Effects of Ignatia amara in mouse behavioural models

Department of Pathology and Diagnostics, University of Verona (I), Italy.
Homeopathy: the journal of the Faculty of Homeopathy (Impact Factor: 0.76). 01/2012; 101(1):57-67. DOI: 10.1016/j.homp.2011.10.001
Source: PubMed
ABSTRACT
Ignatia amara (Ignatia), a remedy made from the Strychnos ignatii seeds, is used for anxiety-related symptoms, but consistent evidence of its activity in reproducible experimental models is lacking. An investigation was performed in order to assess on mice, by means of emotional response models, the activity of homeopathic Ignatia dilutions/dynamizations.
Groups of 8 mice of the CD1 albino strain were treated intraperitoneally for 9 days with 0.3ml of five centesimal (C) dilutions/dynamizations of Ignatia (4C, 5C, 7C, 9C and 30C). Control mice were treated with the same hydroalcoholic (0.3%) solution used to dilute the medicines. Diazepam (1mg/kg) was the positive reference drug. Validated test models for locomotion and emotional response, the Open-Field (OF) and the Light-Dark (LD) tests, were employed. Five replications of the same protocol were carried out, in a randomised way using coded drugs/controls.
In the OF the general locomotion of mice was slightly decreased by Ignatia 4C, but not by Ignatia 5C, 7C, 9C and 30C, indicating the absence of unspecific motor impairment or sedation by these dilutions/dynamizations. Ignatia and diazepam seemed to decrease the number of urine spots released in the OF during 10min, with borderline significance (P=0.083). In the LD the tested medicine showed anxiolytic-like activity (increase of time spent and distance travelled in the lit area), though to a lesser extent than diazepam. The highest and most significant difference with untreated controls (P<0.01) was observed with the 9C dilution/dynamization. Among the 5 replication experiments, the best drug effects were obtained where the baseline anxiety of mice was higher.
Homeopathic Ignatia dilutions/dynamizations (peak at 9C) modify some emotion-related symptoms in laboratory mice without affecting locomotion.

Full-text

Available from: Paolo Bellavite
ORIGINAL PAPER
Effects of Ignatia amara in mouse behavioural
models
Marta Marzotto
1
, Anita Conforti
2
, Paolo Magnani
1
, Maria Elisabetta Zanolin
2
and Paolo Bellavite
1,
*
1
Department of Pathology and Diagnostics, University of Verona (I), Italy
2
Department of Public Health and Community Medicine, University of Verona (I), Italy
Background: Ignatia amara (Ignatia), a remedy made from the Strychnos ignatii seeds,
is used for anxie ty-related symptoms, but consistent evidence of its activity in reproduc-
ible experiment al models is lacking. An investigation was performed in order to assess
on mice, by means of em otional response models, the activity of homeopathic Ignatia
dilutions/dynamizations.
Methods: Groups of 8 mice of the CD1 albino strain were treated intraperitoneally for 9
days with 0.3 ml of five centesimal (C) dilutions/dynamizations of Ignatia (4C, 5C, 7C, 9C
and 30C). Cont rol mice were treated with the same hydroalcoholic (0.3%) solution used
to dilute the medicines. Diazepam (1 mg/kg) was the positive reference drug. Validated
test models for locomotion and emotional response, the Open-Field (OF) and the Lighte
Dark (LD) tests, were employed. Five replications of the same protocol were carried out,
in a randomised way using coded drugs/controls.
Results: In the OF the genera l locomotion of mice was slightly decreased by Ignatia 4C,
but not by Ignatia 5C, 7C, 9C and 30C, indicating the absence of unspecific motor impair-
ment or sedation by these dilutions/dynamizations. Ignatia and diazepam seemed to
decrease the number of urine spots released in the OF during 10 min, with borderline
significance (P = 0.083). In the LD the tested medicine showed anxiolytic-like activity
(increase of time spent and distance travelled in the lit area), though to a lesser extent
than diazepam. The highest and most significant difference with untreated controls
(P < 0.01) was observed with the 9C dilution/dynamization. Among the 5 replication
experiments, the best drug effects were obtained where the baseline anxiety of mice
was higher.
Conclusions: Homeopathic Ignatia dilutions/dynamizations (peak at 9C) modify some
emotion-related symptoms in laboratory mice without affecting locomotion.
Homeopathy (2012) 101,57e67.
Keywords: Strychnos Ignatii; Ignatia; Strychnine; Animal models of anxiety; Open-
Field test; LighteDark test; Homeopathic dilutions/dynamizations; Nanopharmacology
Introductio n
Anxiety and depression are among the symptoms most
frequently reported by patients seeking complementary
or alternative medical treatments, such as homeopathy
and natural remedies.
1e4
However there is a need of
pharmacological studies elucidating their indications,
limitations and mechanisms of action.
5
With conventional
drugs, dosages and adverse reactions are generally studied
in animal models prior to undertaking human trials. In ho-
meopathy, the opposite has been true: trials on humans
have only recently been followed up with tests on animals.
