Activity report of Omnia & Cytonic Food Supplements.

Activity report of Omnia & Cytonic Food Supplements.

Wednesday, Jul 12, 2017 0 comment(s)

Macrophages are key immune effector cells. They display remarkable plasticity and can change their physiology in response to environmental stimuli, giving rise to different populations of cells with distinct functions. Depending on circumstances, activation of macrophages can result in enhanced microbicidal or tumoricidal capacity, wound-healing activity or differentiation into regulatory macrophages'.

Depending on circumstances, activation of macrophages can result in enhanced microbicidal or tumoricidal capacity, wound-healing activity or differentiation into regulatory macrophages'. 

Vitamin D Transport Protein has been shown to play a role in inflammation by activating macrophages and inducing oxidative burst. Oxygen-dependent mechanisms are known to mediate the anti-microbial and anti-tumor activities of macrophages2.

OBJECTIVE

The aim of this experiment was to evaluate whether Cytonic and Omnia compounds displayed immune activity measured upon oxidative stress caused by ROS (reactive oxygen species) generation.

SAMPLES

  • Omnia Drops (10, 100 and 1000-fold dilutions)
  • Chondroitin sulfate (2.5, 0.25 and 0.025 mg/ml) corresponding to concentrations present in respective dilutions of Omnia Drops
  • Oleic acid (1.25, 0.125, 0.0125 mg/ml) corresponding to concentrations present in respective dilutions of Omnia Drops
  • Oleic acid (1.25, 0.125, 0.0125 mg/ml), dissolved in ethanol (final ethanol concentration in the medium C=0.05%).
  • Cytonic protein (3.3, 0.33, 0.033 p.g/m1)

*All dilutions were prepared in PBS buffer, unless indicated otherwise. 

METHOD

Immortalized bone marrow-derived macrophages (BMDM) and murine macrophage-like cell line RAW 246.7 were used as test cells. ROS production was measured with CeIIROX® Green (CRG) probe. CRG exhibits bright green fluorescence upon oxidation by ROS and subsequent binding to DNA, therefore enables oxidative burst measurement.

Cells were stained with CRG and incubated with analyzed compounds, PMA (positive control; ROS inducer) or buffer only (negative control).

After incubation cells were fixed in formaldehyde and stained with DAPI to visualize nuclei. Fluorescence intensity analysis was performed using confocal microscope.

Results were evaluated using statistical significance of Dunn's multiple comparisons test. Blue bars represent average values and error bars represent standard error values.

1Mosser D. and Zhang X.: Activation of Murine Macrophages. Curr Protoc Immunol; 2008

2Gumireddy K.: Mitogen-Activated Protein Kinase Pathway Mediates DBP—maf-Induced Apoptosis in RAW 264.7

Macrophages. J Cell Biochem; 2003

RESULTS

1) Reactive Oxygen Species production upon treatment with Omnia Drops and its components


Fig. la. ROS production in BMDM cells after incubation with Omnia Drops and its components. CS - chondroitin sulfate, OA - oleic acid, PMA - ROS inducer (positive control).


Fig. lb. ROS production in BMDM cells after incubation with ethanol dissolved oleic acid. OA — oleic acid, E - ethanol, PMA - ROS inducer (positive control).


Fig. 2a. ROS production in RAW 246.7 cells after incubation with Omnia Drops and its components. CS - chondroitin sulfate, OA oleic acid, PMA ROS inducer (positive control).


Fig. 2b. ROS production in RAW 246.7 cells after incubation with ethanol dissolved oleic acid. E - ethanol, PMA ROS inducer (positive control).


Fig. 3. ROS production in BMDM cells after incubation with Cytonic protein. CP

Fig. 4. ROS production in RAW 246.7 cells after incubation with Cytonic protein. CP

NOTE

All results were compared to the control sample (buffer only or E 0.05%) that did not contain any of the tested substances, and that was marked as 100% of the fluorescent signal intensity (delineated as a red dashed line across the chart). PMA effect was included in each chart showing the level of fluorescent signal measured for standard ROS inducer.

Oleic acid is insoluble in water and was mixed with ethanol in order to increase its water solubility.

CONCLUSIONS

1. Treatment with Omnia resulted in increased oxidative stress caused by ROS generation in both BMDM and RAW 246.7 cells. Cells treated with Omnia diluted 10 and 100 times displayed higher fluorescence intensity in comparison to PMA (ROS inducer, positive control). Minor stimulation was observed in BMDM cells treated with 1000 times dilution (fluorescence intensity higher than negative control, lower than PMA). No effect was observed in RAW 246.7 cells treated with 1000 times diluted Omnia.

2. Treatment with particular components of Omnia revealed that chondroitin sulfate alone inhibited ROS generation, while oleic acid, both water and ethanol dissolved, stimulated ROS generation:

  • BMDM and RAW 246.7 cells treated with 2.5 and 0.25 mg/ml of chondroitin sulfate displayed lower fluorescence intensity in comparison to cells incubated only with buffer (negative control). No significant effect was observed for 0.025mg/m1concentration.
  • BMDM and RAW 246.7 cells treated with 1.25 and 0.125 mg/ml of oleic acid, both water and ethanol dissolved, displayed higher fluorescence intensity in comparison with PMA (ROS inducer, positive control). No significant effect was observed for 0.0125 mg/m1 concentration, except for RAW 246.7 cells treated with ethanol dissolved oleic acid, where stimulation was observed as well.

3. Treatment with Cytonic protein resulted in increased oxidative stress caused by ROS generation in both BMDM and RAW 246.7 cells. Both cell lines treated with Cytonic at 3.3 and 0.33 ug/ml concentrations displayed higher fluorescence intensity in comparison to PMA (ROS inducer, positive control). Cells treated with Cytonic at 0.033 ug/ml concentration displayed higher fluorescence intensity in comparison to control sample (buffer only), but lower when compared to PMA (ROS inducer, positive control) indicating minor stimulating effect.

4. To sum up, both Omnia Drops and Cytonic stimulate ROS generation in BMDM and RAW 246.7 macrophage cell lines, which may be translated into enhanced immune activity of the cells.

Version: final (audited)

REPORT PREPARED BY: M. Sagan

REPORT VERIFIED BY: R. Kofodzijczyk, PhD

DATE OF RELEASE: 19 APR 2017

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