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

In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from the natural environment. Shoots of Eryngium alpinum L. were multiplied on Murashige and Skoog (MS) medium in various systems, namely on the solid media and in two liquid cultures-stationary and agitated, as well as via regeneration from callus. The biomass increments were closely correlated with the number of shoots arising from one explant, which was connected with the supplementation of the culture media with the studied plant growth regulators. The methanolic extracts from shoots grown in the tested systems were subjected to phenolic acids and flavonoids qualitative and quantitative analysis. Biomass from in vitro shoot cultures accumulated from 19.59 to 32.95 times more phenolic acids [the total content ranged from 272.52 to 458.38 mg/100 g dry weight (DW)] and from 3.02 to 4.43 times more flavonoids (the total content ranged from 100.03 to 146.98 mg/100 g DW), depending on the culture system, than the extracts from basal leaves from the intact plant (13.91 and 33.16 mg/100 g DW, respectively). The phenolics present in shoot cultures include seven phenolic acids-3,4-dihydroxyphenylacetic, caftaric, caffeic, neochlorogenic, chlorogenic, isochlorogenic, and rosmarinic acids, and three flavonoids-isoquercetin, quercitrin and robinin. The best system for shoot proliferation resulting in the highest biomass growth and phenolic acids and flavonoids accumulation was solid culture on MS medium with BAP, IAA, and $GA_{3}$ (each 1.0 mg/l). The aim of this work was to check the effect of various culture systems (stationary and agitated, on solidified and in liquid media) on the production of phenolic compounds in E. alpinum shoots cultured in vitro

Comparative analysis of phenolic acids and flavonoids in shoot cultures of Eryngium alpinum L. : an endangered and protected species with medicinal value

Phenolic acids and flavonoids, important bioactive compounds of polyphenols, play a significant role in plants; their impact, mainly as antioxidants, on human health have been of great interest in recent years. The genetically uniform shoots of Eryngium alpinum L. cultured in vitro, developed via axillary buds and regenerated from callus tissue, maintained on the media supplemented with various plant growth regulators, were subjected to the phenolic acids and flavonoids quantitative analysis applying HPLC-DAD technique. In vitro cultures give the opportunity to perform the phytochemical studies on the protected species without harvesting the plant material from natural environment. Depending on the hormonal supplementation, the biomass from the shoot cultures accumulated from 11.41 to 25 times more phenolic acids [the total content ranged from 158.66 to 1817.96 mg/100 g of dry weight (DW)] and from 6.8 to 11.8 times more flavonoids (the total content ranged from 29.30 to 51.30 mg/100 g DW) than the shoots from the soil-grown plant. The polyphenols present in the shoot cultures include two phenolic acids: 3,4-dihydroxyphenylacetic and caffeic, four depsides: caftaric, neochlorogenic, chlorogenic, isochlorogenic, and rosmarinic acids, and flavonoids: aglycone-isoquercetin and glucoside-quercitrin. Most of them (apart from chlorogenic and rosmarinic acids) were detected for the first time in this species cultured in vitro. To our best knowledge, the present report is the first one that discusses establishment of Eryngium alpinum L. in vitro cultures and the shoot and callus biomass capacity to produce two subgroups of polyphenols i.e. phenolic acids and flavonoids

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

Micropropagation and Production of Somatic Seeds for Short-Term Storage of the Endangered Species Eryngium alpinum L.

