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PHYSICAL INVESTIGATION,

THEORETICAL ANALYSIS
AND SUCCESSFUL BIOPHYSICAL
APPLICATION OF ACTIVATED WATER


Vladimir I.Vysotskii
Kiev National Shevchenko University, Kiev, Ukraine


Outline


1. Investigation of physical properties of activated water and
study of water memory

2. Biohysical action of activated water

3. Possible medical aspects of action of activated water
Water is one of the most mysterious chemical compounds. Anomalous
properties of water became long ago a classical example of the
manifestations of characteristics of a nontrivial system.
In the process of vital activity of any biological object, the most
important chemical compound is water.
1. Water is a base of the intracellular liquid
2. Water forms the transport paths for the transfer of vitally necessary
chemical elements
3. Water forms the necessary state of these elements in the form of
atomic and molecular ions
4. Water is active element of all biochemical molecules and organs in
alive systems
5. Water is the most universal solvent.
6. The spatial structure of water agrees ideally with the secondary
structure of a DNA macromolecule.
At the same time, water is one of the most mysterious chemical
compounds.

Till now there are no adequate answers on the questions about:

1) spatial structure of water on the supermolecular level;

2) numerous paradoxes of long-term water memory.

3) connection between long-term water memory and
biophysical (& biochemibal) action of water

AE
0
W = exp(-AE
0
/k
B
T)

t = 1/e
0
W ~ 10
-11
-

10
-12
c - "standard" duration of water memory

e
0
~ 10
-13
c
-1
, AE
0
~ 0.2 eV

The "standard" model of water (quasicrystal model) does not explain
the experimental facts connected with presence of effects of long-
term water "memory and other abnormal phenomena.
We performed a cycle of complex studies of activated water and
water memory, by using the MRET activator - stationary source of
very low-frequency (e~8 Hz) magnetic field with composite space
structure and very weak amplitude (less 1 Oe).

Photo-polymeric
matrix with alloying
admixtures


Activated water
Source of periodical
optical pulses
N
N
N
S
S
S
N
N
System of constant
magnets

Generation of weak
periodic magnetic field
The scheme of the device used for the activation of water
Frequency of magnetic
field is 7.2-8.2 Hz
(Schumann frequency).
Magnitude of field
is 1 Oe.
INFLUENCE OF WATER ACTIVATION ON LONG-TERM
PHYSICAL-MOLECULAR PROPERTIES OF WATER
1. Dielectric permittivity and conductivity of activated water

Generator
Heat setting

Capaitor
Investigated
water
U(t) I(t)
a)
U(t)
I(t)
t
t
= eAt
T = 2t/e
b)
Novocontrol installation including an analyzer of the impedance (a dielectric
analyzer ALPHA operating in the real time scale in the range of frequencies from
3.10
-6
Hz to

3.10
7
Hz); a measuring cell containing water under study; a
thermostating system for the measuring cell providing the heating and the heat
setting in a very wide interval of temperatures from -160
0
to 400
0
C.
Investigated
water
5 mm
14 mm
Construction of the cell for the measurement of the electrodynamic
characteristics of ordinary or activated water.


10
-2
10
-1
1 10 10
2
10
3
10
4
10
5
10
6
10
7
10
10
2
10
3
10
4
10
5
10
6
10
7
10
8
10
9
4x10
-6
6x10
-6
8x10
-6
10
-5
1,2x10
-5
0 h
0,5 h
1 h
2 h
5 h
frequency [Hz]
c'
o, S/cm
2


10
-2
10
-1
1 10 10
2
10
3
10
4
10
5
10
6
10
7
10
10
2
10
3
10
4
10
5
10
6
10
7
10
8
10
9
10
-5
1,5x10
-5
2x10
-5
2,5x10
-5
3x10
-5
3,5x10
-5
4x10
-5
4,5x10
-5
-
frequency [Hz]
c'
o, S/cm
2
c' o

Conductivity and permittivity of
water activated during 30 minutes.
Water was kept after activation
during 0.5h, 1 h, 2 h and 5 h at
temperature 20.

Dielectric permittivity c' and
conductivity o typical nonactivated
("ordinary") distilled water at 20
0

versus the frequency.

10
-2
10
-1
1 10 10
2
10
3
10
4
10
5
10
6
10
10
2
10
3
10
4
10
5
10
6
10
7
10
8
10
-5
1,5x10
-5
0 min
30 min
1h
2h
frequency [Hz]
c'
c'
o
o, S/cm
2
10
-2
10
-1
1 10 10
2
10
3
10
4
10
5
10
6
10
7
10
10
2
10
3
10
4
10
5
10
6
10
7
10
8
3x10
-6
4x10
-6
5x10
-6
6x10
-6
7x10
-6
8x10
-6
9x10
-6

Frequency [Hz]
c'
c'
o
o, S/cm
2
0.5 h
1 h
5h
Conductivity and permittivity of
water activated during 30 minutes.
Water was kept at temperature 40 .

Conductivity and permittivity of
water activated during 30 minutes.
Water was kept at temperature 5 .

Experimental dependence of the relaxation change of c'(e, t, T) on the
storage duration of activated water after the activation.
Relaxation of the
dielectric permittivity
of activated water
c'(e) versus the
storage duration after
the activation. Three
curves correspond to
three temperatures of
the storage of water
(T =5
0
C, 20
0
C, and
36.6
0
C).
T
W
- duration of
relaxation (duration
of water "memory")
These results correspond to the frequency e = 10 Hz
and are obtained on the direct processing of the
above-discussed spectra obtained for water stored for
the different time intervals at different temperatures
after the activation. Water was activated for 30 min.

0 1.0 2.0 3.0 4.0 5.0 t, h
c'(t)/c'()

T = 5
0
C, T
W
~ 7 d

T = 20
0
C, T
W
~ 12 h

T = 36.6
0
C, T
W
~ 4 h
0.75
0.5
0.25
0.0
c'(t) ~ c'() - Ac'exp(-t/T
W
)
1.0
2. Influence of the activation on water viscosity

In our experiments, the viscosity of water was measured with the help of a
rheometer RS 150 L of Hake firm. The studies were executed on the
basis of a measuring cell. It consisted of two immovable coaxial cylinders
1 and 2. In the region between them, there is a rotor (a rotating coaxial
cylinder 3 coaxial with cylinders 1 and 2).
Water under study is placed in the free region between the surfaces of
immovable cylinders 1 and 2 and the rotor. With the help of this system of
coaxial cylinders, we performed the measurements at low shear stresses.