The past few years have seen an increa se in the number
of pre-clinical (in vitro and animal) studies aimed at eval-
uating the pharmacological activity or efficacy of some
*Correspondence: Paolo Bellavite, Dipartimento di Patologia e
Diagnostica, Universit
a di Verona, Strada Le Grazie 8, 37134
Verona (I), Italy.
E-mail: paolo.bellavite@univr.it
Received 6 May 2011; revised 26 September 2011; accepted 17
October 2011
Homeopathy (2012) 101, 57e67
Ó 2011 The Faculty of Homeopathy
doi:10.1016/j.homp.2011.10.001, available online at http://www.sciencedirect.com
Page 1
homeopathic medicines under potentiall y reproducible
conditions.
6e13
In the field of psychopharmacology,
rodent models are the most frequently used for exploring
various aspects of anxiety making use of defensive
behaviours. Although it cannot be proven that animals
experience anxiety in the same way as human beings, it
is generally undisputed that certain behaviours of rodents
in experimental conditions correspond to forms of central
and peripheral anxiety: Hormonal and neuromediator
variations are common to hum ans and animals and, most
importantly, drug respons es in animals are in many cases
predictive of the response of the average population in
human clinical studies, or can in any case suggest novel
pharmacological approaches.
9
We
14
and others
15,16
have
previously found a statistically significant anxiolytic-like
effect of a homeopat hic medicine, Gelsemium sempervi-
rens, using experimental models in rodents. In the present
work, we employ the same protocol
14
to test another ho-
meopathic remedy that is widely used for anxiety syn-
dromes: Ignatia (also named Ignatia amara), obtained
from the extract of Strychnos ignatii beans.
Strychnos ignatii (Figure 1) is a plant belonging to the
Loganiaceae family, native to South East Asia, with long
branches and pear shaped fruit that contain hard, 2.5 cm
long seeds that are odourless but bitter and very poisonous
due to a high strychnine content. Although it is best known
as a poison, small doses of strychnine were once used in
medicine as a stimulant, as a laxative, and as a treatment
for other stomach ailments.
17
The Jesuits valued the seeds
as a remedy against cholera and named them Ignatius beans
after the Jesuit founder St Ignatius Loyola. Strychnine’s
stimulant effects also led to its use historically for enhanc-
ing performance in sports.
18
The use of strychnine in med-
icine was, however, eventually abandoned due to its high
toxicity and tendency to cause convulsions. Symptoms of
mild strychnine poisoning are drooling, nausea, vomiting,
delusions, involuntary muscle spasms and twitching, and
vertigo.
19
The rationa le for testing Ignatia is both clinically and ex-
perimentally grounded. Ignatia is one of the homeopathic
remedies most commonly used on patients with anxiety
symptoms, depression, manic episodes, emotive urination
and diarrhoea, as well as hyperaesthesia and hypersensitiv-
ity to emotions.
20e22
It is also one of the first remedies to
have been studied in laboratory animals.
19,23,24
However
these works were published in non-indexed journals, and
consistent evidence for efficacy with validated models is
lacking.
9
Recent in vitro studies on the rat spinal cord
and limbic system have shown that synthesis of the
stress-related neurosteroid allopregnanolone is stimulated
by Gelsemium and blocked by strychnine (the latter in
non-homeopathic doses).
16,25
Since strychnine is the
major alkaloid of Ignatia, it was of interest to determine
whether homeopathic dilutions/dynamizations of this
plant have some regulating effects on behavioural models
in laboratory animals.
Ignatia has been previously investigated by some
authors in rodent models, but the resul ts reported are not
always consistent, chiefly due to uncertainty connected
with the methodology and a lack of statistical evaluations.
9
In 1978 Binsard tested the effect of Ignatia 3C, 7C and 30C
in the ‘hole-board and ‘escape’ tests, finding a possible
anxiolytic effect for the 3C dilution/dynamizati on.
23
The
same author reports a subsequent experiment in which
Ignatia 3C, 4C, 5C and Gelsemium 3C, 4C, 5C were simul-
taneously tested in the four-plate test with electric shock,
which creates a state of anxiety, due to the conflict between
the propensity to explore and the fear of the electric
shock.
26
Ignatia 3C and Gelsemium 5C were reported to
have an anxiolytic action (less than that of diaze pam, but
with the same direction of effect), whereas Ignatia 5C
was reported to have a sedative action, in that it diminished
the movements of the animal. Although the experiments
were done in blind, their evidence were treated as prelim-
inary due to the lack of a statistical evaluation of the differ-
ences observed.
In the present work, the mice were treated with a number
of increasing dilutions/dynamizations of Ignatia, represen-
tative of those commonly used in humans for treatment
of chronic ailments including anxiety symptoms: the
dilutions/dynamizations ranged from 4C, which still con-
tains a substantial quantity of molecules of the original ac-
tive substance, to 30C, which is beyond the Avogadro-
Loschmidt limit. Given the extremely low doses used, to
increase the probability of inducing an effect, the animals
were treated using a chronic regimen of one i.p. injection
every day for 9 days. As a positive reference drug to assess
the validity of the test we used diazepam (1 mg/kg, the
most frequently used dosage in mice, given 30 min before
the test), and as negative control we used a treatment con-
sisting of only the solvent. After 1 week of treatment, the
behaviour of the animals was assayed using ‘ethological’
test conditions which elicit a state of anxiety and fear
(for example: being kept isolated rather than with other
mice, being placed in a brightly lit area rather than in
semi-darkness, or finding themselves in a novel environ-
ment different from the usual housing cages), and evaluate
Figure 1 Strychnos ignatii plant (1), flower (2) and fruit (3). Insert
shows the structure of the principal alkaloid strychnine (4) (molec-
ular formula C
21
H
22
N
2
O
2
, molar mass 334.41).
