Simple
plant models in order to obtain
a large data samples testing homeopathic treatments
“Lilli
Kolisko” research group
Simple
plant models in order to obtain a large data samples testing homeopathic
treatments.
Experimental
studies testing homeopathic treatments on biological systems are generally very
difficult due to the complexity of the system itself and its interaction with
the environment. Moreover, as there is no complete theory to explain the action
of homeopathic potencies on living matter, it is very hard to deal with the
contradictory results sometimes obtained. For this reason in our studies,
started in 1990, we sought simple plant models in order to obtain a large data
samples for structured statistical analysis and to relate directly any effect to
the corresponding treatment. Our research group is multidisciplinary, including
biologists, medical doctors, a statistician and a laboratory technician, in
order to take account of different aspects of the research.
As
a general feature of the in vitro
systems, our plant models let overcome main disavantages of clinical trials such
as placebo effect, ethical difficulties, consuming of time, low number of
replications, and high costs. As peculiar and unique advantages, plant-based
bioassays can rely on a very cheap and nearly inexhaustible source of biological
material, which can be easily stored and quickly cultured on simple growth
media, maintaining a high degree of differentiation in
vitro.
The
plant models we have studied are:
·
in
vitro
seed germination and growth (wheat and Arabidopsis thaliana)
·
in
vitro
pollen germination (apple and kiwifruit)
·
tobacco/tobacco
mosaic virus (TMV) interaction
In
all our plant models we studied the effects of various decimal potencies of Arsenicum
album (As2O3), which was choosen because of its well-known toxic effect on
plant growth until 10-6 dilutions. In some experiments, part of the seeds or of
the pollen were previously stressed with a material sublethal dose of As2O3. The
purpose of this preliminary poisoning was to establish whether or not there is
an amplification of the homeopathic treatment effect.
Experiments
on germination and on seedling length were performed. In the first series of
experiments, we tested more than 50,000 seeds of Triticum
durum L., which
were germinated on sterile sand in Petri dishes in order to record germination
values after 4 days. In the second series of experiments, seeds (900 in total)
were germinated into plastic/paper envelops in order to periodically evaluate
root and stem length after 4 to 7 days of growth.
A
certain number of decimal potencies (between 23x and 45x) of As2O3
were used to treat stressed and non-stressed seeds by blind protocol. Four
classes of treatments were performed:
-
Merck H2O (control)
-
potentised H2O
-
highly diluted As2O3
-
highly diluted and potentised As2O3
By
means of different statistical analyses, significant effects of homeopathic
treatment on either wheat germination or shoot/root length were repeatedly
evidenced. In particular, some As2O3 potencies (40x, 42x,
45x) markedly increased germination in both stressed and non-stressed seeds, the
effects being highly significant against control group. By contrast, other As2O3
potencies (27x, 35x) repeatedly induced significant inhibitory effects. Arsenic
30x showed an inhibitory effect in one experiment and a stimulatory effect in
another one. Interestingly, the potentisation of water (30x, 42x, 45x)
induced highly significant positive effects on germination, especially in
the case of stressed seeds, whereas highly diluted As2O3 did
not show any effect at all. When we compared H2O 45x and As2O3
45x, both highly significant vs. control, the difference was generally
highly significant in favour of the arsenic. Experiments on wheat plantlets
confirmed the stimulating effect of As2O3
45x. Stem length, measured after 4 to 7 day from germination
of stressed seeds, resulted in fact significantly enhanced by the homeopathic
treatment against control group in both the experiments performed. Moreover, by
analyzing the average growth on day 7 in relation to different treatments with
potentised water, diluted arsenic,
and potentised arsenic vs. control, an oscillatory trend was observed.
Recently,
we performed experiments to evaluate the influence of temperature and ageing on
As2O3 45x and H2O
45x effectiveness in stimulating germination of stressed wheat seeds. We
have collected 48 sets of data (standard trials), corresponding to 1584 seeds,
for each treatment or control group. We have then compared each treatment with
the control group corresponding to the same temperature (20°C, 40°C, 70°C and
100°C, respectively). We detected a highly significant stimulating effect (p-value
< 0.1%) of As2O3 45x, at 20°C, 40°C and 70°C, and a
slightly significant stimulating effect (p-value
< 0.1%) at 100°C, which seems to be the “critical point”. When
considering potentised water (H2O 45x), we detected a significant
stimulating effect only at 20°C, but less significant than As2O3
at the same temperature. We also observed that the effects are more
evident in the last experiments when excluding the data taken from the first 12
or 24 experiments out of 48, and all the comparisons considered, with As2O3
45x and with H2O 45x, become significant. These data show that
temperature has a greater influence on potentised water than on potentised
arsenic, and that ageing significantly increases the effectiveness of homepathic
treatments.
