1639 Unknown flavonoids 3 F 11498684
[M + H]+ 755.20740 755.20347 5.2 179.3386, 303.0505
58 8.77 [M − H]− 463.08752 463.0882 −1.5 C21H20O12 151.0020, 255.0296, 271.0252, 300.0288, 301.0371, 463.0862 Quercetin-3-O-glucoside 1s F 5280804 1,4
[M + H]+ 465.10287 465.10275 0.3 257.0451, 275.6729, 285.0396, 303.0504, 465.1721
59 8.96 [M − H]− 447.09442 447.09329 2.5 C21H20O11 167.4627, 279.2310, 284.0321, 285.0379, 447.0933 Luteolin 7-O-glucoside 3 F 5280637 2
60 9.01 [M − H]− 245.09334 245.09317 0.7 C13H14N2O3 - N-Acetyltryptophan 3 AA 2002
61 9.02 [M − H]− 521.13037 521.12952 1.6 C24H26O13 135.0531, 161.0233, 179.0336, 197.0438 Rosmarinic acid glucoside 2 HC 132604855 3
62 9.12 [M + H]+ 223.06041 223.0601 1.4 C11H10O5 121.0291, 149.0244, 162.0302, 177.0907, 190.0266, 207.0280 Fraxidin 2 C 3083616
63 9.19 [M + H]+ 609.14673 609.14556 1.9 C27H28O16 303.0503 Unknown flavonoids 3 F -
[M − H]− 607.13054 607.12991 −1.0 151.0025, 178.9976, 255.0293, 271.0252, 300.0273, 463.0867
64 9.19 [M + H]+ 739.21136 739.20856 3.8 C33H38O19 287.0552 Unknown flavonoids 3 F -
[M − H]− 737.20084 737.19291 9.8 151.0023, 255.0289, 284.0325, 285.0395
65 9.23 [M − H]− 505.09988 505.09875 2.2 C23H22O13 151.0018, 271.0246, 300.0292, 301.0322 Quercetin 3-(6-O-acetyl)-hexoside 2 F 10006384
66 9.25 [M − H]− 593.15141 593.15118 0.4 C27H30O15 133.0971, 151.0018, 255.0304, 284.0320, 285.0382 Luteolin-7-O-rhamnohexoside 2 F 5318767 2
[M + H]+ 595.16858 595.16630 2.9 287.0552
67 9.29 [M − H]− 449.10779 449.10730 1.1 C21H20O11 287.0544 Kaempferol-O-hexoside I 2 F 5282149 2
447.09445 447.09329 2.6 150.1186, 196.2700, 227.0370, 255.0304, 284.0321, 285.0380
68 9.55 [M − H]− 515.12018 515.11951 1.3 C25H24O12 135.0437, 161.0230, 179.0334, 191.0546, 353.0867 Dicaffeoylquinic acid 3 HC 13604687 3
[M + H]+ 517.13562 517.13403 3.1 135.0442, 145.0291, 163.0386
69 9.56 [M − H]− 577.16005 577.15625 6.6 C27H30O14 269.0468 Apigenin-7-O-rhamnohexoside 2 F 5282150
[M + H]+ 579.17301 579.17084 3.7 85.0294, 200.8167, 271.0601
70 9.67 [M − H]− 447.09457 447.09329 2.9 C21H20O11 227.0337, 249.0604, 255.0309, 279.2349, 284.0317, 285.0379 Kaempferol-O-hexoside II 2 F 5282102 2
[M − H]− 447.0943 447.09329 2.3 279.2305, 285.0385
71 10.03 [M − H]− 535.10642 535.10879 −4.4 C24H24O14 135.0436, 179.0335, 197.0448 Rosmarinic acid, glucuronide 2 HC - 3
72 10.07 [M − H]− 351.0724 351.07214 0.7 C16H16O9 - 4-Methylumbelliferyl glucuronide 3 C 91553
73 10.13 [M − H]− 191.03415 191.0341 0.3 C10H8O4 - Coumarin base + 1O, 1MeO 3 C -
74 10.22 [M − H]− 359.07709 359.07724 −0.4 C18H16O8 72.9915, 135.0437, 161.0231, 179.0334, 197.0434 Rosmarinic acid 1 s HC 5281792 3
75 10.23 [M − H]− 313.0722 313.07122 3.1 C17H14O6 109.0279, 123.0430, 133.0286, 151.0387, 161.0231 3,4-Dihydroxycinnamoyl-(Z)-2-(3,4-dihydroxyphenyl)ethenol 2 HC 14353342
76 10.61 [M − H]− 431.09967 431.09836 3 C21H20O10 152.9944, 227.0337, 255.0304, 285.0388 Kaempferol-3-O-rhamnoside 2 F 5316673 2
77 10.71 [M − H]− 193.04984 193.05063 −4.1 C10H10O4 121.0280, 133.0775, 148.8996, 161.02284, 177.0188, Ferulic acid 2 HC 445858