Investigated
water
rotor
immovable
cylinders


3


1 2 d 1

Measuring system
of coaxial cylinders.
,
,
| / |
friction
friction
friction shear stress
vS
F
d
F
v
P
S d
d d
v R d dt
q
t q
q t t

=
=
= =

0.04
- nonactivated water
- t
act
= 15 min
- t
act
= 30 min
- t
act
= 45 min
- t
act
= 60 min

q, Pa*S
0.03
0.02
0.01
0.00
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
0 10 20 30 40 50 60 70 80 v, cm/s
t, Pa
Viscosity q of control nonactivated distilled water and four fractions of the same but
activated water in the region of small shear stresses t and small speed v at a
temperature of 36.6
0
.
,
,
friction
friction
friction shear stress
vS
F
d
F
v
P
S d
d
v
q
t q
q t
=
=
=

In the regions of small pressures and small speeds the effect of sharp reduction of both
viscosity and coefficient of friction of activated water by 100-300 times takes place!
Process of water activation lead to formation of "superfluidity" of
activated water!
Such "superfluid" water has extremely small friction with walls
and can penetrate through very small pores, capillaries, and
channels.
With regard for the fact that the small pressure of a liquid is a
characteristic sign of alive systems, such an effect of
"superfluidity" of activated water allows us to explain many
effects of the anomalous influence rendered by activated water on
biological objects.
3. Influence of the activation of water on hydrogen index





0 2 4 6 8 10 12 14 16 t, days
t
act
= 30 min
t
act
= 60 min
t
act
= 15 min
t
act
= 45 min
t
act
= 0
7,6
7,4
7,2
7,0
6,8
6,6
6,4
6,2
6,0
Influence of the water activation duration
on depending of the time of its storage
at a temperature of 20.
pH

0 2 4 6 8 10 12 14 16 t, days
t
act
= 60 min
t
act
= 15 min
t
act
= 0
t
act
= 30 min
t
act
= 45 min
pH
7,6
7,4
7,2
7,0
6,8
6,6
6,4
6,2
6,0
Influence of the duration of activation of
water on depending on the time of its
storage at a temperature of 4 (directly
before the measurements, the water
samples were heated to 20).

Detailed studies showed that so-called "clathrate" model is most close to the
reality. In the final form, this model was developed by Pauling (1959).
The Pauling clathrate model is based on the idea of that the union of
atoms of oxygen and hydrogen is able to create spatial flexible
tetrahedral frames (relative amount of molecules of "frame" water at
room temperature is 20-30% and increases as the temperature
decreases). Outside of this frame, there are quasifree molecules of
"ordinary" isotropic water, whose properties and structure
approximately correspond to the continual model.
Theoretical investigation of water memory
H
+
H
+
O
-2
2o ~ 104
0
27' ~ 104.5
0



108
0
108
0
108
0
108
0
108
0
Window
Microcavities of the frame are joined with the external space by windows of about D
0

~ 2.5 in diameter, which is not much less than the width of a water molecule
(2R ~ 2.76 ).
Finally, each of the microcavities is separated from the "external" amorphous quasifree
water by a ring-like potential barrier of about AR ~ 0.13 in width which borders a
window. The height of the potential barrier is AE
M
~ 1.1 eV.
This barrier is connected with deformation of water molecule.
In the volume of a microcavity, one of the molecules
H
2
O, H
4
, O
2
, or N
2
, for example, can be freely arranged.

108
0
108
0
108
0
108
0
108
0
D
0
2o
O
-2

r
oR
H
+
H
+
In the frame 18% of microcavities are filled by molecules
H
2
O at a temperature of 40 , 38% at the normal
temperature of a man (36.6 ), and about 50% at 55 .
a)
a
b)
a
Clathrate micro cavity
Free molecules of
amorphous water
H
2
O
R
c
a
AW
~
c)
a
R
c
0 R
c
r
2R
AE
M
Directions of
activation
Potential energy of H
2
O
(D
2
O) molecule in
Clathrate micro cavity
Potential energy of
H
2
O molecule in free
(amorphous) water
Process of thermally
stimulated
a) activation and
b) deactivation of
micro cavities of a
clathrate water frame
at increasing and
decreasing
temperatures;
c) structure of
potential energy of
molecules of
amorphous
and linked water in the
volume of a clathrate
micro cavity and
around its boundaries

T
1W
= 1/F
1
= t
0
exp(AE
M
/k
B
T) - time of relaxation of a vacant spot in a
micro cavity (the duration of "the water memory"); t
0
= 1/e
0
~ 10
-13
s,

T
2W
= 1/F
2
= t
0
exp[(AE
M
- AW)/k
B
T) - time of relaxation in
alternative direction (transferring a single molecule of water from the
volume of a micro cavity into the volume of amorphous water).

T
o
C 1 10 20 30 36.6 40 50 60 70 90
T
1W
300
days
49
days
10
days
58
hours
24
hours
15
hours
4.4
hours
1.3
hours
27
min
3
min
T
2W
30
min
14
min
4
min
1.5
min
45
sec
30
sec
12
sec
4
sec
1.5
sec
0.3
sec


These results satisfactorily agree with the results of our experiments!
Dependence of the duration of relaxation of water (the duration of
"the water memory") on its temperature.

