Gelsemium low doses protect against serum deprivation-induced stress on mitochondria in neuronal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Gelsemium dynamized dilutions (GDD) are known as a remedy for a wide range of behavioral and psychological symptoms of depression and anxiety at ultra-low doses, yet the underlying mechanisms of the mode of action of G. sempervirens itself are not well understood. AIM OF THE STUDY: The present study was designed to examine the neuroprotective effects of Gelsemium preparations in counteracting stress-related mitochondrial dysfunctions in neuronal cells. MATERIALS AND METHODS: We started by studying how serum deprivation affects the mitochondrial functions of human neuroblastoma (SH-SY5Y) cells. Next, we looked into the potential of various Gelsemium dilutions to improve cell survival and ATP levels. After identifying the most effective dilutions, 3C and 5C, we tested their ability to protect SH-SY5Y cells from stress-induced mitochondrial deficits. We measured total and mitochondrial superoxide anion radicals using fluorescent dyes dihydroethidium (DHE) and the red mitochondrial superoxide indicator (MitoSOX). Additionally, we assessed total nitric oxide levels with 4,5-diaminofluorescein diacetate (DAF-2DA), examined the redox state using pRA305 cells stably transfected with a plasmid encoding a redox-sensitive green fluorescent protein, and analyzed mitochondrial network morphology using an automated high-content analysis device, Cytation3. Furthermore, we investigated bioenergetics by measuring ATP production with a bioluminescence assay (ViaLighTM HT) and evaluated mitochondrial respiration (OCR) and glycolysis (ECAR) using the Seahorse Bioscience XF24 Analyzer. Finally, we determined cell survival using an MTT reduction assay. RESULTS: Our research indicates that Gelsemium dilutions (3C and 5C) exhibited neuroprotective effects by: - Normalizing total and mitochondrial superoxide anion radicals and total nitric oxide levels. - Regulating the mitochondrial redox environment and mitochondrial networks morphology. - Increasing ATP generation as well as OCR and ECAR levels, thereby reducing the viability loss induced by serum withdrawal stress. CONCLUSIONS: These findings highlight that dynamized Gelsemium preparations may have neuroprotective effects against stress-induced cellular changes in the brain by regulating mitochondrial functions, essential for the survival, plasticity, and function of neurons in depression.

Pollen limitation of native plant reproduction in an urban landscape.

PREMISE: Evidence suggests that bees may benefit from moderate levels of human development. However, the effects of human development on pollination and reproduction of bee-pollinated plants are less-well understood. Studies have measured natural variation in pollination and plant reproduction as a function of urbanization, but few have experimentally measured the magnitude of pollen limitation in urban vs. non-urban sites. Doing so is important to unambiguously link changes in pollination to plant reproduction. Previous work in the Southeastern United States found that urban sites supported twice the abundance of bees compared to non-urban sites. We tested the hypothesis that greater bee abundance in some of the same urban sites translates into reduced pollen limitation compared to non-urban sites. METHODS: We manipulated pollination to three native, wild-growing, bee-pollinated plants: Gelsemium sempervirens, Oenothera fruticosa, and Campsis radicans. Using supplemental pollinations, we tested for pollen limitation of three components of female reproduction in paired urban and non-urban sites. We also measured pollen receipt as a proxy for pollinator visitation. RESULTS: We found that all three plant species were pollen-limited for some measures of female reproduction. However, opposite to our original hypothesis, two of the three species were more pollen-limited in urban relative to non-urban sites. We found that open-pollinated flowers in urban sites received less conspecific and more heterospecific pollen on average than those in non-urban sites. CONCLUSIONS: These results suggest that even when urban sites have more abundant pollinators, this may not alleviate pollen limitation of native plant reproduction in urban landscapes.

Floral Scent Variation in the Heterostylous Species Gelsemium sempervirens.

