WATER AND EFFECT OF BIOLOGICALY
ACTIVE SUBSTANCES IN ULTRA LOW
CONCENTRATIONS.
PALMINA N.P., BELOV V.V.,
CHASOVSKAYA T.E.,
ZHERNOVKOV V.E.,
MALTSEVA E.L.
EMANUEL INSTITUTE of
BIOCHEMICAL PHYSICS
RAS, MOSCOW, RUSSIA.
INTRODUCTION
• Observation of biologically active substances (BAS)
effect in ultra - low concentrations (10-22-10-15M) on
the living systems with different complicity is one of
the most impressive discoveries of the last decades.
• The observed effect was investigated using a number
of BAS: antioxidants and anti-metastatic agents,
radioprotectors, inhibitors and stimulators of the
growth of plants, neurotropic compounds of different
classes, hormones, adaptogens, immunomodulators,
detoxicants, peptides, etc. At the moment more than
100 compounds have demonstrated this property.
THE CHARACTERISTIC FEATURES IN THE EFFECT
OF BAC IN ULTRALOW CONCENTRATIONS.
100
I,% (PK C)
80
60
40
20
0
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
Log [-TPh]
Inhibition of Protein Kinase C Activity in
Dependence on α-Tocopherol
Concentration.
Pal’mina N., Mal’tseva E., Kurnakova
N.,Burlakova E. Biochemistry, 1994
0
1. Nonmonotonous, nonlinear polymodal
concentration-effect dependence. In
most cases, the maximal activity is
observed at certain concentration
ranges, separated from one another by
so-called “dead area”, where the effect
is not expressed.
2. Changes in the sensitivity (usually an
increase) of biological objects to various
agents (both endo- and exogeneous).
3. Manifistation of kinetic paradoxes, i.e.
the possibility of detecting the effect of
BAC at ultralow concentrations when
the same compound is present in the cell
or organism at the concentrations that
exeed the ultralow concentration by
several orders of magnitude.
4. “Segregation” of properties of BACs
upon a decrease in its concentration,
when its activity is still retained, but
side effects disappear.
At the first stage, the main task of the researchers was prove the existence of
the effect of ultra-low doses in various biological systems.
• At the moment the level of biological organization on which the effect of
ultra-low doses (ULD) of BAS became evident, also greatly different – from
macromolecules, cells, organs and tissues to animals, growing organisms and
even populations.
Macromolecules
BAS in ultra-low
doses
Membranes
Cells
Tissues
Organism
Ashmarin et all, 1999; Burlakova et all, 2003;2004
From our point of view the study of this phenomena
on relatively simple models is the most of interest
because in these cases we can exclude the
complicated interconnection and influence of
different metabolic ways and reactions on each
other.
We have used as a model for our research isolated
cell membranes and carried out our experiments IN
VITRO .
WHY
?
CELL MEMBRANES AS A TARGET OF
BIOLOGICALY ACTIVE SUBSTANCES IN ULTRA-LOW
DOSES.
THREE THE MOST IMPORTANT CONTROL SYSTEMS ARE LOCALIZED IN THE
CELL MEMBRANES : CYCLIC NUCLEOTIDES, PHOSPHOINOSITOL CYCLE AND
LIPID PEROXIDATION. CROSS-TALKS TAKE PLACE BETWEEN THEM.
LIPID PEROXIDATION,
STRUCTURE OF DIFFERENT LIPID REGIONS OF
CELL MEMBRANES,
ACTIVITY OF MEMBRANE-BOUND ENZYMES
HAVE BEEN STUDIED UNDER THE EFFECT OF
DIFFERENT BAS IN A WIDE RANGE OF
CONCENTRATIONS.
( α-tocopherol; potassium salt of β-4-hydroxy-3,5-di-tretbutylphenyl-propionic acid; thyroliberin; phorbol esters)
SUBSTANCES, CHOSEN FOR THE STUDY OF THEIR
EFFECT IN ULTRA LOW DOSES.