ACTIVATED WATER AND BIOLOGICAL SYSTEMS

INFLUENCE OF ACTIVATED WATER ON SHAPING
AND GROWTH OF A KALUSS TI SSUE I N VI TRO




We carried out the studies of the influence of activated water on the development of
irregularly growing nondifferentiated vegetative cells in vitro.
Such objects are referred to callus tissues. They are characterized by a nonspecific
growth of nondifferentiated cells.
In these experiments sterile agar-like cultural Murashige-Skoog medium with
initiators of the accelerated cell-like divisions of plants was used. Such plants are
termed kaluss tissue. They are characteristic by nonspecific growing of
nondifferentiable cells of plants.
The experiments on forming of kaluss tissue were carried out on segments of a stalk
of Solanum rickii plant in sterile cultural medium. The process of body height of
these kaluss tissues is similar to body height of oncologic cells or psoriasis.
Petri dishes,
containing
initial segments
of a stalk before
the beginning of
experiments
The enlarged parts of a working field of Petri dishes, containing initial
segments of a stalk before the beginning of experiments on kaluss tissues
cultivation (top) and in two weeks (bottom)

.
t
act
= 1 h t
act
= 0.5 h Control

Repeated experiment on kaluss tissues cultivation in two weeks


Fraction of
water
Initial averaged
weight of one
segment, mg
Final averaged
weight of one
segment, mg
Increase of a
weight of one
segment, mg
Coefficient of
inhibition of
kaluss tissues
growth
t
act
= 1 h 20.2 0.1 20.5 0.1 0.3 0.2 350...1000
t
act
=

0.5 h 20.8 0.1 21.2 0.1 0.4 0.2 300...900
Control 19.6 0.1 193.5 0.1 173.9 0.2 1
Table. Growth of kaluss tissues in activated water and "usual" water

The activation of water suppresses very significantly (by tens and
hundred of times) the development of a callus culture of plant
cells (in particular, Solanum rickii) in the presence of a stimulator
of uncontrollable nonspecific growth.
INVESTIGATION OF INHIBITION ACTIVITY OF
ACTIVATED WATER


The examination of influence of activated water on growth of
Staphylococcus aureus and Escherichia coli microbiological
cultures on nutrient medium meat broth with 1.5% agar was carried
out.

The aim of investigation was definitions of influence of process of
activation of nutrient medium on a survival rate of cells of
pathogenic microbiological cultures, growth of colonies on the
surface of agar during 24 hours.

Cultivation of colonies was produced at t=20 in aerobic conditions
General view of Petri dishes at the beginning of experiments.
Identical very small amount of Escherichia coli K-12 cells was
inoculated on a surface of three fractions of activated nutrient
medium (t
act
=0.5 h, t
act
=1.0 h and control).
Colonies of microorganisms are absent in all dishes.
A) Inhibition activity of activated water on growth of
Escherichia coli microbiological cultures in 29 hours
View of different Petri dishes with grown colonies and statistical
parameters of the colonies in 23 hours after beginning of
experiments
a) Control
(nonactivated medium).
Number of culture
Escherichia coli colonies is
very great.
Number of cells is
N
C
= 1.7.10
8
cells/ml.
Average diameter of grown
colonies is d = 1.1 mm.

b) t
act
= 0.5 hour.
Number of cells is
N
0.5
= 6.4.10
6
cells/ml.
Average diameter of grown
colonies is d = 1.8 mm
c) t
act
= 1.0 hour.
Number of cells is
N
1.0
= 5.2.10
5
cells/ml.
Average diameter of grown
colonies is d = 1.5 mm.

The general view of the dishes with different fractions of activated
nutrient medium in 29 hours after beginning of experiments.
a) Control. Number of cells
is N
C
= 1.7.10
8
cells/ml.
Averaged diameter of grown
colonies is d = 1.2 mm.

b) t
act
= 0.5 h. Number of
cells is
N
0.5
= 6.4.10
6
cells/ml.
Averaged diameter of grown
colonies is d = 2.7 mm.

c) t
act
= 1.0 h. Number of
cells is
N
1.0
= 5.6.10
5
cells/ml.
Averaged diameter of grown
colonies is d = 2.0 mm


0 0.5 1.0 t
act
, h
10
8
10
7
10
6
10
5
Number of pathogenic cells/ml
Bactericidal (bacteriostatic) action of
different fraction of activated water on
culture Escherichia coli growing under
aerobic conditions
It was observed for the first
time that at low initial
concentration of cells of
investigated culture
Escherichia coli in nutrient
medium the MRET water
activated during 0.5 hour and
1 hour have suppressed
culture growth in
N
C
/N
0.5
= 27 and
N
C
/N
1.0
= 303 times.
At the increasing of water
activation time, inhibition of
conditionally pathohenic
strain growth is more
effective.
n
0
=10
2
cell/ml, Control
n
0
=10
2
cell/ml, t
act
= 15 min
n
0
=10
2
cell/ml, t
act
= 30 min
n
0
=10
2
cell/ml, t
act
= 45 min n
0
=10
2
cell/ml, t
act
= 60 min
Influence of duration of
MPA activation on
growth of culture
Staphylococcus aureus
in 24 hours at initial
deluting of suspension
with microorganisms up
to a level
n
0
= 100 cells/ml.

B. Inhibition activity of activated water on growth of
Staphylococcus aureus microbiological cultures in 24 hours

t
act
, min 0 15 30 45 60
N, number of grown colonies
per Petri dishes 300
200
100
0
Bacteriostatic action of different fraction of activated water on culture
Escherichia coli growing during 24 hours under aerobic conditions.
Initial concentration of Staphylococcus aureus cells was n
0
=10
2
cell/ml.
0 10 20 30 40 50 60
0
20
40
60
80
100
t activation, min

Index of bacteriostatic activity
N
0
= 1000 cell/ml, N
0
= 1000 cell/ml,
Control t
act
= 30 min
N
0
= 1000 cell/ml, N
0
= 1000 cell/ml,
t
act
= 45 min t
act
= 60 min
0 10 20 30 40 50 60
0
20
40
60
80
100
t activation, min

Bacteriostatic action of different fraction of activated water on culture
Staphylococcus aureus growing during 24 hours under aerobic conditions.
Initial concentration of Escherichia coli cells was n
0
=1000 cell/ml.
Index of bacteriostatic activity
In activated water the effect of anomalous division of grown cells takes place.
Photos of fragments of the system of cells in one Escherichia coli colony that
were grown on the surfaces of the nonactivated (a) and activated (b - t
act
= 1.0 h)
agarized nutrient media.
These fragments are obtained under a great magnification of separate colonies on
the surfaces of the corresponding Petri dishes by a microscope.
1 - cells with normal division, 2 - cells with anomalous division, 3 non-
separated linear chains of cells, 4 nonseparated branched chains of cells.
a)
b)
The view of paws of a mouse that consumed non-activated water (LEFT)
(reddening of the injected paw) and activated water (RIGHT) (no reddening of
the injected paw) ) in 24 hours after the inoculation of Staphylococcus culture.
The procedure of
inoculation of
Staphylococcus
aureus culture in the
hinder left paw of a
mouse.

