MRET Activated Water Research Results


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VISCOSITY TEST













The anomalous low viscosity of MRET activated water in the area of very low magnitudes of tangent pressure applied to the water was discovered during the experiments conducted at Moscow State University, Russia with the help of Haake equipment Reometer RS 150L in compliance with the standard methodology for this instrumentation. Reometer contains the measurement system of coaxial cylinders and the liquid (water in the described test) between them. The cylinders move inside each other very slowly in order to create the tangent pressure along water surfaces.

In the area of relatively significant tangent pressure τ>0.2Pa there was insignificant difference in the values of viscosity of water samples depending on the time of activation and all measurements were close to the standard dynamic viscosity value of the water η=0.001Pa•s=1cP at about 20°C.
In the area of very low tangent pressure magnitudes close to zero (the tangent pressure τ in the range of 0.004 – 0.005Pa) the value of viscosity of non-activated water in compliance with the phenomenon of adgezia (molecular coupling during the process of movement of surfaces along each other) sharply increases 150 – 250 times, for instance, to η=0.22Pa•s at τ=0.004Pa. In the same area of very low tangent pressure MRET activated water showed the anomalous effect of the decrease of viscosity about 2 times with a slight difference for both times of activation. As a result of such anomalies the level of difference in the values of viscosity for non-activated and MRET activated water in the range of low tangent pressure magnitudes of 0.004 – 0.005Pa was registered at the level of 300 – 500 times for 30 minutes activated water and at the level of 200 – 250 times for 60 minutes activated water.

The anomalous low viscosity of MRET activated water in the area of very low tangent pressure confirms the multilayer molecular structuring of MRET water: the high level of long-range molecular coupling (hydrogen bonding) inside the “layer” and very low level of molecular coupling between the “layers.”

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THE EFFECT OF MRET ACTIVATED MEDIUM ON CELL MORPHOLOGY

The in vitro investigations on normal PMBC cells (peripheral blood mononuclear cell) and on HeLa cancer cells (cell line ATCC # CCL-2 cervical adenocarcinoma) and were conducted under the supervision of Patrick Pezzoli, Ph.D. at AltheaDx Technologies, San Diego, USA. The experiments analyzed: cells lysed at 0 hour, cells cultured for 24 hours in untreated medium and cells cultured for 24 hours in medium treated with MRET activator for 30 minutes. DNA samples from each batch were processed and the resultant data was analyzed using Affymetrix Genotyping Console 3.0 to obtain genotype calls and copy number calls. Cell counts and % viability were obtained using the Trypan Blue exclusion technique.

The experimental data revealed no difference between the zero hour (control), MRET treated and untreated samples in term of genotypes and copy number calls. Thus, MRET activation of water based medium did not induce any changes in cells on genetic level.

The studies showed that in MRET activated medium the viability of normal cells (PBMC) was higher (Table 1), and the viability of cancer cells (HeLa) was lower (Table 2) compared to their viability in untreated medium.

Table 1: PBMC cell counts and % viability
Sample Cell Count % Viability Viable cells
0 hour  3.27x106         91.5     2.99x106
Untreated     8.70x105          83.4     0.73x106
Treated        7.73x105         88.8     0.69x106

Table 2: HeLa cell counts and % viability
Sample Cell Count % Viability Viable cells
0 hour     3x106            92        3.27x106
Untreated       7x106             97        6.79x106
Treated   5x106            92        4.60x106

For normal cells (PBMC) the changes in cell counts were similar for untreated and MRET treated medium (Fig 1). Thus, MRET treatment did not affect the growth of normal cells.

For cancer cells (HeLa) the experimental data revealed significant inhibition of cancer cells growth in MRET treated medium. The growth of viable cancer cells was inhibited by 54% in MRET treated medium compared to untreated medium (Fig 2).















The results of AltheaDx research on HeLa cancer cells in vitro support the results obtained earlier in the investigation regarding the effects of MRET water on tumor resistance in animal model. The study on 500 mice was conducted under the supervision of Professor V. Vysotskii, S. Olishevsky, Ph.D. and Y. Yanish, Ph.D. at Kyiv Institute of Experimental Pathology, Oncology and Radiobiology of Ukrainian Academy of Science. It showed substantial inhibition of growth of viable tumor cells following the consumption of MRET water. In the course of this investigation the groups of mice in “preventive regime” ingested MRET water for 2 weeks prior to the inoculation of Ehrlich carcinoma cancer cells and for 3 weeks after the inoculation. The groups of mice in “therapeutic regime” ingested MRET water only during 3 weeks after the inoculation of Ehrlich carcinoma cancer cells. Following the consumption of MRET water activated for 30 minutes (the optimal time of activation) the growth of viable tumor cells was inhibited by 76% in “preventive regime” and by 55% in “therapeutic regime.”

As a conclusion it is possible to say that the studies conducted at AltheaDx Technology confirmed that MRET activated water did not affect the cells on genetic level; it affected the morphology of normal cells in positive way increasing their viability and promoted significant inhibition of growth of cancer cells.