CH3
• Natural (α-tocopherol – α-ТC) и
synthetic (potassium salt of β-(4hydroxy—3,5-dithretbutyl-phenyl)
propionic acid ––Phenozan -PPS)antioxidants;
• Thyroliberin- releasing hormone
(TRH), which interacts through the
receptor with the adenylate cyclase
system of regulation;
• Phorbol esters, modifiers of
phosphoinositide cycle and having
PK C as a receptor.
HO
H3C
H3C
H3C
HO
CH3
O
CH3
H3C
CH3
CH CH COOH(K+)
2
2
EFFECT OF SYNTHETIC ANTIOXIDANT (PHENOZAN)
ON INITIATED LPO IN MICROSOMAL MEMBRANES.
DB
50
100
INHIBITION, %
INHIBITION, %
DC
40
30
20
10
0
-18-17-16-15-14-13-12-11-10-9 -8 -7 -6 -5 -4
Lg C
•
•
•
80
60
40
20
0 -18-17-16-15-14-13-12-11-10 -9 -8 -7 -6 -5 -4 -3
Lg C
PHENOZAN shows antioxidative effect on the model of initiated LPO estimated on the
basis of dienic conjugates (DC) formation and total content of double bonds (DB)
decreasing.
DEPENDENCE CONCENTRATION - EFFECT HAS NONLINEAR POLYMODAL
CHARACTER.
Pal’mina N. et all, 2004
EFFECT OF PHORBOL ESTER AND PROTEIN KINASE C ON LIPID
PEROXIDATION IN PLASMATIC MEMBRANES ISOLATED FROM
MOUSE BRAIN.
I N H I B I T I O N, %
100
90
80
PKC
70
60
50
TPA
40
30
20
10
-19 -18 -17 -16 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6
Lg C
Palmina N.P., Pinzar Е.I., Kurnakova. N. V.,
Burlakova Е.B., Biological Membranes, 1997
Effect of thyroliberin (TRH), a small neuropeptide (pGlu-His-Pro-NH2 in a wide range of concentrations on
lipid microviscosity of plasmatic membranes IN
VITRO.
· TRH in the 10-4-10-18М dosage interval
results in unspecific total change in the
structural properties of the lipids in the
plasmatic membranes isolated from the
liver and brain cells. The TRH
concentration – effect dependence is
nonlinear and polymodal.
30
A
25
20
15
effect, %
10
5
0
-5
-10
-15
B
-20
-25
0
2
4
6
8
10
12
14
16
18
20
-LOG[TRH]
Change in the of rotary correlation time τС (% to
control) of spin probe 16-DSA in plasmatic
membranes of liver (A) and brain (B) cells in
dependence on ТRH concentration.
Probe
-5
concentration of 5х10 М.
Zhernovkov V., Pal’mina N., Bull ExpBiolMed,
2006
· TRH causes increasing the of the lipid
microviscosity in the area of probe C16 (2022Å) localization in the liver plasmatic
membranes and decreasing of τC in the
brain membranes.
· Used in ultra – low concentrations (10-141016M) TRH had the same effect on
plasmatic membranes as after introduction
in “physiological” doses (10-4-10-6 M).
Dose dependences of the effect of PPS on the microviscosity
(tc, red curve) of deep areas and rigidity (S, blue curve) of
surface areas of plasmatic membranes.
12.5
2.5
10
2
7.5
1.5
5
1
2.5
0.5
-Log[PPS]
0
-2.5
0
4 5 6 7 8 9 10111213141516171819202122
-5
-7.5 tc,Effect, %
-0.5
-1
S,Effect,-1.5
%
Chasovskaya T., Mal’tseva E., Palmina N., Biophysics, 2013
DOSE DEPENDENCE FOR ALL STUDED
SUBSTANCES HAVE MULTIMODAL
CHARACTER.
Effect,%
EFFECT OF α-tocopherol ON LIPID
BILAYER STRUCTURE IN
PLASMATIC MEMBRANES.