The plant model system of Arabidopsis
thaliana seedlings is the most used in plant physiology and plant molecular
biology, due to the unique features of this species such as small genome, small
size, short life cycle and easy handling. We propose this plant as a further
model for research in homeopathy field.
The
first part of the present work consisted in the optimisation of the experimental
protocol in order to obtain reliable data. In fact, experiments were carried out
to define the minimum culture medium (that is containing as few chemical
compounds as possible which could interfere with the various treatments) able to
allow a satisfactory percentage of seed germination. Since the seeds had to be
pre-treated (stressed) with ponderal amounts of the same principle (Arsenicum
album), we showed that As2O3 induced a 31% inhibition
of germination but did not affect seedling development. Under these experimental
conditions, the model system was shown to be statistically reproducible, and
thus the various treatments were performed.
After
the preliminary stress-treatment with As2O3, seeds (10 per
Petri dish) were soaked in different media containing Phytagar and a homeopathic
dilution or Merck water (stressed controls, Cs). A non-stressed control (C) was
also prepared in which the preliminary treatment simply consisted in water. The
treatments were:
Merck
H2O, stressed and non-stressed controls
potentised
H2O (23x, 30x, 45x)
highly
diluted As2O3 (10-23, 10-30, 10-45)
potentised
As2O3 (23x, 30x, 45x)
The
following experimental parameters were evaluated:
a)
percentage of germinated seeds per Petri dish at 4, 5, 6, 7, 8 days;
b)
size of 7 day-old cotyledons.
All
the experiments were performed “blind” on a total of 2070 seeds. As far as
the percentage of seed germination is concerned, statistical analysis indicated
a significant inhibitory effect by As2O3 30x compared with
stressed-controls (Cs). This effect was present throughout the germination
period but exhibited a significantly decreasing trend from day 5 to day 8.
Regarding cotyledon size, the inhibitory effect of As2O3 30x
was confirmed; this treatment in fact caused a significant reduction in the
development of cotyledons compared with Cs. The same effect was observed with
water 30x but not with As2O3 10-30. This
confirms previous reports indicating a greater influence of the dynamisation
compared with the dilution process.
The
As2O3 45x treatment was also highly significant, compared
with Cs, in inhibiting the growth of cotyledons; on the contrary, in the wheat
model system the same dilution always exerted a stimulating effect on seed
germination and growth.
The
pollen model presents some peculiar features which make it well suited to test
homeopathic preparations: easy to obtain, storage at –20°C for several years,
test prerformed in a short lapse of time, very high number (several thousands)
of individuals tested in each trial, wide range of data for statistical analysis.
We are currently using pollen from apple and kiwifruit, performing a liquid mass
culture into Petri dishes and recording percent germination (with a
semi-automatized procedure) after 3 hour incubation. First results showed a
significant stimulatory effect of some As2O3 potencies on
germination of pollen preliminarily stressed with a material dose of As2O3.
An
important difference between this model and others described up to now is that
the stress imposed is a biotic one, that is inoculation with the tobacco mosaic
virus (TMV). Tobacco plants with the N resistance gene exhibit a hypersensitive
response (HR) to TMV that is considered one of the important mechanisms leading
to resistance: instead of spreading systemically, the virus is limited to the
vicinity of the inoculated cells, many of which undergo a controlled process of
cell death (PCD, programmed cell death), resulting in the development of
macroscopically visible necrotic lesions: a lower number of hypersensitive
lesions involves a faster and more effective defence response.
Nicotiana
tabacum
cv. Samsun NN plants were used. After TMV inoculation disks were cut from leaves
and left floating in Petri dishes containing the different treatments. The
working variable was the number of local hypersensitive lesions per leaf disk,
counted three days after virus inoculation. As2O3
treatments 5x and 45x induced a significant decrease in the number of
hypersensitive lesions vs. controls, whereas 5C and 45C did not show significant
effects. These findings seem to agree with the classical point of view of
homeopathy, both hahnemannianan and anthroposophical, according to which the
centesimal potencies are more related to the psychic world than the decimal ones,
so we suppose centesimal potencies could fit better with animal models.
Moreover,
since our results showed repeatable effects of As2O3 potencies
on hypersensitive reaction, we carried out poisoning tests on healthy tobacco
plants with ponderal As2O3 (by analogy with “remedy
proving”). The detected symptoms are quite similar, at a macroscopic level, to
hypersensitive lesions due to TMV inoculation, whereas poisoning with other
inorganic compounds induce very different responses. These preliminary studies,
which will be further investigated by microscopic analyses, seem to confirm the
law of similarity.