Eryngium alpinum L. is a high-value herb and a source of important compounds that include phenolics, triterpenoid saponins, and essential oils. The present report indicates successful micropropagation of this species. In our study, medium supplemented with BAP 2.0 mg/L, IAA 1.0 mg/L, and GA3 1.0 mg/L was found to be the most suitable for long-term culture and for effective proliferation, irrespective of the passage number. Roots induction, without basal callus formation, was observed when individual microshoots were placed on Murashige & Skoog medium augmented with auxin, and formation was the most advantageous in the presence of NAA alone or when combined with IAA or IBA. The encapsulated propagules were tested for their capability to endure different storage periods under low temperature. Therefore, we developed an efficient method for synseeds production by encapsulation of axillary buds in the sodium alginate matrix, storage for 2, 4, and 6 months, as well as the regeneration process. The maximum regeneration rate of 74% ± 2.72% was observed for axillary buds encapsulated in 4% sodium–alginate complexed with 300 mM calcium chloride after 2 months of storage at low temperature. This is the first report on E. alpinum micropropagation and somatic seeds production

Methyl jasmonate, yeast extract and sucrose stimulate phenolic acids accumulation in Eryngium planum L. shoot cultures

Eryngium planum L. has been reported as a medicinal plant used in traditional medicine in Europe. The tissue cultures may be an alternative source of the biomass rich in desired bioactive compounds. The purpose of this study was to investigate the influence of the biotechnological techniques on the selected phenolic acids accumulation in the agitated shoot cultures of E. planum. Qualitative and quantitative analyses of those compounds in 50% aqueous - methanolic extracts from the biomass were conducted by applying the HPLC method. Methyl jasmonate (MeJA), yeast extract (YE) and sucrose (Suc) stimulated accumulation of the phenolic acids: rosmarinic (RA), chlorogenic (CGA) and caffeic (CA) in in vitro shoot cultures. Cultivation of shoots in liquid MS media supplemented with 1.0 mg L-1 6-benzyladenine and 0.1 mg L-1 indole-3-acetic acid in the presence of 100 µM MeJA for 48h was an optimum condition of elicitation and resulted in approximately 4.5-fold increased content of RA + CGA + CA in plant material compared to the control (19.795 mg g-1 DW, 4.36 mg g-1 DW, respectively). The results provide the first evidence that the selected phenolic acids can be synthesized in elicited shoot cultures of flat sea holly in higher amount than in untreated shoots

Essential Oil Composition of the Different Parts and In Vitro Shoot Culture of Eryngium planum L.

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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

Comparative Study of <i>Plantago media</i> Extracts in the Treatment of <i>Acanthamoeba</i> sp. Trophozoites

(1) Background: The aim of the study was to compare the potency of Plantago media L. (Plantaginaceae) extracts on Acanthamoeba sp. trophozoites, which are opportunistic protozoan parasites leading to several dangerous diseases; (2) Methods: The chromatographically (TLC, HPLC-DAD) characterized water fractions of the extracts from biomass from in vitro cultures (shoots and roots), leaves, and inflorescences from field cultivation were used for the study of the acanthamoebic activity in a Thoma haemocytometer chamber; (3) Results: The anti-amoebic effect at the lowest concentration (1.0 mg/mL) was demonstrated only by the extract of the leaves from the cultivation (50.50% inhibition). The remaining samples inhibited the growth of parasites from a concentration of 5.0 mg/mL in the range of 41.36% inflorescences to 63.89% shoots in vitro. Quantitative determinations of phenolic compounds in the tested extracts indicate a tendency to increase the potency of the anti-amoebic effect with the content of a phenylethanoid glycoside—acteoside. The maximum content of this compound was determined in leaves from field cultivation (6.64%) and the minimum in inflorescences (0.65%). This is confirmed by the range of the lowest IC50 values (the strongest biological activity) for the tested samples, 0.95–1.80 mg/mL for leaves from cultivation, and the high values, 9.70–5.30 mg/mL for inflorescences and in-vitro-derived roots. The strength of the biological activity of the extracts correlated with the content of acteoside, which constituted 84–93% of the sum of phenolic compounds determined; (4) Conclusions: The performed investigations proved the anti-acanthamoebic efficacy of Plantago media organs, including those obtainable by biotechnological methods, and indicated phenylethanoid glycosides, their main phenolic constituents, to be responsible for the activity. To our knowledge, this is the first report on the amoebicidal activity of Plantago media extracts from biomass produced by biotechnological methods and organs of an intact plant