0
50
100
150
200
250
1 2 3

weight of spleen
en


weight of spleen
en

weight of thymus
thymus
weight of
lymph nodes
Weight of organs, mg


.
The weight of lymphoid organs in two
weeks of experiment: 1 Control
group; 2 Group #1 consumed
MRET water (preventive for 4
weeks); 3 Group #2 consumed
MRET water (preventive for 2
weeks).

0
10
20
30
40
50
60
70
80
1 2 3
neutrophils
macrophages
Index of Phagocytosis, %
Index of Phagocytosis of neutrophils
and macrophages in two weeks of
experiment (object of phagocytosis
Staphylococcus aureus): 1 Control
group; 2 group #1 (preventive for 4
weeks on MRET water); 3 group #2
(preventive for 2 weeks on MRET
water).
Influence of activated water on the stability of the microbiological culture
Escherichia coli to the action of antibiotics under aerobic conditions
In each Petri dish on the surface of the agarized medium sown
uniformly, 5 sterile paper disks containing 5 different antibiotics
were placed (total 10 different antibiotics) The number of each
antibiotic corresponded to its name from the Table.

Violet resazurin was presented in the medium as the indicator of
metabolic activity. Microorganisms during their growth on the
medium surface decreased the redox-potential. As a result,
resazurin was successively transformed into resorufin (red) and
then into the colorless form(leukoresazurin).
If there was no growth, the redox-potential remained high, and the
color of resazurin was not changed. Inside the growth inhibition
zone (GIZ) (around the disk with an antibiotic), the reductase
activity was absent. As a result, this zone was colored in blue-
violet or red. The stability to the action of antibiotics (SA) was
determined by the diameter (D, mm ) of GIZ. The quantitative
characteristics were determined in 24 and 36 h of the growth of
cultures in the presence of antibiotics.
. Name of antibiotic
1 Clindamycin
2 Kanamycin
3 Cephalexin
4 Ceftriaxone
5 Chloromycetin
6 Ciprofroxacin
7 Ampicillin
8 Tetracycline
9 Cephaclor
10 Carbenicillin
Fig. General view of Petri dishes with
Escherichia coli culture for three fractions
of water after 15 h of the experiment on
the influence of antibiotics on the cultures
. Name of antibiotic
1 Clindamycin
2 Kanamycin
3 Cephalexin
4 Ceftriaxone
5 Chloromycetin
6 Ciprofroxacin
7 Ampicillin
8 Tetracycline
9 Cephaclor
10 Carbenicillin











Fig. General view
of Petri dishes
with culture
Escherichia coli
for three fractions
of water after 24 h
of the experiment.

.Name of antibiotic
1 Clindamycin
2 Kanamycin
3 Cephalexin
4 Ceftriaxone
5 Chloromycetin
6 Ciprofroxacin
7 Ampicillin
8 Tetracycline
9 Cephaclor
10 Carbenicillin
Fig. General view of Petri dishes with
culture Escherichia coli for three fractions
of water after 36 h of the experiment.
.Name of antibiotic
1 Clindamycin
2 Kanamycin
3 Cephalexin
4 Ceftriaxone
5 Chloromycetin
6 Ciprofroxacin
7 Ampicillin
8 Tetracycline
9 Cephaclor
10 Carbenicillin












Diameter of
GIZ, D
0
,mm
t
act
=0;
Duration of
experiment
24 h
Diameter of
GIZ, D
0
, mm
t
act
=0;
Duration of
experiment
36 h
Diameter of
GIZ, D
0
, mm
t
act
=0.5 h;
Duration of
experiment
24 h
Diameter of
GIZ, D
0
,
mm
t
act
=0.5 h;
Duration of
experiment
36 h
Diameter of
GIZ, D
0
, mm
t
act
=1.0 h;
Duration of
experiment
24 h
Diameter of
GIZ, D
0
, mm
t
act
=1.0 h;
Duration of
experiment
36 h
clindamycin,
30
0 0 0 0 0 0
kanamycin, 30
21 12 12 + 12 10 + 0 +
cephalexin, 30
29 13 22 + 15 14 + 0 +
ceftriaxone, 30
36 29 32 + 32 32 + 30
chloromycetin,
30
32 19 8 + 0 + 26 26 |
ciprofroxacin,5
26 23 23 + 23 31 | 31 |
ampicillin, 10
16 11 13 + 11 25 | 25 |
tetracycline, 30
9 0 8 + 0 0 + 0 +
cephaclor, 30
31 22 22 + 20 30 | 30 |
carbenicillin,
100
24 19 19 + 18 29 | 29 |
Table. Stability indices of the microbiological culture Echerichia coli to
various antibiotics in activated media (change of growth inhibition zone)
These results will be discussed below: + - sensitivity | - stability

Ampicillin:
Increasing sensitivity by 230%


Chloromycetin:
Increasing stability
by 19 times!
Kanamycin:
Increasing stability
by 12 times!
t
act
=60 min t
act
=60 min t
act
=30 min
Influence of activated water on the efficiency of action of antibiotics

ANTITUMOR ACTION AND APPLICATION OF MRET
ACTIVATED WATER IN ONCOLOGY

The aim of investigations was to study the effects of activated water on
various transformed cells and immune system cells. For research how
different fractions of activated water affect on antitumor resistance of
organism the following approaches and techniques were used:

