BackgroundThe 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.MethodsThe 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.ResultsThe 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.ConclusionsSecurinine 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.

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 NH 2 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.

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 (IC 50 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.

In the presented studies, plant growth regulators (PGRs) were evaluated for their influence on the shoot development and the content of biologically active indolizidine alkaloids, securinine, and allosecurinine in Phyllanthus glaucus (Euphorbiaceae) in vitro shoot cultures. The experiments included five cytokinins [kinetin, 6-benzylaminopurine (BAP), 2-isopentenyladenine, and thidiazuron] and two auxins [indole-3-acetic acid and indole-3-butyric acid (IBA)] used in a different concentrations. The results were tested for statistical differences. The highest number of shoot per explant (11.5) was achieved on Murashige and Skoog's (MS) medium supplemented with IBA 0.5 mg/L and BAP 1.0 mg/L, and the optimal rooting response (87.50%) was obtained on MS medium-containing IBA 1.0 mg/L. The rooted plantlets were transferred into the soil and the survival rate was 70.0%. The differences in accumulation of particular alkaloids were revealed depending on PGRs present in harvesting medium. The highest total concentration of alkaloids was determined in the shoot culture grown in the presence of BAP 0.5 mg/L [5.82 mg/g dry weight (DW)]. In routine propagation, MS medium supplemented with IBA 0.5 mg/L and BAP 0.5 mg/L was used as a compromise between the relatively high proliferation rate (~ 8 shoots/explant) and the total content of alkaloids (4.73 mg/g DW). The antimicrobial activity of the biomass and isolated securinine was evaluated towards 12 strains of bacteria and one fungal strain. The differences in bacterial susceptibility against P. glaucus extract and securinine were showed. The MBC values range from 0.01 to 5 mg/mL and from 0.063 to 0.250 mg/mL, respectively, for extract and securinine.

Black raspberry (Rubus occidentalis L.) is a plant found in the natural state in eastern North America. In recent years, there has been growing interest in its fruits, mainly due to the results of scientific research, pointing to the health benefits associated with their consumption. Research concerning biological activity of black raspberry fruit includes chemopreventive activity, protective activity against UV radiation, mainly in the context of generating cancerous changes as well as inhibiting their development. Persistent oxidative stress and its accompanying inflammation result in disturbances in the structure and function of healthy cells, and may be related to neoplastic transformation, manifested by DNA damage, increased proliferation, survival, migration and angiogenesis. Numerous in vitro and in vivo studies as well as clinical trials confirm the chemopreventive activity of R. occidentalis fruits in va-rious types of cancer. This activity includes regulation of carcinogenic factors metabolism, inhibition of cell growth, promotion of apoptosis and reduction of expression of inflammation and angiogenesis markers. The chemopreventive activity of R. occidentalis fruit results, among other things, from the high content of anthocyanins, which are one of the strongest antioxidants.