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Introduction: Panax vietnamensis is a valuable medicinal plant and a source of a broad spectrum of biologically active ginsenosides of different structural groups. Overexploitation and low adaptability to planation cultivation have made this species vulnerable to human pressure and prompted the development of cell cultivation in vitro as a sustainable alternative to harvesting wild plants for their bioactive components. Despite high interest in biotechnological production, little is known about themain factors affecting cell growth and ginsenoside biosynthesis of this species under in vitro conditions. In this study, the potential of cell cultures of P. vietnamensis as a biotechnological source of ginsenosides was was assessed. Methods: Six suspension cell lines that were developed from different sections of a single rhizome through a multi-step culture optimization process and maintained for over 3 years on media with different mineral salt base and varying contents of auxins and cytokinins. These cell lines were evaluated for productivity parameters and cytological characteristics. Ginsenoside profiles were assessed using a combination of the reversed-phase ultra-high-performance liquid chromatography-Orbitrap-tandem mass spectrometry (UHPLC-Orbitrap-MS/MS) and ultra-performance liquid chromatography-time of flight-mass spectrometry (UPLC-TOF-MS). Results: All lines demonstrated good growth with a specific growth rate of 0.1-0.2 day-1, economic coefficient of 0.31-0.70, productivity on dry weight (DW) of 0.30-0.83 gDW (L·day)-1, and maximum biomass accumulation varying from 10 to 22 gDW L-1. Ginsenosides of the protopanaxadiol (Rb1, Rb2/Rb3, malonyl-Rb1, and malonyl-Rb2/Rb3), oleanolic acid (R0 and chikusetsusaponin IV), and ocotillol (vinaginsenoside R1) groups and their isomers were identified in cell biomass extracts. Chikusetsusaponin IV was identified in P. vietnamensis cell culture for the first time. Discussion: These results suggest that suspension cell cultures of Vietnamese ginseng have a high potential for the biotechnological production of biomass containing ginsenosides, particularly of the oleanolic acid and ocotillol groups. [ABSTRACT FROM AUTHOR]

Polyscias fruticosa (L.) Harms, or Ming aralia, is a medicinal plant of the Araliaceae family, which is highly valued for its antitoxic, anti-inflammatory, analgesic, antibacterial, anti-asthmatic, adaptogenic, and other properties. The plant can be potentially used to treat diabetes and its complications, ischemic brain damage, and Parkinson's disease. Triterpene glycosides of the oleanane type, such as 3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucuronopyranosyl] oleanolic acid 28-O-β-D-glucopyranosyl ester (PFS), ladyginoside A, and polysciosides A-H, are mainly responsible for biological activities of this species. In this study, cultivation of the cell suspension of P. fruticosa in 20 L bubble-type bioreactors was attempted as a sustainable method for cell biomass production of this valuable species and an alternative to overexploitation of wild plant resources. Cell suspension cultivated in bioreactors under a semi-continuous regime demonstrated satisfactory growth with a specific growth rate of 0.11 day−1, productivity of 0.32 g (L · day)−1, and an economic coefficient of 0.16 but slightly lower maximum biomass accumulation (~6.8 g L−1) compared to flask culture (~8.2 g L−1). Triterpene glycosides PFS (0.91 mg gDW−1) and ladyginoside A (0.77 mg gDW−1) were detected in bioreactor-produced cell biomass in higher concentrations compared to cells grown in flasks (0.50 and 0.22 mg gDW−1, respectively). In antibacterial tests, the minimum inhibitory concentrations (MICs) of cell biomass extracts against the most common pathogens Staphylococcus aureus, methicillin-resistant strain MRSA, Pseudomonas aeruginosa, and Escherichia coli varied within 250–2000 µg mL−1 which was higher compared to extracts of greenhouse plant leaves (MIC = 4000 µg mL−1). Cell biomass extracts also exhibited antioxidant activity, as confirmed by DPPH and TEAC assays. Our results suggest that bioreactor cultivation of P. fruticosa suspension cell culture may be a perspective method for the sustainable biomass production of this species. [ABSTRACT FROM AUTHOR]