1. study of possible antitumor effectiveness of prophylactic administration
of different fractions of activated water; for that mice received activated
water before tumor cell transplantation (prophylactic treatment regime);

2. study of possible antitumor effectiveness of therapeutic administration
of different fractions of activated water; for that mice received activated
water after tumor cell transplantation (therapeutic treatment regime);

3. investigation of functional (cytotoxic) activity of lymphocytes with
natural killer cell cytotoxicity isolated from spleens of normal (without
tumors) mice which received activated water.
5*20=100 mice
Activated
water:
t
act
= 15 min
t
act
= 30 min
t
act
= 45 min
t
act
= 60 min
"Old" activated
water, t
act
=30 min
5*10=50 mice
Analysis of
tumors, 8
th
day
20 mice
Nonactivated
water
20 mice
Nonactivated
water
Prophylaxis
action of water
on mice
within 14 days
Analysis of volume
and composition of
tumors in 7 days after
transplantation
Tumor
inoculation
220 mice
A
C
Therapeutic
action of water
on mice with
tumors.
Analysis of survival
of all mice with
tumors.
5*20=100 mice
Activated
water:
t
act
= 15 min
t
act
= 30 min
t
act
= 45 min
t
act
= 60 min
"Old" activated
water, t
act
=30 min
5*10=50 mice
Analysis of survival
Activated
water:
t
act
= 15 min
t
act
= 30 min
t
act
= 45 min
t
act
= 60 min
"Old" activated
water, t
act
= 30 min
20 mice. Analysis of
tumors, 8
th
day
10 mice
Analysis of survival
Nonactivated water:
Water fraction 11 Control
Water fractions 6 -10 Therapeutic action
Water fractions 1 -5 Prophylaxis action
Second stage
of investigations
First stage
of investigations
Preliminary stages
5*20=100 mice
Nonactivated
water:
20 mice
20 mice
20 mice
20 mice
20 mice
5*10=50 mice
Analysis of
tumors, 8
th
day
B
5*20=100 mice
Activated
water:
t
act
= 15 min
t
act
= 30 min
t
act
= 45 min
t
act
= 60 min
"Old" activated
water, t
act
=30 min
5*10=50 mice
Analysis of survival
Activated
water:
t
act
= 15 min
t
act
= 30 min
t
act
= 45 min
t
act
= 60 min
"Old" activated
water, t
act
= 30 min
General scheme of
antitumor effect
investigation of different
fractions of activated
water (water fractions 1
5 and 610) on mice
with transplanted
tumors. Water fraction
11 of nonactivated water
was used in control
experiments.
Tumor transplantation A
corresponds to study of
prophylactic treatment
with activated water,
tumor transplantation B
study of therapeutic
treatment of activated
water, C control
investigations.
The first series of studies was performed using such experimental
tumor growth model as ascitic Ehrlich carcinoma.
A spontaneous mammary gland tumor appeared in an underbred female
mouse was maintained further as an experimental strain of solid
Ehrlich adenocarcinoma. Ascitic Ehrlich carcinoma was obtained by
the inoculation of tumor cells in the peritoneal cavities of mice.

In the present work, cells of ascitic Ehrlich carcinoma were obtained
from peritoneal cavities of underbred white mice on the 7th8th day of
tumor growth.
In the study, inbred adult male BALB/c mice aged 12 weeks with 21
24 g corporal weight were used.
All mice from 11 groups were inoculated intraperitoneally with 200000
viable cells/mouse of ascitic Ehrlich carcinoma in accordance with the
general scheme presented above.
In 7 days after the tumor transplantation, ascitic fluid from the
peritoneal cavities of a half of all animals was obtained.
The appearance of mice from the
control (A) and prophylactic
treatment groups (the duration of
activation was 30 min) () on the 18th
day after the ascitic Ehrlich carcinoma
cell inoculation.
Dead mice of the control group (on the left side) and alive mice
from the prophylactic treatment group (water activated during 45
min was used). The photo was made on the 21st day after the
inoculation of mice with cells of ascitic Ehrlich carcinoma.
Ascitic fluid samples obtained
from peritoneal cavities of mice
with transplanted ascitic Ehrlich
carcinoma (on the left side, 5
samples from mice after the
prophylactic administration
(prophylactic treatment group)
of water activated during 30
min; on the right side, 5 samples
obtained from control mice
(control group)).
Every test-tube contains ascitic
fluid obtained from one mouse.
Measurement of the ascitic
fluid volume in mice with
transplanted ascitic sarcoma 37
of the prophylactic treatment
group (on the left side)
received water activated during
15 min and control group (on
the right side).
3.0
2.5
2.0
1.5
1.0
0.0
15 min 30 min 45 min 60 min Control
30 min
"Old water"
<V>, ml
1
6
2
7
3
8
4
9
5
10
0.5
250
200
150
100
50
0
15 min 30 min 45 min 60 min Control
30 min
"Old water"
</V>, ml
-1
(*10
6
viable cells)
1
6
2
7
3
8
4
9
5
10
600
500
400
300
200
100
0
15 min 30 min 45 min 60 min Control
30 min,
"Old water"
700
<>, (*10
6
viable cells)
1
6
2
7
3
8
4
9
5
10
Fig. 3. Effect of prophylactic (15) and therapeutic (6
10) application of activated water on average volume of
ascitic fluid obtained from mice inoculated
intraperitoneally with tumor cells of Ehrlich carcinoma.


Fig. 4. Effect of prophylactic (15) and therapeutic
(610) application of activated water on average
number of viable cells in 1 ml of ascitic fluid obtained
from mice inoculated intraperitoneally with tumor
cells of Ehrlich carcinoma.

Fig. 5. Effect of prophylactic (15) and therapeutic
(610) application of activated water on average
number of viable cells in an ascitic tumor, obtained
from mice inoculated intraperitoneally with tumor
cells of Ehrlich carcinoma.




Ehrlich carcinoma
Influence of different fractions of activated water on the investigated
parameters of tumour of ascitic Ehrlich carcinoma.

