IN VITRO EVALUATION OF BIOLOGICAL ACTIVITIES OF PISTACIA LENTISCUS AQUEOUS EXTRACT

Objective: The aim of this study was to study the antioxidant potential and the antibacterial activity of leaves aqueous extract of Pistacia Lentiscus as well as the protective effect of this extract against the haemolysis in hypotonic condition, in oxidative stress and in the existence of saponin injury.Methods: We studied the antioxidant capacity through the DPPH assay, H2O2 scavenging activity, Ferric-reducing power (FRAP) assay, total antioxidant assay and the antibacterial activity using the disc diffusion method. We also investigated the haemolytic activity with the spectrophotometric method.Results: The result showed that the aqueous extract had a good antioxidant capacity, which was calculated as IC50. IC50 of the aqueous extract was found to be 9.89±0.7µg/ml for DPPH scavenging, 200±18.02µg/ml for H2O2 scavenging assay, 54.06±12.66µg/ml for Ferric-reducing power (FRAP) and 500±22.3 µg/ml for total antioxidant capacity. The aqueous extract also inhibited the growth of the tested bacterial strains with a maximum inhibition zone of 30.33±5.5 mm observed on Pseudomonas aeruginosa for wood-seed and a moderate activity against all other strain. The haemolytic analysis showed that the aqueous extract is not toxic for the human erythrocytes and protects them against the oxidative and osmotic stress and also against saponin injury.Conclusion: The results of our study suggest that the aqueous extract of leaves of Pistacia lentiscus possess potent anti-haemolytic activity, are a good source of natural antioxidant.Â

Antioxidant and anti-inflammatory activities of Arbutus unedo aqueous extract

Objective: To evaluate the antioxidant and anti-inflammatory activities of aqueous extract of Arbutus unedo (A. unedo) leaves. Methods: In this context, the in vitro antioxidant activity was demonstrated by 2,2-diphenyl-1-picrylhydrazyl, hydroxyl radical and H2O2 radical scavenging, ferrous ion chelating, ferric reducing power, total antioxidant capacity and by the protection against peroxidation of β-carotene-linoleic acid in emulsion. The anti-inflammatory activity was evaluated first by studying the membrane of human red blood cells against different hypotonic concentrations of NaCl and against heat, inhibiting the denaturation of albumin. Results: Total phenolic and flavonoid content were found respectively [(207.84 ± 15.03) mg gallic acid equivalent/g, and (13.070 ± 0.096) mg quercetin equivalent/g]. The extract displayed significant scavenging activity of some radicals such as 2,2-diphenyl-1-picrylhydrazyl [IC50 at (7.956 ± 0.278) μg/mL], ·−OH [IC50 = (1015.74 ± 46.35 μg/mL)], H2O2 [IC50 = (114.77 ± 16.86) μg/mL] and showed a good antioxidant activity through ferrous ion chelating activity [IC50 = (1014.30 ± 36.21) μg/mL], ferric reducing power [IC50 = (156.55 ± 17.40) μg/mL], total antioxidant capacity [IC50 = (461.67 ± 4.16) μg/mL] and β-carotene-linoleic acid protection against peroxidation [I% = (87.04 ± 1.21)% at 1 000 μg/mL]. Conclusions: A. unedo showed in vitro anti-inflammatory activity by inhibiting the heat induced albumin denaturation and red blood cells membrane stabilization. Our results show that aqueous leaf extract of A. unedo has good antioxidant activity and interesting anti-inflammatory properties. A. unedo aqueous extract can be used to prevent oxidative and inflammatory processes

Mitotic arrest and apoptosis in breast cancer cells induced by Origanum majorana extract: upregulation of TNF-α and downregulation of survivin and mutant p53.

BACKGROUND: In the present study, we investigated the effect of Origanum majorana ethanolic extract on the survival of the highly proliferative and invasive triple-negative p53 mutant breast cancer cell line MDA-MB-231. RESULTS: We found that O. majorana extract (OME) was able to inhibit the viability of the MDA-MB-231 cells in a time- and concentration-dependent manner. The effect of OME on cellular viability was further confirmed by the inhibition of colony growth. We showed, depending on the concentration used, that OME elicited different effects on the MDA-MB 231 cells. Concentrations of 150 and 300 µg/mL induced an accumulation of apoptotic-resistant population of cells arrested in mitotis and overexpressing the cyclin-dependent kinase inhibitor, p21 and the inhibitor of apoptosis, survivin. On the other hand, higher concentrations of OME (450 and 600 µg/mL) triggered a massive apoptosis through the extrinsic pathway, including the activation of tumor necrosis factor-α (TNF-α), caspase 8, caspase 3, and cleavage of PARP, downregulation of survivin as well as depletion of the mutant p53 in MDA-MB-231 cells. Furthermore, OME induced an upregulation of γ-H2AX, a marker of double strand DNA breaks and an overall histone H3 and H4 hyperacetylation. CONCLUSION: Our findings provide strong evidence that O. majorana may be a promising chemopreventive and therapeutic candidate against cancer especially for highly invasive triple negative p53 mutant breast cancer; thus validating its complementary and alternative medicinal use

<i>O. majorana</i> induces apoptosis by activation of caspase 8 and upregulation of TNF-α.