Gelsemium sempervirens (L.) W.T. Aiton, a distylous woody vine of the family Gelsemiaceae, produces sweetly fragrant flowers that are known for the toxic alkaloids they contain. The composition of this plant's floral scent has not previously been determined. In this study, the scent profiles of 74 flowers obtained from six different wild and cultivated populations of G. sempervirens were measured by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). There were 81 volatile organic compounds identified and characterized as benzenoids, terpenoids, fatty acid derivatives, and yeast associated compounds. The most abundant compound was benzaldehyde (23-80%) followed by ethanol (0.9-17%), benzyl benzoate (2-15%), 4-anisaldehyde (2-11%), (Z)-α-ocimene (0-34%), and α-farnesene (0.1-16%). The impacts of geographic location, population type (wild or cultivated), and style morph (L = long, S = short) on scent profile were investigated. The results showed no relationship between geographic location or population type and volatile organic compounds (VOC) profile, but did show a significant scent profile difference between L and S morphs based on non-metric multidimensional scaling (NMDS) using Bray-Curtis similarity indices. The L morphs contained higher amounts of benzenoids and the S morphs contained higher amounts of terpenoids in their scent profiles. The L morphs also produced a higher total abundance of scent compounds than the S morphs. This study represents the first floral scent determination of G. sempervirens finding significant variation in scent abundance and composition between style morphs.

Gene Discovery in Gelsemium Highlights Conserved Gene Clusters in Monoterpene Indole Alkaloid Biosynthesis.

Genome mining is a routine technique in microbes for discovering biosynthetic pathways. In plants, however, genomic information is not commonly used to identify novel biosynthesis genes. Here, we present the genome of the medicinal plant and oxindole monoterpene indole alkaloid (MIA) producer Gelsemium sempervirens (Gelsemiaceae). A gene cluster from Catharanthus roseus, which is utilized at least six enzymatic steps downstream from the last common intermediate shared between the two plant alkaloid types, is found in G. sempervirens, although the corresponding enzymes act on entirely different substrates. This study provides insights into the common genomic context of MIA pathways and is an important milestone in the further elucidation of the Gelsemium oxindole alkaloid pathway.

Experimental neuropharmacology of Gelsemium sempervirens: Recent advances and debated issues.

Gelsemium sempervirens L. (Gelsemium) is traditionally used for its anxiolytic-like properties and its action mechanism in laboratory models are under scrutiny. Evidence from rodent models was reported suggesting the existence of a high sensitivity of central nervous system to anxiolytic power of Gelsemium extracts and Homeopathic dilutions. In vitro investigation of extremely low doses of this plant extract showed a modulation of gene expression of human neurocytes. These studies were criticized in a few commentaries, generated a debate in literature and were followed by further experimental studies from various laboratories. Toxic doses of Gelsemium cause neurological signs characterized by marked weakness and convulsions, while ultra-low doses or high Homeopathic dilutions counteract seizures induced by lithium and pilocarpine, decrease anxiety after stress and increases the anti-stress allopregnanolone hormone, through glycine receptors. Low (non-Homeopathic) doses of this plant or its alkaloids decrease neuropathic pain and c-Fos expression in mice brain and oxidative stress. Due to the complexity of the matter, several aspects deserve interpretation and the main controversial topics, with a focus on the issues of high dilution pharmacology, are discussed and clarified.

Nuclear Magnetic Resonance characterization of traditional homeopathically manufactured copper (Cuprum metallicum) and plant (Gelsemium sempervirens) medicines and controls.