·
Plant
model systems used in our laboratories resulted to be sensitive to homoeopathic
treatments.
·
The
effect of treatments depended on the model, and the dose-response curve showed
an oscillatory trend.
·
We
succeded in obtaining:
-
low
internal variability of our systems
-
good
reproducibility of the results
-
a
large data base necessary to perform a powerful statistical analysis
-
quickness
in performing the trials (4-8 days for seeds; 3 h for pollen; 3 days for leaf
disks)
Another
aspect which seems particularly interesting came out considering for wheat and
tobacco/TMV models the variability parameter: Certain potencies of As2O3
showed a surprisingly constant effect on wheat germination and tobacco
hypersensitive response in comparison with control or water potencies. This
result can open new perspectives in the interpretation of homeopathic phenomena.
Under
the effect of As2O3 potencies, in particular As2O3
D45, both wheat and tobacco/TMV models showed a decreasing of variability of the
system. This surprising result seems to be due to a “dilution” of the effect
on the “whole” system rather than an effect on the single elements under
analysis. If this result will be confirmed, it could be the basis for a
“theory of living systems”, more specific and adequate than a theory based
on quantum-mechanic models.
1)
Betti L., M. Brizzi, D.
Nani, M. Peruzzi, 1994. A
pilot statistical study with homoeopathic potencies of Arsenicum album in wheat
germination as a simple model. Br. Hom. J. , 83, 195-201.
2)
Betti L., M. Brizzi, D.
Nani, M. Peruzzi, 1997. Effect
of high dilutions of Arsenicum album on wheat seedlings from seed poisoned with
the same substance. Br. Hom. J. , 86, 86-89.
3)
Nani
D., L Betti, M. Brizzi, M. Peruzzi, 1997. High
dilution effect in an Arsenicum album/ wheat model: experimental results and
hints for basic research. In:
Der
Merkurstab, Beitrage zu einer Erweiterung der Heilkunst. Tagungsband
Wissenschaftlicher Kongreb,
Goetheanum, Dornach, 13-15 Juni 1997, pp 46.
4)
Brizzi M., L. Betti, D.
Nani, M. Peruzzi, 1997. An
overall analysis of a series of experiments based on high dilutions in an Arsenicum
album/wheat model. In:
Omeomed’97, Urbino (Italy), 25-28 Sett. 1997, Abstract Book, pp. 12-13.
5)
Brizzi M., L. Betti, D.
Nani, M. Peruzzi, 1998. A
statistical overview of some recent studies regarding the effects of Arsenicum
album high dilutions on wheat germination.
In: Unconventional medicine at the beginning of the third millennium, Pavia
(Italy), 4-6 Giugno 1998, Abstract Book, pp.136.
6)
Betti
L, Brizzi M, Calzoni G L, Speranza A, Scaramagli S, Torrigiani P, Vanti M,
Viglino D, Cuffiani M, Nani D, Peruzzi M, 1999. Il problema dell’efficacia
dell’omeopatia: modelli vegetali complementari agli studi clinici. Cahiers
de Bioterapie,
2: 17-26.
7)
Brizzi M., L. Betti, 1999. Using
statistics for evaluating the effectiveness of homeopathy: analysis of a large
collection of data from simple plant models. Atti
III Congresso Nazionale della Società Italiana di Biometria, Roma 7-9
luglio 1999, pp. 74-76.
8)
Brizzi M., D. Nani, M.
Peruzzi, Betti L., 2000. Statistical
analysis of the effect of high dilutions of arsenic in a large dataset from a
wheat germination model. Br. Hom. J. , 89: 63-67.
9)
Betti L., R. Chattat, A. Brusa, F. Borghini, 2000. L’omeopatia in
farmacia. Risposte teorico-sperimentali ad un campione statistico di
informazione professionale. Atti Omeopatia italiana a convegno, Milano
18-20 Febbraio 2000, pp. 12-14.
10)
Borghini F., L. Betti et al., 2000.
L’omeopatia in farmacia. Presupposti metodologici, teorici e
sperimentali alla pratica professionale. Atti Omeopatia italiana a convegno,
Milano 18-20 Febbraio 2000, pp. 19-30.
11)
Betti L, Biondi S, Brizzi M, Calzoni G L, Lazzarato L, Nani D, Peruzzi M,
Scaramagli S, Speranza A, Torrigiani P, Trebbi G, 2000. Modelli sperimentali di
studio degli effetti biologici delle diluizioni omeopatiche. Atti del convegno:
Il medicinale omeopatico-Tra tradizione e realtà farmacoterapica. Milano,
mercoledì 27 settembre 2000, 24-48.