Ignatia and emotional responses in mice
M Marzotto et al
58
Homeopathy
Page 2
the movements and behavioural responses of the animals in
a standardised manner, through assignment of point scores.
In order to rule out any possible bias due to experimenter
interventions or cage effects, a procedure was introduced
using randomised and blind conditions. Two validated an-
imal models, the Open-Field test (OF) and the Light-Dark
choice test (LD), were used to acquire various behavioural
parameters widely employed in neuropsychopharmacol-
ogy for drug screening.
27,28
Methods
Animals
All the experiments were performed at the Faculty of
Medicine, Verona University, Italy, as previously de-
scribed,
14
with minor modifications. Male mice 4e5 weeks
old of the CD1 strain were purchased from Cha rles River
Laboratories (Lecco, Italy), and allowed to acclimate for
2 weeks before testing, in a controlled animal facility (tem-
perature 22 2
C, humidity 55% 5%). The mice were
randomly distributed, two per cage, in plastic cages (size:
25 14 12 cm) and housed with food and water avail-
able ad libitum, except for during the brief testing periods.
Lights were on between 7 a.m. an d 7 p.m.
A minimum required sample size of n = 34 subjects for
each treatment group was determined on the basis of previ-
ous experiences with the LD test on CD1 mice,
14
and
assuming a pre-established statistical power of 0.8 and an
alpha level of 0.05. In order to compare treatment and con-
trol groups of sufficient sample size (i.e. a total of 40 ani-
mals per treatment group), the study was conducted with
5 separate replications of the same protocol: in each repli-
cation experiment, mice were randomised into 8 groups of
8 animals; 5 groups were treated with different Ignatia
dilutions/dynamizations, one group with diazepam, and
two groups with the vehicle. Two groups of control
vehicle-treated animals were employed for each replica-
tion to obtain highly consistent values, which were used
as the reference for calculating percentage effects in the
treated groups. Therefore , the total sample size was 40
mice for each drug-treated group and 2 40 = 80 mice
for the vehicle-treated, control group. The mice belonging
to each experimental group were rand omly distributed
among different cages, and the order in which cages were
placed in the rack and the mice injected and tested was bal-
anced between all cages and all experimental groups . Each
animal was used only once in the same test to avoid the
confounding effects of learning and habituation.
Drugs and treatments
The medicines were produced by Boiron Laboratoires,
Lyon (F), according to the European pharmacopoeia
(Monograph 01/2008:0672Ph.Eu), starting from a Mother
Tincture (MT) consisting of a hydroalcoholic extract
(60% ethanol/distilled water v/v) of dried and powdered
Strychnos ignatii seeds. The content of strychnine e the
principal alkaloid of Ignatia (Figure 1) e in the MT was
0.166% (w/v), corresponding to a concentr ation of
4.9 10
3
moles/l. The MT was diluted 100 times in
30% ethanol/dis tilled water to obtain the 1C dilution/dy-
namization. Subsequent serial 100 dilutions/dynamiza-
tions up to 29C were then made up in the same solvent,
using glass bottles. After each dilution/dynamization, the
bottle was vigorously agitated using a mechanical shaker.
The control solution (vehicle) consisted of the same batch
of 30% ethanol/distilled water solution used to prepare the
Ignatia dilutions/dynamizations. All solutions were stored
in the dark at room temperature.
Before being used in each experiment, aliquot s of the
medicines and of control solutions were furt her diluted
by 1:100 in sterile, apyrogenic distilled water and shaken
with 20 strokes by hand. In this way, the ethanol concentra-
tion of the solutions administered to the mice was reduced
to 0.3%. In order to blind the operators with respect to the
tested solutions, all the samples were then coded by an in-
dependent person and the codes recorded on a sheet that
was kept sealed inside an envelope until all the tests and
calculations were completed. The solutions were distrib-
uted in 15-ml sterile Falcon plastic tubes (3.5 ml/tube),
wrapped in aluminium foil and stored at +4
C until the
day of use. Prior to administration each tube was again
manually shaken with 20 strokes. The drug and control
solutions were admini stered in the morning for 9 consecu-
tive days (including on the last two days, when the tests
were carried out) by intraperitoneal (i.p.) injection
(0.3 ml) using 1-ml (insulin) syringes. The allopathic
drug group (diazepam) received 0.3 ml of control solvent
solution (0.3% ethanol/distilled water) for the first 7 days
and 0.3 ml of solution containing diazepam (Valium,
Roche, final dose 1 mg/kg bw) in the days of experiments
only to avoid the well known tolerance to benzodiaze-
pines.
29
Diazepam was administered 30 min before the
test, due to its short half-li fe.
30
The treatments were admin-
istered row by row, and all the behavioural tests were
started 30 min after the injections.
