Vladimir I.vysotskii ActivatedWater Bulgaria 2013

8/13/2019 Vladimir I.vysotskii ActivatedWater Bulgaria 2013

1/71

PHYSICAL INVESTIGATION,THEORETICAL ANALYSIS

AND SUCCESSFUL BIOPHYSICAL

APPLICATION OF ACTIVATED WATER

Vladimir I.Vysotskii

Kiev National Shevchenko University, Kiev, Ukraine


2/71

Outline

1.Investigation of physical properties of activated water andstudy of water memory

2. Biohysical action of activated water

3. Possible medical aspects of action of activated water


3/71

Water is one of the most mysterious chemical compounds. Anomalousproperties of water became long ago a classical example of themanifestations of characteristics of a nontrivial system.

In the process of vital activity of any biological object, the mostimportant chemical compound is water.

1. Waterisabase of the intracellular liquid

2. Waterforms the transport paths for the transfer of vitally necessarychemical elements3. Waterforms the necessary state of these elements in the form ofatomic and molecular ions4. Wateris active element of all biochemical molecules and organs in

alive systems5. Wateristhe most universal solvent.6. The spatial structure of wateragrees ideally with the secondarystructure of a DNA macromolecule.


4/71

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


5/71

E0

W = exp(-E0/kBT)

= 1/0W 10-11- 10-12c - "standard" duration of water memory

0 10-13

c-1

, E0 0.2 eV

The "standard" model of water (quasicrystal model) does not explainthe experimental facts connected with presence of effects of long-term water "memory and other abnormal phenomena.


6/71

We performed a cycle of complex studies of activated water andwater memory, by using the MRET activator - stationary source ofvery low-frequency (8 Hz) magnetic field with composite spacestructure and very weak amplitude (less 1 Oe).


7/71

Photo-polymeric

matrix withalloyingadmixtures

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 magneticfield is 7.2-8.2 Hz

(Schumann frequency).Magnitude of field

is 1 Oe.


8/71

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

= t

T = 2/

b)

Novocontrol installation including an analyzer of the impedance (a dielectricanalyzer ALPHAoperating in the real time scale in the range of frequencies from3.10-6Hz to3.107Hz); a measuring cell containing water under study; athermostating system for the measuring cell providing the heating and the heatsetting in a very wide interval of temperatures from -160 0to 4000C.


9/71

Investigatedwater

5 mm

14 mm

Construction of the cell for the measurement of the electrodynamic

characteristics of ordinary or activated water.


10/71

10-2 10-1 1 10 102 103 104 105 106 10710

102

103

104

105

106

107

108

109

4x10-6

6x10-6

8x10-6

10-5

1,2x10-5

0 h0,5 h1 h2 h5 h

frequency [Hz]

, S/cm

2

10-2 10-1 1 10 102 103 104 105 106 10710

102

103

104

105

106

107

108

109

10-5

1,5x10-5

2x10-5

2,5x10-5

3x10-5

3,5x10-54x10-5

4,5x10-5-

frequency [Hz]

, S/cm2

Conductivity and permittivity ofwater activatedduring 30 minutes.Water was kept after activationduring 0.5h, 1 h, 2 h and 5 h attemperature 20.

Dielectric permittivity andconductivity typical nonactivated("ordinary") distilled waterat 200versus the frequency.


11/71

10-2

10-1

1 10 102

103

104

105

106

10

102

103

104

105

106

107

108

10-5

1,5x10-5

0 min30 min1h2h

frequency [Hz]

, S/cm2

10-2 10-1 1 10 102 103 104 105 106 10710

102

103

104

105

106

107

10

8

3x10-6

4x10-6

5x10-6

6x10-6

7x10-6

8x10-6

9x10-6

Frequency [Hz]

, S/cm

2

0.5 h

1 h

5h

Conductivity and permittivity ofwater activatedduring 30 minutes.Water was kept at temperature 40 .

Conductivity and permittivity ofwater activatedduring 30 minutes.Water was kept at temperature 5 .


12/71

Experimental dependence of the relaxation change of (, t, T) on thestorage duration of activated water after the activation.

Relaxation of thedielectric permittivity

of activated water

() versus the

storage duration after

the activation. Threecurves correspond to

three temperatures of

the storage of water

(T =50C, 20

0C, and

36.60C).TW- duration of

relaxation (duration

of water "memory")

These results correspond to the frequency = 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

(t)/()

T = 50C, TW 7 d

T = 200C, TW 12 h

T = 36.60C, TW 4 h0.75

0.5

0.25

0.0

(t) () - exp(-t/TW)

1.0


13/71

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 thebasis of a measuring cell. It consisted of two immovable coaxial cylinders1 and 2. In the region between them, there is a rotor (a rotating coaxialcylinder 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 ofcoaxial cylinders, we performed the measurements at low shear stresses.

