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Complementary
Therapies
in
Medicine
(2014)
22,
333—340
Available
online
at
www.sciencedirect.com
ScienceDirect
jo
ur
nal
home
p
ag
e:
www.elsevierhealth.com/journals/ctim
Droplet
evaporation
method
as
a
new
potential
approach
for
highlighting
the
effectiveness
of
ultra
high
dilutions
Maria
Olga
Kokornaczyka,∗,
Grazia
Trebbia,
Giovanni
Dinellia,
Ilaria
Marottia,
Valeria
Bregolaa,
Daniele
Nanib,
Francesco
Borghinic,
Lucietta
Bettia,∗
aDepartment
of
Agricultural
Sciences,
University
of
Bologna,
Viale
Fanin
42,
40127
Bologna,
Italy
bItalian
National
Health
System,
Lombardy
District,
ASL,
Corso
Italia
19,
20122
Milan,
Italy
cDepartment
of
Medical
Therapy,
Faculty
of
Medicine,
Chieti
University,
Via
dei
Vestini
31,
66013
Chieti,
Italy
Available
online
24
February
2014
KEYWORDS
Droplet
evaporation
method;
Fractal
dimension;
Fluctuating
asymmetry;
Wheat
seedling
growth;
Arsenic
trioxide;
Ultra
high
dilutions;
Polycrystalline
structures
Summary
Objective:
This
study
sought
to
verify
whether
the
droplet
evaporation
method
(DEM)
can
be
applied
to
assess
the
effectiveness
of
ultra-high
dilutions
(UHDs).
We
studied
the
shape
char-
acteristics
of
the
polycrystalline
structures
formed
during
droplet
evaporation
of
wheat
seed
leakages.
Methods:
The
experimental
protocol
tested
both
unstressed
seeds
and
seeds
stressed
with
arsenic
trioxide
5
mM,
treated
with
either
ultra-high
dilutions
of
the
same
stressor
substance,
or
with
water
as
a
control.
The
experimental
groups
were
analyzed
by
DEM
and
in
vitro
growth
tests.
DEM
patterns
were
evaluated
for
their
local
connected
fractal
dimension
(measure
of
complexity)
and
fluctuating
asymmetry
(measure
of
symmetry
exactness).
Results:
Treatment
with
arsenic
at
UHD
of
both
stressed
and
non-stressed
seeds
increased
the
local
connected
fractal
dimension
levels
and
bilateral
symmetry
exactness
values
in
the
polycrystalline
structures,
as
compared
to
the
water
treatment.
The
results
of
in
vitro
growth
tests
revealed
a
stimulating
effect
of
arsenic
at
UHD
vs.
control,
and
a
correlation
between
the
changes
in
growth
rate
and
the
crystallographic
values
of
the
polycrystalline
structures
was
observed.
Conclusions:
The
results
indicate
that
polycrystalline
structures
are
sensitive
to
UHDs,
and
so
for
the
first
time
provide
grounds
for
the
use
of
DEM
as
a
new
tool
for
testing
UHD
effectiveness.
DEM
could
find
application
as
a
treatment
pre-selection
tool,
or
to
monitor
sample
conditions
during
treatment.
Moreover,
when
applied
to
biological
liquids
(such
as
saliva,
blood,
blood
serum,
etc.),
DEM
might
provide
information
about
UHD
effectiveness
on
human
and
animal
health.
©
2014
Elsevier
Ltd.
All
rights
reserved.
∗Corresponding
authors.
Tel.:
+39
051
2096734;
fax:
+39
051
2096770.
E-mail
addresses:
maria.kokornaczyk@unibo.it
(M.O.
Kokornaczyk),
lucietta.betti@unibo.it
(L.
Betti).
http://dx.doi.org/10.1016/j.ctim.2014.02.005
0965-2299/©
2014
Elsevier
Ltd.
All
rights
reserved.
Author's personal copy
334
M.O.
Kokornaczyk
et
al.