The treatment and testing procedures were indepen-
dently approved by the Animal Ethical Committee of the
Interdepartmental Centre for Animal Research (CIRSAL)
of Verona University, and by the Italian Health Ministry.
Aside from the treatment injections and testing, the ani-
mals were not subjected to pain or other forms of emotional
or physical stress.
Behavioural tests
All testing procedures were performed between 9 a.m.
and 3 p.m. The animals were tested individually in 4 sepa-
rate devices, allowing a complete set of up to 64 mice to be
tested during a 5e6 h experimental session. The experi-
ments were performed in the following order: OF explora-
tion test on 8th day of solution administration, LD choice
test on the following day (9th day of solution administra-
tion). Immediately before testing, the animals were al-
lowed to acclimate to the room inside their cages for
3 min, after being moved there from their customary hous-
ing area. The operators stayed outside the testing room dur-
ing recording of the experimental sessions. The test arenas
were cleaned thoroughly with tap water between trials.
Ignatia and emotional responses in mice
M Marzotto et al
59
Homeopathy
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The OF behaviour test involves placing an animal in an
unknown environment consisting of a 50 50 cm black-
painted wood platform with 25-cm high surrounding walls,
illuminated with white light (100 lux).
31e33
The total
distance travelled in the arena reflects general exploratory
activity that could be changed by locomotor ability, and is
reduced in case of sedation, paralysis, or impairment of
movements, and conversely increased in case of excitation.
The arena is divided virtually into two parts, with a square
central zone having an area corresponding to 25% of the
total area. The percentage time spent in the central zone is
considered indicative of exploratory behaviour, and could
reflect a decrease in anxiety, although this OF parameter is
not sensitive to all anxiolytics and may not model features
of anxiety disorders.
33
Measurement of various parameters
are electronically taken in the same test at two time points,
namely 5 and 10 min; at the end of the trial the number of
urine spots > 1 cm diameter and the number of defecations
are recorded.
The LD exploration test is based on the innate aversion
of rodents to brightly lit areas, and their spontaneous
exploratory behaviour in response to mild stressors such
as novel environments and light.
34e36
Mice tend to prefer
dark, enclosed spaces to large, well-lit areas, and the
amount of time they spend in the dark zone is sensitive
to benzodiazepines and to the agonists of serotonergic
receptors, in a manner that correlates well with clinical
efficacy in humans.
37
The test apparatus consists of a small,
secure dark compartment (15 30 cm) and a large, aver-
sive illuminated compartment (30 30 cm). The two com-
partments are separated by a partition with an opening
(4 4 cm) through which the anima l can pass from one
compartment to the other. The open arena is brightly illu-
minated with 200 lux, and the mice are left to explore the
space. Measurement of various parameters is taken after
5 and 10 min. An increase in the amount of time spent in
the lit compartment is an indicator of decreased anxiety,
and the number of light-dark transitions has been reported
to be an indicator of activity and of exploration over time.
Classic anxiolytics (benzod iazepines) as well as the newer
anxiolytic-like compounds (e.g. serotonergic drugs) and
natural compounds can be tested using this paradigm.
35,36
All the sessions were recorded with a video-tracking
camera (GZ-MG135, JVC, Japan) and stored on DVD.
A software program (‘Smart’ VTS system from PanLab,
Barcelona, E) was used to automatically trace the position
and movements of the animals and calculate the time spent
in different zones and the distance travelled. All the above
described behavioural measurements, including the transi-
tions between compart ments in the LD, and the number of
urine spots and stools in the OF, were considered as pri-
mary outcomes and done with the observer unaware of
the treatment group assignment of the mice.
Statistics
Analyses were performed using the SPSS software, ver-
sion 17 (SPSS Inc., Chicago, IL, USA: http://www.spss.
com). The effect of the drugs on each mouse was calculated
as a percentage relative to the mean values of the controls
(vehicle-treated) in each replication experiment, taken as
zero effect, according to the formula:
½ðTest value of each mouse =
Mean test value of control miceÞ1100
This standardizat ion allowed the effects observed in all
the experiments to be pooled and compared. All data are
represented as mean SEM values. The ShapiroeWilk
test showed that the data collected were normally distrib-
uted. The effects of the various dilutions/dynamizations
tested in the different experiments, expressed in standar-
dised form as a percen tage of the internal control values
for untreated animals in the sa me experiment, were
analysed by two-way analysis of variance, using the treat-
ment group and the experiment as factors, thus correcting
for the possible confounding effect of the latter. Post-hoc
t-tests were performed assuming equal variances with
least significant difference (LSD) corrections to adjust
for multiple c omparisons, as suggested by a consensus
report for basic research in high-dilution/dynamization
pharmacology.
38
Results
Open-Field test
Several different behavioural parameters were evaluated
in the OF test (Figures 2 and 3). Figure 2, panels AeD,
reports the effects of diazepam and Ignatia dilutions/
dynamizations on the total distance travelled by mice in
the open field arena and on their walking speed. These
measures were computed from software movement traces
after 5 and 10 min of testing. These indexes do not reflect
changes in anxiety level, but are important for evaluating
the locomotor activity and speed of the animals during
the trial.