Investigated

water

rotor

immovablecylinders3

1 2 d 1

Measuring system

of coaxial cylinders.

,

,

| / |

friction

friction

friction shear stress

vSF

d

F vPS d

d d

v R d dt


14/71

0.04 - nonactivated water- tact= 15 min

- tact= 30 min

- tact= 45 min

- tact= 60 min

, 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, Pa

Viscosity of control nonactivated distilled waterand four fractionsof the same but

activated waterin the region of small shear stresses and small speed v at a

temperature of 36.60.

,

,

friction

friction

friction shear stress

vSF

d

F vPS d

d

v

In the regions of small pressures and small speeds the effect of sharp reduction of bothviscosity and coefficient of friction of activated water by 100-300 times takes place!


15/71

Process of water activation lead to formation of "superfluidity" ofactivated water!

Such "superfluid" water has extremely small friction with wallsand can penetrate through very small pores, capillaries, andchannels.

With regard for the fact that the small pressure of a liquid is acharacteristic sign of alive systems, such an effect of"superfluidity" of activated water allows us to explain manyeffects of the anomalous influence rendered by activated water on

biological objects.


16/71

3. Influence of the activation of water on hydrogen index

0 2 4 6 8 10 12 14 16 t, days

tact= 30 min

tact= 60 min

tact= 15 min

tact= 45 min

tact= 0

7,6

7,4

7,2

7,0

6,8

6,6

6,4

6,2

6,0

Influence of the water activation durationon depending of the time of its storage

at a temperature of 20.

pH

0 2 4 6 8 10 12 14 16 t, days

tact= 60 min

tact= 15 min

tact= 0

tact= 30 min

tact= 45 min

pH7,6

7,4

7,2

7,0

6,8

6,6

6,4

6,2

6,0

Influence of the duration of activation ofater on depending on the time of its

storage at a temperature of 4 (directly

before the measurements, the water

samples were heated to 20).


17/71

Detailed studies showed that so-called "clathrate" model is most close to thereality. In the final form, this model was developed by Pauling (1959).

The Pauling clathrate model is based on the idea of that the union ofatoms of oxygen and hydrogen is able to create spatial flexible

tetrahedral frames (relative amount of molecules of "frame" water atroom temperature is 20-30% and increases as the temperaturedecreases). Outside of this frame, there are quasifree molecules of"ordinary" isotropic water, whose properties and structureapproximately correspond to the continual model.

Theoretical investigation of water memory

H+H+

O-2

21040

27104.50

108

108 108

108 108


18/71

Window

Microcavities of the frame are joined with the external space by windows of about D02.5 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 quasifreewater by a ring-like potential barrierof about R 0.13 in width which borders awindow. The height of the potential barrier is EM 1.1 eV.

This barrier is connected with deformation of water molecule.In the volume of a microcavity, one of the moleculesH2O, H4, O2, or N2, for example, can be freely arranged.

108

108 108

108 108

D0

2

O-2

r

RH+ H+

In the frame 18% of microcavities are filled by molecules

H2O at a temperature of 40 , 38% at the normaltemperature of a man (36.6 ), and about 50% at 55 .


19/71

a) b)

Clathrate micro cavity

Free molecules of

amorphous water

H2O

Rc

W

c)

Rc 0 Rc

2R

EM

Directions of

activation

Potential energy of H2O(D2O) molecule in

Clathrate micro cavity

Potential energy ofH2O molecule in free

(amorphous) water

Process of thermallystimulateda) activation and

b)deactivation ofmicro cavities of aclathrate water frameat increasing anddecreasingtemperatures;c) structure ofpotential energy ofmolecules ofamorphousand linked water in the

volume of a clathratemicro cavity andaround its boundaries


20/71

T1W= 1/F1= 0exp(EM/kBT) - time of relaxation of a vacant spot in a

micro cavity (the duration of "the water memory"); 0= 1/010-13s,

T2W= 1/F2= 0exp[(EM - W)/kBT) - 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).

ToC 1 10 20 30 36.6 40 50 60 70 90

T1W 300days

49days

10days

58hours

24hours

15hours

4.4hours

1.3hours

27min

3min

T2W 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.


21/71

ACTIVATED WATER AND BIOLOGICAL SYSTEMS

INFLUENCE OF ACTIVATED WATER ON SHAPING

AND GROWTH OF A KALUSS TI SSUEI N VITRO

We carried out the studies of the influence of activated water on the development ofirregularly growing nondifferentiated vegetative cellsin vitro.Such objects are referred tocallus tissues.They are characterized by a nonspecific

growth of nondifferentiated cells.