10.75 [M + H]+ 195.06569 195.06519 2.6 C10H10O4 135.0446,145.0289, 149.0605, 163.0385, 177.0550
78 11.08 [M − H]− 191.03412 191.03499 −4.6 C10H8O4 147.0439, 149.0238, 175.0114, 191.0345 Scopoletin 2 C 5280460 2
11.08 [M + H]+ 193.05025 193.04953 3.7 C10H8O4 -
79 11.4 [M − H]− 1251.60254 1251.60100 1.2 C59H96O28 589.4164, 633.4128, 733.4748, 751.4918, 865.5422, 883.5467 927.5394, 957.5598, 1045.6177, 1089.5382, 1251.6044 Triterpenoid saponin 3 TT -
80 11.63 [M − H]− 1119.55419 1119.55874 −4.1 C54H88O24 589.4158, 633.4137, 733.4717, 751.4915, 777.4700, 795.4831, 913.5570, 957.5609, 1119.5493 Triterpenoid saponin (Eryngioside C) 3 TT - 5
81 11.74 [M − H]− 373.09302 373.09235 1.8 C19H18O8 123.0437, 135.0438, 149.0595, 160.0153, 175.0390, 179.0340, 193.0498, 197.0448 Methyl rosmarinate 3 HC 6479915
82 11.97 [M − H]− 285.04071 285.04047 0.8 C15H10O6 68.2225, 164.8401, 171.4909, 175.0386, 285.0380 Luteolin 2 F 5280445 6
83 11.99 [M + H]+ 303.05045 303.04993 1.7 C15H10O7 144.9594, 160.2895, 303.0504 Quercetin 1s F 5280343 1
11.99 [M − H]− 301.03513 301.03537 −0.8 C15H10O7 151.0019, 178.9968, 215.4516, 243.6472, 301.0371
84 12.22 [M − H]− 1103.52 1103.52744 −6.7 C53H84O24 - Triterpenoid saponin 3 TT -
85 13.63 [M − H]− 285.04074 285.04047 0.9 C15H10O6 176.8428, 187.0409, 285.0426 Kaempferol 1s F 5280863 6
86 13.84 [M − H]− 209.11772 209.11832 −2.9 C12H18O3 59.0122, 97.0641, 165.1275, 209.1182 Jasmonic acid 2 O 5281166
87 14.05 [M − H]− 161.02324 161.02387 −3.9 C9H6O3 133.0287, 161.0232 Umbelliferone 2 C 5281426 2
14.05 [M + H]+ 163.03908 163.03897 0.7 C9H6O3 119.0368, 121.0656, 137.3907, 145.0288, 163.0385
88 14.87 [M − H]− 1117.5357 1117.54309 −6.6 C54H86O24 583.3667, 715.4624, 743.4582, 937.5284, 985.5461, 1027.5735, 1075.5281, Triterpenoid saponin 3 TT -
89 15.2 [M − H]− 1203.53925 1203.54348 −3.5 C57H88O27 645.4126, 715.4597, 743.4602, 1099.5226, 1159.5333 Triterpenoid saponin 3 TT -
90 15.27 [M − H]− 1071.4957 1071.50122 −5.2 C52H80O23 436.7717, 746.7781, 772.1053 Triterpenoid saponin 3 TT -
91 15.31 [M − H]− 1159.54309 1159.5384 4 C52H88O28 436.1860, 587.3589, 715.4593, 1099.5171 Triterpenoid saponin 3 TT -
92 17.3 [M − H]− 1099.52958 1099.53252 −2.7 C54H84O23 734.0673, 890.0820 Triterpenoid saponin (Eryngioside J) 3 TT - 5
93 17.52 [M + H]+ 177.05464 177.05463 0.1 C10H8O3 93.0346, 119.0863, 121.0391, 135.0801, 145.0289, 177.0549 7-Methoxycoumarin 2 C 5280567 2
94 17.71 [M + H]+ 969.51023 969.50591 4.5 C49H76O19 - Triterpenoid saponin (3-O–β-d-glucopyranosyl-(1 → 2)-β-d glucuronopyranosyl-
22-O-angeloyl-R1-barrigenol) 		3 TT - 7
95 18.22 [M − H]− 1187.54376 1187.54857 −4 C57H88O26 - Triterpenoid saponin 3 TT -
96 18.31 [M + H]+ 911.50453 911.50043 4.5 C47H74O17 - Triterpenoid saponin (3-O-β-dglucopyranosyl-(1 → 2)-β-d-glucuronopyranosyl-22-O-angeloyl-A1- barrigenol) 3 TT - 7
97 18.4 [M − H]− 1041.58291 1041.57868 4.1 C57H86O17 489.3563, 502.9471, 583.3742, 603.3950, 639.4351, Triterpenoid saponin 3 TT -
98 21.07 [M + H]+ 293.2114 293.21112 1 C18H28O3 - OPDA 3 O 656750
99 23.01 [M − H]− 471.34903 471.34799 2.2 C30H48O4 - Triterpenoid sapogenin 3 TT -
Open in a new tab

