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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.

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 as a sustainable alternative to harvesting wild plants for their bioactive components. Despite high interest in biotechnological production, little is known about the main factors affecting cell growth and ginsenoside biosynthesis of this species under conditions. In this study, the potential of cell cultures of as a biotechnological source of ginsenosides was was assessed. 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). All lines demonstrated good growth with a specific growth rate of 0.1-0.2 day , economic coefficient of 0.31-0.70, productivity on dry weight (DW) of 0.30-0.83 gDW (L·day) , and maximum biomass accumulation varying from 10 to 22 gDW L . 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 cell culture for the first time. 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.

Using Panax japonicus as research material, callus induction and culture were carried out, and high-yielding cell lines were screened to establish a suspension culture system that promotes callus growth and the accumulation of the “total saponins” (total content of triterpenoid glycosides or ginsenosides). Using the root as an explant, the medium for callus induction and proliferation was optimized by adjusting culture conditions (initial inoculation amount, carbon source, shaking speed, hormone concentration, culture time) and a high-yielding cell line with efficient proliferation and high total saponins content was screened out. The conditions of suspension culture were refined to find out the most suitable conditions for the suspension culture of callus, and finally, the suspension culture system was established. We found that the lowest (5%) contamination rate was achieved by disinfecting the fresh roots with 75% alcohol for 60 s, followed by soaking in 10% NaClO for 15 min. The highest induction rate (88.17%) of callus was obtained using the medium MS + 16.11 μmol·L−1 NAA + 13.32 μmol·L−1 6-BA + 30.0 g·L−1 sucrose + 7.5 g·L−1 agar. The callus was loose when the callus subcultured on the proliferation medium (MS + 5.37 μmol·L−1 NAA + 13.32 μmol·L−1 6-BA + 30.0 g·L−1 sucrose + 3.8 g·L−1 gellan gum) for 21 days. The callus growth was cultured in a liquid growth medium (MS + 5.37 μmol·L−1 NAA + 13.32 μmol·L−1 6-BA + 30.0 g·L−1 sucrose) with an initial inoculation amount of 40 g·L−1, a shaking speed of 110 r/min and darkness. Cell growth was fastest with a culture period of 21 days. We replaced the growth medium with the production medium (MS + 5.37 μmol·L−1 NAA + 13.32 μmol·L−1 6-BA + 30.0 g·L−1 glucose) for maximum accumulation of total saponins. [Conclusion] A callus induction and suspension culture system for the root of P. japonicus was established. In this way, we can promote the accumulation of total saponins in callus cells and provide a basis for large-scale cell culture and industrial production of medicinal total saponins.

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.

Callus and suspension cell cultures were successfully developed from Sutherlandia frutescens (Fabaceae), an endemic medicinal plant of South Africa. Two callus cell lines, originating from hypocotyl and cotyledon explants of in vitro seedlings under both dark and light conditions, showed intensive fresh weight accumulation with growth index ranging from 4.6 to 5.9. Suspension cell cultures induced from two callus lines had similar growth profiles and their growth index (15–18), specific growth rate (0.15–0.16 day −1 ), productivity (0.83–0.96 g/(l day)) and maximum biomass accumulation (16–18 g/l) remained relatively high for Fabaceae cell cultures during 27 sub-cultivations. Callus and suspension cell cultures showed similar profiles of secondary metabolites that were, however, different from leaves of greenhouse plants. Isoflavones were predominant in both callus and suspension cell cultures while flavonoids (sutherlandins) and triterpene glycosides of the cycloartane group (sutherlandiosides) were mostly found in leaves. Nineteen fatty acids (FA), both short- and very-long-chained (up to C 25:0 ), were found in cell cultures. Linoleic and α-linolenic FA together comprised 60–64% out of total FA content in cell cultures followed by palmitic acid (18–25%). Extracts of suspension cell biomass exhibited antimicrobial activity against Staphylococcus aureus but were not effective against Pseudomonas aeruginosa. To the best of our knowledge, this is the first report on the induction, phytochemical composition and antimicrobial activity screening of S. frutescens suspension cell cultures which opens the door for their biotechnological application.