22/71

In these experiments sterile agar-like cultural Murashige-Skoog medium withinitiators of the accelerated cell-like divisions of plants was used. Such plants aretermed kaluss tissue.They are characteristic by nonspecific growing ofnondifferentiable cells of plants.

The experiments on forming of kaluss tissuewere carried out on segments of a stalkof Solanumrickiiplant in sterile cultural medium. The process of body height ofthese kaluss tissuesis similar to body height of oncologic cells or psoriasis.

Petri dishes,

containinginitial segmentsof a stalk beforethe beginning ofexperiments


23/71

The enlarged parts of a working fieldof Petri dishes, containing initialsegments of a stalk before the beginning of experiments on kaluss tissues

cultivation (top) and in two weeks (bottom)


24/71

tact= 1 h tact= 0.5 h Control

Repeated experiment on kaluss tissuescultivation in two weeks

T bl G th f k l ti i ti t d t d " l" t


25/71

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

tact = 1 h 20.2 0.1 20.5 0.1 0.3 0.2 350...1000

tact =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 tissuesin 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 r icki i) in the presence of a stimulatorof uncontrollable nonspecific growth.


26/71

INVESTIGATION OF INHIBITION ACTIVITY OF

ACTIVATED WATER

The examination of influence of activated water on growth ofStaphylococcus aureusand Escher ichia colimicrobiologicalcultures on nutrient medium meat broth with 1.5% agar was carriedout.

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

) i i i i i f i f


27/71

General view of Petri dishes at the beginning of experiments.

Identical very small amount ofEscherichia coli K-12cells wasinoculated on a surface of three fractions of activated nutrient

medium (tact=0.5 h, tact=1.0 h and control).

Colonies of microorganisms are absent in all dishes.

A) Inhibition activity of activated water on growthof

Escher ichia colimicrobiological cultures in29 hours


28/71

View of different Petri dishes with grown colonies and statisticalparameters of the colonies in 23 hours after beginning ofexperiments

a) Control(nonactivated medium).

Number of cultureEscherichia coli colonies isvery great.Number of cells isNC= 1.7.10

8cells/ml.Average diameter of grown

colonies is d = 1.1 mm.

b) tact= 0.5 hour.Number of cells is

N0.5= 6.4.106

cells/ml.Average diameter of growncolonies is d = 1.8 mm

c) tact= 1.0 hour.Number of cells is

N1.0= 5.2.105

cells/ml.Average diameter of growncolonies is d = 1.5 mm.


29/71

The general view of the dishes with different fractions of activatednutrient medium in 29 hours after beginning of experiments.

a) Control. Number of cellsis NC= 1.7.10

8cells/ml.Averaged diameter of growncolonies is d = 1.2 mm.

b) tact

= 0.5 h. Number ofcells is

N0.5= 6.4.106cells/ml.

Averaged diameter of growncolonies is d = 2.7 mm.

c) tact= 1.0 h. Number ofcells is

N1.0= 5.6.105cells/ml.

Averaged diameter of growncolonies is d = 2.0 mm


30/71

0 0.5 1.0 tact, h

108

107

106

105

Number of pathogenic cells/ml

Bactericidal (bacteriostatic) action of

different fraction of activated water oncultureEscherichia coligrowing under

aerobic conditions

It was observed for the firsttime that at low initialconcentration of cells of

investigated cultureEscherichia coliin nutrientmedium the MRET wateractivated during 0.5 hour and1 hour have suppressedculture growth in

NC/N0.5= 27 and

NC/N1.0= 303 times.

At the increasing of wateractivation time, inhibition ofconditionally pathohenicstrain growth is moreeffective.

B Inhibition acti it of acti ated ater on gro th of


31/71

n0=102cell/ml, Control n0=10

2cell/ml, tact= 15 min n0=102cell/ml, tact= 30 min

n0=102cell/ml, tact= 45 min n0=102cell/ml, tact= 60 min

Influence of duration ofMPA activation ongrowth of culture

Staphylococcus aureusin 24 hours at initialdeluting of suspensionwith microorganisms upto a level

n0= 100 cells/ml.

B. Inhibition activity of activated water on growthof

Staphylococcus aureusmicrobiological cultures in24 hours


32/71

tact, min0 15 30 45 60

N, number of grown colonies

per Petri dishes300

200

100

0

Bacteriostatic action of different fraction of activated water on cultureEscherichia coligrowing during 24 hours under aerobic conditions.Initial concentration of Staphylococcus aureuscells was n0=10

2cell/ml.