Introduction
The
droplet
evaporation
method
(DEM)
is
based
on
the
phenomenon
by
which
particles
suspended
in
fluids
self-
organize
to
form
nano-
and
micro-structures
during
droplet
evaporation,1and
has
been
studied
by
researchers
work-
ing
in
a
variety
of
fields.
The
DEM
has
found
applications
in
DNA/RNA2—4 and
protein
microarray
deposition,5DNA
molecule
stretching,6drug
discovery,7inkjet
printing,8and
the
manufacture
of
novel
electronic
and
optical
materials,8,9
including
thin
films
and
coatings.10 There
have
also
been
studies
investigating
possible
applications
of
DEM
in
medical
diagnostics.11—18 As
we
have
previously
reported
in
detail,19
pattern
formation
during
droplet
evaporation
is
a
complex
process,
dependent
on
the
phase
transitions
and
differ-
ent
flow
dynamics
which
occur
during
evaporation.1,20—22
Those
phenomena
are
in
their
turn
dependent
on
condi-
tions
external
to
the
droplet
(e.g.,
temperature,
relative
humidity,
pressure),7,20,23,24 as
well
as
on
conditions
inter-
nal
to
the
droplet
(e.g.,
liquid
composition,
viscosity).23,25
However,
for
droplets
that
evaporate
under
the
same
exter-
nal
conditions,
any
variations
in
the
phase
transitions
—
and
the
resultant
variations
in
patterns
—
will
depend
exclu-
sively
on
differences
in
the
droplets’
internal
conditions.23
This
sensitivity
to
liquid
composition
suggests
a
wide
range
of
potential
applications
for
DEM,
for
example
as
a
tool
for
qualitative
analysis
of
agricultural
products
and
foods.
Recently,
our
research
team
investigated
using
DEM
for
the
quality
analysis
of
wheat,
and
found
that
different
wheat
cultivars,19 or
different
specimens
of
the
same
cul-
tivar,
some
untreated
and
some
treated
with
a
chemical
stressor,25 show
significant
differences
in
their
evaporation
patterns.
We
also
found
that
those
differences
correlated
with
the
vigor
of
the
analyzed
seeds.
In
that
study,
the
quality
indicators
which
we
used
to
objectively
evaluate
the
patterns
were:
(i)
the
pattern
complexity,
measured
as
the
local
connected
fractal
dimension
(LCFD)
characterizing
local
variations
in
image
complexity,
and
(ii)
the
bilateral
symmetry
exactness
of
the
polycrystalline
structures
(PCS)
expressed
as
fluctuating
asymmetry
(FA).
Both
those
param-
eters
have
been
previously
shown19,25 to
correlate
with
seed
vigor
expressed
as
germination
rate.
Our
purpose
in
the
present
work
was
to
evaluate
whether
DEM
is
a
suitable
tool
for
studying
the
effectiveness
of
ultra
high
dilutions
(UHDs),
a
form
of
treatment
commonly
used
in
homeopathy
and,
in
recent
years,
also
in
agriculture.26 UHDs
are
prepared
through
a
process
of
repeated
dilution
and
rhythmic
shaking
(succussion),
starting
from
a
mother
tinc-
ture.
Dilutions
are
usually
performed
on
a
decimal
(1:10)
or
centesimal
(1:100)
scale,
designated
with
the
letters
x
and
c,
respectively.
The
biological
effectiveness
of
UHDs
remains
controversial:
since
their
dilution
levels
are
beyond
the
Avo-
gadro
limit,
the
probability
that
such
‘‘solutions’’
contain
molecules
of
the
original
substance
is
close
to
zero.27 There-
fore,
according
to
the
conventional
scientific
paradigm
of
the
‘‘molecule/receptor’’
model,
any
biological
activity
is
highly
unlikely.