Investigated parameters

Types of treatment and
fractions of used water

Average
volume of
ascitic fluid in
one tumour,
<V>, ml
Average number of
viable tumor cells in
1 ml of ascitic fluid,
</V>, ml
-1

(*10
3
cells)
Average number of
viable tumor cells in
peritoneal cavity of
one mouse
<>,(*10
6
cells)


Index of
tumor
growth
inhibition
, D, %
Control, t
act
= 0
2,85 0,2 235724 44915
672 -
Prophylactic, t
act
=15 min
1,6 0,1 117354 35134
188 43,6
Prophylactic, t
act
=30 min
1,4 0,07 111268 23714
156 50,9
Prophylactic, t
act
=45 min
1,5 0,1 120068 11711
180 47,4
Prophylactic, t
act
=60 min
2,2 0,08 181868 36784
400 22,8
Prophylactic, t
act
=30 min
"Old activated water",
1,9 0,1 161166 13774
306 33,3
Therapeutic, t
act
=15 min
2,2 0,1 193231 32144
425 22,8
Therapeutic, t
act
=30 min
2,0 0,07 151283 30561
303 30,0
Therapeutic, t
act
=45 min
2,3 0,1 150014 11301
345 19,3
Therapeutic, t
act
=60 min
2,5 0,08 210067 23677
525 12,3
Therapeutic, t
act
=30 min
"Old activated water",
2,2 0,1 166541 23454
366 22,8

Ehrlich carcinoma
0
10
20
30
40
50
60
70
80
90
100
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
t, days
S
u
r
v
i
v
a
l

o
f

a
n
i
m
a
l
s
,

%

0
10
20
30
40
50
60
70
80
90
100
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
t, days
S
u
r
v
i
v
a
l

o
f

a
n
i
m
a
l
s
,

%

Fig. Survival dynamic of tumor-
bearing mice which received different
types of activated water in
prophylactic treatment regime.


Fig. Survival dynamic of tumor-
bearing mice which received
different types of activated water in
therapeutic treatment regime

t
act
= 30 min
t
act
= 30 min
Control
Control
Ehrlich carcinoma (Survival dynamic)







Percentage increase in life span, K,%
60
50
40
30
20
10
15 30 45 60 30
"Old water"
Prophylactic treatment regime.
0
15 30 45 60 30
"Old water"
Therapeutic treatment regime

Change of percentage increase in life span of tumor-bearing mice with ascitic
Ehrlich carcinoma which received different types of activated water in
prophylactic treatment and therapeutic treatment regimes.
The digits near the charts correspond to duration of water activation in minutes.

Ehrlich carcinoma (Survival dynamic)

<>, (*10
6
viable cells)
600
500
400
300
200
100
0
1



6



2



7



3



8



4



9



5



10



Fig. 6.16. Effects of activated water on the average number
of viable cells in one tumor obtained from mice transplanted
intraperitoneally with sarcoma 37 and treated with activated
water in the prophylactic (15) and therapeutic (610)
modes of application.

15 min



30 min



45 min



60 min



Control



30 min
"Old water"



Ascitic sarcoma 37 (average number of viable cells in one tumor )
0
10
20
30
40
50
60
70
80
90
100
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
t , days
S
u
r
v
i
v
a
l

o
f

a
n
i
m
a
l
s
,

%

Fig. Survival dynamic of tumor-
bearing mice which received different
types of activated water in
prophylactic treatment regime.


0
10
20
30
40
50
60
70
80
90
100
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
t , days
S
u
r
v
i
v
a
l

o
f

a
n
i
m
a
l
s
,

%
t
act
= 30 min
t
act
= 30 min
Fig. Survival dynamic of tumor-
bearing mice which received
different types of activated water in
therapeutic treatment regime

Ascitic sarcoma 37 (Survival dynamic )

Percentage increase in the lifetime, K,
%
50
40
30
20
10
15 30 45 60 30
"Old water"

"Prophylactic treatment " mode




Percentage increase in the lifetime of tumor-bearing mice with
ascitic sarcoma 37 which received different types of activated water
in the prophylactic treatment and therapeutic treatment modes.
The numbers near the charts correspond to the water activation
duration in minutes.

0
15 30 45 60 30
"Old water"

Therapeutic treatment mode



Ascitic sarcoma 37 (Survival dynamic )
Research of the influence of activated water on the cytotoxic
activity of murine lymphocytes
In order to understand the possible mechanism of antitumor effects
of activated water, the studies of changes in the cytotoxic activity of
lymphocytes of mice treated with different fractions of activated
water were carried.
Natural killer (NK) cells are the important cells of immune systems.
Based on their defining function of spontaneous cytotoxicity
without prior immunization, NK cells have been thought to play a
critical role in immune surveillance and cancer therapy. NK cells
that infiltrate tumors may protect against the tumor spread.

The following procedure and the scheme of investigation were used.





Isolation,
cleaning, and
separation of
lymphocyte
fraction
enriched with
natural killer
cells
Healthy mice receive activated water
Isolation of tumor target cells from peritoneal cavities of mice
transplanted with ascitic Ehrlich carcinoma
7 days

Test for the
cytotoxic activity
of lymphocytes

Procedure of the study of the influence of activated water on
the cytotoxic activity of lymphocytes.

Prophylactic
treatment mode
5 groups of mice
received different
fractions of activated
water for 21 days

Control mode

1 group of mice
received nonactivated
water for 21 days
Short Prophylactic
treatment mode
5 groups of mice
received different
fractions of activated
water for 14 days
Scheme of studies of the effects of activated water on the cytotoxic
activity of lymphocytes.
Lymphocyte fraction enriched with natural killer cells (NK cells)
was isolated from spleens of all groups of mice.

Cytotoxicity assays were performed using 96-well plates.
NK cells were incubated in vitro with tumor target cells for 16 h.

Tumor target cells were obtained from peritoneal cavities of mice
transplanted with ascitic Ehrlich carcinoma.