0 10 20 30 40 50 60

0

20

40

60

80

100

t activation, min

Index of bacteriostatic activity


33/71

N0= 1000 cell/ml, N0= 1000 cell/ml,Control tact = 30 min

N0= 1000 cell/ml, N0= 1000 cell/ml,tact = 45 min tact = 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 aureusgrowing during 24 hours under aerobic conditions.Initial concentration ofEscherichia coli cells was n0=1000 cell/ml.

Index of bacteriostatic activity


34/71

In activated water the effect of anomalous division of grown cells takes place.Photos of fragments of the system of cells in one Escher ichia colicolony thatwere grown on the surfaces of the nonactivated (a) and activated (b - tact= 1.0 h)agarized nutrient media.These fragments are obtained under a great magnification of separate colonies onthe surfaces of the corresponding Petri dishes by a microscope.1 - cells with normal division, 2 - cells with anomalous division, 3non-

separated linear chains of cells, 4nonseparated branched chains of cells.

a) b)


35/71

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 Staphylococcusculture.

The procedure of

inoculation of

Staphylococcus

aureus culture in thehinder left paw of a

mouse.


36/71

0

50

100

150

200

250

1 2 3

weight of spleen

weight of spleen

weight of thymus

weight oflymph nodes

Weight of organs, mg

.

The weight of lymphoid organs in two

weeks of experiment: 1Controlgroup; 2Group #1 consumedMRET water (preventive for 4weeks); 3Group #2 consumedMRET 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 neutrophilsand macrophages in two weeks ofexperiment (object of phagocytosisStaphylococcus aureus): 1Controlgroup; 2group #1 (preventive for 4weeks on MRET water); 3group #2(preventive for 2 weeks on MRET

water).


37/71

Influence of activated water on the stability of the microbiological culture

Escherichia col ito 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 antibioticswere placed (total10 different antibiotics) The number of eachantibiotic corresponded to its name from the Table.

Violet resazurin was presented in the medium as the indicator ofmetabolic activity.Microorganisms during their growth on themedium surface decreased the redox-potential. As a result,resazurin was successively transformed into resorufin (red)andthen into the colorl ess form(leukoresazurin).If there was no growth, the redox-potential remained high, and thecolor of resazurin was not changed. Inside the growth inhibition

zone (GIZ)(around the disk with an antibiotic), the reductaseactivity was absent. As a result, this zone was colored in blue-violet or red. The stability to the action of antibiotics (SA) wasdetermined by the diameter (D, mm ) of GIZ. The quantitativecharacteristics were determined in 24 and 36 h of the growth of

cultures in the presence of antibiotics.

.Name of antibiotic1 Clindamycin2 Kanamycin3 Cephalexin4 Ceftriaxone5 Chloromycetin

6 Ciprofroxacin7 Ampicillin8 Tetracycline9 Cephaclor10 Carbenicillin


38/71

Fig. General view of Petri dishes withEscherichia coliculture for three fractions

of water after 15 h of the experiment onthe influence of antibiotics on the cultures

.Name of antibiotic1 Clindamycin2 Kanamycin3 Cephalexin

4 Ceftriaxone5 Chloromycetin6 Ciprofroxacin7 Ampicillin8 Tetracycline

9 Cephaclor10 Carbenicillin


39/71

Fig. General viewof Petri disheswith culture

Escherichia colifor three fractionsof water after 24 hof the experiment.

.Name of antibiotic1 Clindamycin2 Kanamycin3 Cephalexin4 Ceftriaxone

5 Chloromycetin6 Ciprofroxacin7 Ampicillin8 Tetracycline9 Cephaclor

10 Carbenicillin


40/71

Fig. General view of Petri dishes withcultureEscherichia colifor three fractionsof water after 36 h of the experiment.

.Name of antibiotic1 Clindamycin2 Kanamycin3 Cephalexin

4 Ceftriaxone5 Chloromycetin6 Ciprofroxacin7 Ampicillin8 Tetracycline9 Cephaclor10 Carbenicillin

Table. Stability indices of the microbiological cultureEcherichia coli to


41/71

Diameter ofGIZ, D0,mmtact=0;

Duration of

experiment

24 h

Diameter of

GIZ, D0, mm

tact=0;

Duration of

experiment

36 h

Diameter of

GIZ, D0, mm

tact=0.5 h;

Duration of

experiment

24 h

Diameter of

GIZ, D0,

mm

tact

=0.5 h;

Duration of

experiment

36 h

Diameter of

GIZ, D0, mm

tact=1.0 h;

Duration of

experiment

24 h

Diameter of

GIZ, D0, mm

tact=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 tovarious antibiotics in activated media (change of growth inhibition zone)

These results will be discussed below: - sensitivity - stability


42/71

Ampicillin:

Increasing sensitivity by 230%

Chloromycetin:Increasing stability

by 19 times!