To
tackle
this
problem,
experimental
studies
of
high
methodological
quality
have
been
carried
out
in
dif-
ferent
fields
of
basic
research
on
UHDs,
providing
empirical
evidence
of
specific
UHD
activity
in
vitro28—30 and
in
vivo31,32
and
suggesting
a
possible
mechanism
of
action.33—36 These
contributions
hypothesize,
based
on
physicochemical
mea-
surements,
that
the
technique
used
to
prepare
UHDs
(i.e.,
repeated
dilution
and
succussion
steps)
may
cause
structural
alterations
in
the
aqueous
solvent
which
in
their
turn
trigger
the
formation
of
molecular
aggregates
of
water
molecules.37
In
previous
works38—41 our
group
has
demonstrated
stimu-
lating
effects
of
arsenic
trioxide
(As2O3)
UHDs
on
wheat
seeds
previously
stressed
(poisoned)
with
As2O35
mM.
In
these
types
of
models,
referred
to
as
‘‘isopathic’’,
a
biolog-
ical
subject
is
first
poisoned
with
some
agent
in
molecular
concentration,
and
then
an
attempt
is
made
to
counteract
the
toxicity
by
applying
a
UHD
of
the
same
agent.42,43 We
adopted
this
approach
because
our
previous
research
has
always
detected
an
‘‘isopathic
sensitization’’,
meaning
a
notable
increase
in
UHD
effectiveness
when
working
with
seeds
previously
stressed
with
the
same
substance.
Recently,
a
crystallographic
approach
(biocrystallization
method)
has
been
successfully
integrated
into
a
seedling
test
system
for
UHDs,
yielding
results
which
show
crys-
tallographic
structures
to
be
treatment-specific.44 The
biocrystallization
method,
developed
around
a
hundred
years
ago45 and
mostly
used
for
quality
analysis
of
foods
and
agricultural
products,46,47 involves
the
formation
of
evaporation-induced
patterns
from
a
watery
extract
pre-
pared
by
mixing
the
analyzed
sample
with
dihydrate
copper
chloride.
The
aim
of
the
present
study
was
to
investigate
whether
DEM
might
serve
as
a
test
to
evaluate
the
effectiveness
of
As2O3UHDs
applied
to
both
not-stressed
and
stressed
wheat
seeds
(ns-
and
s-seeds,
respectively);
the
stress
consisted
in
pre-treating
the
seeds
with
As2O35
mM.
In
particular,
we
focused
on
the
following
research
questions:
(i)
whether
DEM
patterns
produced
out
of
ns-
and
s-seeds
show
signif-
icant
differences;
(ii)
whether
the
UHD
treatment
applied
to
both
ns-
and
s-seeds
may
induce
changes
in
DEM
pat-
terns
and,
if
so,
(iii)
whether
these
changes
reflect
the
seed
viability
revealed
by
the
in
vitro
growth
tests.
For
what
con-
cerns
the
pattern
shapes,
a
further
aim
of
this
study
was
to
investigate
whether
the
same
parameters,
LCFD
and
bilat-
eral
symmetry
exactness,
already
applied
in
our
previous
works,19,25 are
sensitive
to
seed
viability
changes
due
to
UHD
treatments.
Finally,
we
would
like
to
underline
that
this
study
was
carried
out
taking
into
account
the
present
guidelines
for
basic
research
investigations
with
homeopathic
remedies.48
Materials
and
methods
Plant
material,
classes
of
treatments
and
experimental
protocol
Seeds
of
the
common
wheat
(Triticum
aestivum
L.)
culti-
var
‘‘Pandas’’,
from
organic
farming,
were
used
after
being
selected
for
integrity
and
uniformity
of
size,
shape
and
color.
A
portion
of
the
seeds
was
stressed
(s-seeds)
by
immersion
for
30
min
in
an
As2O35
mM
water
solution
(As2O3,
Sigma-
Aldrich,
Milan,
Italy;
H2O
p.a.
Merck,
Darmstadt,
Germany),
and
then
rinsed
in
tap
water
for
60
min,
dried
in
ambient
air
until
the
seeds
reached
12%
moisture
content,
and
stored
in
the
dark
at
room
temperature
until
use.