Ignatia did not change these values as compared with the
untreated control animals, except for the 4C dilution/dy-
namization which produced a small but statistically signif-
icant decrease in speed during the first 5 min (panel C)
(P < 0.05). Diazepam caused a significant increase in
both total distance travelled and walking speed during
the entire test time (P < 0.01), suggesting a possible stimu-
lation of movement or unspecific excitation effect of this
drug at the dosage used. There were significant differences
between the replications of the experiment performed, but
no interaction between groups and experiments.
In the OF test, additional behavioural variables were
evaluated (Figure 3), consisting of the percentage time
spent in the central zone of the field (panels AeB) and
the number of urine spots and defecations detected in the
field at the end of the 10-min trial (panels CeD). The
experiments did not reveal any clear and reproducible
dose-dependence relation with the time spent in the central
zone, and the standard drug diazepam likewise had no
effect on this parameter, suggesting that this OF variable,
in these experimental conditions, fails to detect standard
anxiolytic drug effect. Also for these features, there was
significant variability between experiments but no
Ignatia and emotional responses in mice
M Marzotto et al
60
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interaction between experiments and groups. The number
of urine spots decreased in treated groups as compared
with controls, with an almost significant P-value of 0.083
in global ANOVA for groups. Even if a post-hoc analysis
could not be performed due to the absence of global AN-
OVA significance, these studies indicated the Ignatia 5C
and 9C as the most active dilutions/dynamizations. The
number of stools (panel D) was unaffected by any treat-
ment.
LighteDark test
Ignatia and diazepam treatment caused a markedly sig-
nificant increase in the parameters of the LD paradigm
(global ANOVA values for groups P < 0.01), whose cumu-
lative evaluations and post-hoc statistics are reported in
Figure 4. With respect to the percentage time spent in the
illuminated compartment (panels AeB), this was increased
by diazepam and by Ignatia, in a significant way by the 9C
dilution/dynamization (P < 0.01). This Ignatia solution
changed the animals’ behaviour in the direction of reduced
anxiety, by 40e50%.
The percent effects of both diazepam and Ignatia were
more evident in the first 5 min of the trial (panels A and
CofFigure 4), in keeping with the hypothesis that they
are really due to a decrease in the anxiety elicited by the
novel environment into which the animal is suddenly
placed, which would be expected to decrease over time
due to habituation. A very similar trend was apparent for
the computation of distance travelled in the light compart-
ment (panels CeD). The calculation of the number of
transitions between compartments showed a significant
stimulating effect with diazepam, while all the Ignatia
dilutions/dynamizations had only a slight stimulating
effect, that however did not reach the threshold of statisti-
cal significance (e.g. P = 0.06 with Ignatia 7C) (data not
shown).
Also in this tes t, there were significant differences
between groups, and a significant interaction between
group (treatment) and experimental replications emerged
from global statistical analysis for time spent in the lit
area (Figure 4A). So, we then analysed all the 5 replications
of the LD test, considering the time spent in the lit area dur-
ing the first 5 min of the trial, which appeared to be the
most significant parameter for the purposes of this study.
Figure 5 shows the results in both absolute value (left col-
umn panels) and percentage terms (right column panels).
Considering the absolute values of time spent in the lit
area (which is inversely proportional to the level of anxi-
ety), the untreated control groups exhibited widely differ-
ing behaviour in the series of experiments, showing more
anxiety in experiments no. 1, 3 and 4 than in experiments
2 and 5. The time spent in the lit area by diazepam-
treated animals was higher than for the controls in all ex-
periments, thus confirming the validity of the test and the
high significance of the global analysis (Figure 4A), but
the difference relative to the control, and thus the
Figure 2 Effects of diazepam and Ignatia on the OF locomotion parameters, expressed as percentages S.E.M. relative to the mean values
for the vehicle-treated, control animals. N = 40 mice in diazepam (Diaz.) and Ignatia treated groups, N = 80 mice in the control group. The mean
absolute values for these parameters in control mice were: panel A 3220 cm/5 min; panel B 6228 cm/10 min; panel C 15.6 cm/s; panel D
14.8 cm/s. Global ANOVA values for groups and experiments were P < 0.01 in all test systems. The reported P values (*P < 0.05;
**P < 0.01) are from LSD post-hoc analysis by two-ways ANOVA, comparing drug-treated groups with the mean of the corresponding control
group.
Ignatia and emotional responses in mice
M Marzotto et al
61
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Page 5
percentage effect, was variable in the 5 replications of the
protocol. The strongest effects of diazepam occurred in ex-
periments no. 1, 3, 4, and in one experiment (no. 5) the drug
was almost ineffective. Ignatia showed a positive trend
(anxiolytic-like) in experiments 1, 3 and 4, while in the
other experiments the effects were essentially undetectable
or even slightly negative for some dilutions/dynamizations.
Since the strongest effects of Ignatia were noted in those
experiments where diazepam was also most active, this
analysis lends support to the hypothesis that the effects
of the homeopathic dilutions/dynamizations are due to
a true anxiolytic-like activity and not to chance.