Kanamycin:

Increasing stability

by 12 times!

tact=60 min tact=60 mintact=30 min

Influence of activated water on the efficiency of action of antibiotics

ANTITUMOR ACTION AND APPLICATION OF MRET


43/71

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 howdifferent fractions of activated water affect on antitumor resistance oforganism the following approaches and techniques were used:

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

3. investigation of functional (cytotoxic) activityof lymphocytes withnatural killer cell cytotoxicity isolated from spleens of normal (without

tumors) mice which received activated water.

Water fractions 1 -5 Prophylaxis action


44/71

5*20=100 mice

Activatedwater:

tact= 15 mintact= 30 mintact= 45 mintact= 60 min

"Old" activatedwater, tact=30 min

5*10=50 miceAnalysis of

tumors, 8thday

20 miceNonactivatedwater

20 miceNonactivated

water

Prophylaxisaction of water

on micewithin 14 days

Analysis of volumeand composition of

tumors in 7 days aftertransplantation

Tumorinoculation220 mice

A

C

Therapeuticaction of wateron mice with

tumors.

Analysis of survivalof all mice with

tumors.

5*20=100 mice

Activatedwater:



5*10=50 miceAnalysis of survival

Activatedwater:


"Old" activatedwater, tact= 30 min

20 mice. Analysis oftumors, 8thda 10 miceAnalysis of survival

Nonactivated water:

Water fraction 11 Control

Water fractions 6 -10 Therapeutic action

p y

Second stageof investigations

F ir st stageof investigations

Prelimin ary stages

5*20=100 mice

Nonactivated

water:20 mice20 mice20 mice20 mice20 mice

5*10=50 miceAnalysis of

tumors, 8thday

B

5*20=100 mice

Activated

water:tact= 15 mintact= 30 mintact= 45 mintact= 60 min


5*10=50 miceAnalysis of survival

Activated

water:tact= 15 mintact= 30 mintact= 45 mintact= 60 min

"Old" activatedwater, tact= 30 min

General scheme ofantitumor effectinvestigation of differentfractions of activated

water (water fractions 15 and 610) on micewith transplantedtumors. Water fraction11 of nonactivated water

was used in controlexperiments.Tumor transplantation Acorresponds to study ofprophylactic treatmentwith activated water,tumor transplantation B

study of therapeutictreatment of activatedwater, Ccontrolinvestigations.


45/71

The first series of studies was performed using such experimentaltumor growth model as asciticEhrlich carcinoma.A spontaneous mammary gland tumor appeared in an underbred female

mouse was maintained further as an experimental strain of solidEhrlich adenocarcinoma. AsciticEhrlich carcinomawas obtained bythe inoculation of tumor cells in the peritoneal cavities of mice.

In the present work, cells of asciticEhrlich carcinomawere obtained

from peritoneal cavities of underbred white mice on the 7th8th day oftumor growth.

In the study, inbred adult male BALB/c mice aged 12 weeks with 2124 g corporal weight were used.

All mice from 11 groups were inoculated intraperitoneally with 200000viable cells/mouse of asciticEhrlich carcinomain accordance with thegeneral scheme presented above.In 7 days after the tumor transplantation, ascitic fluid from the

peritoneal cavities of a half of all animals was obtained.


46/71


47/71

Ascitic fluid samples obtainedfrom peritoneal cavities of mice

with transplanted asciticEhrlichcarcinoma(on the left side, 5samples from mice after the

prophylactic administration(prophylactic treatment group)

of water activated during 30min; on the right side, 5 samplesobtained from control mice(control group)).Every test-tube contains asciticfluid obtained from one mouse.

Measurement of the asciticfluid volume in mice withtransplanted asciticsarcoma 37of the prophylactic treatmentgroup (on the left side)received water activated during

15 min and control group (onthe right side).

, ml l

-1(*10

6i bl ll )Ehr li ch carcinoma


48/71

3.0

2.5

2.0

1.5

1.0

0.0

15 min 30 min 45 min 60 minControl 30 min"Old water"

1

6

2

7

3

84

9

5

10

0.5

250

200

150

100

50

0

15 min 30 min 45 min 60 minControl 30 min

"Old water"

, ml1(*10

6viable 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 minControl 30 min, "Old water"

700

, (*10 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 ofascitic fluid obtained from mice inoculatedintraperitoneally with tumor cells of Ehrlich carcinoma.

Fig. 4. Effect of prophylactic(15) and therapeutic

(610) application of activated water on averagenumber of viable cells in 1 ml of ascitic fluid obtainedfrom mice inoculated intraperitoneally with tumorcells of Ehrlich carcinoma.