This
sub-lethal
poisoning
protocol
was
selected
on
the
basis
of
previous
Author's personal copy
Droplet
evaporation
method
335
Figure
1
General
experimental
design.
experiments.40,49 Non-stressed
seeds
(ns-seeds),
used
as
a
control,
were
pre-treated
in
the
same
way,
but
using
dis-
tilled
water
instead
of
As2O35
mM.
The
treatment
classes
for
testing
were:
-
Distilled
water
(control,
0)
-
As2O3at
the
45th
decimal
dilution/succussion
level
(As
45×,
T).
Both
test
substances
were
freshly
prepared
from
the
same
batch
of
water
and
supplied
by
Laboratoires
Boiron,
Sainte-Foy-lès-Lyon,
France.
The
As
45×
solution
was
prepared
following
the
Hahnemann
multi-vial-method
in
accordance
with
the
European
Pharmacopoeia,
by
a
process
of
repeated
serial
dilutions
at
decimal
scale
(starting
from
a
mother
tincture
of
As2O30.01
M)
followed
by
mechanical
shaking
(dynamization)
at
each
dilution
step.
This
procedure
was
continued
until
the
45×
dilution/dynamization
level
was
reached.
To
reduce
microbial
growth,
Pyrex
glass
bottles
were
stored
at
a
cool
temperature
(4 ◦C)
until
use.
All
treat-
ments
were
letter-coded
(blinded)
by
a
person
not
involved
in
the
experiments,
and
the
codes
were
kept
by
independent
people
until
the
time
of
disclosure.
As
shown
in
Fig.
1,
the
following
four
experimental
groups
were
formed:
(1)
ns-seeds
treated
with
distilled
water
(ns
+
0)
(2)
ns-seeds
treated
with
As
45×
(ns
+
T)
(3)
s-seeds
treated
with
distilled
water
(s
+
0)
(4)
s-seeds
treated
with
As
45×
(s
+
T)
These
four
experimental
groups
were
analyzed
by
both
DEM
and
in
vitro
growth
tests.
Droplet
evaporation
method
and
pattern
evaluation
The
experimental
procedure,
drawn
from
Kokornaczyk
et
al.,19 is
briefly
described
here.
From
each
set
of
seed
sam-
ples
(s-seeds
and
ns-seeds)
we
prepared
two
sub-samples,
each
consisting
of
five
entire
kernels
selected
for
integrity
and
uniformity
of
size,
shape
and
color.
The
kernels
were
weighed,
cleaned
by
rinsing
them
in
distilled
water,
and
placed
in
four
test
tubes.
Tw o
of
the
four
sub-samples
(one
from
s-seeds
and
one
from
ns-seeds)
were
watered
with
distilled
water
and
the
other
two
with
As
45×,
in
w/v
pro-
portion
1:20.
The
test
tubes
were
slightly
shaken
by
hand
and
left
at
room
temperature.
After
1
h,
leakage
drops
were
collected
using
a
micropipette,
placed
on
a
clean
micro-
scope
slide,
and
allowed
to
dry
in
a
thermostat
at
25 ◦C
and
under
UV
light
PHILIPS
TL-D
18W
BLB
1SL,
Monza,
Italy.
The
residues
were
then
photographed
under
a
dark
field
microscope
MT4300H,
MEIJI
Techno,
Saitama,
Japan,
with
a
connected
CMOS
Camera
UK1175-C,
EHD
imaging
GmbH,
Damme,
Germany,
in
QXGA
(2048
×
1536)
resolution,
and
magnifications
25×
and
100×.
The
experiment
was
repeated
four
times
to
obtain
a
total
of
240
droplet
residues
(2
sam-
ples,
2
treatments,
3
replicates,
5
droplet
residues
per
replicate,
4
experimentation
days).