a Metabolite identification level according to Metabolite Standards Initiative recommendation (1—identified metabolites, 2—putatively annotated compounds, 3—putatively characterized compound classes, 4—unknown compounds); b Group of compounds: AA—amino acids, BA—benzoic acid derivates, C—coumarins, HC——hydroxycinnamic acid derivates, F—flavonoids, N—nucleotide, TT—triterpenoids, O—other compounds; c CID—Compound ID, PubChem; s Compounds identified with using commercial standards.

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, C6H10O5) and the quinic acid (characteristic fragment m/z 191.0195, C7H11O6−). 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 C16H17O9− and gave the fragmentation pattern 191.0546, 179.0334, 161.0226, 135.0438 characteristic for caffeoylquinic acids. Compound 68 was detected in negative ion mode as a pseudo-molecular ion 515.11951, corresponded with the molecular formula C25H23O12−, and was annotated as dicaffeoylquinic acid. Compound 39 (RT = 7.14 min) was detected in negative ion mode as a coumaroylquinic acid (m/z 337.09219, C16H17O8−). The ion was observed at three different retention times (RT = 6.49, 7.76, and 8.43 min) at m/z 367.10380 (C17H19O9−), which suggested the existence of three isomeric forms of feruloylquinic acid. The conjugates of choline were detected in positive ion mode and assigned as caffeoylcholine and coumaroylcholine. The most intense peak at RT = 10.22 min was exhibited by [M − H]− at m/z 359.07709 (C18H15O8−) and by a complex ion [2M − H]− at m/z 719.15418. This compound was identified as rosmarinic acid by the exact mass, the fragmentation pattern and the comparison with the external standard (Sigma-Aldrich). Rosmarinic acid was previously described by Le Claire et al. [4] and Kikowska et al. [6,7]. In accordance with the literature, rosmarinic acid glucoside (521.13037, C24H25O13−) and glucuronide (535.10642, C24H23O14−) were found in negative ion mode [17]. Furthermore, 3,4-dihydroxycinnamoyl-(Z)-2-(3,4-dihydroxyphenyl)ethanol (313.0722, C17H13O6−) and methyl rosmarinate (373.09302, C19H18O8−) were recognized in the extracts. 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 in the samples as esculin, scopoletin-7-O-dihexoside, and scopolin. The pseudo-molecular ions were observed at two different retention times at m/z 223.0601 (C11H11O5+) and were tentatively assigned as isofraxidin and fraxidin.

