Grazia Trebbi

Use of homeopathic preparations in experimental studies with healthy plants

BACKGROUND The last comprehensive review of experimental research on effects of homeopathic treatments on plants was published in 1984, and lacked formal predefined criteria to assess study quality. Since then several new studies with more advanced methods have been published. OBJECTIVES To compile a review of the literature on basic research in homeopathy with healthy plants with particular reference to studies investigating specific effects of homeopathic remedies. METHODS The literature search included English, French, German, Italian, Portuguese and Spanish publications from 1920 to April 2009, using predefined selection criteria. We included experiments with healthy whole plants, seeds, plant parts and cells. The outcomes had to be measured by established procedures and statistically evaluated. We developed a Manuscript Information Score (MIS) and included only publications which provided enough information for proper interpretation (MIS>or=5). A formalised Study Methods Evaluation Procedure (SMEP) was used to evaluate these studies, and the subgroup of studies with adequate controls to identify specific effects. RESULTS A total of 86 studies in 79 publications was identified, 43 studies included statistics, 29 had MIS>or=5, and 15 studies investigated the specificity of homeopathic preparations. Specific effects of decimal, centesimal and fifty millesimal potencies were found including dilution levels far beyond the Avogadro number. In consecutive series of potencies only some of the tested potencies showed effects. There were many individual studies with diverse methods and very few reproduction trials. CONCLUSIONS Healthy plant models seem an useful approach to investigate basic research questions about the specificity of homeopathic preparations. More investigations with more advanced methods are recommended, especially in the sectors of potentisation techniques, effective potency levels and conditions for reproducibility. Systematic negative control experiments should become a routine procedure to control the stability of the experimental systems.

Use of homeopathic preparations in phytopathological models and in field trials: a critical review.

BACKGROUND The literature on the applications of homeopathy for controlling plant diseases in both plant pathological models and field trials was first reviewed by Scofield in 1984. No other review on homeopathy in plant pathology has been published since, though much new research has subsequently been carried out using more advanced methods. OBJECTIVES To conduct an up-to-date review of the existing literature on basic research in homeopathy using phytopathological models and experiments in the field. METHODS A literature search was carried out on publications from 1969 to 2009, for papers that reported experiments on homeopathy using phytopathological models (in vitro and in planta) and field trials. The selected papers were summarized and analysed on the basis of a Manuscript Information Score (MIS) to identify those that provided sufficient information for proper interpretation (MIS>or=5). These were then evaluated using a Study Methods Evaluation Procedure (SMEP). RESULTS A total of 44 publications on phytopathological models were identified: 19 papers with statistics, 6 studies with MIS>or=5. Publications on field were 9, 6 with MIS>or=5. In general, significant and reproducible effects with decimal and centesimal potencies were found, including dilution levels beyond the Avogadros number. CONCLUSIONS The prospects for homeopathic treatments in agriculture are promising, but much more experimentation is needed, especially at a field level, and on potentisation techniques, effective potency levels and conditions for reproducibility. Phytopathological models may also develop into useful tools to answer pharmaceutical questions.

Exogenous spermidine, arsenic and β-aminobutyric acid modulate tobacco resistance to tobacco mosaic virus, and affect local and systemic glucosylsalicylic acid levels and arginine decarboxylase gene expression in tobacco leaves.

The polyamine spermidine and the metalloid arsenic increased resistance responses in the well-known pathosystem NN tobacco/tobacco mosaic virus (TMV). Both the hypersensitive response to TMV in a leaf disk model system (inoculated disks floating in the 0.1mM treatments) and systemic acquired resistance (SAR) in whole plants were significantly affected. In the latter case, 1mM foliar sprays of spermidine and arsenic were as effective as TMV and dl-beta-aminobutyric acid (BABA), both taken as positive controls, in improving the plants response to subsequent challenge inoculation with TMV. Moreover, this phenotypic response was correlated with changes in the endogenous concentration of the SAR-related molecule salicylic acid and in transcript levels of some pathogenesis/stress-related genes (pathogenesis-related proteins PR-1a and PR-2 and arginine decarboxylase (ADC)). Concentrations of free salicylic acid and of 2-O-beta-d-glucosylsalicylic acid and mRNA amount of PR-1a, PR-2 and ADC were analyzed in plants treated with either spermidine or arsenic, and compared with those from untreated plants and from positive (TMV-inoculated or BABA-treated) controls. Conjugated salicylic acid content and ADC transcripts were found to significantly increase, at both the local and systemic levels, relative to untreated controls.

Use of homeopathic preparations in experimental studies with abiotically stressed plants.