The
PCS
were
evaluated
for
their
LCFD
and
FA
using
the
software
Image
J
for
microscopy
1.43
m,50 as
described
in
detail
in
Kokornaczyk
et
al.19,25 The
evaluation
was
per-
formed
on
pattern
images
in
100×
magnification.
The
LCFD
measurement
was
done
on
images
converted
to
binary
using
the
installed
fractal
analysis
plug-in
FRAC-LAC
2.5.51 The
analysis
was
designed
to
avoid
any
experimenter
bias.
FA,
a
parameter
inversely
correlated
to
bilateral
symmetry
(BS),
was
measured
only
on
images
containing
structures
that
exhibited
BS
(BS
structures;
an
example
is
depicted
in
Fig.
2).
BS
structures
were
preselected
on
the
basis
of
clearly
defined
end-points
of
the
first-order
ramifications.
Subse-
quently,
in
up
to
three
randomly
chosen
BS
structures
per
Author's personal copy
336
M.O.
Kokornaczyk
et
al.
Figure
2
Example
of
a
BS
structure
from
an
evaporated
droplet
of
wheat
seed
leakage
and
the
FA
measurements:
let-
ters
indicate
the
measured
lengths
of
both
symmetric
branch
pairs
(L1and
L
1,
L2and
L
2)
(Kokornaczyk
et
al.25).
image,
the
first-order
ramification
lengths
were
measured
with
the
segmented
line
tool:
L1,
length
of
the
upper
left
branch;
L2,
length
of
the
lower
left
branch;
L
1,
length
of
the
upper
right
branch;
and
L
2,
length
of
the
lower
right
branch
(Fig.
2).
The
FA
value
was
calculated
for
each
structure,
as
follows:
FA
=|L1−
L
1|
L1+
L
1
+|L2−
L
2|
|L2+
L
2|
.
In
vitro
growth
test
Following
a
procedure
described
in
previous
papers,38,41
each
of
the
seeds
(280
s-
and
280
ns-seeds)
was
attached
to
the
top
of
a
piece
of
filter
paper
(Perfecte
2-extrarapid,
Cordenons,
Pordenone,
Italy)
with
clay
(0.20
±
0.05
g).
The
filter
paper
was
inserted
into
a
transparent
polyethylene
envelope
(12
cm
×
20
cm)
which
was
in
its
turn
placed
in
a
larger
black
cardboard
envelope,
in
such
a
way
as
to
allow
the
shoots
to
develop
in
natural
light
and
the
roots
in
the
dark.
Each
seed
was
treated
by
wetting
the
filter
paper
with
3.2
ml
of
distilled
water
or
As2O345×.
The
cardboard
envelopes
(80
envelopes,
20
for
each
experimental
group)
were
fixed,
following
a
completely
randomized
design,
to
a
wooden
support
(85
cm
×
100
cm),
positioned
one
beside
the
other,
hanging
vertically.
The
experiment
was
repeated
7
times
and
carried
out
in
a
glasshouse
at
a
temperature
of
20
±
1◦C,
in
diffuse
natural
light,
following
a
natural
day—night
rhythm.
A
total
of
140
seedlings
for
each
treat-
ment
was
analyzed.
The
main
variable
considered
was
the
shoot
length
on
day
7
of
growth,
determined
by
scanning
the
whole
plants
(Epson
Perfection
2480
Photo)
and
measuring
the
length
of
coleoptiles
using
the
Assess
2.0
software.52
Statistical
analysis
All
data
were
processed
by
two-way
analysis
of
variance;
the
separation
of
means
was
performed
using
Fisher’s
least
significant
difference
test
at
a
significance
level
of
p
≤
0.05.
Correlations
between
average
shoot
growth
data
and
LCFD
or
FA
were
evaluated
by
r
Pearson
coefficient.
To
the
sta-
tistical
analysis
the
CoStat
software
(version
6.002,
Cohort
Software,
Monterey,
CA,
USA)
was
used.