The effects of methyl jasmonate (MeJ) on growth and taxoid formation in the cell culture of Taxus wallichiana were investigated to elucidate the specifics of phytohormone action in dedifferentiated plant cells in vitro. The characteristics of the same suspension cell culture were compared in 2017 (the «young» culture) and in 2022 (the «old» culture)—1.5 or 6 years after culture induction, respectively. MeJ (100 µM) is added to the cell suspension at the end of the exponential growth phase. Cell culture demonstrated good growth (dry weight accumulation 10–18 g/L, specific growth rate µ = 0.15–0.35 day−1) regardless of its «age», cultivation system, and MeJ addition. UPLC-ESI-MS analysis revealed the presence of C14-hydroxylated taxoids (yunnanxane, taxuyunnanine C, sinenxane C, and sinenxane B) in the cell biomass. The content of C14-OH taxoids increased from 0.2–1.6 mg/gDW in «young» culture to 0.6–10.1 mg/gDW in «old» culture. Yunnanxane was the main compound in «young» culture, while sinenxane C predominated in «old» culture. Without elicitation, small amounts of C13-OH taxoids (<0.05 mg/gDW) were found only in «young» cultures. MeJ addition to «young» culture had no effect on the content of C14-OH taxoids but caused a 10-fold increase in C13-OH taxoid production (up to 0.12–0.19 mg/gDW, comparable to the bark of yew trees). By contrast, MeJ added to «old» culture was not beneficial for the production of C13-OH taxoids but notably increased the content of C14-OH taxoids (1.5–2.0 times in flasks and 5–8 times in bioreactors). These findings suggest that hormonal signaling in dedifferentiated yew cells grown in vitro is different from that in plants and can be affected by the culture's age. This might be a result of the high level of culture heterogeneity and constant auto-selection for intensive proliferation, which leads to the predominant formation of C14-OH taxoids versus C13-OH taxoids and a modified cell response to exogenous MeJ treatment. [ABSTRACT FROM AUTHOR]

Plant cell cultures of various yew species are a profitable source of taxoids (taxane diterpenoids) with antitumor activity. So far, despite intensive studies, the principles of the formation of different groups of taxoids in cultured in vitro plant cells have not been fully revealed. In this study, the qualitative composition of taxoids of different structural groups was assessed in callus and suspension cell cultures of three yew species (Taxus baccata, T. canadensis, and T. wallichiana) and two T. × media hybrids. For the first time, 14-hydroxylated taxoids were isolated from the biomass of the suspension culture of T. baccata cells, and their structures were identified by high-resolution mass spectrometry and NMR spectroscopy as 7β-hydroxy-taxuyunnanin C, sinenxane C, taxuyunnanine C, 2α,5α,9α,10β,14β-pentaacetoxy-4(20), 11-taxadiene, and yunnanxane. UPLC–ESI-MS screening of taxoids was performed in more than 20 callus and suspension cell lines originating from different explants and grown in over 20 formulations of nutrient media. Regardless of the species, cell line origin, and conditions, most of the investigated cell cultures retained the ability to form taxane diterpenoids. Nonpolar 14-hydroxylated taxoids (in the form of polyesters) were predominant under in vitro culture conditions in all cell lines. These results, together with the literature data, suggest that dedifferentiated cell cultures of various yew species retain the ability to synthesize taxoids, but predominantly of the 14-OH taxoid group compared to the 13-OH taxoids found in plants. [ABSTRACT FROM AUTHOR]

Cell culture of Panax spp. is a potent sustainable source of biologically active triterpene glycosides (ginsenosides) specific for this genus. In this study, growth and biosynthetic profiles were investigated for P. japonicus suspension cell culture maintained for over 20 years by periodic subcultures in flasks. Cell culture demonstrated intensive growth evidenced by high accumulation of dry weight (DW, 9.8 gL−1), viability (80–90%) and specific growth rate (0.12 day−1) that were comparable to those recorded at the year of culture induction. Nineteen structurally different ginsenosides were identified in cell biomass using UPLC-Q-Exactive-Orbitrap HRMS and HPLC-ESI-Q-MS. Ginsenoside R 0 and malonyl-ginsenosides (MalGs) accounted for over 80% of the total ginsenoside content in cells during the subculture cycle while the sum of neutral ginsenosides of protopanaxatriol (PPT: Re, Rg 1 , Rf) and protopanaxadiol (PPD: Rb 1 , Rb 2 , Rc, Rd) groups constituted less than 20%. Ginsenoside contents were maximized during growth decline phase as 35.3 mg/gDW for R 0 , 48.7 mg/gDW for MalGs, 10.4 mg/gDW for PPD and 5.2 mg/gDW for PPT. These data confirmed that 20-year-old cell culture of P. japonicus retained the ability for intensive growth and accumulation of wide spectrum of ginsenosides which opens the door for wide application of this culture. • 20-year-old suspension cell culture of Panax japonicus shows stable growth and biosynthetic characteristics. • UPLC-Q-Exactive-Orbitrap HRMS was used for metabolite identification. • Cell culture accumulates 19 structurally diverse ginsenosides. • Major compounds in cell culture are malonyl-ginsenosides (47.8 mg/g dry weight) and R 0 (35.3 mg/g dry weight). [ABSTRACT FROM AUTHOR]

Highlights: [•] Malonyl-ginsenosides in Panax japonicus cell culture were found for the first time. [•] Ginsenosides accumulation during growth of P. japonicus cell culture was studied. [•] 20(S)-protopanaxadiol-type ginsenosides were represented by their malonylated forms. [ABSTRACT FROM AUTHOR]