Paolo Magnani

Homeopathic Doses of Gelsemium sempervirens Improve the Behavior of Mice in Response to Novel Environments

Gelsemium sempervirens is used in homeopathy for treating patients with anxiety related symptoms, however there have been few experimental studies evaluating its pharmacological activity. We have investigated the effects of homeopathic doses of G. sempervirens on mice, using validated behavioral models. Centesimal (CH) dilutions/dynamizations of G. sempervirens, the reference drug diazepam (1 mg/kg body weight) or a placebo (solvent vehicle) were intraperitoneally delivered to groups of mice of CD1 strain during 8 days, then the effects were assessed by the Light-Dark (LD) choice test and by the Open-Field (OF) exploration test, in a fully blind manner. In the LD test, the mean time spent in the illuminated area by control and placebo-treated animals was 15.98%, for mice treated with diazepam it increased to 19.91% (P = .047), while with G. sempervirens 5 CH it was 18.11% (P = .341, non-significant). The number of transitions between the two compartments increased with diazepam from 6.19 to 9.64 (P < .001) but not with G. Sempervirens. In the OF test, G. sempervirens 5 CH significantly increased the time spent and the distance traveled in the central zone (P = .009 and P = .003, resp.), while diazepam had no effect on these OF test parameters. In a subsequent series of experiments, G. sempervirens 7 and 30 CH also significantly improved the behavioral responses of mice in the OF test (P < .01 for all tested variables). Neither dilutions of G. sempervirens affected the total distance traveled, indicating that the behavioral effect was not due to unspecific changes in locomotor activity. In conclusion, homeopathic doses of G. sempervirens influence the emotional responses of mice to novel environments, suggesting an improvement in exploratory behavior and a diminution of thigmotaxis or neophobia.

Testing homeopathy in mouse emotional response models: pooled data analysis of two series of studies.

Two previous investigations were performed to assess the activity of Gelsemium sempervirens (Gelsemium s.) in mice, using emotional response models. These two series are pooled and analysed here. Gelsemium s. in various homeopathic centesimal dilutions/dynamizations (4C, 5C, 7C, 9C, and 30C), a placebo (solvent vehicle), and the reference drugs diazepam (1 mg/kg body weight) or buspirone (5 mg/kg body weight) were delivered intraperitoneally to groups of albino CD1 mice, and their effects on animal behaviour were assessed by the light-dark (LD) choice test and the open-field (OF) exploration test. Up to 14 separate replications were carried out in fully blind and randomised conditions. Pooled analysis demonstrated highly significant effects of Gelsemium s. 5C, 7C, and 30C on the OF parameter “time spent in central area” and of Gelsemium s. 5C, 9C, and 30C on the LD parameters “time spent in lit area” and “number of light-dark transitions,” without any sedative action or adverse effects on locomotion. This pooled data analysis confirms and reinforces the evidence that Gelsemium s. regulates emotional responses and behaviour of laboratory mice in a nonlinear fashion with dilution/dynamization.

Effects of Ignatia amara in mouse behavioural models

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS Homeopathic Ignatia dilutions/dynamizations (peak at 9C) modify some emotion-related symptoms in laboratory mice without affecting locomotion.

Response to a comment by Luigi Cervo and Valter Torri on: “Dose–effect study of Gelsemium sempervirens in high dilutions on anxiety-related responses in mice” (Magnani P. et al., Psychopharmacology, 2010)

Dear Editor, We are glad that our Psychopharmacology paper, published on May 2010, is still raising interest with promise of a large impact. As a result of a challenging and pioneering work on this medicinal plant, we published three papers that do nothing more than report and discuss the experimental findings, as these were consecutively obtained. These papers contain all the necessary explanations, so that an unbiased perusal of them should be enough to confirm the overall validity of our findings. In the limited space at our disposal, we are forced to respond to only a few key points (a detailed rebuttal of the letter is available on request). The charge of non-reproducibility by Cervo and Torri is unfounded because in both series of experiments Gelsemium sempervirens worked in two well-validated models to the same direction, even with different statistical significance. In the cited preliminary study, we report the significant effects of G. sempervirens 5C, 7C and 30C in an open field (OF), and the Psychopharmacology paper reports a similar result, albeit with lower statistical significance (p = 0.060, Fig. 2). In light–dark test (LD), the anxiolytic-like effect of G. sempervirens was highly significant in the second paper, while in the first one, it was present in 5C and 30C, albeit in a non-significant way. The differences may be due to the variability of animal responses, well known in behavioural research, and to some changes in protocols (animal vendor, type of housing in cages and sequence of tests) that we describe in the paper and in the cited review, as recognised also by Cervo and Torri. For further confirmation, we also performed a pooled analysis of the two papers’ results and found a highly significant effect of G. sempervirens 5C, 7C and 30C in OF parameters (permanence in centre area) and of G. sempervirens 5C, 9C and 30C in LD parameters (time spent in light and number of light–dark transitions) (Bellavite et al. 2011). Regarding the lack of activity of buspirone and diazepam in OF parameters, as reported also by others (references in the Psychopharmacology discussion), this may suggest that the effect of G. sempervirens in OF concerns the exploratory behaviour and the decrease in neophobia, instead of the anxiolytic-like effect. G. sempervirens reproducibly did not alter the locomotion assessed in OF in any series, indicating that the effect was not sedative. The analysis of variance (ANOVA) was conducted appropriately since we compared each treatment group, composed of 48 mice, against 96 untreated mice in order to have a larger control group, and this is correct. In the ANOVA, the usual procedure is for each treatment group to be compared with a single control group, which is what we did. Comparing each treatment with each of the two controls would have engendered more problems of multiple comparisons for no useful purpose and resulted in loss of power. Moreover, the two control groups showed similar variability and could therefore be pooled without qualms. For what concerns the alleged lack of a dose–response effect, this is not at all uncommon in behavioural pharmacology and there can be a host of possible reasons. The objection arises from a misunderstanding of high-dilution effects and hormesis, where non-linear phenomena and possible physicochemical changes of the solvent come into play, as discussed and referenced in the paper (page 542, paragraph 3). These are not “post hoc” interpretations but rather up-to-date working models (Bellavite et al. 2010). This criticism is therefore also unjustified. Our research has reported for the first time the effect of G. sempervirens on two highly validated behavioural paradigms in laboratory mouse. Yours sincerely, Bellavite et al.