Results
All
DEM
patterns
consisted
of
a
border
line
(BL),
a
rather
structure-free
peripheral
zone
(PZ),
and
polycrystalline
structures
(PCS)
placed
in
the
middle
zone
(MZ)
of
the
residue
(Fig.
3).
The
centrally
placed
PCS
were
the
only
elements
that
varied,
and
so
our
analysis
focused
only
on
these
structures.
As
can
be
seen
in
Fig.
4,
the
patterns
obtained
from
ns-seeds
treated
with
water
(ns
+
0)
consist
of
ramified
PCS
surrounded
by
single
spots.
These
PCS
are
all
centered,
and
their
complexity
ranges
from
simple
rami-
fied
structures
to
well-structured
and
complex
ramification
networks
(Fig.
4a).
For
ns-seeds
treated
with
As
45×
(ns
+
T)
we
see
that
the
complexity
and
regularity
of
the
PCS
struc-
tures
is
increased
compared
to
those
for
ns
+
0
(Fig.
4b).
The
s-seeds
treated
with
water
(s
+
0)
created
only
non-centered
patterns
with
no
PCS
and
a
large
number
of
separate
spots;
the
patterns
were
partially
dissolved
and
contained
amor-
phous
agglomerates
in
their
MZ
and
PZ
(Fig.
4c).
For
s-seeds
treated
with
As
45×
(s
+
T)
we
instead
notice
a
partial
recov-
ery
of
the
structured
patterns,
characterized
by
an
increase
in
the
amount,
size
and
complexity
of
PCS
with
respect
to
s
+
0
(Fig.
4d).
For
the
pattern
evaluation
(Table
1)
we
measured
the
LCFD
on
60
images
for
each
experimental
group,
whereas
for
the
FA
analysis
we
only
considered
those
patterns
that
contained
BS
structures
(these
amounted
to
201,
or
84%,
out
of
the
total
of
240
patterns).
More
specifically,
the
per-
centages
of
patterns
analyzed
for
FA
for
each
experimental
group
were:
96.7%
for
ns
+
0;
95.0%
for
ns
+
T;
70.0%
for
s
+
0;
and
73.3%
for
s
+
T.
As
regards
the
effects
induced
by
As
45×
treatment,
we
found
that
both
ns-
and
s-seeds
(ns
+
T
and
s
+
T,
respectively)
showed
a
significant
LCFD
increase
—
denoting
a
significant
increase
in
pattern
complexity
(Fig.
4b
and
d)
—
compared
to
the
same
seeds
treated
with
water
(ns
+
0
and
s
+
0,
respectively).
The
As
45×
treatment
also
produced
a
decreasing
trend
in
the
FA
values
of
the
PCS,
cor-
responding
to
a
recovery
of
more
exact
bilateral
symmetry,
to
a
significant
extent
for
s-seeds
(s
+
T).
The
effects
of
stress
with
As2O35
mM,
evaluated
on
the
s
+
0
group,
amounted
to
a
significant
decrease
in
LCFD
(reduced
PCS
complex-
ity)
and
increase
in
FA
(reduced
bilateral
symmetry)
with
respect
to
the
ns
+
0
group,
as
also
shown
in
Fig.
4c.
The
two-
way
analysis
of
variance
with
independent
factors
treatment
and
experiment
number
(experimentation
day)
performed
on
LCFD
and
FA
data
showed
no
significance
neither
for
the
day
factor
nor
for
the
interaction
between
treatment
and
experimentation
day.
For
what
concerns
the
results
of
the
in
vitro
growth
test
(Table
1),
the
shoot
length
of
the
s-seeds
treated
with
water
(s
+
0)
was
significantly
less
than
for
the
ns
+
0
group,
evincing
the
effect
of
the
stress
on
seedling
growth.
Following
the
As
45×
treatment,
both
ns-
and
s-seeds
(ns
+
T
and
s
+
T,
respec-
tively)
showed
a
significant
shoot
length
increase
compared
to
ns
+
0
and
s
+
0,
respectively.