Discussion
Use of homeopathic remedies in single-component or
complex formulations is frequent among patients seeking
relief for anxiety-related symptoms in a variety of condi-
tions
1e3,39,40
and in supportive cancer care,
4,41
but there
is currently little evidence for the efficacy of homeopathy
in the treatment of those disorders.
42e44
The clinical
use of anxiolytic drugs is not without its drawbacks,
particularly due to the risk of side effects such as
development of tolerance, cognitive and memory
changes, physical dependence, and withdrawal reaction
on discontinuation. Natural remedies possessing the same
efficacy as conventional drugs, but with fewer side effects,
would thus be a valuable addition to the treatment options
for anxiety-related disorders.
As part of a line of research we have been pursuing for
years, our group has recently shown that high-dilutions/
dynamizations of Gelsemium sempervirens are able to
modulate some emotional responses of laboratory
mice.
14
The present study was designed to explore the
effects of the homeopathic remedy Ignatia on animal be-
haviour, using sufficient sample sizes to detect small differ-
ence effects, with validated models and double blinding.
According to traditional homeopathic Materia Medica,
20
Ignatia has a marked action on various mental conditions
such as moodiness, anxiety, anguish, depres sion, disap-
pointment, melancholy, which can be caused by the ill ef-
fects of bad news, fright, grief, anger, disappointed love, or
suppressed sexual desire. Other typical symptoms for
which Ignatia is indicated as a remedy are neuralgic
pain, twitching of the face or lip muscles, spasmodic
cough, excessive flatulenc e, griping pains in the abdomen,
diarrhoea due to fright, and profuse and watery urine.
Ailments which are sensitive to Ignat ia are typically wors-
ened after drinking coffee or smoking, and relieved by
lying on the painful side or by profuse urination.
Figure 3 Effects of diazepam and Ignatia on the time spent in the centre of the OF (panels AeB), on the number of urine spots (panel C) and
on the number of defecations (panel D), expressed as percentages S.E.M. relative to the mean values for the vehicle-treated, control an-
imals. N of animals per group as in legend of Figure 2. The mean absolute values for these parameters in control mice were: panel A
43.6 s/5 min; panel B 106.2 s/10 min; panel C 1.33 urine spots/10 min; panel D 2.99 defecations/10 min. Global ANOVA values for groups
and experiments respectively were: panel A P = 0.622, P < 0.05; panel B P = 0.807, P < 0.05; panel C P = 0.083, P = 0.273; panel D
P = 0.831, P < 0.01.
Ignatia and emotional responses in mice
M Marzotto et al
62
Homeopathy
Page 6
The findings of the present study show, in a rigorous
manner and using a large group of animals, that Ignatia
reduced anxiety and fear in a manner comparable to,
though quantitatively lower than, the effects of diazepam.
The strongest statistical effect (P < 0.01) was obtained
with the 9C dilution/dynamization, which contained ex-
tremely low doses of strychnine in molecular terms. Since
the mother tincture contained 4.9 10
3
moles/l of strych-
nine, the theoretical concentration of this compound in the
9C solution was 4.9 10
21
moles/l and 4.9 10
24
moles/ml, i.e. near the Avogadro-Loschmidt limit. Since
the mice received 0.3 ml/day (approximately 1 molecule
of strychnine in the 9C dilutions/dynamization), this calcu-
lation allows to suggest that the pharmacological action
was not attributable to direct action of strychnine mole-
cules on putative receptors but on ‘homeopathic’ effects in-
volving the participation of ‘imprinted’ solvent, which
remain to be clarified.
45,46
No significant anxiolytic-like
effects were observed using low dilutions/dynamizations,
so there was no evidence of hormetic mechanisms of
very low doses of active principl es acting on sensible re-
ceptors in the ‘molecular’ range of concentrations. Includ-
ing non-succussed Ignatia dilutions would be a decisive
step toward demonstrating the mechanism of action, but
due to technical limitations on the number of animals
housed and tested, this additional control couldn’t be
done in our conditions.
Interestingly, the only statistically significant effect of
low dilutions/dynamizations (Ignatia 4C) was observed in
speed in the OF, suggesting this might be a ‘pathogenetic’
effect, possibly arising from the direct action of strychnine
on nerve or muscular function; however it should be pointed
out that this Ignatia 4C affected total motility (locomotion)
and not the anxiety-like responses, which are evaluated ac-
cording to different parameters. From these results the inter-
esting hypothesis emerges that low dilutions/dynamizations
may act at a physical level while high dilutions/dynamiza-
tions may regulate behaviour at an emotional level. The
stimulating effect of diazepam on locomotion remains
unexplained and appears paradoxical, and has also been
reported on rare occasions in humans.
