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

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

Structure of securinine (A) and allosecurinine (B).

<p>Structure of securinine (A) and allosecurinine (B).</p

IC₅₀ values (μg/ml) of the <i>Phyllanthus</i> compounds, fraction and extract based on the xCELLigence system (RTCA) and MTT assay.

<p>IC₅₀ values (μg/ml) of the <i>Phyllanthus</i> compounds, fraction and extract based on the xCELLigence system (RTCA) and MTT assay.</p

ecurinine treatment induced cell cycle arrest in S and G2/M phases in HeLa cells.

<p>The cells were stimulated with the compound at concentrations of 10.0 (1) and 20.0 μg/ml (2) and incubated for 24 h and 48 h (*). The percentage of the cells in each phases was measured by flow cytometry and determined in comparison to the untreated cells (ctrl) and DMSO control. Each sample was run in triplicate.</p

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

Abstract 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

Securinine from <i>Phyllanthus glaucus</i> Induces Cell Cycle Arrest and Apoptosis in Human Cervical Cancer HeLa Cells

<div><p>Background</p><p>The <i>Securinega</i>-type alkaloids occur in plants belonging to <i>Euphorbiaceae</i> family. One of the most widely distributed alkaloid of this group is securinine, which was identified next to allosecurinine in <i>Phyllanthus glaucus</i> (leafflower). Recently, some <i>Securinega</i>-type alkaloids have paid attention to its antiproliferative potency towards different cancer cells. However, the cytotoxic properties of allosecurinine have not yet been evaluated.</p><p>Methods</p><p>The cytotoxicity of the extract, alkaloid fraction obtained from <i>P</i>. <i>glaucus</i>, 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.</p><p>Results</p><p>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 <i>Phyllanthus</i> 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.</p><p>Conclusions</p><p>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.</p></div

Securinine induced chromatin condensation in HeLa cells.

<p>The cells nuclei were stained with Hoechst 33342 dye after treating the cells with 0.5% DMSO (A) and securinine at concentrations of 1.0 (B), 5.0 (C), 10.0 (D), 15.0 (E), 20.0 (F), 30.0 (G) and 50.0 μg/ml (H). The cells exposed to securinine showed condensed chromatin in comparison to the control sample (DMSO). Arrows represent nuclear changes in the apoptotic cells.</p

Securinine-induced changes in transmembrane mitochondrial potential in HeLa cells.

<p>The cells were exposed to 0.5% DMSO (A) and securinine at concentrations of 1.0, 5.0, 10.0 (B), 15.0, 20.0(C, D), 30.0 (E, F) and 50.0 μg/ml (G, H). The cells were treated for 5 h (A, B, C, E, G) and 8 h (A, B, D, F, H) and the extent of mitochondrial cell depolarization was determined in comparison to the DMSO control (I). Each sample was run at least in triplicate. Error bars represent standard deviations. Significant differences relative to the control are marked with an “*” (p<0.05).</p