<p>(A) <i>O. majorana</i> extract induces an activation of caspase 8 but not caspase 9 in MDA-MB-231 cells. MDA-MB-231 cells were incubated with various concentrations of the extract for 24 h. The caspase activation induced by the OME was assayed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and methods</a>. The relative caspase 8 and 9 activity was normalized to the number of viable cells per well and is expressed as fold of induction compared to the control. (B) Western blot analysis showing an increase in cellular TNF-α protein in the MDA-MB-231 cells treated with OME. Whole cell protein were extracted from OME-treated cells or vehicle (ethanol)-treated cells and subjected to Western blot analysis, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>, for TNF-α and β-actin (loading control) proteins. (C) Immunofluorescence staining for TNF-α in OME-treated MDA-MB- 231cells. Cells were treated with 150 and 300 µg/mL of the extract for 24 h, fixed, permeabilized, and then processed for immunofluorescence using antibodies against TNF-α protein. DAPI was used as a nuclear stain. (*<i>p</i><0.05 and **<i>p</i><0.005).</p

<i>O. majorana</i> induces hyperacetylation of histone H3 (AcH3) and H4 (AcH4).

<p>(A) Protein levels of Ac-H3 and Ac-H4, extracted from OME-treated cells, were detected by western blot using antibodies specific for the modified histone (Ac-H3) and Ac-H4. β-actin was used as loading control. (B) Immunofluorescence staining of Ac-H3 and Ac-H4 in MDA-MB231 cells treated with 150, 300 and 450 µg/mL of OME or equal volume of vehicle (ethanol) as control for 24 h. Cells were fixed, permeabilized, and then processed for immunofluorescence using antibodies against the indicated modified histones. DAPI was used as a nuclear stain.</p

Inhibition of colony growth by <i>O. majorana</i> extract.

<p>Inhibition of colony growth was assessed by measuring the size of the colonies obtained in vehicle (ethanol)- and OME-treated plates. Data were compared with those obtained for the 2 weeks colonies. Two types of colonies were counted and depending on their diameter were categorized as large (≥200 µm) and small (<200−≥50 µm).</p

The proposed signal pathways on <i>O. majorana-</i>induced cell cycle arrest, at low concentrations, and apoptosis, at high concentrations, in the triple negative mutant p53 human breast cancer MDA-MB-231 cells.

<p>The proposed signal pathways on <i>O. majorana-</i>induced cell cycle arrest, at low concentrations, and apoptosis, at high concentrations, in the triple negative mutant p53 human breast cancer MDA-MB-231 cells.</p

Induction of G2/M cell cycle arrest and apoptosis by <i>O. majorana</i> extract in MDA-MB-231 cells.

<p>(A) MDA-MB-231 cells (1.8×10⁶) seeded on 100 mm culture dish were exposed various concentrations of <i>O. majorana</i> extract or equal volume of vehicle (ethanol) as control for 24 h. Following treatment, cells were harvested, fixed, stained with propidium iodide, and analyzed for cell cycle distribution by flow cytometry. Data represent the mean of three independent experiments. The percentage of cells in sub-G1 (apoptosis), G1, S and G2/M appears at the upper right of each graph. (B) Expression of cell cycle regulator in OME-treated MDA-MB-231. Western blot analysis of phospho(ser10)-H3, and cyclin B1 in MDA-MB231 cells exposed for 24 h to ethanol or indicated concentrations of OME. (C) Stimulation of caspase 3/7 activity in MDA-MB-231 cells after exposure to OME (0–600 µg/mL) for 24 h and 48 h, relative to a similar amount of viable ethanol-treated cells. The relative caspase 3/7 activity was normalized to the number of viable cells per well and is expressed as fold of induction compared to the control. (D) Concentration-dependent induction of PARP cleavage in OME-treated MDA-MB231 cells. Cells were treated with or without increasing concentrations of the extract and proteins were extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>. Western blot analysis was carried out using anti-PARP antibodies. (*<i>p</i><0.05, **<i>p</i><0.005 and ***<i>p</i><0.0005).</p

Differential regulation of survivin expression by <i>OME</i> in MDA-MB-231 cells.

<p>Western blot analysis showing a differential effect on survivin expression by different concentrations of OME in MDA-MB-231 cells. Whole cell protein were extracted from OME or vehicle (ethanol)-treated cells and subjected to Western blot analysis, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>, for survivin and β-actin (loading control) proteins.</p

<i>O. majorana</i> extract induces a dose-dependent activation of γH2AX, a marker of DNA double-strands breaks, in MDA-MB 231 cells.

<p>(A) Western blot analysis of phosphor-H2AX (ser 139) in MDA-MB231 cells exposed for 6 and 24h with the indicated concentrations of OME or equal volume of vehicle (ethanol) as control. (B) Immunofluorescence staining for γH2AX in OME-treated MDA-MB 231cells. Cells were treated with vehicle or 150, 300 and 450 µg/mL extract for 24 h, fixed, permeabilized, and then processed for immunofluorescence using antibodies against p-H2AX (ser 139) protein. DAPI was used as a nuclear stain.</p