9
MECHANISMS OF α-tp EFFECTS:
1. The area of physiological
concentrations (10-4M-10-9M) –
restriction of conformational
mobility of lipids as a result of αtp incorporation into the
membrane;
A
Effect, %
7
5
3
1
-1 3
5
7
9
11
13
15
17
19
21
23
-Lg[alpha-tocopherol]
3
B
2
1
0
3
5
7
9
11 13 15 17 19
-1
-Lg[alpha-tocopherol]
21 23 25
25
2. The area of ultra low doses(109M1017M) – specific interaction
with binding sites on the
membrane (e.g. protein kinase-C
(PKC), or inducing by α-tp
formation of microdomains in
the membrane (e.g. rafts,
indirect
evidence
is
the
appearance
of
additional
transition
at
physiological
temperature);
3. The area of “apparent”
concentrations (<10-17M)-
?
STRUCTURE OF WATER AS A TARGET OF ULD
EFFECTS.
Macromolecules
Membranes
BAS in ultra-low
doses
WATER
Cells
Tissues
Organism
 A set of investigations of diluted BAS aques solution properties using IR-spectroscopy
methods was carried out [ Zubareva, Kargapolov,2003; Fesenko, Terpugov, 1999; Yamskov,
Yamskova, 1999; Havinson, 2003]. All of them have observed the instability of the spectrum
parameters of water thin layers during the time, for example, transmittance fluctuations in IR
spectrum of water.
 The physical basis of the process can be connected with formation of a huge regular clusters
(~1 mkm), [Smirnov, Siroeshkin,2003]. Such kind of fluctuations can be one of the characteristic
feature of water dynamics and structure under the certain conditions and different effects, for
example BAS.
IS IMPORTANT THE
SOLVENT POLARITY IN
EFFECT OF ULD?
ROLE of SOLVENT POLARITY in the MECHANISM of ACTION of
BAS in ULTRALOW CONCENTRATIONS.
THERE ARE 3 AREAS OF CONCENTRATIONS ON
THE CURVES:
4
C16
2
3
Effect, %
2
PHYSIOLOGICAL CONCENTRATIONS (10-4-10-9M);
1
1
ULTRA-LOW DOSES (10-9-10-18M);
0
-1
“APPARENT” CONCENTRATIONS < 10-18M
-2
2
4
6
8
10
12
14
16
18
20
22
24
26
-lg [alfa-tocopherol])
CONCLUSIONS:
1). α-TL exhibits its effect using both as in polar as in
nonpolar solusions in the areas of physiological and
ultra low concentrations.
14
12
C5
10
8
Effect, %
6
1
4
2) only aqueous solutions of α-TL were effective at the
area of “apparent” concentrations and shown a
significant effect on the structural parameters of
membrane lipids.
2
0
-2
2
-4
-6
-8
2
4
6
8
10
12
14
16
18
20
22
24
26
-lg [alfa-tocopherol]
The effect of -TP in polar (1) and nonpolar (2) solvents on
the order parameter – S of spin probe C5 and microviscosity
value - c of spin probe C16 localized in the surface lipids (a)
and hydrophobic lipid regions (b) of membranes
correspondingly.
Belov V., Mal’tseva E., Pal’mina N., Burlakova
E., DAN 2004
SOLVENT POLARITY HAS A PRINSIPAL
IMPORTANCE IN MECHANISM OF
TRANSMISSION OF “INFORMATION”
ABOUT COMPOUNDS.
The Study of BAS solutions in the middle IRspectrum range (800-3500sm-1)
1 3500 - 3200 sm-1
2 3085 – 2832 sm-1
3 2120 – 1880 sm-1
4 1710 – 1610 sm-1
5 1600 – 1535 sm-1
6 1543 – 1425 sm-1
7 1430 – 1210 sm-1
8 1127 – 1057 sm-1
9 1067 - 930 sm-1
sm
sm
The spectrum of water in the middle
range of IR – irradiation.