The
two-way
analysis
of
vari-
ance
showed
no
significance
for
the
day
factor,
whereas
the
interaction
between
treatment
and
experimentation
day
Author's personal copy
Droplet
evaporation
method
337
Figure
3
Examples
of
patterns
from
the
droplet
evaporation
method:
(a)
a
pattern
with
a
non-centered
polycrystalline
structure,
and
(b)
a
pattern
with
a
centered
polycrystalline
structure.
BL:
border
line,
PZ:
peripheral
zone,
MZ:
middle
zone,
SP:
single
spots,
and
PCS:
polycrystalline
structure.
resulted
significant
(p
<
0.01)
indicating
that
the
experimen-
tation
day
influenced
the
seedling
growth.
The
results
of
the
growth
test
correlated
against
the
crystallographic
values
of
DEM
patterns:
we
found
a
positive
correlation
(r
=
0.91)
with
LCFD,
and
a
negative
correlation
(r
=
−0.89)
with
FA.
Discussion
First
of
all
the
conformation
of
DEM
patterns
obtained
in
this
experiment
coincided
with
the
pattern-type
we
reported
in
our
previous
studies19,25 and
can
be
considered
typical
for
wheat
seed
leakages.
As
far
as
the
PCSs
are
concerned,
partially
dissolved
patterns
without
connected
structures
(Fig.
4c)
were
obtained
from
s-seed
leakages,
reflecting
the
destructive
effect
of
the
arsenic
treatment
on
seedling
vigor.
Conversely,
the
growth-stimulating
effect
of
arsenic
at
UHD
positively
correlated
with
an
increased
complexity
and
bilateral
symmetry
exactness
of
both
ns-
and
s-seeds
(Fig.
4b
and
d,
respectively).
In
our
previous
studies19,25 we
showed
that
complexity
and
bilateral
symmetry
exactness
in
DEM
patterns
prepared
from
wheat
seed
leakages
reflected
the
seeds
viability
differences
due
to
cultivar
characteristics
and
chemical
stress
influence.
The
present
study
is
consis-
tent
with
our
previous
results
and
highlights
that
the
same
pattern
characteristics
(LCFD
and
FA)
might
be
sensitive
also
to
UHD
treatment.
The
UHD
effect
was
higher
on
s-seeds
than
on
ns-seeds,
confirming
the
‘‘isopathic
sensitization’’,
i.e.,
the
notable
increase
in
treatment
effectiveness
when
applied
to
previously
stressed
seeds.39,49
It
is
worth
pointing
out
that
in
the
present
experimen-
tation
undiluted/unsuccussed
water
was
used
as
a
control
instead
of
dynamized
water,
as
recommended
in
the
REHBaR
guidelines.48 As
demonstrated
by
Witt
et
al.,53 during
the
succussions
applied
to
the
liquid
in
the
dynamization
pro-
cess
some
trace-elements
(inter
alia
Na,
Si,
Mg,
Al,
Cu,
Rh,
and
Li)
pass
from
the
vessel
walls
into
the
liquid
changing
its
composition
in
confront
to
the
undynamized
liquid.
In
accor-
dance
to
this
study
the
concentration
of
trace-elements
is
expected
to
be
higher
in
dynamized
As
45×
than
in
undy-
namized
water
and
the
differences
in
DEM
patterns
could
be
ascribed
to
unspecific
physicochemical
alterations.54,55
This
aspect
should
be
deeply
investigated
in
follow-up
tri-
als.
Nevertheless,
in
a
pilot
study56 we
examined
by
means
of
DEM
the
effects
of
As
45×
and
dynamized
water
45×,
prepared
with
a
different
number
of
strokes
between
the
dilution
steps,
with
respect
to
undiluted/unsuccussed
water.
The
data
showed
that
LCFD
of
DEM
patterns
take
higher
values
for
the
As
45×
in
comparison
to
water
45×
for
all
investigated
stroke
numbers.