BACKGROUND: NMR proton relaxation is sensitive to the dynamics of the water molecule H2O, through the interaction of the spin of the proton (1H) with external magnetic and electromagnetic fields. METHODS: We measured dilution and potentization processes through measurements of 1H spin-lattice T1 and spin-spin T2 relaxation times. In order to interpret the recorded fluctuations in T1- or T2-values, experimental data were linearized by investigating how the area under a fluctuating time = f(dilution) curve (dilution integral or DI) changes with dilution. Two kinds of fitting procedures were considered: chi-square fitting with a goodness-of-fit probability, and least absolute deviations criterion with Pearson's linear correlation coefficient. RESULTS: We showed that fluctuations are not attributable to random noise and/or experimental errors, evidencing a memory effect quantifiable by the slope of the DI = f(dilution) straight line. For all experiments, correlation coefficients were found to lie above 0.9999, against 0.999 for random noise. The discrimination between experimental slopes and slopes associated with random noise data was very good at a five-sigma level of confidence (i.e. probability 3 × 10-7). Discrimination between experimental slopes at a five-sigma level was possible in most cases, with three exceptions: gelsemium aqua pura v gelsemium dilution (four-sigma); copper aqua pura v gelsemium aqua pura (four-sigma) and copper simple dilution v gelsemium simple dilution (three-sigma). All potentized samples show very good discrimination (at least nine-sigma level) against aqua pura, lactose or simple dilution. It was possible to transform the associated relaxation times into a molecular rotational correlation time τc and an average spin-spin distance d. Our experiments thus point to a considerable slowing down of molecular movements (τc > 1300 ps or T = 224-225 K) around water molecules up to a distance of 3.7 Å, values. It was also possible to rule out other possible mechanisms of relaxation (diffusive motion, 17O-1H relaxation or coupling with the electronic spin, S = 1, of dissolved dioxygen molecules). CONCLUSION: There is clear evidence that homeopathic solutions cannot be considered as pure water as commonly assumed. Instead, we have evidenced a clear memory effect upon dilution/potentization of a substance (water, lactose, copper, gelsemium) reflected by different rotational correlation times and average H⋯H distances. A possible explanation for such a memory effect may lie in the formation of mesoscopic water structures around nanoparticles and/or nanobubbles mediated by zero-point fluctuations of the vacuum electromagnetic field as suggested by quantum field theories. The existence of an Avogadro's 'wall' for homeopathically-prepared medicines is not supported by our data. Rather it appears that all dilutions have a specific material configuration determined by the potentized substance, also by the chemical nature of the containers, and dissolved gases and the electromagnetic environment. This sensitivity of homeopathically-prepared medicines to electromagnetic fields may be amplified by the highly non-linear processing routinely applied in the preparation of homeopathic medicines. Future work is needed in such directions. The time is now ripe for a demystification of the preparation of homeopathic remedies.

Geographic variation in resistance to nectar robbing and consequences for pollination.

PREMISE OF THE STUDY: Floral evolution is frequently ascribed to selection by pollinators, but may also be shaped by antagonists. However, remarkably few studies have examined geographic mosaics in resistance to floral antagonists or the consequences for other floral interactions. METHODS: Gelsemium sempervirens experiences frequent nectar robbing in northern Georgia, but rarely in southern Georgia. We conducted common-garden experiments in both locations using genotypes from each region and measured robbing, pollinator attraction, floral attractive and defensive traits, and plant reproduction. KEY RESULTS: Nectar robbing was more than four times higher in the north vs. south, and pollinator visits did not differ between gardens. Across both gardens, northern genotypes were half as likely to be nectar-robbed but received half as many pollinator visits as southern genotypes, suggesting evolution of resistance to robbing at a cost of reduced pollinator attraction. Plant-level traits, such as height and number of flowers, were more closely associated with resistance to robbing than floral size, shape, or chemistry. Northern genotypes had lower female and estimated male reproduction compared to southern genotypes at both locations, which could be due to costs of resistance to nectar robbing, or costs of adaptations to other biotic or abiotic differences between regions. CONCLUSIONS: Our study indicates that geographic variation can play a strong role structuring interactions with floral antagonists and mutualists and provides evidence consistent with the hypothesis that local resistance to nectar robbing imposes costs in terms of decreased pollinator attraction and reproduction.

Reconstituted mother tinctures of Gelsemium sempervirens L. improve memory and cognitive impairment in mice scopolamine-induced dementia model.

ETHNOPHARMACOLOGICAL RELEVANCE: Gelsemium sempervirens (L.) J.St.-Hil is a herb used for the treatment of various neuroses in both homeopathic and Ayurvedic systems. The present study examines whether Gelsemium reconstituted tincture can protect against scopolamine induced cognitive discrepancies in amnesic mouse model. In order to investigate the protective mechanism of Gelsemium against dementia, in vitro acetyl cholinesterase and ß-secretase enzyme inhibition and estimation of glutathione level in mouse brain were carried out. MATERIALS AND METHODS: The inhibition study on acetyl cholinesterase and ß-secretase enzyme was conducted on brain homogenate supernatant spectrophotometrically using specific substrate. Cognitive enhancement activity was assessed by elevated plus maze and passive avoidance study in scopolamine induced dementia mouse model. Glutathione, an anti-oxidant, was measured spectrophotometrically from scopolamine induced amnesic mice brain supernatant using 5,5'-dithiobis 2-nitrobenzoic acid in the presence and absence of Gelsemium tincture. RESULTS: Significant inhibition was found with Gelsemium on AChE and ß-secretase enzyme with an IC50 of 9.25 and 16.25 µg/ml, respectively, followed by increasing glutathione levels in comparison to the untreated dementia group. The effect of Gelsemium of scopolamine-induced cognitive deficits was determined by measuring the behavioral parameters and the antioxidant status of the brain after scopolamine (1mg/kg i.p.) injected amnesic mice. Gelsemium significantly demonstrated in vivo anti-dementia activity (60% protection) and increased exploratory behavior. CONCLUSION: Our investigations indicated that alkaloid, iridoids and coumarin enriched reconstituted Gelsemium tincture extract displays promising cognitive enhancement in adult mice after short-term oral treatment. Hence, Gelsemium can be a promising anti-dementia agent, mediating the protection against amnesia, attention disorders and learning dysfunctions through dual inhibition of both acetyl cholinesterases (no false positive effect was shown), ß-secretase and antioxidant activity.