In the first stage of
investigation, mice of the
experimental and control
groups received activated
water for different time
intervals. Mice of the
prophylactic treatment
and short prophylactic
treatment groups received
activated water,
respectively, for 21 days
and for 14 days. When the
treatment with activated
water was finished,
mononuclear lymphocyte
fractions enriched with NK
cells were isolated from
spleens of mice of
experimental groups.
In the second stage, the cytotoxic activity of NK cells incubated with tumor target cells
obtained from the peritoneal cavities of mice with transplanted ascitic Ehrlich
carcinoma was studied.
In the study inbred adult male BALB/c mice at 12 weeks of age, with 21 24 g
corporal weight were used. All mice were randomly divided into 11 groups with 5
animals in each group depending on activated water application regime as follows:

a) control group mice received non-activated distilled water during 14 days;
b) prophylactic treatment 15, 30 45 and 60 min groups of mice received water
activated directly before application during 15, 30, 45 and 60 min;
c) prophylactic treatment 30 min ("old activated water") group of mice
received during 21 days water activated during 30 min before experiment beginning
and stored in refrigerator; that water was used up to the end of experiment;
d) therapeutic treatment 15, 30, 45 and 60 min groups of mice received water
activated directly before application during 15, 30, 45 and 60 min;
e) "therapeutic treatment 30 min" ("old activated water") group of mice received
during 14 days water activated during 30 min before experiment beginning and stored
in refrigerator; that water was used up to the end of experiment.

Animals of 10 experimental groups received appropriate fraction of activated water
(according to the daily rate of water intake) whereas, control group mice received
usual (non-activated) water.

The subject of the investigation was fraction of lymphocytes enriched with NK
cells.
The subject of the investigation was the fraction of lymphocytes enriched with NK
cells.
Splenocytes were obtained by the homogenization of spleens resected from mice.
Mononuclear lymphocytes were isolated by the standard Ficoll-Verografin technique.
The final concentration of lymphocytes was 7.5 10^6 cell/ml.
Ascitic Ehrlich carcinoma cells were isolated from the peritoneal cavities of white
mice aged 89 weeks on the 8th day after the tumor cell inoculation and were used in
the cytotoxic test as NK-resistant target cells (TC). Tumor cells were suspended in the
cultivation medium to a concentration of 2.510^6 cell/ml, and their number and
viability were determined in the microscopic supravital test with trypan blue. The cell
viability was about 98%.
All cytotoxicity tests were performed in 96-microwell round-bottomed plates.
After the incubation at 37C for 18 h in the humidified athmosphere with 5% CO
2
,
microplates were gently centrifuged (400 g, 5 min). The numbers of viable and dead
TC in control and experimental wells were determined using the microscopic test with
supravital staining with trypan blue. The cytotoxic activity of NK cells was expressed
as the cytotoxicity index (CI, %) and was calculated as follows:
CI = [(NTC -NTT+NK)/NTC]100%
where NTC the number of viable tumor cells in wells with only TC;
NTC+NK the number of viable tumor cells in wells with TC and NK cells.
Based on this definition, CI for basal experiments was equal zero.
Types of treatment and
fractions of used water
Average number of
viable tumor cells in
microwells, *10
3

Cytotoxicity
index (CI, %)
Change of cytotoxicity
index (in relation to
nonactivated water), %
Basal tumor cells 235.0 0 -16
Control, t
act
= 0 197.5 16 0
Prophylactic, t
act
=15 min 197.5 16 0
Prophylactic, t
act
=30 min 188.0 20 4.0
Prophylactic, t
act
=45 min 200.0 15 -1.0
Prophylactic, t
act
=60 min 200.0 15 -1.0
Prophylactic, "Old activated
water", t
act
=30 min
195.0 17 1.0
Short Prophylactic, 15 min 202.0 14 -2.0
Short Prophylactic, 30 min 195.0 17 1.0
Short Prophylactic, 45 min 200.0 15 -1.0
Short Prophylactic, 60 min 202.5 13.8 -2.2
Short Prophylactic, "Old
activated water",
t
act
=30 min
200.0 15 -1.0

Effects of activated water on the cytotoxic activity of splenic
mononuclear lymphocytes with NK-activity.

Cytotoxic index change (ACI)
4.0
3.0
2.0
1.0
0
-1.0
45 60

15 30 30
"Old
water"



"Prophylactic treatment " mode
( 21 )

-2.0
15 45 60

30

30
"Old
water"

Short prophylactic treatment mode
( 14 )
Changes of the cytotoxic activity of mononuclear lymphocytes of mice received
different types of activated water in comparison to the results of the application of
nonactivated water. Numbers at the diagrams corresponds to the water activation
duration in minutes.
The data obtained demonstrate that the immunostimulatory potential
of activated water is dependent on both the duration of activation and
the duration of storage of activated water before the treatment of
mice.
The application of optimally activated water allows one to potentiate
the cytotoxic activity of lymphocytes without any adverse effects.
The application of this water in the prophylactic treatment mode
resulted in the increase of the cytotoxicity index by 20% as compared
with control values obtained after the application of nonactivated
water.
It is possible that the prolongation of the treatment period of activated
water will cause a more expressed augmentation of the NK cell
cytotoxic activity.
CONCLUSIONS AND RECOMMENDATIONS

The performed detailed studies have shown that activated water has a
number of special or anomalous properties
Essential features of MRET activated water:
1. very great change (reduction by 5 and more times) of the dielectric permittivity
and the conductivity in the area of low and ultralow frequencies;

2. very significant reduction of both the viscosity and the coefficient of friction at
small speeds of water movement;

3. the temporal nonmonotone behavior of the hydrogen index pH during many
hours and days;

4. anomalous properties of activated water are preserved during a large time interval
which can last many hours and days;

5. the duration of preservation of the anomalous properties of activated water
depends strongly on its temperature (it increases as the temperature drops and
decreases as the temperature grows).
Conducted experiments has shown the essential influence of activated
water (or a medium on the basis of activated water) on metabolism and
internal stability of the following six levels of the organization of the
living matter:

1-st level microbial cultures Escherichia coli and Staphylococcus
aureus;
2-nd level microbial syntrophic associations including the maximum
number of species and physiological groups of microorganisms, being
in the state of symbiosis and natural synergism;
3-rd level culture of higher plants "in vitro" (callus cells);
4-th level full-value higher plants "in vitro" (sterile plants on the
agarized nutrient medium) and "in vivo" (plants in soil),
5-th level - animal cells "in vitro" (as-separated tumoral cells of the
ascitic form of inoculated Ehrlich carcinoma separated from abdominal
cavities of mice),
6-th level - animals with inoculated cancer cells of different lines
(Ehrlich carcinoma and Sarcoma 37).
Activated water changes some very important biochemical and biophysical processes
which proceed in alive systems. It renders the essential influence on the processes of
cell division and ionic transport and on the interaction between biological
macromolecules, cells, viruses, leukocytes, etc.