47e49
In the OF, the percentage time spent in the centre of the
arena was not affected either by diazepam or by Ignatia,
indicating that, in the experimental conditions of this inves-
tigation, this test may be insuffi ciently sensitive for detect-
ing drug-related anxiolytic effects. An overview of the
literature on the action elicited by effective or putative an-
xiolytics in animals subjected to this procedure indicates
that some compounds (triazolobenzodiazepines such as
adinazolam and alprazolam, selective serotonin reuptake
inhibitors) that have different spectra of therapeutic effi-
cacy in anxiety disorders such as panic attacks, generalised
anxiety disorder or obsessive-compulsive disorder, are
poorly effective as anxiolytics in the OF test, suggesting
Figure 4 Effects of diazepam (Diaz.) and Ignatia on the LD parameters, expressed as percentages S.E.M. relative to the mean values for
the vehicle-treated, control animals. N of animals per group as in legend of Figure 2. The mean absolute values for these parameters in con-
trol mice were: panel A 45.0 s/5 min; panel B 135.2 s/10 min; panel C 561 cm/5 min; panel D 1495 cm/10 min. Global ANOVA values for
these evaluations were: panel A for groups P < 0.01, for experiments P < 0.01, interaction p = 0.025; panel B for groups P < 0.05, for exper-
iments P < 0.01, interaction P = 0.053; panel C for groups P < 0.01, for experiments P < 0.01, interaction P = 0.105; panel D for groups
P < 0.01, for experiments P < 0.01, interaction P = 0.085. The P values (* and **) are as in Figure 2.
Ignatia and emotional responses in mice
M Marzotto et al
63
Homeopathy
Page 7
that this paradigm may not model all behaviour disorders.
33
In a previous work, we found a lack of effect also for the
partial 5HT-agonist buspirone.
14
A finding not previously reported concerns the urine spot
count, which was reduced by both diazepam and all the
Ignatia dilutions/dynamizations, although with low statisti-
cal significance at the global ANOVA test, which precluded
a post-hoc analysis (P = 0.083). This parameter has been
linked by other authors to changes in the emotional behaviour
of rodents, with higher urine counts occurring when the
animals are placed in a novel or stressful environment
(Henderson and others).
50
Interestingly, in the traditional
Figure 5 Time spent in the lit area of the LD test in control and drug-treated mice (left column panels) and percent effects of diazepam and
Ignatia (right column panels) in the 5 replication experiments performed. Values are seconds S.E.M. spent in the lit area during the first
5 min of the trial (left column panels) or percentages S.E.M. relative to the mean values for the vehicle-treated, control animals of the
same experiment (right column panels). For each replication experiment, N = 8 mice per group in diazepam and Ignatia treated mice,
N = 16 animals per group in the control mice.
Ignatia and emotional responses in mice
M Marzotto et al
64
Homeopathy
Page 8
homeopathic literature there are reports which include fre-
quent urination among the symptoms that may be cured by
Ignatia.
20
Therefore, this preliminary evidence, if confirmed,
may represent a bridge between the traditional literature
(most of which is still awaiting confirmation by statistical
evidence) and modern experimental pharmacology.
There was significant variability between experiments.
This problem, which affected all the test parameters and
has also been noted in previous studies,
14
is possibly attrib-
utable to the high sensitivity of the mice and behavioural
test systems to peripheral factors like the season, weather,
animal breed, etc. We
9,10,51
and others
8,13,52
have
previously reported and discussed the methodological
aspects of research and theory in the high-dilution/
dynamization pharmacology field, particularly with respect
to the problem of reproducibility. If a treatment acts by
influencing the complex natural healing dynamics of the
treated subject as a whole, by means of small doses or highly
diluted administrations, this action could, at least in theory,
be highly sensitive to even small changes in experimental
conditions.
51
This concept is in agreement with our data,
which show that the best drug effects in the LD test were ob-
tained in the three experiments where baseline anxiety was
higher. This is relevant in the light of homeopathic theory,
according to which significant effects of highly diluted
drugs are to be expected only in sensitive subjects or in bi-
ological systems with primed, upregulated receptors.
51,53
It
is also worth noting that our protocol included in the
statistical analysis the complete sequence of replications,
irrespective of the basal level of anxiety and of the effect
of positive control (diazepam). However our results
suggest that in future it might be interesting to consider
including only those experiments in which the basal level
of anxiety is high and the effect of the positive control is
higher than a certain predetermined threshold.
On the other hand, the effects of diazepam in all five rep-
etitions also seem to be roughly proportional to baseline
anxiety (Figure 5), suggesting that a common environmen-
tal factor may be influencing the behavioural responses. In
our laborat ory, only unconditioned ‘ethological’ models
and spontaneous reactions to non-painful stimuli were
used, both for ethical reasons and becaus e our aim was to
approximate the natural conditions under which behaviour
is influenced by emotional states of fear, curiosity and anx-
iety. Ethological models are highly sensitive to any influ-
ences, and exhibit individual differences and variable
behavioural baseline levels.
28
An investigation of neuroen-
docrineeimmune interactions in rats has shown that com-
munication between animals in different cages, by means
of noise or body odours, can alter their performance.
54
We were unable to determine such factor(s), despite taking
the greatest care to reproduce the experimental conditions
of the protocol. In our conditions, cage effects can be ruled
out because, in each of the 5 repetitions, 8 animals per
treatment group were randomly distributed in 4 cages,
which were uniformly distributed in the holding rack.
These questions could be further elucidated by future
research on these models, repeating the experiments in
different settings and rooms.
The mechanisms of action of Ignatia on the central
nervous system could involve the centres which control
pain and anxiety, possibly through interaction with glyci-
nergic receptors, because strychnine at high doses
has been found to bind to those receptors. Some authors
report indirect evidence for the binding of Ignatia with re-
ceptors for glycine.
