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Licorice root is traditionally used in medicine due to its content of saponins and flavonoids. Licorice leaves are not used as pharmacopoeial raw materials, although their chemical composition and biological activity have been studied recently, which makes it possible to evaluate the possibilities of using this raw material as a medicinal one. Active harvesting of licorice can jeopardize its natural populations, so urgent tasks are to cultivate cells of this plant in systems in vitro and to study the composition of cell culture metabolites. In our study, the material for obtaining a callus culture of licorice leaves was plants from the collection of the Botanical Garden of the Ural Branch of the Russian Academy of Sciences. To select optimal conditions for growing calli, nine combinations of phytohormones were compared. The best growth of calli was found on Murashige–Skoog medium with a combination of phytohormones 1 mg/L BAP and 10 mg/L NAA. Under these conditions, flavonoids accumulated in callus in quantities comparable to their content in intact leaves and roots. The content of phenolic compounds was comparable to their amount in the roots. Ethanol extracts obtained from callus culture had pronounced antioxidant activity comparable to extracts from the intact plant and standards of rutin, gallic, and ascorbic acids. When assessing the effect of extracts on animal cell cultures in the MTT test, it was shown that all obtained extracts increased the metabolic activity of both normal human cells and the HeLa line. At the same time, the extract obtained from the leaves showed the maximum effect, while that from the callus showed the minimum and differed slightly from the root extract. Thus, calli from leaf explants can be considered as a new raw material for obtaining dietary supplements with antioxidant activity. [ABSTRACT FROM AUTHOR]

Taxane diterpenoids (taxoids) are found only in representatives of the Taxaceae family (different yew species); however, the unique structure and therapeutic properties of taxoids, which are in demand in medicine, have made these compounds one of the most studied secondary metabolites of higher plants. In this work, for the first time, a detailed study is performed into the structural diversity of polyesters of 14-hydroxylated taxoids in Taxus canadensis a yew species, for intact plants of which nonpolar 14-hydroxylated taxoids have not been previously described. At the first stage of the work, it was shown using chromato-mass spectrometry that polyesters of 14-hydroxylated taxoids (yunnanxan, taxuyunnanin C, sinenxan B, sinenxan C) are the dominant diterpenoid secondary metabolites in T. canadensis callus cell culture. Based on these results, as well as the similarity of the metabolism of cultivated in vitro plant cells and root cells in planta, it was suggested that polyesters of 14-hydroxylated taxoids will preferentially accumulate in the roots in intact T. canadensis plants. The validity of this hypothesis was confirmed using liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). According to chromato-mass-spectrometric screening in T. canadensis needles, polyesters of 14-hydroxylated taxoids are actually found only in trace amounts, while they are one of the major (quantitatively) diterpenoids in the roots. One of the main 14-hydroxylated taxoids of T. canadensis roots, yunnanxan, was isolated preparatively and uniquely identified using NMR spectroscopy and high-resolution mass spectrometry. This work is the first report on the presence of polyesters of 14-hydroxylated taxoids in intact Canadian yew plants. [ABSTRACT FROM AUTHOR]

The work aimed on studying the effect of synthetic phytohormones on growth and biosynthetic parameters of a suspension culture of Tribulus terrestris L., and the influence of cytokinins on these characteristics. In addition to the previously obtained results about alternative action of two types of auxins, 2,4-D and α-NAA (the first of them activates cell growth in vitro but inhibits the formation of steroidal glycosides, while the second causes cytodifferentiation, leading to a slowdown and arrest of culture growth, but activates the formation of secondary compounds), it was shown that the type of cytokinin and its concentration can modulate its action. It was found that, after transferring a culture from a "growth" medium (containing 2,4-D) to a medium with α-NAA and a different composition of cytokinins (BAP or kinetin in various concentrations), it retains the ability to actively grow for at least three growing cycles. Then, with a suboptimal combination of phytohormones, the cell culture stops growing and dies. However, with a certain composition of growth regulators in a nutrient medium (1 or 2 mg/L α-NAA + 1 mg/L kinetin), after a long lag phase (12–18 days), culture growth resumes and its long-term cultivation is subsequently possible. Suspension on a medium with 2 mg/L α-NAA and 1 mg/L kinetin T. terrestris were maintained for 70 growing cycles without deterioration of growth characteristics (the maximum level of accumulation of dry biomass Mmax was 12–16 g/L, growth index I was 13–18, specific growth rate μ was 0.18–0.28 days–1). In this case, the content of steroidal glycosides in cells did not fall below 0.2–0.3% of dry biomass. Using the UPLC-ESI-MS method, the presence in the cell biomass of at least four steroidal glycosides of the furostanol type—derivatives of hydroxy-diosgenin and tigogenin/neotigogenin—was shown. The results obtained confirm the general patterns of development of cell populations in vitro and are important for optimizing the growth and biosynthetic characteristics of plant cell cultures. [ABSTRACT FROM AUTHOR]