Water activated during 30 min enhances the reductase activity of microbial syntrophic
associations under anaerobic conditions. Such water entering the organism of warm-
blooded animals and a man (with food and drink) can render the essential influence on
the metabolic processes of the microflora of macroorganisms.


Activation of the water based nutrient medium with suspended Escherichia coli and
Staphylococcus cultures leads to very high bacteriostatic activity of such nutrient
medium which depends on the time duration of activation and the initial concentration
of culture cells. The bacteriostatic activity increases following the increase of time of
activation and decrease of initial concentration of the suspension of staphylococcal
culture! The activation of water suppresses by many tens of times the growth of
colonies and reproduction of staphylococcal microorganisms in vitro.


The activation of water suppresses very significantly (by tens and hundred of
times) the development of a callus culture of plant cells (in particular, Solanum
rickii) in the presence of a stimulator of uncontrollable nonspecific growth.

MRET-activated water influences essentially the stability of pathogenic
microflora (e.g. the culture of Escherichia coli) to the action of different types of
antibiotics. Influence of activated water on the efficiency of action of antibiotics
can be very strong.
For example, on the use of water activated during 30 min, the stability of the
culture of Escherichia coli to chloromycetin increases by 19 times! On the use
of water activated during 60 min, the stability to kanamycin and cephalexin
increases by 12 13 times.
In a contrast to this, on the use of the same type of activated water, the sensitivity
of the culture of Escherichia coli to the action of ampicillin increases by 2.3
times.
The determination and use of modes of the activation resulting in the maximal
increase of the sensitivity or stability of specific microbiological cultures to the
action of antibiotics can lead to the overturn in clinical medical microbiology and
a significant reduction of the dosage of antibiotics and can sharply weaken the
negative side effects accompanying the standard methods of the use of
antibiotics.

The application of optimally activated water renders the very significant positive
influence on the prophylaxis and treatment of the tumoral process on the basis of
Ehrlich carcinoma and Sarcoma 37 in organisms of animals.

Water activated during 30 min in the mode of the prophylactic action (on its
intake before the appearance of the oncological process) inhibits the growth rate
of Ehrlich carcinoma by 2 times and reduces the number of alive cancer cells in
the volume of a tumor more than by 4 times. Such water also increases the
lifetime of mice infected by Ehrlich carcinoma by 61.7 %.
The growth inhibition of tumors which is close by efficiency and the increase of
the lifetime were observed also under the intake of water activated during 15 and
45 min.
The same water inhibits the development of a tumor on the basis of cancer cells
of Sarcoma 37 by 67 % and reduces the number of alive oncological cells in the
volume of a tumor by 3 times. In this case, the lifetime of sick animals grew by
50%.
Experiments have shown that water activated during 30 min is optimum on the
therapeutic mode of treatment (on the intake after the beginning of the oncological
process). The efficiency of the therapeutic action of activated water turned out 2
times worse than that in the case of the prophylactic use of this water.

Summing up, we note that as-prepared activated water with the duration of
activation of 30 min which is used for the prophylaxis is a very effective means for
the growth inhibition of tumors caused by cells of Ehrlich carcinoma.

The efficiency of the prophylactic action of such water approaches the efficiency
of preparations of chemotherapy but, unlike chemotherapy, the application of
activated water does not result in negative side effects.

In conclusion, application of activated water can induce significant activation of
NK cell cytotoxic potential. In this scenario, it is apparent that application of
activated water in tumor-bearing immunocompetent hosts would result in cytotoxic
activation of NK cells to destroy tumor cells. Thus, obtained results may be
important for future therapeutic approaches that implicate activated water.
List of participants
Prof. V.I.Vysotskii Kiev National Shevchenko University
Dr. A.A.Kornilova - Lomonosov Moscow State University
Dr. A.B. Tashirev - Institute of Microbiology and Virology, Kiev
Dr. N.A. Matveeva - Institute of Cell Biology and Gene Engineering, Kiev
Dr. Yu.V. Yanish - Kavetsky Institute of Experimental Pathology,
Oncology and Radiology, Kiev
Dr. S. Olishevsky - Kavetsky Institute of Experimental Pathology,
Oncology and Radiology, Kiev
Prof. L.S.Kholodna - Kiev National Shevchenko University, biologist
Dr. N.D. Gavrilova - Lomonosov Moscow State University
Dr. E. Malyshkina - Lomonosov Moscow State University
Detailed investigation of the nature, models, parameters and concrete
mechanism of memory of different kinds of activated water and
biophysical properties of activated watew were conducted and
published in our works:
1. Vysotskii V.I., Kornilova A.A.. Physical foundation of long-time water memory//
Moscow University Physics Bulletin, v.59, no.3 (2004) pp.58-62.

2. Vysotskii V.I., Smirnov I.V., Kornilova A.A.. I ntroduction to the Biophysics of
Activated Water, Universal Publishiers, USA, 2005.
3. Vysotskii V.I., Kornilova
A.A., Smirnov I.V. Applied
Biophysics of Activated
Water: the physical properties,
biological effects and medical
applications of mret activated
water, World Scientific
Publishing, 2009

Vysotskii V.I ., Smirnov I .V., Kornilova
A.A. Introduction to the Biophysics of
Activated Water, Universal
Publishiers, Roca Raton, USA, 2005
Vysotskii V.I ., Kornilova A.A., Smirnov
I .V. Applied biophysics of activated
water, World Scientific Publishing,
Singapore, London, 2009

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