We have used for our research IRspectrometer
IKAR,
new
programmed hardware set designed
in Tver Medical Academy. This set
allows
quick
quantitative
determination of transmittence
fluctuantions in nine ranges of IR
spectrum. The record rate is 1
measurment by 9 channels per
second. The range width was defined
by optical parameters of the
appropriate interference filter. The
measurement was carried out in
cuvettes from KRS-5 20 µm thick.
For IKAR system, the measurement
error of transmittance is below
±0.3%.Experimental conditions gave
us possibility to determine the
transmittence fluctuations which are
connected in accordance with
literature data with the specific
changes in water clusters of studied
solutions.
The influence of α-TL in different concentrations (10-4М –
1025М) on the structure of water estimated by
Mahalonobis criterion.
9
Belov V.,Belyaeva I., Zubareva G.,Pal’mina N., DAN, 2011
8
A
7
channel number
350
250
5
4
α-TL has no absorbtion in these channels
3
2
1
200
0,0
0,5
1,0
1,5
2,0
2,5
relation of dispersions
150
9
8
100
50
0
4
6
8
10
12
14
16
18
20
22
24
26
-Lg[-TL]
The significant changes in the structural parameters of water
have been observed under the effect both as ULD(10-14-10-16M)
as “apparent”(10-18-10-21M) α-TL concentrations which
induced the great deviations in cell membrane structure.
number of channels
Mahalanobis distance
300
6
7
B
6
5
4
3
α-TL has no absorbtion in these
channels
2
1
0,0
0,5
1,0
1,5
2,0
2,5
relation of dispersions
Dispersions of transmission coefficients of
α-TL solutions : 10-15 M (A) and 10-20M (B)
These data confirmed our suggestion concerning the role of in relation to etalon in 9 ranges of
water in ULD effect.
spectrum.
THE STUDY OF ULTRA –LOW TRH CONCENTRATION.
TRH16
9
8
7
chanals
6
5
4
3
TRH HAS NO ABSORBANCE IN THE CHANNEL
2
1
0.0
0.5
1.0
1.5
2.0
2.5
variance sample/variance control
Ratios of dispersions for TRH solution in concentration 10-16M.
chanal3
1,3
Changes in Mahalanobis distance in dependence
on TRH concentrations.
1,2
1,1
1,0
variance
0,9
0,8
0,7
0,6
0,5
0,4
0,3
-2
0
2
4
6
8
10
12
14
16
18
-LOG[TRH]
The changes of dispersion relatios for
channel 3.
20
22
ULD of TRH (10-16M) induced
a big changes in Mahalanobis
distance, ratios of
transmittance coefficient in
channel 3 and its dispersions
Zhernovkov V., Roschina I.,Zubareva G.,Pal’mina
N., Water,2010
The changes of absorbance coefficient in dependence on TRH
concentration in water solution for 9 chosen regions 5550, 5950, 6900,
7130, 7227, 8000, 8500, 9500, 10300 sм-1, where the maximal deviations
have been found. The effect was calculated by the formula:[(absorption
index for TRH solution) / (absorption index for control sample – 1)] x
100.
5550 sm
-1
5950 sm
1,0
-1
6900 sm
0,3
-1
0,5
0,2
0,5
0,1
0,0
effect, %
0,0
0,0
-0,5
-0,5
-0,1
-1,0
-1,0
-1,5
-0,2
-1,5
2
4
6
8
1,0
10
12
14
7130 sm
16
18
20
22
-1
-0,3
2
4
6
8
10
2,0
12
14
7227 sm
16
18
20
22
2
4
6
8
10
2
-1
1,5
12
14
8000 sm
16
18
20
22
16
18
20
22
16
18
20
22
-1
1
0,5
effect, %
0,0
-0,5
1,0
0
0,5
-1
0,0
-2
-0,5
-3
-1,0
-4
-1,5
-5
-1,0
-1,5
-2,0
2
4
6
8
10
12
14
16
18
20
22
-6
2
4
6
8
10
12
14
16
18
20
22
2
4
6
8
10
12
14
X Axis Title
8500 sm
5
-1
9500 sm
8
4
-1
-1
6
3
4
4
2
2
2
1
effect, %
10300 sm
8
6
0
0
0
-2
-1
-2
-2
-4
-4
-3
-6
-6
-4
-8
-8
-10
-10
-5
-6
2
4
6
8
10
12
-LOG[TRH]
14
16
18
20
22
2
4
6
8
10
12
-LOG[TRH]
14
16
18
20
22
2
4
6
8
10
12
-LOG[TRH]
14
Zhernovkov V.,
Lokshin B.,
Pal’mina N., in
press
The changes of absorption coefficient in dependence on
TRH concentration for the frequency 9500 sм-1
For all spectrum regions, the effect
dependence on TRH dose is nonlinear
with the minimum for high doses and
general return to the control level for
low
ones,
except
of
TRH
concentration of 10-15M, which is
statistically different from the effect
of higher doses. For high TRH
concentrations (10-6-10-9M) causing a
strong impact on absorbtion index
and reducing it down to 10%, the
greatest quantitative changes are
observed.