Rhizogenic, callus, and suspension cultures in vitro were obtained for Digitalis lanata and their growth, cytophysiological and biochemical characteristics were investigated. The obtained cultures were characterized by good growth characteristics (growth indexes I in the range of 5–13). Suspension cell culture had a specific growth rate μ within 0.2–0.3 days–1 and it was characterized by a two-phase growth curve (growth retardation during the exponential phase). In the obtained cultures, a study of the qualitative and quantitative composition of secondary metabolites by UPLC-ESI-MS and HPLC-ESI-MS showed the absence of cardiac glycosides. At the same time, phenylethanoids and steroidal glycosides of the furostanol type were found in all studied cultures. The total content of phenylethanoids in callus and suspension cultures was approximately 0.5% of the dry biomass. Based on the results of mass spectrometry, ten phenylethanoid structures, including digiciliside A, digiciliside B, maxoside, purpureaside E, and their methyl derivatives and isomers, and also seven furostanol glycosides with aglycones tigogenin and gitogenin were identified. It has been shown that the composition of secondary metabolites depends on the degree of cell differentiation: furostanol glycosides were prevalent in a rhizogenic culture consisting mainly of differentiated cells, while the diversity of phenylethanoids significantly increases in callus and suspension cell cultures consisting of dedifferentiated cells. The results of the study confirm the hypothesis put forward in our previous works about the specificity of secondary metabolism and its high intensity in plant cell cultures. [ABSTRACT FROM AUTHOR]

A detailed UPLC-ESI-MS study was performed on secondary metabolites in the biomass of a suspension plant cell culture of Turkmenian mandrake (Mandragora turcomanica Mizgir.), which has been maintained for over 30 years. Both compounds widespread in plants (amides of hydroxycinnamic acids with putrescine and feruloyltyramine) and very rare metabolites (glycosides of phenylethylamides of hydroxycinnamic acids) have been identified. The identification of compounds was carried out using ultra-performance liquid chromatography combined with high-resolution mass spectrometry with electrospray ionization (UPLC-ESI-MS) and detection of positive and negative ion modes. Structural analysis of glycosides of phenylethylamides of ferulic acid was carried out on the basis of interpreting MS spectra obtained by fragmentation of protonated molecular ions [M + H]+ of these compounds in the ionization source. Based on the obtained results, the presence of five hexosides of ferulic acid, three of which were with tyramine residues and one each with methoxytyramine and octopamine residues, respectively, in the M. turcomanica cells cultivated in vitro was revealed. One of the detected glycosides belongs to a very rare group of plant metabolites: feruloyltyramine dihexosides. The obtained results confirm the concept developed in the authors' works on the change in specialized metabolism of plant cells cultivated in vitro and indicate that dedifferentiated proliferating cells retain the ability to form a complex set of secondary metabolites, which contradicts the prevailing ideas about the loss or decrease in the intensity of specialized metabolism in plant cell cultures. [ABSTRACT FROM AUTHOR]

Features of the growth and qualitative composition of secondary metabolites in two lines of suspension cell cultures of Phlojodicarpus sibiricus (Steph. ex Spreng.) K.-Pol., an endangered endemic species of Eastern Siberia, have been studied. A suspension cell culture of leaf origin demonstrated the best growth characteristics, which included growth indices for different criteria (dry and crude biomass and cell concentration): I = 10–14; specific growth rate μ = 0.3–0.4 day–1; the maximum dry biomass accumulation М = 9.6 g/L and economic coefficient Y = 0.29. A plant cell culture derived from a hypocotyl was characterized by lower growth parameters: I = 3.6–4.9, μ = 0.12–0.18 day–1, M = 6.6 g/L, Y = 0.16. Differences in the growth of the studied cultures correlated with the cell aggregation level: the "leaf" culture consisted mainly of small-size aggregates (10–30 cells), whereas the "hypocotyl" culture was presented by large aggregations (50 cells or more). The instrumental cultivation of the small-aggregated suspension cell culture of leaf origin was carried out using two types of laboratory bioreactors (bubble column and stirred tank). Cultivation in a bubble column reactor improved the basic growth characteristics of the cell culture: the growth index for the dry biomass I = 12.7; dry biomass productivity P = 0.78 g/L day, μ = 0.18 day–1, М = 15.8 g/L, Y = 0.49. In the case of a stirring tank reactor, all growth parameters were decreased, which was probably connected with the cell damage with stirring devices. Additionally, a phytochemical analysis of the secondary metabolite composition in the studied cell cultures was carried out in comparison with the root cells of intact Ph. sibiricus plants. Significant differences in the composition of phenolic compounds were revealed between in vitro cell cultures and plant roots. In the case of cell cultures, polar (hydrophilic) compounds belonging to phenolic derivatives (coumarin glycosides and benzofurans) prevailed. In roots, the main components were more hydrophobic (khellactone ethers). The obtained results confirmed the earlier developed concept of differences in the secondary metabolism of in vitro and in vivo plant cells. [ABSTRACT FROM AUTHOR]