BACKGROUND Experimental research on the effects of homeopathic treatments on impaired plants was last reviewed in 1990. OBJECTIVES To compile a systematic review of the existing literature on basic research in homeopathy with abiotically stressed plants using predefined criteria. METHODS The literature search was carried out on publications that reported experiments on homeopathy using abiotically stressed whole plants, seeds, plant parts and cells from 1920 to 2010. Outcomes had to be measured by established procedures and statistically evaluated. Using of a Manuscript Information Score (MIS) we identified those publications that provided sufficient information for proper interpretation (MIS≥5). A further evaluation was based on the use of adequate controls to investigate specific effects of homeopathic preparations and on the use of systematic negative control experiments. RESULTS A total of 34 publications with abiotically stressed plants was identified, published between 1965 and 2010. The 34 publications described a total of 37 experimental studies. Twenty-two studies included statistics, 13 had a MIS≥5, 8 were identified with adequate controls and 4 with negative control experiments. Significant and reproducible effects with decimal and centesimal potencies were found, including dilution levels beyond Avogadros number. One experimental model was independently assessed by another research team and yielded inverted results compared to the original trial. CONCLUSIONS Abiotically stressed plant models seem to be a useful approach to investigate homeopathic basic research questions, but more experimentation and especially more independent replication trials are needed. Systematic negative control experiments should be implemented on a routine basis to exclude false-positive results.

The Efficacy of Ultramolecular Aqueous Dilutions on a Wheat Germination Model as a Function of Heat and Aging-Time

This study evaluates the effects of temperature and aging on the efficacy of As2O3 at the 45th decimal potency in a wheat germination model, compared against a control and potentized H2O 45×. Each treatment-temperature combination was tested on seeds (Triticum aestivum L.) of Pandas variety, using six Petri dishes (33 seeds/dish) per trial, performing eight trials. Seeds were pre-treated by poisoning with 0.1% As2O3 solution to reduce germination, to allow a better evaluation of homeopathic treatment effects. The outcome variable was the number of non-germinated seeds after 96 h. Temperature effect was investigated by heating each treatment in a water bath for 30 min (at 20, 40 or 70°C), or for 5 min (at 100°C), and that of aging by dividing experimental data, collected over a period of nearly five months, into two groups: early and late experiments. Results seem to show that the efficacy of As2O3 45× is unaltered at 20 and 40°C, increases at 70°C and decreases at 100°C. As regards aging, a notable difference was found between early trials, with no significant efficacy, and late trials, where As2O3 45× exhibits a repeated significant effect versus control, except at 100°C. A reduction in variability was observed for As2O3 45× at 20°C versus control, confirming the findings of previous work. The main conclusion suggested by this experiment is that the efficacy of As2O3 45× on wheat germination may be influenced by heating degree and seems to have an increasing trend as a function of aging.

Models with Plants, Microorganisms and Viruses for Basic Research in Homeopathy

Most criticism about homeopathy concerns the lack of a scientific basis and theoretical models. In order to be accepted as a valid part of medical practice, a wellstructured research strategy for homeopathy is needed. This is often hampered by methodological problems as well as by gross underinvestment in the required academic resources. Fundamental research could make important contributions to our understanding of the homeopathic and high dilutions mechanisms of action. Since the pioneering works of Kolisko on wheat germination (Kolisko, 1923) and Junker on growth of microorganisms (paramecium, yeast, fungi) (Junker, 1928), a number of experiments have been performed either with healthy organisms (various physiological aspects of growth) or with artificially diseased organisms, which may react more markedly to homeopathic treatments than healthy ones. In the latter case, the preliminary stress may be either abiotic, e.g. heavy metals, or biotic, e.g. fungal and viral pathogens or nematode infection. Research has also been carried out into the applicability of homeopathic principles to crop growth and disease control (agrohomeopathy): because of the extreme dilutions used, the environmental impact is low and such treatments are well suited to the holistic approach of sustainable agriculture (Betti et al., 2006). Unfortunately, as Scofield reported in an extensive critical review (Scofield, 1984), there is little firm evidence to support the reliability of the reported results, due to poor experimental methodology and inadequate statistical analysis. Moreover, since there is no agricultural homeopathic pharmacopoeia, much work is required to find suitable remedies, potencies and dose levels.