The representative flavonoids were mostly recognized as quercetin, kaempferol, and luteolin derivates in positive and negative ion mode. The MS/MS spectra showed the typical fragmentation pattern for O-flavonoids with hexose (−162.0539, C6H10O5), rhamnose (−146.0656, C6H11O4), rutinose (−308.1105, C12H20O9), or dihexose (−324.1061, C12H20O10) losses in negative ion mode. Consequently, quercetin-3-O-rutinose, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin, and kaempferol were verified by means of the reference standards (Sigma-Aldrich). Other flavonoids were tentatively assigned as kaempferol-O-rhamnodihexoside, kaempferol-3-O-dihexoside, kaempferol-3-O-rutinoside, kaempferol-O-hexoside (I and II), kaempferol-3-O-rhamnoside, luteolin-7-O-rhamnohexoside, luteolin-7-O-hexoside, quercetin-3-O-dihexoside-7-O-rhamnoside, quercetin-3-O-glucoside-7-O-rhamnoside, and quercetin-3-O-dihexoside and verified with the literature data for the described Eryngium species [19,20]. Moreover, luteolin-C-hexoside, apigenin-7-O-rhamnohexoside, and quercetin 3-(6-O-acetyl)-hexoside were found in the extracts. Peak 59 showed a precursor ion at m/z 447.09238 (C21H19O11−) and was tentatively annotated as a luteolin-C-hexoside according to the MS/MS analysis, which corresponded to the loss of fragments−90 and −120, characterizing the break of C-hexoside. Compound 65 was assigned as quercetin 3-(6-O-acetyl)-hexoside (505.09988, C23H21O12−) whereas compound 69 was characterized as an apigenin-7-O-rhamnohexoside (577.16005, C27H29O20−). The product ion spectra demonstrated a fragment ion at m/z 269.0468 corresponding to apigenin aglycone. Five compounds (46, 56, 57, 63, 64), which corresponded with ions at m/z: 753.18783 (C33H37O20−), 609.14679 (C27H29O16+), 753.21992 (C33H37O20−), 607.13054 (C27H28O16−), and 739.21136 (C33H39O19+), were recognized as unknown flavonoids. The MS/MS spectrum of compounds 55, 65, 66 and 72 showed a major fragment at m/z 301.03711 (C15H9O7−) in negative ion mode, which could be quercetin aglycone. Furthermore, a fragment at m/z 285.03955 (C15H9O6−) was found in the MS/MS spectrum of compound 64 and corresponded with the tetrahydroxyflavone moiety.

Several triterpenoid saponins were found in the samples. Similar to Ożarowski et al. [17], we observed precursors and the fragmentation pattern as in the related species, E. planum. The pseudo-molecular ion at m/z 471.34903 corresponded with the molecular formula C30H47O4− and was recognized as a major sapogenin in the extract. Twelve precursors were tentatively assigned as triterpenoid saponins. The pseudo-molecular ions 1119.55419 (C54H87O24−) and 1099.52958 (C54H83O23−) could be annotated as eryngioside C and eryngioside J according to the exact mass and fragmentation [21]. Moreover, ions 969.51023 (C49H77O19+) and 911.50453 (C47H75O17+) could be putatively assigned as 3-O–β-d-glucopyranosyl-(1→2)-β-d-glucuronopyranosyl-22-O-angeloyl-R1-barrigenol and 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucuronopyranosyl-22-O-angeloyl-A1-barrigenol – saponins observed in E. planum and E. maritimum [22]. This is the first report on triterpenoid saponins detection in E. alpinum. However, further works are required for identification of the saponin structures in this species.

In line with the literature, hydroxybenzoates were found in the extracts of Eryngium [20]. Glucosyringic acid (359.09950, C15H19O10−), trimethoxybenzoic acid (213.07613, C10H13O5+), dihydroxybenzoic acid hexoside (315.0725, C13H15O9−), vanillic acid hexoside (329.08841, C14H17O9−), vanillic acid (167.0350, C8H7O4−), hydroxyphenyllacetic acid (181.05003, C9H9O4−), dimethoxybenzaldehyde (167.07097, C9H11O3+), and glucovanillin (313.09305, C14H17O8−) were 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 (C8H8NO4−) and 218.10310 (C9H16NO5−) 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 (Table 3, Table 4 and Table 5; Figure 4 and Figure 5).

Table 3.

The effect of the extract from shoots from in vivo plant of Eryngium alpinum L. [0.5 mg/mL, 2.5 mg/mL, 5 mg/mL] on inhibition of Acanthamoeba trophozoites during four days of treatment.

Extract Concentration
[mg/mL]		 Duration of Treatment [Days]
2nd Day 3rd Day 4th Day
MN ± SD MN ± SD MN ± SD
control 5.62 ± 1.96 11.93 ± 2.33 17.06 ± 2.95
0.5 5.00 ± 2.26 11.83 ± 2.73 17.06 ± 2.50
2.5 4.62 ± 1.50 11.54 ± 3.66 16.44 ± 6.61
5 2.67 ± 1.88 * 5.33 ± 3.98 * 6.69 ± 3.08 *
Open in a new tab

MN—mean number of trophozoites. * p < 0.05 statistically significant difference in comparison with the control during the same time interval; n = 18.