Effect of synthetic analogs of auxins—2.4-dichlorophenoxyacetic (2.4-D) and α-naphthylacetic (α-NAA) acids—on growth characteristics and accumulation of steroidal glycosides was investigated in suspension cell culture of Tribulus terrestris L. It was found that the substitution of α-NAA for 2.4-D in the nutrient medium brought about a rise in the content of steroidal glycosides in the cultured cells (up to six times) and broadened their structural diversity (nine identified oligofurostanosides when growing culture on the medium with α-NAA vs. five compounds on the medium with 2.4-D). Positive influence of α-NAA exerted on biosynthetic characteristics of T. terrestris suspension cell culture was accompanied by changes in cell morphology (cytodifferentiation), the extent of their aggregation, and gradual decrease (during 3–4 cycles of growing) in culture viability up to its extinction. Simultaneous presence of both synthetic analogs of auxins (α-NAA and 2.4-D) in the growing medium also caused a rise in the content of steroidal glycosides in the cells of T. terrestris, although it was less pronounced (up to three times); however, the culture showed a relatively steady growth and great viability in this case. It was concluded that these two auxin analogs differently influenced the growth of plants' cells in vitro and biosynthesis of substances of specialized metabolism, 2.4-D promoted cell proliferation, whereas α-NAA induced cytodifferentiation and activated the production of secondary compounds. This conclusion is corroborated by the data concerning plant cell cultures of other species of medicinal plants accumulating secondary metabolites from other groups. For instance, in the experiments with suspension cell culture of Panax ginseng C.A. Mey., substitution in the growth medium of α-NAA for 2.4-D brought about a rise in the content and diversity of triterpene glycosides (ginsenosides) associated with an elevated cell aggregation and deterioration of culture's viability. Comparison of production of steroidal glycosides in plant cell cultures of T. terrestris and Dioscorea deltoidea Wall. that is grown for more than 40 years has shown that both cultures accumulated only furostanol (rather than spirostanol) glycosides that promoted cell proliferation. In suspension cell culture of Dioscorea, a high level of oligofurostanosides (up to 12% of dry biomass) was accumulated when growing culture on the medium with 2.4-D. Thus, investigation into the production of furostanol glycosides in plant cultured cells points to a multifactor system of regulation of secondary metabolism in vitro. Different synthetic auxin analogs may exert alternative influences on growth and biosynthetic processes. At the same time, prolonged culturing leads to an autoselection of cells with the properties promoting proliferation, specifically, with a high content of furostanol glycosides. [ABSTRACT FROM AUTHOR]

Callus and suspension plant cell cultures of Tribulus terrestris L., a valuable medicinal plant producing steroidal glycosides, were obtained. The seeds from an American population of T. terrestris were used as explants. Regulation of the production and growth of cell cultures, as well as the biosynthetic characteristics of the cell lines, were studied. The combination of phytohormones of 2,4-D (2.0 mg/L) and BAP (1.0 mg/L) was found to be optimal for callus induction and cultivation. Suspension cell culture obtained in liquid medium of the same composition showed such high growth characteristics during prolonged cultivation (more than 2 years) as a maximum accumulation of dry biomass of 13 g/L, specific growth rate at exponential phase of 0.24 day-1, and economical coefficient of 0.39. A semicontinuous mode of cultivation was used to grow the plant cell suspension in a lab-scale bioreactor. Screening of the steroidal glycosides in the obtained cell cultures was carried out. Steroidal glycosides were not found in the callus cultures. However, as was demonstrated by TLC and UPLC ESI MS methods, the suspension culture contained furostanol glycosides, and their amount increased during the cultivation process. These results support the hypothesis of the autoselection of cultivated cells containing compounds promoting their proliferation in vitro. [ABSTRACT FROM AUTHOR]

This paper reports for the first time about the detection and identification of ginsenoside malonyl-Rg (the rare 20( S)-protopanaxatriol-type ginsenoside) in the biomass of plant cell suspension culture of Japanese ginseng ( Panax japonicus C.A. Mey. var. repens). Ginsenosides were analyzed by means of high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC-ESI-MS) in positive-ion mode. Malonyl-Rg1 was identified as a result of interpretation of MS spectra obtained upon fragmentation of protonated molecular ion ([M + H]) of this compound in an ionization source. Chromatographic analysis and MS spectra showed that the cells of P. japonicus var. repens cultivated in vitro contain several isomers of malonyl-Rg. Thus, we ascertained for the first time that, in addition to malonyl ginsenosides of 20(S)-protopanaxadiol group, the plant cell culture of ginseng P. japonicus var. repens can accumulate glycosides of 20( S)-protopanaxatriol group acylated with a malonic acid residue. The obtained results showed that, in the cells of ginseng cultivated in vitro for a long time (for 10 years and more), the assortment of secondary metabolites (ginsenosides) may be as wide as in intact plants. [ABSTRACT FROM AUTHOR]

This is the first study to show that the formation of 14β-hydroxylated derivatives of taxa-4(20),11-diene is a specific feature of in vitro cultured dedifferentiated yew cells that distinguishes them from intact plant cells. This may be due to a lower toxicity of the 14-OH taxoids for proliferating plant cells compared to the 13-OH derivatives. [ABSTRACT FROM AUTHOR]