Effects of Gelsemium sempervirens L. on pathway-focused gene expression profiling in neuronal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Gelsemium sempervirens L. is a traditional medicinal plant mainly distributed in the southeastern of the United States, employed in phytotheraphy and homeopathy as nervous system relaxant to treat various types of anxiety, pain, headache and other ailments. Although animal models showed its effectiveness, the mechanisms by which it might operate on the nervous system are largely unknown. This study investigated for the first time by a real-time PCR technique (RT-PCR Array) the gene expression of a panel of human neurotransmitter receptors and regulators, involved in neuronal excitatory signaling, on a neurocyte cell line. MATERIALS AND METHODS: Human SH-SY5Y neuroblastoma cells were exposed for 24h to Gelsemium sempervirens at 2c and 9c dilutions (i.e. 2 and 9-fold centesimal dilutions from mother tincture) and the gene expression profile compared to that of cells treated with control vehicle solutions. RESULTS: Exposure to the Gelsemium sempervirens 2c dilution, containing a nanomolar concentration of active principle gelsemine, induced a down-regulation of most genes of this array. In particular, the treated cells showed a statistically significant decrease of the prokineticin receptor 2, whose ligand is a neuropeptide involved in nociception, anxiety and depression-like behavior. CONCLUSIONS: Overall, the results indicate a negative modulation trend in neuronal excitatory signaling, which can suggest new working hypotheses on the anxiolytic and analgesic action of this plant.

Effects of high-dilutions in behavioural models: a commentary on critical issues, from reproducibility to plausibility

As part of a rigorous investigation into the effects of Gelsemium sempervirens on laboratory mice, we performed two complete series of experiments and published three scientific papers. A recent commentary has, however, called into question the reproducibility and validity of these findings. In this article we discuss the major issues raised by this critique within the framework of methodological aspects and the interpretation of results of high-dilution and homeopathic research. The charge of non-reproducibility is shown to be unfounded, because a same homeopathic medicine displayed the same direction of effects in two well-validated models (light-dark and open-), albeit with nonlinear patterns. The double-blind protocols and statistics by means of ANOVA were performed appropriately and the difference between dilutions of Gelsemium (5cH, 7cH, 9cH and 30cH with variations according to model) and placebo was statistically highly significant. Our investigations brought to light some problems related with the lack of activity of buspirone and diazepam (conventional anxiolytic drugs used as control) on some behavioural parameters, suggesting that Gelsemium may have broader action, and raising doubts as to the reliability of benzodiazepines as positive controls for homeopathic treatments. Concerning the plausibility of experiments in this , disputed on the grounds of alleged lack of dose-response effect, we note that the latter is not at all uncommon, and can be accounted for by a host of possible reasons. In conclusion, our research line showed reproducible and consistent effects of Gelsemium in laboratory mice.(AU)

