Suspension cell cultures of Panax vietnamensis as a biotechnological source of ginsenosides: growth, cytology, and ginsenoside profile assessment.

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 the main 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.

Suspension Cell Culture of Polyscias fruticosa (L.) Harms in Bubble-Type Bioreactors-Growth Characteristics, Triterpene Glycosides Accumulation and Biological Activity.

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-[ß-D-glucopyranosyl-(1→4)-ß-D-glucuronopyranosyl] oleanolic acid 28-O-ß-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.

Profiling of Taxoid Compounds in Plant Cell Cultures of Different Species of Yew (Taxus spp.).

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 7ß-hydroxy-taxuyunnanin C, sinenxane C, taxuyunnanine C, 2α,5α,9α,10ß,14ß-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.

The Hypoglycemic and Hypocholesterolemic Activity of Dioscorea deltoidea, Tribulus terrestris and Panax japonicus Cell Culture Biomass in Rats with High-Fat Diet-Induced Obesity.

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.

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.

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.

Malonyl-ginsenoside content of a cell-suspension culture of Panax japonicus var. repens.

The presence of large amounts of ginsenosides malonyl-Rb1, -Rc, -Rb2, and -Rd in a suspension culture of Panax japonicus var. repens cells was demonstrated for the first time. Identification of ginsenoside malonyl-Rb1 was based on chromatographic, chemical, and spectroscopic evidence. Ginsenosides malonyl-Rc, -Rb2, and -Rd were identified on the basis of chromatographic and chemical data. Content and composition of the individual ginsenosides (Rg1, R0, malonyl-Rb1, Rb1, Rc, Rb2, and Rd) were monitored in the suspension culture over 4 years. The RP-HPLC-UV analysis showed that Rg1, R0, and malonyl-Rb1 accounted for more than 75% of the total pool of ginsenosides. In accordance with this result, and data analysis reported in the literature, we propose that ginsenoside formation in the cells of P. japonicus var. repens in vitro is closely related to the cellular compartmentation of these substances. In particular, the accumulation of the 20(S)-protopanaxadiol ginsenosides (especially Rb1) is strongly dependent on their pattern of malonylation, which likely targets them for transport into the vacuole.

Expression of acyl-lipid Delta12-desaturase gene in prokaryotic and eukaryotic cells and its effect on cold stress tolerance of potato.

We report the expression profile of acyl-lipid Delta12-desaturase (desA) gene from Synechocystis sp. PCC6803 and its effect on cell membrane lipid composition and cold tolerance in prokaryotic (Escherichia coli) and eukaryotic (Solanum tuberosum) cells. For this purpose, a hybrid of desA and reporter gene encoding thermostable lichenase (licBM3) was constructed and used to transform these cells. The expression of this hybrid gene was measured using qualitative (Petri dish test, electrophoregram and zymogram) and quantitative methods (spectrometry and gas liquid chromatography assays). The maximum level of linoleic acid in the bacterial cells containing hybrid gene was 1.9% of total fatty acids. Cold stress tolerance assays using plant damage index and growth parameters showed that cold tolerance was enhanced in primary transgenic lines because of increased unsaturated fatty acid concentration in their lipids. The greatest content of 18:2 and 18:3 fatty acids in primary transgenic plants was observed for lines 2 (73%) and 3 (41%). Finally, our results showed that desaturase could enhance tolerance to cold stress in potato, and desaturase and lichenase retain their functionality in the structure of the hybrid protein where the enzymatic activity of target gene product was higher than in the case of reporter lichenase gene absence in the construction.

Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants.

The desC gene for the acyl-lipid Delta9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus was introduced into Nicotiana tabacum under control of the 35S promoter. Expression of the desaturase was confirmed by Western blotting. Lipid analysis revealed that lipid content and the extent of fatty acid unsaturation significantly increased in leaves of transgenic plants. Chilling tolerance of those plants also increased, as estimated by the electrolyte leakage from the tissues damaged by cold treatments. Seeds of plants that expressed the desC gene imbibed at low temperatures demonstrated higher chilling tolerance than those of the control plants. The results demonstrate that the cyanobacterial thermophilic acyl-lipid desaturase was efficiently expressed in tobacco at ambient temperatures, and its expression resulted in the enhanced chilling tolerance of the transgenic plants.