Formulation and characteristic parameters have been confirmed organoleptically.
2. Protein concentration
Protein concentration of unprocessed VDTP preparation has been determined by Bradford assay (IB-16.05.00).
After processing, VDTP final solutions were prepared in concentrations:
167 ng/ml; 1,000 ng/mI and 3,334 ng/mI (±10%) in relation to initial concentration.
3. Protein identity has been confirmed by mass spectrometry
Accuracy of measurement is ±1%, therefore result of 50,969 Da is acceptable for VDTP (51,243 Da ).
Protein purity has been evaluated with densitometry.
The amounts of 0.5, 1 and 2 ktg of VDTP protein were loaded on SDS-PAGE gel. After electrophoretic separation, gel was stained with Coomassie Brilliant Blue. Absorbance intensity analysis was performed using Image Lab 5.2.1 software.
5. Endotoxin level
Endotoxin level in VDTP has been evaluated using Limulus Amebocyte Lysate (LAL) assay.
Total aerobic microbial count has been evaluated by inoculating Trypone Soya Agar with 100 [..t1 of VDTP and incubation at 37°C for 5 days. No colonies were observed.
Total combined yeast and mold count has been evaluated by inoculating Sabouraud (+ dextrose + chloramfenikol) with 100 µl of VDTP and incubation at 37°C for 5 days. No colonies were observed.
Both tests were done in triplicates.
Absence of E.coli in 10g has been confirmed by inoculating McConkey Broth with 10g of VDTP and incubation at 37°C for 5 days. No turbidity was observed.
Mycoplasma presence in VDTP has been evaluated by NAT (Nucleic Acids Amplification Techniques). Result was negative.
Retrovirus presence in VDTP has been evaluated by PERT (Product Enhanced Reverse Transcriptase assay). Result was negative.
pH of the VDTP solution has been determined using Hanna Instruments HI2212 pH-meter. Measurement was repeated three (3) times and average value was calculated:
Anticancer efficacy of Vitamin D Transport Protein (VDTP) can be ascribed at least to three biological properties of the molecule:
1) activation of tumoricidal macrophages;
2) inhibition of tumor-induced angiogenesis and
3) direct inhibition of cancer cell proliferation, migration and metastatic potential.
Reference: Pacini S, Punzi T, Morucci G, Gulisano M and Ruggiero M: Effects of vitamin D-binding protein-derived macrophage activating factor on human breast cancer cells. Anticancer Res 32: 45-52, 2012
In order to test whether Cytonic proteins (CP) inhibits cancer cells proliferation, we treated MCF-7 cells (cell line derived from human breast cancer) with various concentrations of proteins.
It has been showed that Cytonic proteins directly inhibits breast cancer cells proliferation in dose- dependent manner.
Fig. 1: Breast cancer cells viability after treatment with various concentration of Cytonic proteins (CP). After 72h incubation with Cytonic proteins viability reduction in dose-dependent manner was observed. The effect was the most significant for 400 ng/ml, where cell viability is reduced to ≈ 59.6% with respect to control cells.
Inactive and Active Immune Cells
Immature immune cells are created in our bone marrow, lymph glands and spleen. Then they are released into our blood plasma where they circulate with the blood looking for a specific protein to become fully active and functional immune cells. The protein required to activate all of our immune cells is Vitamin D Transport Protein (VDTP)
In the first 10 seconds of the above video you will see an ‘inactive’ immune cell, it is consuming just enough energy to stay alive but cannot function as a mature immune cells until it is activated.
From 10 seconds onwards you will see different types of active immune cells in action, despite their differences they carry out similar functions and that is to ‘seek and destroy’. We can see the active immune cells using their pseudopods to check red blood cells for any proteins which may indicate an infection. If a ‘suspect’ cell is discovered the the immune cell will consume it then use oxidise bursts to breakdown the infected cell into amino acids and proteins which are recycled into new and healthy red cells.
At 36 seconds we observe an immune cell homing in an a ‘Helmut Cell’ half a red blood cell that served no purpose in the body and therefore must be eliminated and recycled.
ACTIVITY REPORT OF OMNIA & CYTONIC FOOD SUPPLEMENTS.
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.
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.
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.
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
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
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.
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.