24,55
Glycine is an amino acid that
functions as a neurotransmitter with inhibitory effect
because it stimulates biosynthesis of the neurosteroid
allopregnanolone, a pathway that was proposed as
a putative mechanism of action also for Gelsemium
sempervirens.
9,25
Strychnine is known to block the
glycinergic receptors, but it could be hypothesised that in
extremely low doses (or at high-dilutions/dynamizations)
it might instead hormetically stimulate them, thereby
acting in a manner leading to increa sed neurosteroid
synthesis. The hypothesis appears in the recently
re-evaluated framework of theories and evidence of a rela-
tion between hormesis and high-dilution /dynamization
effects.
56,57
The development of interventions that
activate hormetic signalling pathways in neurons is
a promising new approach for the prevention and
treatment of a range of neurological disorders.
58
In conclusion, Ignatia has been shown to be able to mod-
ulate the emotional responses of mice in the LD paradigm,
and possibly to have some effect in regulating the urination
behaviour of the animals. Since the maximum anxiolytic-
like activity was exhibited by solutions where the theoret-
ical concentration of strychnine is about 1 molecule/ml,
these results are compatible with the hypothesis of specific
physicochemical changes of the solvent during dilution/
dynamization and suggest non-linear mechanisms of regu-
lation in the animals’ central nervous system.
Conflictsofinterest
The authors declare that they have no competing inter-
ests.
Acknowledgements
Our work was supported by a scientific collaboration
between University of Verona (Department of Pathology)
and Laboratoires Boiron.
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  • Source
    • "Bellavite [22] has reported that Gelsemium has reduced anxiety and fear in murine, in a manner quantitatively comporable to the effects of anxiolytic drugs without provoking any sedation side-effects. It has been also reported that Ignatia reduced anxiety and fear in mouse [23] . In our study, we also applied Ignatia and/or Gelsemium to the horses and our results imply that the same effects occur in the horses as in other animals. "
    Preview · Article · Jan 2016 · Kafkas Üniversitesi Veteriner Fakültesi Dergisi
  • Source
    • "Moreover, others have shown that adverse effects of homeopathic drugs are exceedingly rare and these therapies are well tolerated. Direct evidence from placebo-controlled studies of Gelsemium sempervirens (Bellavite et al. 2011b; Magnani et al. 2010) and of Ignatia amara (Marzotto et al. 2012) showed that used dilutions have anxiolytic-like properties without weakening locomotion and without adverse or sedative effects. The Authors concluded their reply hoping that the difficult-to-solve technical issues of high dilutions, hormesis, and paradoxical reversal of the effects of drugs in future be addressed not through subjective opinions and jeering, but rather on the experiential ground, through patient and critical comparison of data and results (Bellavite,P., M.Marzotto, and A.Conforti. "
    [Show abstract] [Hide abstract] ABSTRACT: The group of P. Bellavite working at Verona university (Institute of Chemistry and Clinical Microscopy, then Department of Pathology and Diagnostics) has been involved in studies on leukocyte metabolism, inflammation, pharmacology of natural products for over twenty years. Part of its studies were dedicated to complementary medicine (phytotherapy, acupuncture, homeopathy, diet), as an Observatory of Complementary Medicine organized by Bellavite and other Verona University researchers was recognized by Institute of Chemistry and Clinical Microscopy since 1997. Starting from y. 2011, studies of the Verona group were the object of some critical opinions, as “letters” or “commentaries”, most of which from the same person, who was former collaborator of the same group until the end of y. 2010. As principal investigator in a series of studies conducted in collaboration with colleagues of Verona University I have the duty to clarify the questions raised. No experiment invalidating experimental findings was ever reported, while the effect of Gelsemium in neurological and behavioural models was reported by several independent laboratories. Even if the whole scientific interest of this provoked discussion could be scarce, its key points are reported here in the interest of completeness and precision of documentation. It should be noted that Bellavite and coworkers never critiqued the experimental work of others, and replied only to criticisms that were considered as inappropriate or affecting Verona university. The same person recently posted in RG website further personal opinions including redundant criticisms (to which we had previously carefully responded), personal attacks and even bizarre statements that by their very nature require no reply. If anyone would need further explanations she/he could contact directly the author (paolo.bellavite@univr.it). Needless to say, the concept of "reproducibility" is not applicable to this synthesis of a literature debate.
    Full-text · Technical Report · Dec 2014
    • "Despite this complex picture, much people suffering from anxiety usually turn to homeopathy to solve this ailment, due to the simple fact that diagnosed anxiety may include a more complex psychiatric pattern and because of a significant proportion of patients failing to respond to first-line pharmacotherapy agents [16]. Nevertheless, according to recent papers [1,1718192021222324, homeopathy does work, at least in anxiety, although some homeopaths have raised criticisms, as a comprehensive search should demonstrate that the evidence on the benefit of homeopathy in anxiety and anxiety disorders is limited [25]. At least in humans, a number of studies of homeopathy in such conditions were located but the randomised controlled trials report contradictory results, are underpowered or provide insufficient details of methodology. "
    No preview · Dataset · Feb 2013
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