8
6
4
эффект,%
2
0
-2
-4
-6
-8
-10
2
4
6
8
10
12
14
16
18
20
-LOG[ТРГ]
TRH itself had no absorption in the near IRirradiation. We can conclude that observed
effect is due to the structure of water under TRH
action.
By amplitude of changes, 2 bands are
separated: 9 500 and 10 300 sm-1,
where the absorbtion index changed
from minus 10% for high TRH doses
(10-6-10-9M) to 5% for 10-15M.
Effect of PPS on the surface lipid area of the PM and
liposomes (probe C5, the parameter S)
Chasovskaya T., Plaschina I., Pal’mina N., DAN, 2013
5.0
4.5
LIPOSOMES
S,EFFECT,%
4.0
3.5
3.0
PM
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
2
4
6
8
10
12
14
16
18
20
22
-Lg [ PPS]
Lipids are the primary target
of the PPS.
There are the next special features of the
curve for the liposomes compared with the
curve for the PM: the absolute value of the
effect of PPS on liposomes was or equal to the
effect on the PM (in the range of SMD), or
superior to its more than 1.5 times (in the
range of high concentrations), a maximum of
“physiological" concentrations shifted to their
increase, which may be associated with PPS
interaction with other components of the
membrane, which is not in the liposomes. In
the range of ULD maxima of the parameter S
in the PM and the liposomes almost
completely coincide. In our view,
correspondence received by the dose
dependency for the effects of PPS on the PM
and the liposomes, especially the coincidence
of maximum in the range of SMD shows that
PPS acts directly on the lipid component of the
membrane.
200 µm
As one of the mechanisms of the effect of
BAS in the ULD, some authors consider
the transfer of information through the
water system [Zubareva, Fesenko,
Yamskov,
Lobyshev,
Voeikov
].
Previously, we also found that the effect
on biological membranes of certain active
substances (tocopherol, thyrotropinreleasing hormone) in the ULD was
correlated with the properties of aqueous
solutions of biologically active substances
in the infra-red spectral region, using
solutions of tocopherol in vaseline oil
effect of the drug in the ULD would
disappear. In the studies Pollack develop
a view of the presence of hydrophilic
surfaces and particles, which can be
considered as a membrane, and
"surface" layers of water, differing from
the "bulk" water on the viscosity, density,
dielectric constant and conductivity.
Academician AI Konovalov and colleagues found that many
biologically active substances formed in aqueous solutions
nanoassociates of about 200 nm, and the concentration dependence of
the size and nanoassociate conductivity solutions are polymodal and
interconnected.
Experiments with dilute solutions of PPS carried out under the
direction of A.I.Konovalov and I.S. Ryzhkina showed that, indeed, an
antioxidant creates with the water molecules nanoassociates. Size and
their charge, as well as the electrical conductivity of solutions have
nonlinear change depending on the concentration of solutions.
Relationship between the change in the order parameter (S) of lipids in
the surface areas of the lipid bilayer of plasma membranes vs. diameter of
nanoassociates and specific electrical conductivity of α-tocopherol
solutions in the concentrationrange of 1 × 10–3–1 × 10–20 mol/L.