Fig. 5. Effect of prophylactic(15) and therapeutic(610) application of activated water on averagenumber of viable cells in an ascitic tumor, obtainedfrom mice inoculated intraperitoneally with tumor

cells of Ehrlich carcinoma.

Ehr li ch carcinoma

Ehr li ch carcinoma


49/71

Influence of different fractions of activated water on the investigated

parameters of tumour of ascitic Ehr li ch carcinoma.

Investigatedparameters

Types of treatment and

fractions of used waterAverage

volume of

ascitic fluid in

one tumour,

, ml

Average number of

viable tumor cells in

1 ml of ascitic fluid,

, ml-1

(*103cells)

Average number of

viable tumor cells in

peritoneal cavity of

one mouse

,(*106cells)

Index of

tumor

growth

inhibition

,D

, %Control, tact= 0 2,85 0,2 235724 44915 672 -

Prophylactic, tact=15 min 1,6 0,1 117354 35134 188 43,6




Prophylactic, tact=30 min

"Old activated water",1,9 0,1 161166 13774 306 33,3

Therapeutic, tact=15 min 2,2 0,1 193231 32144 425 22,8




Therapeutic, tact=30 min

"Old activated water",

2,2 0,1 166541 23454 366 22,8

Ehr li ch carcinoma

Ehr li ch carcinoma (Survival dynamic)


50/71

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

Survivalofanimals,

%

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

Survivalofa

nimals,

%

Fig. Survival dynamic of tumor-bearing mice which received differenttypes of activated water inprophylactic treatmentregime.

Fig. Survival dynamic of tumor-bearing mice which receiveddifferent types of activated water intherapeutic treatment regime

tact = 30 min

tact = 30 min

Control

Control

Ehr li ch carcinoma(Survival dynamic)

Ehr li ch carcinoma (Survival dynamic)


51/71

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 treatmentand therapeutic treatmentregimes.

The digits near the charts correspond to duration of water activation in minutes.

Ehr li ch carcinoma(Survival dynamic)


52/71

, (*106viable cells)

600

500

400

300

200

100

0

1

6

2

7

3

84

9

5

10

Fig. 6.16. Effects of activated water on the average number

of viable cells in one tumor obtained from mice transplantedintraperitoneally withsarcoma 37and treated with activated

water in the prophylactic (15) and therapeutic (610)

modes of application.

15 min 30 min 45 min 60 minControl 30 min

"Old water"

Ascitic sarcoma 37(average number of viable cells in one tumor )

Ascitic sarcoma 37 (Survival dynamic )


53/71

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

Survivalofanim

als,

%

Fig. Survival dynamic of tumor-bearing mice which received different

types of activated water inprophylactic treatmentregime.

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

Survivalofanimals,

% tact = 30 min

tact = 30 min

Fig. Survival dynamic of tumor-bearing mice which receiveddifferent types of activated water intherapeutic treatment regime

Ascitic sarcoma 37(Survival dynamic )

Ascitic sarcoma 37(Survival dynamic )


54/71

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 37which 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 treatmentmode

( y )

R h f th i fl f ti t d t th t t i


55/71

Research of the influence of activated water on the cytotoxic

activity of murine lymphocytes

In order to understand the possible mechanism of antitumor effectsof activated water, the studies of changes in the cytotoxic activity oflymphocytes of mice treated with different fractions of activatedwater were carried.

Natural killer (NK) cells are the important cells of immune systems.Based on their defining function of spontaneous cytotoxicitywithout prior immunization, NK cells have been thought to play acritical role in immune surveillance and cancer therapy. NK cellsthat infiltrate tumors may protect against the tumor spread.

The following procedure and the scheme of investigation were used.

Procedure of the study of the influence of activated water on


56/71

Isolation,cleaning, andseparation oflymphocyte

fraction

enriched withnatural killercells

Healthy mice receive activated water

Isolation of tumor target cells from peritoneal cavities of micetransplanted with asciticEhrlich carcinoma

7 days

Test for thecytotoxic activityof lymphocytes

Procedure of the study of the influence of activated water on

the cytotoxic activity of lymphocytes.

Prophylactic

t t t d C t l d

Short Prophylactic

t t t d

In the first stage of

i i i i f h


57/71

treatment mode5 groups of mice

received different

fractions of activated

water for 21 days

Control mode

1 group of mice

received nonactivated

water for 21 days

treatment mode5 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 s leens of all rou s of mice.

Cytotoxicity assays were performed using 96-well plates.

NK cells were incubated in vitrowith tumor target cells for 16 h.