Como parte da investigação rigorosa sobre os efeitos do Gelsemium sempervirens em camundongos de laboratório, realizamos dois conjuntos completos de experimentos o que deu origem a três artigos sobre o tema. Porém, uma crítica recentemente publicada questionou a reprodutibilidade e validade desses resultados. Neste artigo, discutimos os pontos principais levantados pelos críticos, com base em aspectos metodológicos e a interpretação de resultados de pesquisas em altas diluições e homeopatia. Mostramos que a crítica sobre a não reprodutibilidade é infundada porque um mesmo medicamento sempre gerou efeitos na mesma direção, em dois modelos bem validados (claro-escuro e campo aberto), embora com padrões não-lineares. Os protocolos duplo-cegos e a estatística ANOVA foram devidamente realizados e as diferenças estatísticas entre as diluições de Gelsemiun semprevirens (5cH, 7cH, 9cH e 30cH, com variações de acordo com o modelo) e o placebo foram altamente significativa. Nossas pesquisas trouxeram a tona alguns problemas relacionados com a atividade da buspirona e diazepam (drogas anxiolíticas convencionais usadas como controle) em alguns parâmetros comportamentais, sugerindo que Gelsemiun pode ter uma ação mais ampla, levantando dúvidas sobre o uso de benzodiazepinas como controle positivo para tratamentos homeopáticos. Em relação a plausividade dos experimentos nessa area e a alegada falta de efeito dose-resposta, notamos que estas não são de todo incomuns, e podem ser explicadas por uma série de possíveis razões. Em conclusão, nossa linha de pesquisa mostrou reprodutibilidade e efeitos consistentes, para os efeitos do Gelsemiun em camundongos de laboratório.(AU)

Como parte de una rigurosa investigación de los efectos de Gelsemium sempervirens en ratones de laboratorio, realizamos dos series completas de experimentos y publicamos tres artículos científicos. Sin embargo, un comentario reciente critica la reproductibilidad y validad de nuestros resultados. En este artículo discutimos los aspectos principales de esta crítica en relación a los aspectos metodológicos e interpretativos de la investigación en homeopatía y altas diluciones. La acusación de falta de reproductibilidad carece de fundamentos, pues los efectos de un mismo medicamento homeopático mostraron una misma dirección en dos modelos bien validados (luz/oscuridad y campo abierto), empero, con respuestas no lineares. Los protocolos doble ciego y cálculos estadísticos mediante ANOVA fueron ejecutados correctamente y la diferencia entre diluciones de Gelsemium (5cH, 7cH, 9cH y 30cH según el modelo) y placebo fueron notablemente significativas. Nuestra rigurosa investigación evidenció problemas relacionados con la inactividad de buspirona y diazepam (drogas ansiolíticas convencionales utilizadas como control) en algunos parámetros conductuales, lo que indica que Gelsemium puede tener una acción más amplia y que la confiabilidad de las benzodiazepinas como controles positivos de tratamientos homeopáticos debe ser cuestionada. Con respecto a la plausibilidad de los experimentos en este campo, cuestionados con base en la supuesta ausencia de efecto dosis-respuesta, recordamos que no se trata de nada poco común y que puede ser explicada por innúmeros factores. Concluyendo, nuestra línea de investigación demostró que Gelsemium causa efectos reproducibles y consistentes en ratones de laboratorio.(AU)

Effects of high-dilutions in behavioural models: a commentary on critical issues, from reproducibility to plausibility / Efeitos de altas diluições em modelos comportamentais: comentários sobre temas críticos, da reprodutibilidade a plausibilidade / Efectos de las altas diluciones em modelos comportamentales: comentários respecto a temas críticos, da reprodutibilidad a la plausibilidad

As part of a rigorous investigation into the effects of Gelsemium sempervirens on laboratory mice, we performed two complete series of experiments and published three scientific papers. A recent commentary has, however, called into question the reproducibility and validity of these findings. In this article we discuss the major issues raised by this critique within the framework of methodological aspects and the interpretation of results of high-dilution and homeopathic research. The charge of non-reproducibility is shown to be unfounded, because a same homeopathic medicine displayed the same direction of effects in two well-validated models (light-dark and open-), albeit with nonlinear patterns. The double-blind protocols and statistics by means of ANOVA were performed appropriately and the difference between dilutions of Gelsemium (5cH, 7cH, 9cH and 30cH with variations according to model) and placebo was statistically highly significant. Our investigations brought to light some problems related with the lack of activity of buspirone and diazepam (conventional anxiolytic drugs used as control) on some behavioural parameters, suggesting that Gelsemium may have broader action, and raising doubts as to the reliability of benzodiazepines as positive controls for homeopathic treatments. Concerning the plausibility of experiments in this , disputed on the grounds of alleged lack of dose-response effect, we note that the latter is not at all uncommon, and can be accounted for by a host of possible reasons. In conclusion, our research line showed reproducible and consistent effects of Gelsemium in laboratory mice.(AU)

