Development of an elicitation strategy on enhanced accumulation of oleanolic acid in suspension cultures of Ocimum tenuiflorum L.

Ocimum tenuiflorum Linn. is an important aromatic medicinal plant which produces several secondary metabolites responsible for diverse pharmacological activities. The present study focuses on enhanced production of biomass as well as oleanolic acid (OA), an anticancer and antioxidant compound, in suspension cell cultures of O. tenuiflorum upon elicitation with different elicitors. Leaf explants derived friable calli were inoculated intol iquid Murashige and Skoog (MS) media containing plant growth regulators [0.25 mg/L of α-naphthaleneacetic acid (NAA) and 0.5 mg/L of 6-benzyl amino purine (BAP)] for the establishment of suspension cultures. Influence of several factors such as, age of the suspension cultures, different concentrations, and exposure times of various elicitors such as yeast extract (YE), methyl jasmonate (MeJ) and salicylic acid (SA) respectively were analysed for cell biomass production and accumulation of OA during this study. Among the elicitors tested, YE at 50 mg/L was found to be the most efficient in terms of increased biomass production and accumulation of OA in the cultures. The highest increase in OA production (13.16-fold in elicited cultures compared to untreated cultures) was noted on 17-day-old suspension cultures when exposed to 50 mg/L of YE for four days. Enhancement of 2.72-fold in OA content was also recorded in 17-day-old cultures when treated with MeJ (60 mg/L) during two days of exposure. SA was not efficient in inducing accumulation of OA in 17- and 22-day-old suspension cultures at any concentration and exposure time. Furthermore, it was observed that the effect of different elicitors on biomass production and OA content depended on concentration and the duration of exposure times. Therefore, utilization of elicitation method could be a promising tool to enhance cell growth and OA accumulation in the suspension cell cultures of O. tenuiflorum.

Putrescine Promotes Betulin Accumulation in Suspension Cell Cultures of Betula platyphylla by Regulating NO and NH4+ Production

Putrescine (Put) can enhance secondary metabolite production, but its intrinsic regulatory mechanism remains unclear. In this study, Put treatment promoted betulin production and gene expression of lupeol synthase (LUS), one of betulin synthetic enzymes. The maximum betulin content and gene expression level of LUS was 4.25 mg·g−1 DW and 8.25 at 12 h after 1 mmol·L−1 Put treatment, approximately two- and four-times that in the control, respectively. Put treatment increased the content of nitric oxide (NO) and its biosynthetic enzyme activity of nitrate reductase (NR) and NO synthase (NOS). Pretreatment of the birch suspension cells with NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline- 1-oxyl-3-oxide (cPTIO), NR inhibitor sodium azide (NaN3), and NOS inhibitor NG-nitro-L-Arg methyl ester (L-NAME) decreased Put-triggered NO generation and blocked Put-induced betulin production. Put treatment improved the content of NH4+ and its assimilation enzyme activity of glutamate synthase and glutamate dehydrogenase. NH4+ supplementation also promoted NO and betulin production. Thus, the above data indicated that Put-induced NO was essential for betulin production. NO derived from NR, NOS, and NH4+ mediated betulin production in birch suspension cell cultures under Put treatment.

Physicochemical properties and biological activity of extracts of dried biomass of callus and suspension cells and in vitro root cultures

Introduction. One of the urgent problems of medicine and biology is the use of plant objects as industrial producers of target metabolites in vitro. In vitro cells can be used as pharmaceutical preparations. Study objects and methods. The present research featured medicinal plants that grow in the Siberian Federal district and are a popular source of medicinal raw materials. The physicochemical properties, e.g. total ash content in extracts, the content of heavy metals, the content of organic solvents in the extracts, and the mass loss upon drying was determined by standard methods. The antimicrobial properties of in vitro extracts were determined by the diffusion method and the method based on optical density measurement. The list of opportunistic and pathogenic test strains included the following microorganisms: E. coli ATCC 25922, S. aureus ATCC 25923, P. vulgaris ATCC 63, P. aeruginosa ATCC 9027, and C. albicans EMTC 34. The number of viable cancer cells was determined using the MTT colorimetric method. Results and discussion. The paper describes the physicochemical properties, safety indicators, antioxidant activity, antimicrobial activity, and antitumor properties of extracts of a complex of biologically active substances obtained in vitro from the dried biomass of callus and suspension cell cultures and root cultures. The root extracts proved to have the maximum antimicrobial and cytotoxic properties. They could reduce the survival rate of cancer cells to 24.8–36.8 %. Conclusion. The research featured extracts obtained from the dried biomass of callus and suspension cell cultures and root cultures in vitro of safflower leuzea (Leuzea carthamoides L.), Rhodiola rosea (Rhodiola rosea L.), various sorts of skullcap (Scutellaria baicalensis L., Scutellaria andrachnoides L., Scutellaria galericulata L.), Potentilla alba (Potentilla alba L.) and ginseng (Panax L.). The results showed that the extracts can be used for the production of pharmaceuticals and biologically active additives with antitumor, antimicrobial, and antioxidant properties.

Stimulation of phenolic nature secondary metabolites biosynthesis in Echinacea purpurea L. Moench suspension cell cultures under influence yeast extract elisitors

Тhe ability of 50 ‒500 mg / L yeast extract as complex biotic elicitor to induce an increase in the accumulation of secondary phenolic metabolites in Echinacea purpurea L. Moench suspension cell cultures initiated from calli of leaf and root origin was studied. The yeast extract stimulating effect is more pronounced for a weakly aggregated suspension culture of leaf origin compared with a root origin culture of highly aggregated type. The increase of phenylpropanoids (2.5 times) and flavonoids (2.0 times) content in aqueous-alcoholic extracts from leaf culture as a result of 2-day exposure to 100 ‒500 mg/L of yeast extract correlates with their antiradical activity increase in model system for 2,2ʹ-diphenyl-1-picrylhydrazyl radicals inhibition. The exposure of suspension cell cultures of leaf and root origin in the presence of 250 ‒500 mg/L yeast extract leads 2.1‒2.7 and 1.2‒1.3 times increase L-phenylalanine ammonium lyase activity as key enzyme of phenolic compounds biosynthesis.