Tumor target cells were obtained from peritoneal cavities of mice

transplanted with ascitic Ehrlich carcinoma.

investigation, mice of theexperimental and controlgroups received activatedwater for different time

intervals. Mice of theprophylactic treatmentand short prophylactictreatmentgroups receivedactivated water,

respectively, for 21 daysand for 14 days. When thetreatment with activatedwater was finished,mononuclear lymphocyte

fractions enriched with NKcells were isolated fromspleens of mice ofexperimental groups.

In the second stage, the cytotoxic activity of NK cells incubated with tumor target cellsobtained from the peritoneal cavities of mice with transplanted asciticEhrlich

carcinomawas studied.

In the study inbred adult male BALB/c mice at 12 weeks of age, with 2124 gl i h d All i d l di id d i 11 i h 5


58/71

corporal weight were used. All mice were randomly divided into 11 groups with 5animals in each group depending on activated water application regime as follows:

a) control groupmice received non-activated distilled water during 14 days;

b) prophylactic treatment 15,30 45 and 60 mingroups of mice received wateractivated directly before application during 15, 30, 45 and 60 min;c) prophylactic treatment 30 min("old activated water")group of micereceived during 21 days water activated during 30 min before experiment beginningand stored in refrigerator; that water was used up to the end of experiment;

d) therapeutic treatment 15, 30, 45 and 60 mingroups of mice received wateractivated directly before application during 15, 30, 45 and 60 min;e) "therapeutic treatment 30 min" ("old activated water")group of mice receivedduring 14 days water activated during 30 min before experiment beginning and storedin 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 receivedusual (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


59/71

j g y p ycells.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 Ehr li ch carcinomacells were isolated from the peritoneal cavities of whitemice aged 89 weeks on the 8th day after the tumor cell inoculation and were used inthe cytotoxic test as NK-resistant target cells (TC). Tumor cells were suspended in thecultivation medium to a concentration of 2.510^6 cell/ml, and their number andviability were determined in the microscopic supravital test with trypan blue. The cellviability 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% CO2,microplates were gently centrifuged (400 g, 5 min). The numbers of viable and deadTC in control and experimental wells were determined using the microscopic test with

supravital staining with trypan blue. The cytotoxic activity of NK cells was expressedas the cytotoxicity index (CI, %) and was calculated as follows:CI = [(NTC -NTT+NK)/NTC]100%

whereNTCthe number of viable tumor cells in wells with only TC;NTC+NKthe number of viable tumor cells in wells with TC and NK cells.

Based on this definition, CI for basal experiments was equal zero.

Effects of activated water on the cytotoxic activity of splenicmononuclear lymphocytes with NK activity


60/71

Types of treatment andfractions of used water

Average number ofviable tumor cells inmicrowells, *103

Cytotoxicityindex (CI, %)

Change of cytotoxicityindex (in relation tononactivated water), %

Basal tumor cells 235.0 0 -16

Control, tact= 0 197.5 16 0

Prophylactic, tact=15 min 197.5 16 0

Prophylactic, tact=30 min 188.0 20 4.0

Prophylactic, tact=45 min 200.0 15 -1.0Prophylactic, tact=60 min 200.0 15 -1.0

Prophylactic, "Old activatedwater", tact=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, "Oldactivated water",tact=30 min

200.0 15 -1.0

mononuclear lymphocytes with NK-activity.


61/71

Cytotoxic index change (CI)

4.0

3.0

2.0

1.0

0

-1.0

45 60

15 30 30

"Old

water"

"Prophylactic treatment " mode

-2.0

15 45 60

30

30

"Old

water"

Shortprophylactic treatmentmode

Changes of the cytotoxic activity of mononuclear lymphocytes of mice receiveddifferent types of activated water in comparison to the results of the application ofnonactivated water. Numbers at the diagrams corresponds to the water activationduration in minutes.

The data obtained demonstrate that the immunostimulatory potential


62/71

The data obtained demonstrate that the immunostimulatory potentialof activated water is dependent on both the duration of activation andthe duration of storage of activated water before the treatment ofmice.

The application of optimally activated water allows one to potentiatethe 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 comparedwith control values obtained after the application of nonactivatedwater.

It is possible that the prolongation of the treatment period of activated

water will cause a more expressed augmentation of the NK cellcytotoxic activity.

CONCLUSIONS AND RECOMMENDATIONS


63/71

The performed detailed studies have shown that activated water has anumber 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 manyhours and days;

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

5. the duration of preservation of the anomalous properties of activated waterdepends 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


64/71

Co duc ed e pe e s s s ow e esse ue ce o c v edwater (or a medium on the basis of activated water) on metabolism andinternal stability of the following six levels of the organization of theliving matter:

1-st levelmicrobial culturesEscherichia coliand Staphylococcusaureus;2-nd levelmicrobial syntrophic associations including the maximum

number of species and physiological groups of microorganisms, beingin the state of symbiosis and natural synergism;3-rd levelculture of higher plants "in vitro"(callus cells);4-th levelfull-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 theascitic form of inoculatedEhrlich carcinomaseparated from abdominalcavities of mice),6-th level- animals with inoculated cancer cells of different lines

(Ehrlich carcinomaand Sarcoma 37).

