Effect of Synthetic Auxin Analogs (2.4-D and α-NAA) on Growth and Biosynthetic Characteristics of Suspension Cell Culture of Tribulus terrestris L.

Abstract

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.

Effect of Phytopreparations Based on Bioreactor-Grown Cell Biomass of Dioscorea deltoidea, Tribulus terrestris and Panax japonicus on Carbohydrate and Lipid Metabolism in Type 2 Diabetes Mellitus

“…Growth characteristics for cell cultures are presented in Table 2. Since the cell cultures of three species were maintained in our laboratory of the long time (over 40 years for D. deltoidea, over 20 years for P. japonicus and five years for T. terrestris), their biochemical profiles have been investigated by using a variety of methods and reported earlier [21,[28][29][30][31]. The results of these biochemical investigation of the cell cultures are summarized in Table 3.…”

“…Furostanol-type steroidal glycosides were previously identified in the suspension cell culture of T. terrestris grown in flasks [27,31]. However, according to the results of spectrophotometry, these compounds are present in the cell culture grown in the bioreactor only in trace amounts, below 0.1% of dry weight.…”

“…Dioscorea deltoidea, Tribulus terrestris and Panax japonicus are medicinal plants that are known to exhibit a large spectrum of pharmacological activities; however, their natural resources are dwindling [23][24][25]. Suspension cell cultures with the ability to produce triterpene and steroidal glycosides have been developed in our laboratory as a sustainable source of biomass and bioactive ingredients of these species [26][27][28][29][30], and their biochemical profiles have been investigated by using various methods [21,[28][29][30][31]. Recently, Lagunin et al [32] suggested high antihypoxic activity of phytopreparations based on P. japonicus, D. deltoidea and T. terrestris cell cultures that, in the case of P. japonicus and T. terrestris, exceeded the effect of the reference drug Mexidol.…”

“…Cell culture of P. japonicus accumulates mostly "acidic" ginsenosides (malonyl derivatives of dammarane ginsenosides and glycosides of oleanolic acid) by contrast to plant roots where neutral ginsenosides are prevailing [28]. Phenylethanoids are major compounds in the cell culture of T. terrestris, but only trace amounts of these chemicals are usually found in plants [31]. Therefore, the mechanisms of biological actions that have been demonstrated for plant extracts cannot be directly translated to cell cultures of the same species.…”

“…Identification of metabolites was performed based on fragmentation patterns of protonated molecular ions in the ionization source [29]. Quantitative evaluation of total content of furostanol-type steroidal glycosides in cell biomass of T. terrestris was performed by using spectrophotometry, as described in Reference [31].…”