16
360
3.0
340
14
320
2.5
2.5
300
12
2
1.5
260
240
1.0
EFFECT S,%
280
D,nm
EFFECT,S, %
2.0
220
200
0.5
1
180
0.0
2.0
10
1.5
2
1.0
6
0.5
160
-0.5
-26
-22
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
-0.5
3.0
2.0
2.5
S EFFECT,%
2.5
1.5
1.0
0.5
0.0
220
240
2
-26
r = 0.761; p = 0.0228
200
260
280
4
0
Lg[а-ТФ]
3.0
S EFFECT,%
-24
1
0.0
140
8
300
-24
-22
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Lg[a-ТФ]
R= 0.8191; p=0.0002
2.0
1.5
1.0
320
D,nm
Ryzhkina I., Kiseleva Yu, Murtazina L., Pal’mina N, Belov V.,
Mal’tseva L.,Sherman E., Timosheva A., and Konovalov A.,
DAN, 2011
0.5
0.0
4
5
6
7
8
9
10
11
ELECTROCONDUCTIVITY
12
ELECTROCONDUCTIVITY
3.0
Comparison of data on the electrical conductivity of PPS dilute
solutions and their influence on the structure PM and liposomes.
5
σ, μS/ sм
Effect,S,%
40
1.8
35
4
-18
3
25
2
20
EFFECT, S, %
1.6
30
15
1
r = 0,738; p = 0,058
1.4
1.2
1.0
0.8
0.6
0.4
0.2
10
0.0
0
3
-1
-12
PM ( 10 -10 M)
5
7
9
11
13
15
17
19
21
- Lg [PPS]
In the range of SMD found a statistically significant
correlation between changes in the conductivity of
dilute solutions of PPS and their influence on the
surface layers of lipids in the PM and in liposomes.
6
5
0
σ,
μS/
sм
8
10
12
14
16
18
σ, μS/ sм-11
3.0
Liposomes (10
-10
20
22
24
26
-18
М)
2.5
r=0.705; p=0.076
2.0
1.5
1.0
0.5
N. P. Palmina, T. E. Chasovskaya, I. S. Ryzhkina,
L. I. Murtasina, and A. I. Konovalov, DAN, 2009
0.0
6
8
10
12
14
16
18
EFFECT,S,%
20
22
24
26
Influence of External Electromagnetic Field
on the Self-organization of Solutions with
Low Concentrations of the Antioxidant of
Potassium Phenozan and their Effect on the
Microviscosity of the Lipid Membranes in
Vitro
A
500
2
400
D,nm
1
300
200
100
0
-20 -18 -16 -14 -12 -10
-8
-6
-4
-2
NO MAGNETIC FIELD –
Lg[ PhK]
NO NANOASSOCIATES –
[Cexp - C contr/C contr]x 100%
14
B
12
10
NO BIOLOGICAL EFFECTS.
1
8
6
4
2
2
0
-2
-20
-18
-16
-14
-12
-10
-8
-6
-Lg[PhK]
-4
-2
0
Alexander I. Konovalov, Elena L.Mal’tseva , Irina S.
Ryzhkina*,
Lyaisan I.Murtazina, Yuliya V. Kiseleva,
2
Valery V. Kasparov, Nadezhda P. Pal’mina, in press
The concentration dependence of changes in the size (D) nanoassociates formed
in the PP solutions (A) and the membrane lipid microviscosity of the
synaptosomes (B) using dilute the PPS prepared under common conditions
(curves 1) and kept in the container permalloy (curve 2) for 24 hours.
ULD EFFECT.
BAS in ultra-low
doses
?
Macromolecules
Membranes
WATER
Cells
Tissues
Organism
1. We know what happens in cell membranes.
2. We know what happens in diluted solutions.
3. We don’t know the way of nanoaccociates interaction with
cell membranes. We shall know it.
THANK YOU FOR
ATTENTION!