Como parte da investigação rigorosa sobre os efeitos do Gelsemium sempervirens em camundongos de laboratório, realizamos dois conjuntos completos de experimentos o que deu origem a três artigos sobre o tema. Porém, uma crítica recentemente publicada questionou a reprodutibilidade e validade desses resultados. Neste artigo, discutimos os pontos principais levantados pelos críticos, com base em aspectos metodológicos e a interpretação de resultados de pesquisas em altas diluições e homeopatia. Mostramos que a crítica sobre a não reprodutibilidade é infundada porque um mesmo medicamento sempre gerou efeitos na mesma direção, em dois modelos bem validados (claro-escuro e campo aberto), embora com padrões não-lineares. Os protocolos duplo-cegos e a estatística ANOVA foram devidamente realizados e as diferenças estatísticas entre as diluições de Gelsemiun semprevirens (5cH, 7cH, 9cH e 30cH, com variações de acordo com o modelo) e o placebo foram altamente significativa. Nossas pesquisas trouxeram a tona alguns problemas relacionados com a atividade da buspirona e diazepam (drogas anxiolíticas convencionais usadas como controle) em alguns parâmetros comportamentais, sugerindo que Gelsemiun pode ter uma ação mais ampla, levantando dúvidas sobre o uso de benzodiazepinas como controle positivo para tratamentos homeopáticos. Em relação a plausividade dos experimentos nessa area e a alegada falta de efeito dose-resposta, notamos que estas não são de todo incomuns, e podem ser explicadas por uma série de possíveis razões. Em conclusão, nossa linha de pesquisa mostrou reprodutibilidade e efeitos consistentes, para os efeitos do Gelsemiun em camundongos de laboratório.(AU)

Como parte de una rigurosa investigación de los efectos de Gelsemium sempervirens en ratones de laboratorio, realizamos dos series completas de experimentos y publicamos tres artículos científicos. Sin embargo, un comentario reciente critica la reproductibilidad y validad de nuestros resultados. En este artículo discutimos los aspectos principales de esta crítica en relación a los aspectos metodológicos e interpretativos de la investigación en homeopatía y altas diluciones. La acusación de falta de reproductibilidad carece de fundamentos, pues los efectos de un mismo medicamento homeopático mostraron una misma dirección en dos modelos bien validados (luz/oscuridad y campo abierto), empero, con respuestas no lineares. Los protocolos doble ciego y cálculos estadísticos mediante ANOVA fueron ejecutados correctamente y la diferencia entre diluciones de Gelsemium (5cH, 7cH, 9cH y 30cH según el modelo) y placebo fueron notablemente significativas. Nuestra rigurosa investigación evidenció problemas relacionados con la inactividad de buspirona y diazepam (drogas ansiolíticas convencionales utilizadas como control) en algunos parámetros conductuales, lo que indica que Gelsemium puede tener una acción más amplia y que la confiabilidad de las benzodiazepinas como controles positivos de tratamientos homeopáticos debe ser cuestionada. Con respecto a la plausibilidad de los experimentos en este campo, cuestionados con base en la supuesta ausencia de efecto dosis-respuesta, recordamos que no se trata de nada poco común y que puede ser explicada por innúmeros factores. Concluyendo, nuestra línea de investigación demostró que Gelsemium causa efectos reproducibles y consistentes en ratones de laboratorio.(AU)

Effects of high-dilutions in behavioural models: a commentary on critical issues, from reproducibility to plausibility / Efeitos de altas diluições em modelos comportamentais: comentários sobre temas críticos, da reprodutibilidade a plausibilidade / Efectos de las altas diluciones em modelos comportamentales: comentários respecto a temas críticos, da reprodutibilidad a la plausibilidad