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Fig 2: Viable HeLa cell counts after 24 hours of incubation
Fig 1: Viable PBMC cell counts after 24 hours of incubation
THE EFFECT OF MRET ACTIVATED WATER ON STAPHYLOCCOCAL INFECTION IN VIVO IN ANIMAL MODEL (ON THE CELLS OF IMMUNE SYSTEM)

The research was conducted at Kiyv National Shevchenko University, Ukraine on 400 mice. The mice-male of line BALB in the age of 11 – 13 weeks and of the weight 18 – 21 grams were used. In the process of investigation all mice were divided in three groups. Prior to the inoculation of Staphylococcus aureus Wood-46 culture one group of mice consumed MRET water during 4 weeks (Group 1), another group consumed MRET water during 2 weeks (Group 2), the control group consumed non-activated ordinary distilled water. During the following 2 weeks of experiment the first two groups continued to consume MRET water and the control group consumed ordinary distilled water. The first line of experiments was conducted on 225 mice in order to analyze the persistence of pathogen in homogenate of kidneys of mice comparing 5 groups of mice (two Group 1 and two Group 2 on 15 minutes and 30 minutes MRET activated water and Control). After preliminary experiments the optimal 30 minutes MRET activated water (distilled) was chosen for the main line of investigation.

RESULTS:
1.  Significant protective properties of MRET water.
The significant protective properties of MRET water were confirmed by substantial decrease of Staphylococcus CFU (colony forming units) in homogenate of kidneys of mice on MRET water compare to control group of mice following the intra-peritoneal staphylococcal infection after the first 24 hours. The analysis of data in the beginning of experiments leads to the conclusion that significant decrease of pathogen’s colonies in homogenate of kidneys of mice on MRET water begins only after 24 hours following the inoculation of Staphylococcus culture. The results on 30 minutes activated water were much better than on 15 minutes activated water and all further experiments were conducted on 30 minute activated water.

2.  The consumption of MRET water eliminated the death rate from 30% (control group) to 0% (MRET group) during the first 9 days of experiment.
There was no case of animal death in all investigated groups within the first 24 hours after intra-peritoneal inoculation of Staphylococcus culture, which is a pretty standard result. During the next 8 days 30% of animals died in control group which also is a standard result. But there were no death cases in both groups of mice that ingested MRET activated water and it is a remarkable result. Nevertheless, the main consequences of Staphylococcus infection do not manifest in death of animals as in case of oncology diseases. Staphylococcus virus affects the live systems and organs of the body. These pathogenic microorganisms cause inflammations, suppurations, abscesses, furuncles, quinsy, cepsical conditions, etc. That’s why a detailed investigation of the process of stimulation by MRET water of phagocytes and of lymphoid organs of immune system of mice infected with Staphylococcus aureus culture was conducted and is presented in this report.

3. The development of the local acute inflammation is essentially suppressed in case of ingestion of MRET activated water.
The local inflammation was induced with the help of the inoculation of Staphylococcus aureus culture into the hinder left paw. The ordinary inflammatory reaction was observed in the group of mice on non-activated water: the intensive reddening of the hinder left paw (Fig 1). Both groups of mice on MRET water did not develop any reddening the hinder left paw inoculated with Staphylococcus aureus culture (Fig 2). The results of this experiment confirm the fact of the substantial inhibition of inflammatory infection in case of the regular consumption of MRET water.




















4. The consumption of MRET water stimulates the activity of phagocytic system and the level of natural resistance of animals to pathogenic microorganisms.
For the following series of experiments the inoculation of Staphylococcus aureus Wood-46 was conducted intra-peritoneal in dose LD30 in order to spread infection all over the body.

The phagocytic system is one of the main factors of natural non-specific cellular resistance to infections and inflammations. It is the first line of protection of an organism against penetration and reproduction of pathogenic microorganisms. The protective role of phagocytic cells is based on their capacity to identify, engulf and utilize the alien agents penetrated into internal environment of a macro-organism. Phagocytosis is the main mechanism of natural resistance especially at the first stage of contagious process; it is a regular part of formation of the specific immune response.

The most common methodology applied in the studies of the functional activity of phagocytes is the examination of their phagocytic (engulfing of alien cells) and oxygen-dependent bactericidal activity. Phagocytic activity of neutrophils and macrophages is estimated based on Index of Phagocytosis (percentage of the phagocytes which engulfed test-bacteria) and on Phagocytic Number (average number of test-bacteria engulfed by one phagocyte). The cultures of Staphylococcus aureus and Latex are usually used as test-bacteria. The oxygen-dependent bactericidal activity of phagocytes is studied with the help of NBT-test: an oxygen-dependent reduction of Nitro Blue Tetrazolium into an insoluble Diformazan of Nitro Blue Tetrazolium derivative by phagocytes. With the help of NBT-test it is possible to distinguish the activated phagocytes from the non-activated ones.

MRET water stimulated the phagocytic capacities of neutrophils of a peripheral blood and peritoneal macrophages increasing their phagocytic activity, particularly Phagocytic Indexs (Fig 3A) and Phagocytic Number (Fig 3B). It also stimulated their oxygen-dependent bactericidal activity, particularly the increase of quantity of NBT-positive phagocytes (Fig 4).