Table 4.

The effect of the extract from in vitro shoot cultures of Eryngium alpinum L. [0.5 mg/mL, 2.5 mg/mL, 5 mg/mL] on inhibition of Acanthamoeba trophozoites during four days of treatment.

Extract Concentration
[mg/mL]		 Duration of Treatment [Days]
2nd Day 3rd Day 4th Day
MN ± SD MN ± SD MN ± SD
control 5.62 ± 1.96 11.53 ± 2.33 17.06 ± 2.95
0.5 2.53 ± 1.88 * 7.46 ± 3.00 * 13.45 ± 3.58 *
2.5 1.89 ± 1.37 * 6.89 ± 2.33 * 12.69 ± 3.46 *
5 1.06 ± 1.35 * 3.76 ± 2.31 * 5.57 ± 2.02 *
Open in a new tab

MN—mean number of trophozoites. * p < 0.05 statistically significant difference in comparison with the control during the same time interval; n = 18.

Table 5.

Determination of IC50 [mg/mL] for the studied extracts of Eryngium alpinum L.

Plant Material IC50 2nd Day IC50 3rd Day IC50 4th Day
Shoots from in vivo plants 4.80 mg/ml 4.80 mg/ml 4.60 mg/ml
Shoots from in vitro cultures 0.35 mg/ml 3.50 mg/ml 4.15 mg/ml
Open in a new tab

Figure 4.

Figure 4

Open in a new tab

The effect of the extract from shoots of in vivo plant Eryngium alpinum L. [0.5 mg/mL, 2.5 mg/mL, 5 mg/mL] on inhibition of Acanthamoeba trophozoites proliferation in the culture medium.

Figure 5.

Figure 5

Open in a new tab

The effect of the extract from in vitro shoot cultures of Eryngium alpinum L. [0.5 mg/mL, 2.5 mg/mL, 5 mg/mL] on inhibition of Acanthamoeba trophozoites proliferation in the culture medium.

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].

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 leaves of Origanum spp., Salvia spp., Melia azedarach, Ricinus communis, Pastinaca armenea, Inula oculuscristi; aerial parts of Croton spp., Pterocaulon polystachyum, flowers, roots and leaves of Rubus chamaemourus, Pueraria lobata, Solidago spp., flowers, roots, leaves and bark of Ipomoea sp., Kaempferia galanga, Cananga odorata, leaves and calluses of Passiflora spp., leaves and roots of Eryngium planum [9,11,23,24,25,26,27].

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. longibracteolatum [30], Origanum syriacum, O. laevigatum [31], Buddleia cordata at 32 mg/mL [32], and Trigonella foenum-graecum at 400 mg/mL [33].

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 EC50 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.

3. Materials and Methods

3.1. The Plant Material Origin

The fragments of the cuttings of Eryngium alpinum L. obtained from the Botanical Garden of Adam Mickiewicz University in Poznań in 2017 (52°25′13.1′′N 16°52′44.9′′E) were used for the initiation of in vitro cultures. The voucher specimens were deposited at the Department of Pharmaceutical Botany and Plant Biotechnology of PUMS under the following number: H‒AP‒2017‒102.

3.2. 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 GA3 (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/m2s 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).

3.3. 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].

3.4. 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.

3.5. 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.

4. 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.

Author Contributions

Conceptualization, M.K.; methodology, M.K.; D.K. and M.D.; software, M.K.; D.K. and M.D.; formal analysis, M.K.; D.K. and M.D.; investigation, M.K.; D.K. and M.D.; data curation, M.K.; D.K. and M.D.; writing—original draft preparation, M.K. and D.K.; writing—review and editing, M.K.; B.T.; D.K., E.H. and M.D.; visualization, M.K.; D.K. and M.D.; supervision, E.H. and B.T.; project administration, M.K.; funding acquisition, M.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Science Centre, Poland, the grant number 2019/03/X/NZ7/01846.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Samples of biomass are available from the authors.