Obesity, and its consequences for human health, is a huge and complicated problem that has no simple solution. The constant search for natural and safe compounds with systemic action that can be used for obesity prophylactics and treatment is hampered by the limited availability and variable quality of biomass of wild medicinal plants. Plant cell biotechnology is an alternative approach for the sustainable production of vegetative biomass or individual phytochemicals with high therapeutic potential. In this study, the suspension cell biomass of the medicinal plants, Dioscorea deltoidea Wall., Tribulus terrestris L., and Panax japonicus (T. Nees) C.A. Mey, produced in 20 L and 630 L bioreactors, were tested for therapeutic effects in rat models with alimentary-induced obesity. Three-month intake of water infusions of dry cell biomass (100 mg/g body weight) against the background of a hypercaloric diet reduced weight gain and the proportion of fat mass in the obese animals. In addition, cell biomass preparation reduced the intracellular dehydration and balanced the amounts of intra- and extracellular fluids in the body as determined by bioimpedance spectroscopy. A significant decrease in the glucose and cholesterol levels in the blood was also observed as a result of cell biomass administration for all species. Hypocholesterolemic activity reduced in the line P. japonicus > D. deltoidea > T. terrestris/liraglutide > intact group > control group. By the sum of parameters tested, the cell culture of D. deltoidea was considered the most effective in mitigating diet-induced obesity, with positive effects sometimes exceeding those of the reference drug liraglutide. A safety assessment of D. deltoidea cell phytopreparation showed no toxic effect on the reproductive function of the animals and their offspring. These results support the potential application of the biotechnologically produced cell biomass of medicinal plant species as safe and effective natural remedies for the treatment of obesity and related complications, particularly for the long-term treatment and during pregnancy and lactation periods when conventional treatment is often contraindicated.

In the present study, we explored the therapeutic potential of bioreactor-grown cell cultures of the medicinal plant species Dioscorea deltoidea, Tribulus terrestris and Panax japonicus to treat carbohydrate metabolism disorders (CMDs) in laboratory rats. In the adrenaline model of hyperglycemia, aqueous suspensions of cell biomass pre-administered at a dose of 100 mg dry biomass/kg significantly reduced glucose level in animal blood 1–2.5 h (D. deltoidea and T. terrestris) or 1 h (P. japonicus) after adrenaline hydrochloride administration. In a streptozotocin-induced model of type 2 diabetes mellitus, the cell biomass of D. deltoidea and T. terrestris acted towards normalization of carbohydrate and lipid metabolism, as evidenced by a significant reduction of daily diuresis (by 39–57%), blood-glucose level (by 46–51%), blood content in urine (by 78–80%) and total cholesterol (25–36%) compared to animals without treatment. Bioactive secondary metabolites identified in the cell cultures and potentially responsible for their actions were deltoside, 25(S)-protodioscin and protodioscin in D. deltoidea; furostanol-type steroidal glycosides and quinic acid derivatives in T. terrestris; and ginsenosides and malonyl-ginsenosides in P. japonicus. These results evidenced for high potential of bioreactor-grown cell suspensions of these species for prevention and treatment of CMD, which requires further investigation.

Callus and suspension cell cultures of rare foxglove species, Digitalis ciliata and D. grandiflora, were induced from cotyledons and hypocotyls of in vitro seedlings, and their growth and phytochemical profiles were investigated. In both species, callus induction was more efficient from leaf explants (60–80%) than from hypocotyl explants (15–35%). Callus cultures of both species grew well with growth indices ranged from 5 to 10 depending on the genotype. Suspension culture growth profiles also differed between the two species with a 10–11-days lag-phase observed for D. grandiflora and a bi-phasic growth curve without lag-phase recorded for D. ciliata. The main growth characteristics of the D. grandiflora suspension cell culture (maximum biomass accumulation ~ 14 g/L, growth index ~ 10, economic coefficient ~ 0.42, biomass productivity ~ 0.53 g/(L × day)) were 1.5–3 times higher than those for D. ciliata. Ten compounds were identified in cell biomass using UPLC-ESI-Q-TOF-MS: phenylethanoids digiciliside A, digiciliside B, maxoside, purpureaside E, their methyl derivatives and isomers, and two furostanol glycosides with aglycone tigogenin. Phenylethanoid glycosides were major compounds and comprised 0.8–1.1% of dry weight. During the two-years cultivation, suspension cultures retained the ability to accumulate most of the identified compounds evidencing for stability of species-specific secondary metabolism in cultured foxglove cells during this period.Key messagePhenylethanoid glycosides (1.1% DW) and steroidal furostanol glycosides were identified in callus and suspension cell cultures of rare medicinal species Digitalis ciliata and D. grandiflora developed for the first time.