Fig 3A: Phagocytic Index of neutrophils and macrophages in two weeks of experiment (object of phagocytosis - Staphylococcus aureus): 1 – Control group; 2 – Mice on MRET water (preventive for 4 weeks); 3 – Mice on MRET water (preventive for 2 weeks).















Fig 3B: Phagocytic Number of neutrophils and macrophags in two weeks of experiment (object of phagocytosis – Staphylococcus aureus): 1 – Control group; 2 – Mice on MRET water (preventive for 4 weeks); 3 – Mice on MRET water (preventive for 2 weeks).














Fig 4: Oxygen-depended bactericidal activity (NBT-test) of neutrophils and macrophages in two weeks of experiment: 1 – Control group; 2 – Mice on MRET water (preventive for 4 weeks); 3 – Mice on MRET water (preventive for 2 weeks).

These experiments confirmed the increase of effective potentials of phagocytes which constitute one of the main factors of natural protection of an organism and initiate the immune response.

The analysis of data in the beginning of experiments leads to the conclusion that significant intensification of phagocytic and bactericidal activity of macrophages and neutraphils of mice on MRET water begins only after 24 hours following the intra-peritoneal inoculation of Staphylococcus culture. At the end of two weeks of experiment the mean values of studied parameters in both groups of mice on MRET water substantially increased compare to the control group. The differences in mean values of the parameters of functional activity of phagocytes of groups of mice consuming MRET water compare to the control group of mice on non-activated water were statistically significant with p<0.05 (for Phagocytic Index and NBT-test). These results confirm the significant intensification of phagocytic and bactericidal activity and of immune system response following the consumption of MRET water.

The differences in mean values of studied parameters for the groups of mice on MRET water compare to each other were statistically insignificant, which confirms the similarity of the level of beneficial effect of MRET water in both groups. This fact also confirms that the regular consumption of MRET water provides health benefits in rather short time (2 weeks in case of the animal mice model).

5.   The consumption of MRET water substantially enhances the immune activity of lymphoid organs.
By the end of another series of experiments in both groups of mice on MRET water was observed substantial statistically significant (p<0.05) increase of the weight and the cellularity (quantity of cells) of a spleen and lymph nodes as well as the insignificant increase of weight and cellularity of thymus (Fig 5 and Fig 6).













Fig 5: The weight of lymphoid organs in two weeks of experiment: 1 – Control group; 2 – Mice on MRET water (preventive for 4 weeks); 3 – Mice on MRET water (preventive for 2 weeks).













Fig 6: The cellularity of lymphoid organs in two weeks of experiment: 1 – Control group; 2 – Mice on MRET water (preventive for 4 weeks); 3 – Mice on MRET water (preventive for 2 weeks).

These results confirm the fact of significant intensification of immune system response in animals on MRET water subject to Staphylococcus infection. The difference in studied parameters between the groups of mice on MRET water (4 weeks and 2 weeks of preventive consumption of MRET water) was insignificant which confirms quite fast beneficial effect of MRET water on the immune activity of lymphoid organs.

In the beginning of experiment the cellularity and the weight of lymphoid organs in MRET groups did not show the distinct tendency to modifications. It is reasonable to admit that the consumption of MRET water affects the weight and the cellularity of lymphoid organs only during the infection period.

CONCLUSIONS:
The consumption of MRET activated water significantly enhances the factors of natural resistance of the body which constitute the first line of protection of an organism against the penetration and reproduction of pathogenic microorganisms.

The analysis of data in the beginning of experiment leads to the conclusion that significant changes in all studied parameters of mice on MRET water (decrease of pathogen colonies in homogenate of kidneys, increase of the weight and the cellularity of lymphoid organs, intensification of the phagocytic and bactericidal activity of macrophages and neutraphils) begins only after 24 hours following the inoculation of Staphylococcus culture. Another words the consumption of MRET water increases the potentials of immune capacities of the body to counteract the infections without any changes in the vital parameters of immune organs and functions prior to the penetration of infectious pathogens in the body.

At the end of two weeks of experiment the mean values of studied parameters in both groups of mice on MRET water (preventive for 4 and 2 weeks respectively) significantly increased compare to the control group. The differences in mean values of the studied parameters of the groups of mice consuming MRET water compare to the control group of mice on non-activated water were statistically significant with p<0.05 (for most of the parameters). These results confirm the significant intensification of phagocytic activity and of immune system response following the consumption of MRET water.

The differences in mean values of studied parameters for the groups of mice on MRET water compare to each other were statistically insignificant, which confirms the similarity of the level of the beneficial effect of MRET water in both groups. This fact also confirms that the regular consumption of MRET water provides health benefits in rather short period of time (2 weeks in case of the animal mice model).
Fig 1: The view of paws of a mouse on non-activated water (reddenning of the injection paw) in 24 hours after the injection of Staphylococcus culture.
Fig 2: The view of paws of a mouse on MRET activated water (no reddenning of the injection paw) in 24 hours after the injection of Staphylococcus culture.
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