References

  • 1.Cullen J., Alexander J.C.M., Brady A., Brickell C.D., Green P.S., Heywood V.H., Jörgensen P.M., Jury S.L., Knees S.G., Leslie A.C., et al. The European Garden Flora. Volume V. Cambridge University Press; London, UK: 1997. pp. 396–398. [Google Scholar]
  • 2.Andrello M., Devaux C., Quetier F., Till-Bottraud I. Paying for conservation: A bioeconomic analysis of the effects of land management options on the viability of an endangered species, Eryngium alpinum. J. Appl. Ecol. 2017;55:1940–1950. doi: 10.1111/1365-2664.13112. [DOI] [Google Scholar]
  • 3.Crowden R.K., Harborne J.B., Heywood V.H. Chemosystematics of the Umbelliferae—A general survey. Phytochemistry. 1969;8:1963–1984. doi: 10.1016/S0031-9422(00)88084-X. [DOI] [Google Scholar]
  • 4.Le Claire E., Schwaiger S., Banaigs B., Stuppner H., Gafner F. Distribution of a new rosmarinic acid derivative Eryngium alpinum L. and other Apiaceae. J. Agric. Food Chem. 2005;53:4367–4372. doi: 10.1021/jf050024v. [DOI] [PubMed] [Google Scholar]
  • 5.Dunkic V., Vuko E., Bezic N., Kremer D., Ruscic M. Composition an antiviral activity of the essential oils of Eryngium alpinum and E. amethystinum. Chem. Biodivers. 2013;10:1894–1902. doi: 10.1002/cbdv.201300061. [DOI] [PubMed] [Google Scholar]
  • 6.Kikowska M., Thiem B., Szopa A., Klimek-Szczykułowicz M., Rewers M., Sliwinska E., Ekiert H. Comparative analysis of phenolic acids and flavonoids in shoot cultures of Eryngium alpinum L.—An endangered and protected species with medicinal value. Plant Cell Tiss. Organ Cult. 2019;139:167–175. doi: 10.1007/s11240-019-01674-8. [DOI] [Google Scholar]
  • 7.Kikowska M., Thiem B., Szopa A., Ekiert H. Accumulation of valuable secondary metabolites: Phenolic acids and flavonoids in different in vitro systems of shoot cultures of the endangered plant species—Eryngium alpinum L. Plant Cell Tiss. Organ Cult. 2020 doi: 10.1007/s11240-020-01795-5. (in press) [DOI] [Google Scholar]
  • 8.Verpoorte R., Contin A., Memelnik J. Biotechnology for the production of plant secondary metabolites. Phytochem. Rev. 2002;1:13–25. doi: 10.1023/A:1015871916833. [DOI] [Google Scholar]
  • 9.Derda M., Hadaś E., Thiem B. Plant extracts as natural amoebicidal agents. Parasitol. Res. 2009;104:705–708. doi: 10.1007/s00436-008-1277-9. [DOI] [PubMed] [Google Scholar]
  • 10.Niyyati M., Dodangeh S., Lorenzo-Morales J. A review of the current research trends in the application of medicinal plants as a source for novel therapeutic agents against Acanthamoeba infections. Iran. J. Pharm. Res. 2016;15:893–900. [PMC free article] [PubMed] [Google Scholar]
  • 11.Wei C.E., Yeng C.Y., Mahboob T., Ling L.C., Raju C.S., Barusrux S., Nissapatron V. Natural products: Alternative therapeutic compounds against Acanthamoeba spp. Asian J. Pharmacogn. 2019;3:29–38. doi: 10.13140/RG.2.2.15011.40482. [DOI] [Google Scholar]
  • 12.Derda M., Hadaś E. The use of phytotherapy in diseases caused by parasitic protozoa. Acta Parasitol. 2015;60:1–8. doi: 10.1515/ap-2015-0001. [DOI] [PubMed] [Google Scholar]
  • 13.Padzik M., Hendiger E.B., Szaflik J.P., Chomicz L. Amoebae of the genus Acanthamoeba—Pathological agents in humans. Post. Mikrobiol. 2017;56:429–439. [Google Scholar]
  • 14.Thiem B., Kikowska M., Krawczyk A., Więckowska B., Sliwinska E. Phenolic acid and DNA contents of micropropagated Eryngium planum L. Plant Cell Tiss. Organ Cult. 2013;114:197–206. doi: 10.1007/s11240-013-0315-1. [DOI] [Google Scholar]