This
result
suggests
that
Table
1
Results
of
the
pattern
evaluation
and
shoot
length
measurements
for
the
four
experimental
groups.
Experimental
thesis
LCFD
FA
Shoot
length
N
Mean
CI
N
Mean
CI
N
Mean
CI
ns
+
0
60
1.7
(b)
1.64—1.76
58
0.20
(c)
0.18—0.22
140
25.10
(b)
22.67—27.53
ns
+
T
60
1.8
(a)
1.70—1.90
57
0.15
(c)
0.11—0.19
140
31.40
(a)
27.83—34.97
s
+
0
60
1.2
(d)
1.10—1.30
42
0.48
(a)
0.44—0.52
140
19.70
(d)
17.74—21.66
s
+
T
60
1.5
(c)
1.42—1.58
44
0.30
(b)
0.28—0.32
140
22.40
(c)
20.24—24.56
Different
letters
indicate
significant
differences
at
p
<
0.05;
LCFD
local
connected
fractal
dimension,
FA
fluctuating
asymmetry,
N
number
of
samples,
CI
confidence
interval
95%.
Author's personal copy
338
M.O.
Kokornaczyk
et
al.
Figure
4
Examples
of
patterns
from
evaporated
droplets.
(a)
ns-seeds
treated
with
water,
(b)
ns-seeds
treated
with
As
45×,
(c)
s-seeds
treated
with
water,
and
(d)
s-seeds
treated
with
As
45×
(magnification
100×).
the
differences
might
be
correlated
to
the
action
of
the
dynamized
substance
and
not
only
to
differences
in
ion
con-
tent
due
to
succussion.
Furthermore,
the
here
presented
method
should
be
tested
for
its
reproducibility,
one
of
the
main
problems
in
research
with
UHD
preparations,
by
repeated
investigations
with
independently
prepared
homeopathic
remedy
produc-
tion
lots,
different
wheat
cultivars,
several
seed
harvest
lots
as
well
as
different
dilution/succussion
levels.
In
order
to
assess
the
stability
of
the
method,
also
laboratory
external
reproduction
trials
should
be
performed.
Conclusions
Based
on
the
ability
of
crystallization
methods
to
reflect
sample
quality
by
tracking
differences
in
evaporation
patterns,
we
have
here
for
the
first
time
used
DEM
as
a
complementary
and
rapid
tool
for
evaluating
UHD
effective-
ness.
Our
findings
show
that
this
method
can
be
considered
a
promising
approach
to
study
the
viability
changes
in
wheat
seeds
due
to
chemical
stress
and
UHD
treatments,
and
we
hypothesize
that
it
might
also
be
useful
for
testing
the
effects
of
UHDs
on
other
crops.
In
fact,
since
DEM
is
Author's personal copy
Droplet
evaporation
method
339
a
simple
and
time-saving
method,
it
could
be
used
as
a
treatment
pre-selection
tool.
Furthermore,
because
of
its
noninvasiveness,
DEM
could
also
be
used
to
monitor
sample
conditions
even
before
completing
the
treatment.
Finally,
DEM
might
be
applied
to
biological
liquids
(such
as
saliva,
blood,
blood
serum,
etc.),
to
also
provide
information
about
the
effectiveness
of
UHDs
on
human
and
animal
health.
Conflict
of
interest
statement
All
the
authors
declare
no
financial/commercial
conflicts
of
interest.
Acknowledgements
The
authors
would
like
to
thank
Demeter
Italy
for
funding
this
research,
and
Dr.
Antonello
Russo
and
Dr.
Edda
Sanesi
for
their
precious
support
and
encouragement.
Moreover,
the
authors
wish
to
thank
Laboratoires
Boiron,
Italy,
and
in
particular
Dr.
Silvia
Nencioni
and
Dr.
Luigi
Marrari,
for
their
technical
assistance.
The
sponsors
had
no
influence
whatso-
ever
upon
the
design,
conduct,
evaluation
and
manuscript
of
this
investigation.
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