12)
Betti L., S. Biondi, M. Brizzi, G.L. Calzoni, L. Lazzarato, D. Nani, M.
Peruzzi, S. Scaramagli, A. Speranza, P. Torrigiani, G. Trebbi, 2000. Modelli
vegetali per studiare le basi scientifiche dell’omeopatia. Atti Medicina
alternativa e medicina scientifica, Padova 28 Ottobre 2000, in press
13)
Betti L., S. Biondi, M.
Brizzi, G.L. Calzoni, L. Lazzarato, D. Nani, M. Peruzzi, S. Scaramagli, A.
Speranza, P. Torrigiani, G. Trebbi, 2000. Plant
model systems to study the biological effects of homeopathic dilutions.
Proceedings General Meeting European Commitee Homeopathy, Bruxelles 10-12
November 2000, pp. 10-21.
14)
Betti L., S. Biondi, M. Brizzi, G.L. Calzoni, L. Lazzarato, D. Nani, M.
Peruzzi, S. Scaramagli, A. Speranza, P. Torrigiani, G. Trebbi, 2000. Ricerca di
base in omeopatia: studio degli effetti biologici di soluzioni estremamente
diluite e dinamizzate su modelli vegetali. Atti Medicine non convenzionali.
Evidenze clinico-sperimentali, aspetti legislativi e diffusione in Italia,
Bologna 2 Dicembre 2000, in press
15)
Betti L., S. Biondi, M.
Brizzi, G. L. Calzoni, A. Canova, L. Lazzarato, D. Nani, M. Peruzzi, S.
Scaramagli, P. Torrigiani, G. Trebbi, 2001. Plant
model systems to study the biological effects of ultra high dilutions.
International scientific conference on Complementary, alternative and
integrative medicine research, San Francisco 17-19 May 2001, pubbl. in Alternative
Therapies in Health and Medicine, 7(3): S5
16)
Betti L., M. Brizzi,
G. L. Calzoni, L. Lazzarato, G. Trebbi, D. Nani, M. Peruzzi, 2001. Effetti di
preparati omeopatici su modelli vegetali. Atti
convegno: Agricoltura biodinamica: perché. Sabaudia 29 Novembre-1
Dicembre 2001, in press
17)
Betti L., Brizzi M.,
Calzoni G.L., Lazzarato L., Nani D., Peruzzi M., 2001. Testing
“Similarity Law Like” And Quantitative Effects Of Ultra High Dilutions On
Plant Model Systems. General Meeting European Commitee Homeopathy, Parigi
10 December 2001
18)
Brizzi M., C. Biondi,
L. Lazzarato, L. Betti, 2002. Analisi esplorativa dell’effetto di soluzioni
ultramolecolari di triossido di arsenico sullo sviluppo vegetativo in vitro
di plantule di grano. Rivista statistica, in press.
19)
Betti L., Nani D.,
Brizzi M., Lazzarato L., Peruzzi M., Trebbi G., Calzoni G.L., 2002.
An
analysis of the “similarity law like” and quantitative effects of ultra high
dilutions on plant model systems. International
scientific conference on Complementary, alternative and integrative medicine
research, Boston 12-14 April 2002
20)
Betti L., Calzoni
G.L., Brizzi M., Lazzarato L.,
Trebbi G., Nani D., 2002. Effetti biologici di diluizioni-dinamizzazioni di As2O3
su diversi modelli vegetali. Atti convegno: Ricerca e sperimentazione in
agricoltura biologica. Cesena, 9 maggio 2002, pp. 11-13.
Research
Group
Betti
Lucietta, Lazzarato Lisa, Trebbi Grazia
Dipartimento
di Scienze e Tecnologie Agroambientali, Università di Bologna
Brizzi
Maurizio
Dipartimento
di Scienze Statistiche “Paolo Fortunati”, Università di Bologna
Calzoni
Gian Lorenzo, Scaramagli Sonia, Biondi Stefania, Torrigiani Patrizia
Dipartimento
di Biologia Evoluzionistica Sperimentale, Università di Bologna
Nani
Daniele (Medical
Doctor), Peruzzi Maurizio (Laboratory technician)
Gruppo
Medico Antroposofico Italiano, Milano
Borghini
Francesco (Medical
Doctor)
Tutor For. Com., Consorzio interuniversitario, Roma
per
maggiori informazioni
scrivere a:
info@omeopatia-kolisko.com