  • 15.Kikowska M., Thiem B., Sliwinska E., Rewers M., Kowalczyk M., Stochmal A., Długaszewska J. Micropropagation of Eryngium campestre L. via shoot culture provides valuable uniform plant material with enhanced content of phenolic acids and antimicrobial activity. Acta Biol. Cracov. Bot. 2016;58:43–56. doi: 10.1515/abcsb-2016-0009. [DOI] [Google Scholar]
  • 16.Kikowska M., Thiem B., Sliwinska E., Rewers M., Kowalczyk M., Stochmal A., Oleszek W. The effect of nutritional factors and plant growth regulators on micropropagation and production of phenolic acids and saponins from plantlets and adventitious root cultures of Eryngium maritimum L. J. Plant Growth Regul. 2014;33:809–819. doi: 10.1007/s00344-014-9428-y. [DOI] [Google Scholar]
  • 17.Ożarowski M., Thiem B., Mikołajczak P.L., Piasecka A., Kachlicki P., Szulc M., Kaminska E., Bogacz A., Kujawski R., Bartkowiak-Wieczorek J., et al. Improvement in long-term memory following chronic administration of Eryngium planum root extract in scopolamine model: Behavioral and molecular study. Evid.-Based Complement. Alternat. Med. 2015;2015:145140. doi: 10.1155/2015/145140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Wang P., Su Z., Yuan W., Deng G., Li S. Phytochemical constituents and pharmacological activities of Eryngium L. (Apiaceae) Pharm. Crops. 2012;3:99–120. doi: 10.2174/2210290601203010099. [DOI] [Google Scholar]
  • 19.Erdem S.A., Nabavi S.F., Orhan I.E., Daglia M., Izadi M., Nabavi S.M. Blessings in disguise: A review of phytochemical composition and antimicrobial activity of plants belonging to the genus Eryngium. DARU. 2015;23:1–22. doi: 10.1186/s40199-015-0136-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Cádiz-Gurrea M., Fernandez-Arroyo S., Joven J., Segura-Carretero A. Comprehensive characterization by UHPLC-ESI-Q-TOF-MS from an Eryngium bourgatii extract and their antioxidant and anti-inflammatory activities. Food Res. Intern. 2013;50:197–204. doi: 10.1016/j.foodres.2012.09.038. [DOI] [Google Scholar]
  • 21.Zhang Z., Li S., Ownby S., Wang P., Yuan W., Zhang W., Beasley R.S. Phenolic compounds and rare polyhydroxylated triterpenoid saponins from Eryngium yuccifolium. Phytochemistry. 2008;69:2070–2080. doi: 10.1016/j.phytochem.2008.03.020. [DOI] [PubMed] [Google Scholar]
  • 22.Kowalczyk M., Masullo M., Thiem B., Piacente S., Stochmal A., Oleszek W. Three new triterpene saponins from roots of Eryngium planum. Nat. Prod. Res. 2014;28:653–660. doi: 10.1080/14786419.2014.895722. [DOI] [PubMed] [Google Scholar]
  • 23.Derda M., Hadaś E., Cholewiński M., Skrzypczak Ł., Grzondziel A., Wojtkowiak-Giera A. Artemisia annua L. as a plant with potential use in the treatment of acanthamoebiasis. Parasitol. Res. 2016;115:1635–1639. doi: 10.1007/s00436-016-4902-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Hadaś E., Derda M., Nawrot J., Nowak G., Thiem B. Evaluation of the amoebicidal activities of Centaurea bella, Centaurea daghestanica, Rhaponticum pulchrum and Tanacetum vulgare against pathogenic Acanthamoeba spp. Acta Pol. Pharm. 2017;74:1827–1832. [Google Scholar]
  • 25.Tanveer T., Bin-Dukhyil A.A., Nawaz S., Matin A. Therapeutic potential of folk plants used against pathogenic and opportunistic free-living amoeba (Acanthamoeba, Balamuthia and Naegleria): A review. Inter. J. Cell Sci. Mol. Biol. 2018;4:555628. doi: 10.19080/IJCSMB.2018.04.555628. [DOI] [Google Scholar]
  • 26.Hadaś R., Ożarowski M., Derda M., Thiem B. The use of extracts from Passiflora spp. in helping the treatment of acanthamoebiasis. Acta Pol. Pharm. 2017;74:921–928. [PubMed] [Google Scholar]
