Biotechnological strategies for controlled accumulation of flavones in hairy root culture of Scutellaria lateriflora L.

Accumulation of medicinally important flavones and acteoside was evaluated in Scutellaria lateriflora hairy root cultures subjected to different experimental strategies - feeding with precursors of phenolics biosynthesis (phenylalanine, cinnamic acid, and sodium cinnamate), addition of elicitors (chitosan, jasmonic acid) and Amberlite XAD-4 and XAD-7 resins and permeabilization with dimethyl sulfoxide (DMSO) and methanol. The production profile of S. lateriflora cultures changed under the influence of the applied strategies. Hairy roots of S. lateriflora were found to be a rich source of wogonoside or wogonin, depending on the treatment used. The addition of sodium cinnamate (1.0 mg/L) was the most effective approach to provide high production of flavonoids, especially wogonoside (4.41% dry weight /DW/; 566.78 mg/L). Permeabilization with DMSO (2 µg/ml for 12 h) or methanol (30% for 12 h) resulted in high biosynthesis of wogonin (299.77 mg/L and 274.03 mg/L, respectively). The obtained results provide new insight into the selection of the optimal growth conditions for the production of in vitro biomass with a significant level of flavone accumulation. The data may be valuable for designing large-scale cultivation systems of hairy roots of S. lateriflora with high productivity of bioactive compounds - wogonin or wogonoside.

Phyllanthus amarus shoot cultures as a source of biologically active lignans: the influence of selected plant growth regulators.

This is the first comprehensive study of the influence of plant growth regulators (PGRs) on the development of shoots and accumulation of biologically active lignans-phyllanthin and hypophyllanthin, in the shoot culture of P. amarus Schum. & Thonn. (Euphorbiaceae) obtained by direct organogenesis. The following PGRs were included in the experiments-cytokinins: kinetin (Kin), 6-benzylaminopurine (BAP), 2-isopentenyladenine (2iP), 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea, thidiazuron (TDZ) and auxin, indole-3-butyric acid (IBA) and used at various concentrations. Depending on PGRs and their concentrations, differences in the culture response and lignan accumulation were observed. The highest content of the investigated compounds was found in the shoot culture grown on Murashige and Skoog's (MS) medium supplemented with Kin 0.25 mg/L. The sum of phyllanthin and hypophyllanthin was ~ 10 mg/g of dry weight (DW), which was similar or even higher than that in the plant material obtained from natural conditions. The results of the research provide new data on the selection of the optimal growth medium for the production of plant material with a significant level of phyllanthin and hypophyllanthin biosynthesis. The obtained data may also be valuable in designing systems for large-scale cultivation of P. amarus shoots with high productivity of hepatoprotective lignans.

Cytotoxic activity of standardized extracts, a fraction, and individual secondary metabolites from fenugreek seeds against SKOV-3, HeLa and MOLT-4 cell lines.

CONTEXT: Trigonella foenum-graecum L. (Fabaceae) has many therapeutic properties and anticancer potential. OBJECTIVE: The cytotoxic activities of standardized extracts and a fraction from fenugreek seeds and their compounds (sapogenins, flavone C-glycosides, alkaloid trigonelline) against human cancer SKOV-3, HeLa and MOLT-4 cells were evaluated. MATERIALS AND METHODS: Fenugreek seeds were extracted with 70% methanol (A) or water (B). Furthermore, the seeds were purified with petroleum ether and chloroform and next extracted with methanol to obtain fraction (C). The quantitative analysis of saponins and flavonoids in the extracts was done with HPLC methods. The extracts (5-120 µg/mL) and compounds (1-50 µg/mL) were tested on the cells by MTT assay and RTCA system. The effect of a fraction on ROS production, mitochondrial membrane potential and caspase-3/7 activity in HeLa and SKOV-3 cells was also evaluated by flow cytometry. RESULTS: The strongest cytotoxic activity on cancer cells showed the fraction C (IC50 was 3.91 ± 0.03 for HeLa, 3.97 ± 0.07 for SKOV-3, and 7.75 ± 0.37 for MOLT-4) with the highest content of steroidal saponins (163.18 ± 11.03 µg/mg) and flavone C-glycosides (820.18 ± 0.05 µg/mg). The fraction significantly increased ROS production (up to four times higher than in keratinocytes as control) and caspases activity in the cells. The examined flavonoids did not exhibit the cytotoxic activity in contrast to yamogenin, tigogenin, and diosgenin. CONCLUSIONS: The obtained results complement the data on the cytotoxic activity of Foenugraeci Semen and synergistic effect of flavonoids and saponins complex contained in the plant.

TLC-densitometric analysis of allantoin in Symphytum officinale L. roots.

A TLC-densitometric method for determination of allantoin in Symphytum officinale root was developed. Densitometric quantification of allantoin was carried out on TLC Si60 plates with butanol-50 % methanol/formic acid, 66.5:33.2:0.3 (V/V/V) as developing solvent, at a wavelength of 190 nm. The method was preliminarily validated in terms of specificity, linearity, precision, limit of detection, limit of quantification, recovery and robustness. The results of TLC quantification were compared with HPLC analysis carried out on a HILIC Luna NH2 100A column, with mobile phase consisting of acetonitrile/water 80:20 (V/V) and UV detection at 190 and 210 nm. Allantoin content was determined in two herbal products and it varied from 0.94 to 2.09 %, depending on the producer, and was in agreement with literature reports.

Securinine from Phyllanthus glaucus Induces Cell Cycle Arrest and Apoptosis in Human Cervical Cancer HeLa Cells.

BACKGROUND: The Securinega-type alkaloids occur in plants belonging to Euphorbiaceae family. One of the most widely distributed alkaloid of this group is securinine, which was identified next to allosecurinine in Phyllanthus glaucus (leafflower). Recently, some Securinega-type alkaloids have paid attention to its antiproliferative potency towards different cancer cells. However, the cytotoxic properties of allosecurinine have not yet been evaluated. METHODS: The cytotoxicity of the extract, alkaloid fraction obtained from P. glaucus, isolated securinine and allosecurinine against HeLa cells was evaluated by real-time xCELLigence system and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by annexin V and 7-amino-actinomycin (7-AAD) staining and confirmed with fluorescent Hoechst 33342 dye. The assessment of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, the level of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), caspase-3/7 activity and cell cycle analysis were measured by flow cytometry. The enzymatic activity of caspase-9 was assessed by a luminometric assay. The expression of apoptosis associated genes was analyzed by real-time PCR. RESULTS: The experimental data revealed that securinine and the alkaloid fraction were significantly potent on HeLa cells growth inhibition with IC50 values of 7.02 ± 0.52 µg/ml (32.3 µM) and 25.46 ± 1.79 µg/ml, respectively. The activity of allosecurinine and Phyllanthus extract were much lower. Furthermore, our study showed that the most active securinine induced apoptosis in a dose-dependent manner in the tested cells, increased the percentage of ROS positive cells and depolarized cells as well as stimulated the activity of ERK1/2, caspase-9 and -3/7. Securinine also induced cell cycle arrest in S phase. Real-time PCR analysis showed high expression of TNFRSF genes in the cells stimulated with securinine. CONCLUSIONS: Securinine induces apoptosis and activates cell cycle checkpoints in HeLa cells which is associated with oxidative stress. The results indicate that the mitochondrial pathway is involved in the programmed cell death.