As part of a rigorous investigation into the effects of Gelsemium sempervirens on laboratory mice, we performed two complete series of experiments and published three scientific papers. A recent commentary has, however, called into question the reproducibility and validity of these findings. In this article we discuss the major issues raised by this critique within the framework of methodological aspects and the interpretation of results of high-dilution and homeopathic research. The charge of non-reproducibility is shown to be unfounded, because a same homeopathic medicine displayed the same direction of effects in two well-validated models (light-dark and open-), albeit with nonlinear patterns. The double-blind protocols and statistics by means of ANOVA were performed appropriately and the difference between dilutions of Gelsemium (5cH, 7cH, 9cH and 30cH with variations according to model) and placebo was statistically highly significant. Our investigations brought to light some problems related with the lack of activity of buspirone and diazepam (conventional anxiolytic drugs used as control) on some behavioural parameters, suggesting that Gelsemium may have broader action, and raising doubts as to the reliability of benzodiazepines as positive controls for homeopathic treatments. Concerning the plausibility of experiments in this , disputed on the grounds of alleged lack of dose-response effect, we note that the latter is not at all uncommon, and can be accounted for by a host of possible reasons. In conclusion, our research line showed reproducible and consistent effects of Gelsemium in laboratory mice.

Como parte da investigação rigorosa sobre os efeitos do Gelsemium sempervirens em camundongos de laboratório, realizamos dois conjuntos completos de experimentos o que deu origem a três artigos sobre o tema. Porém, uma crítica recentemente publicada questionou a reprodutibilidade e validade desses resultados. Neste artigo, discutimos os pontos principais levantados pelos críticos, com base em aspectos metodológicos e a interpretação de resultados de pesquisas em altas diluições e homeopatia. Mostramos que a crítica sobre a não reprodutibilidade é infundada porque um mesmo medicamento sempre gerou efeitos na mesma direção, em dois modelos bem validados (claro-escuro e campo aberto), embora com padrões não-lineares. Os protocolos duplo-cegos e a estatística ANOVA foram devidamente realizados e as diferenças estatísticas entre as diluições de Gelsemiun semprevirens (5cH, 7cH, 9cH e 30cH, com variações de acordo com o modelo) e o placebo foram altamente significativa. Nossas pesquisas trouxeram a tona alguns problemas relacionados com a atividade da buspirona e diazepam (drogas anxiolíticas convencionais usadas como controle) em alguns parâmetros comportamentais, sugerindo que Gelsemiun pode ter uma ação mais ampla, levantando dúvidas sobre o uso de benzodiazepinas como controle positivo para tratamentos homeopáticos. Em relação a plausividade dos experimentos nessa area e a alegada falta de efeito dose-resposta, notamos que estas não são de todo incomuns, e podem ser explicadas por uma série de possíveis razões. Em conclusão, nossa linha de pesquisa mostrou reprodutibilidade e efeitos consistentes, para os efeitos do Gelsemiun em camundongos de laboratório.

Como parte de una rigurosa investigación de los efectos de Gelsemium sempervirens en ratones de laboratorio, realizamos dos series completas de experimentos y publicamos tres artículos científicos. Sin embargo, un comentario reciente critica la reproductibilidad y validad de nuestros resultados. En este artículo discutimos los aspectos principales de esta crítica en relación a los aspectos metodológicos e interpretativos de la investigación en homeopatía y altas diluciones. La acusación de falta de reproductibilidad carece de fundamentos, pues los efectos de un mismo medicamento homeopático mostraron una misma dirección en dos modelos bien validados (luz/oscuridad y campo abierto), empero, con respuestas no lineares. Los protocolos doble ciego y cálculos estadísticos mediante ANOVA fueron ejecutados correctamente y la diferencia entre diluciones de Gelsemium (5cH, 7cH, 9cH y 30cH según el modelo) y placebo fueron notablemente significativas. Nuestra rigurosa investigación evidenció problemas relacionados con la inactividad de buspirona y diazepam (drogas ansiolíticas convencionales utilizadas como control) en algunos parámetros conductuales, lo que indica que Gelsemium puede tener una acción más amplia y que la confiabilidad de las benzodiazepinas como controles positivos de tratamientos homeopáticos debe ser cuestionada. Con respecto a la plausibilidad de los experimentos en este campo, cuestionados con base en la supuesta ausencia de efecto dosis-respuesta, recordamos que no se trata de nada poco común y que puede ser explicada por innúmeros factores. Concluyendo, nuestra línea de investigación demostró que Gelsemium causa efectos reproducibles y consistentes en ratones de laboratorio.