  • 27.Derda M., Thiem B., Budzianowski J., Wojt W.J., Wojtkowiak-Giera A. The evaluation of the amoebicidal activity of Eryngium planum extracts. Acta Pol. Pharm. 2013;70:1027–1034. [PubMed] [Google Scholar]
  • 28.Malatyali E., Tepe B., Degerli S., Berk S. In vitro amoebicidal activities of Satureja cuneifolia and Melissa officinalis on Acanthamoeba castellanii cysts and trophozoites. Parasitol. Res. 2012;110:2175–2180. doi: 10.1007/s00436-011-2744-2. [DOI] [PubMed] [Google Scholar]
  • 29.Goze I., Alim A., Dag S., Tepe B. In vitro amoebicidal activity of Salvia staminea and Salvia caespitosa on Acanthamoeba castellani and their cytotoxic potentials on corneal cells. J. Ocul. Pharmacol. Ther. 2009;25:293–298. doi: 10.1089/jop.2008.0132. [DOI] [PubMed] [Google Scholar]
  • 30.Malatyali E., Tepe B. In-vitro amoebicidal activity of four Peucedanum species on Acanthamoeba castellanii cysts and trophozoites. Parasitol. Res. 2012;110:167–174. doi: 10.1007/s00436-011-2466-5. [DOI] [PubMed] [Google Scholar]
  • 31.Degerli S., Tepe B., Celiksoz A., Berk S. In-vitro amoebicidal activity of Origanum syriacum and Origanum laevigatum on Acanthamoeba castellanii cysts and trophozoites. Exp. Parasitol. 2012;131:20–24. doi: 10.1016/j.exppara.2012.02.020. [DOI] [PubMed] [Google Scholar]
  • 32.Rodríguez-Zaragoza S., Ordaz C., Avila G., Muñoz J.L. In-vitro evaluation of the amebicidal activity of Buddleia cordata (Loganiaceae, H.B.K.) on several strains of Acanthamoeba. J. Ethnopharmacol. 1999;66:327–334. doi: 10.1016/S0378-8741(98)00186-X. [DOI] [PubMed] [Google Scholar]
  • 33.Dodangeh S., Niyyati M., Kamalinejad M. Antiacanthamoeba activities of chloroformic fractions of Trigonella foenum graecum (Seed) and their cytotoxity on mice macrophage cell. Novel. Biomed. 2001;82:237–246. [Google Scholar]
  • 34.Mahboob T., Azlan A.-M., Tan T.-C., Samudi C., Sekaran S.D., Nissapatorn V., Wiart C. Anti-encystment and amoebicidal activity of Lonicera japonica Thung. And its major constituent chlorogenic acid in vitro. Asian Pac. J. Trop. Med. 2016;9:866–871. doi: 10.1016/j.apjtm.2016.07.008. [DOI] [PubMed] [Google Scholar]
  • 35.Bittner Fialová S., Kello M., Čoma M., Slobodníková L., Drobná E., Holková I., Garajová M., Mrva M., Zachar V., Lukáč M. Derivatization of rosmarinic acid enhances its in vitro antitumor, antimicrobial and antiprotozoal properties. Molecules. 2019;24:1078. doi: 10.3390/molecules24061078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Anwar A., Ting E.L.S., Anwar A., ul Ain N., Faizi S., Shah M.R., Khan N.A., Siddiqui R. Antiamoebic activity of plant-based natural products and their conjugated silver nanoparticles against Acanthamoeba castellanii (ATCC 50492) AMB Express. 2020;10:24. doi: 10.1186/s13568-020-0960-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Mennai I., Hanfer M., Esseid C., Benayache S., Ameddah S., Menad A., Benayache F. Chemical composition, in vitro antiparasitic, antimicrobial and antioxidant activities of Frankenia thymifolia Desf. Nat. Prod. Res. 2019;24:1–6. doi: 10.1080/14786419.2018.1561685. [DOI] [PubMed] [Google Scholar]
  • 38.Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 1962;15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x. [DOI] [Google Scholar]
  • 39.Thugawa H., Nakabayashi R., Mori T., Yamada Y., Takahashi M., Ray A., Sugiyama R., Yamamoto H., Nakaya T., Yamazaki M., et al. A chemoinformatics approach to characterize metabolomes in stable-isotope-labeled organism. Nat. Meth. 2009;16:295–298. doi: 10.1038/s41592-019-0358-2. [DOI] [PubMed] [Google Scholar]

Articles from Molecules are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

RESOURCES