Activated water changes some very important biochemical and biophysical processeswhich proceed in alive systems It renders the essential influence on the processes of


65/71

which proceed in alive systems. It renders the essential influence on the processes ofcell division and ionic transport and on the interaction between biologicalmacromolecules, cells, viruses, leukocytes, etc.

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

Activation of the water based nutrient medium with suspendedEscherichia coliandStaphylococcusculturesleads to very high bacteriostatic activity of such nutrientmedium which depends on the time duration of activation and the initial concentrationof culture cells. The bacteriostatic activity increases following the increase of time of

activation and decrease of initial concentration of the suspension of staphylococcalculture! The activation of water suppresses by many tens of times the growth ofcolonies and reproduction of staphylococcal microorganismsin vitro.

The activation of water suppresses very significantly (by tens and hundred of


66/71

times) the development of a callus culture of plant cells (in particular,Solanumrickii) in the presence of a stimulator of uncontrollable nonspecific growth.

MRET-activated water influences essentially the stability of pathogenicmicroflora (e.g. the culture ofEscherichia coli) to the action of different types ofantibiotics. Influence of activated water on the efficiency of action of antibioticscan be very strong.For example, on the use of water activated during 30 min, the stabilityof theculture ofEscherichia colito chloromycetin increases by 19 times! On the useof water activated during 60 min, the stabilityto kanamycin and cephalexinincreases by 1213 times.In a contrast to this, on the use of the same type of activated water, the sensitivityof the culture ofEscherichia colito the action of ampicillin increases by 2.3times.

The determination and use of modes of the activation resulting in the maximalincrease of the sensitivity or stability of specific microbiological cultures to theaction of antibiotics can lead to the overturn in clinical medical microbiology anda significant reduction of the dosage of antibiotics and can sharply weaken thenegative side effects accompanying the standard methods of the use of

antibiotics.


67/71

The application of optimally activated water renders the very significant positiveinfluence on the prophylaxis and treatment of the tumoral process on the basis ofEhrl ich carcinomaand Sarcoma 37in organisms of animals.

Water activated during 30 min in the mode of the prophylactic action (on itsintake before the appearance of the oncological process) inhibits the growth rateofEhrlich carcinomaby 2 times and reduces the number of alive cancer cells inthe volume of a tumor more than by 4 times. Such water also increases the

lifetime of mice infected byEhrlich carcinomaby 61.7 %.The growth inhibition of tumors which is close by efficiency and the increase ofthe lifetime were observed also under the intake of water activated during 15 and45 min.The same water inhibits the development of a tumor on the basis of cancer cells

of Sarcoma 37by 67 % and reduces the number of alive oncological cells in thevolume of a tumor by 3 times. In this case, the lifetime of sick animals grew by50%.

Experiments have shown that water activated during 30 min is optimum on the


68/71

Experiments have shown that water activated during 30 min is optimum on thetherapeutic mode of treatment (on the intake after the beginning of the oncologicalprocess). The efficiency of the therapeutic action of activated water turned out 2times 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 ofactivation of 30 min which is used for the prophylaxis is a very effective means forthe growth inhibition of tumors caused by cells ofEhrlichcarcinoma.

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

In conclusion, application of activated water can induce significant activation ofNK cell cytotoxic potential. In this scenario, it is apparent that application ofactivated water in tumor-bearing immunocompetent hosts would result in cytotoxicactivation of NK cells to destroy tumor cells. Thus, obtained results may beimportant for future therapeutic approaches that implicate activated water.

Li t f ti i t


69/71

List of participants

Prof. V.I.VysotskiiKiev National Shevchenko University

Dr. A.A.Kornilova - Lomonosov Moscow State UniversityDr. 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 concretemechanism of memory of different kinds of activated water and


70/71

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 f oundation 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. AppliedBiophysics of Activated

Water: the physical properties,

biological effects and medical

applications of mret activated

water,World Scientific

Publishing, 2009

Vysotski i V.I ., Smirnov I .V., Kornilova


71/71

A.A.Introduction to the Biophysics ofActivated Water, UniversalPublishiers, Roca Raton, USA, 2005

Vysotski i V.I ., Korni lova A.A., Smirnov

I .V.Applied biophysics of activatedwater World Scientific Publishing

Vladimir I.vysotskii ActivatedWater Bulgaria 2013

Documents

Transcript of Vladimir I.vysotskii ActivatedWater Bulgaria 2013