Transglutaminase activity changes during the hypersensitive reaction, a typical defense response of tobacco NN plants to TMV

The occurrence of glutamyl polyamines (PAs) and changes in activity and levels of transglutaminase (TGase, EC 2.3.2.13), the enzyme responsible for their synthesis, are reported during the progression of the hypersensitive reaction (HR) of resistant NN tobacco plants (Nicotiana tabacum L. cv. Samsun) to tobacco mosaic virus (TMV). Mature leaves of tobacco were collected over 0-72 h after inoculation with TMV or phosphate buffer (mock). In vivo synthesis of polyamine glutamyl derivatives (glutamyl PAs), catalyzed by TGase activity, was evaluated after supplying labeled putrescine (Pu, a physiological substrate of TGase) to leaves. Results show that, starting from 24 h, mono-(gamma-glutamyl)-Pu and bis-(gamma-glutamyl)-Sd were recovered in TMV-inoculated samples but not in mock-inoculated ones; 2 days later, in the former, the amount of glutamyl derivatives further increased. An in vitro radiometric assay showed that, in TMV-inoculated leaves, TGase activity increased from 24 h onwards relative to mock controls. An immunoblot analysis with AtPng1p polyclonal antibody detected a 72-kDa protein whose amount increased at 72 h in TMV-inoculated leaves and in the lesion-enriched areas. A biotin-labeled cadaverine incorporation assay showed that TGase activity occurred in S1 (containing soluble proteins), S2 (proteins released by both cell walls and membranes) and S3 (membrane intrinsic proteins) fractions. In S3 fraction, where changes were the most relevant, TGase activity was enhanced in both mock-inoculated and TMV-inoculated samples, but the stimulation persisted only in the latter case. These data are discussed in the light of a possible role of TGase activity and glutamyl PAs in the defense against a viral plant pathogen.

Droplet evaporation method as a new potential approach for highlighting the effectiveness of ultra high dilutions

OBJECTIVE This study sought to verify whether the droplet evaporation method (DEM) can be applied to assess the effectiveness of ultra-high dilutions (UHDs). We studied the shape characteristics of the polycrystalline structures formed during droplet evaporation of wheat seed leakages. METHODS The experimental protocol tested both unstressed seeds and seeds stressed with arsenic trioxide 5mM, treated with either ultra-high dilutions of the same stressor substance, or with water as a control. The experimental groups were analyzed by DEM and in vitro growth tests. DEM patterns were evaluated for their local connected fractal dimension (measure of complexity) and fluctuating asymmetry (measure of symmetry exactness). RESULTS Treatment with arsenic at UHD of both stressed and non-stressed seeds increased the local connected fractal dimension levels and bilateral symmetry exactness values in the polycrystalline structures, as compared to the water treatment. The results of in vitro growth tests revealed a stimulating effect of arsenic at UHD vs. control, and a correlation between the changes in growth rate and the crystallographic values of the polycrystalline structures was observed. CONCLUSIONS The results indicate that polycrystalline structures are sensitive to UHDs, and so for the first time provide grounds for the use of DEM as a new tool for testing UHD effectiveness. DEM could find application as a treatment pre-selection tool, or to monitor sample conditions during treatment. Moreover, when applied to biological liquids (such as saliva, blood, blood serum, etc.), DEM might provide information about UHD effectiveness on human and animal health.

A Review of Three Simple Plant Models and Corresponding Statistical Tools for Basic Research in Homeopathy

In this paper, we review three simple plant models (wheat seed germination, wheat seedling growth, and infected tobacco plants) that we set up during a series of experiments carried out from 1991 to 2009 in order to study the effects of homeopathic treatments. We will also describe the set of statistical tools applied in the different models. The homeopathic treatment used in our experiments was arsenic trioxide (As2O3) diluted in a decimal scale and dynamized. Since the most significant results were achieved with the 45th decimal potency, both for As2O3 (As 45x) and water (W 45x), we here report a brief summary of these results. The statistical analysis was performed by using parametric and nonparametric tests, and Poisson distribution had an essential role when dealing with germination experiments. Finally, we will describe some results related to the changes in variability, which seems to be one of the targets of homeopathic treatment effect.

Weak Extremely High Frequency Microwaves Affect Pollen-Tube Emergence and Growth in Kiwifruit: Pollen Grain Irradiation and Water-Mediated Effects

OBJECTIVE This study was designed to evaluate the effects of weak-intensity extremely high frequency (EHF) microwaves in a model system-the plant organism pollen grain-lacking the placebo effect, available in large populations, to ensure accurate statistical analysis, and whose sensitivity is closely relevant to animal and human biology. DESIGN This study was blinded using an in vitro pollen germination technique. SUBJECTS AND STUDY INTERVENTIONS: Pollen of kiwifruit (Actinidia deliciosa) was either directly irradiated or grown in a medium prepared with irradiated water, using a CromoStim 2000, (PromoPharma, Republic of San Marino) designed for EHF microwave resonance therapy (MRT). It produces weak intensity EHF radiations (40-78 GHz), either continuous wave (cw) or modulated, at a 10 Hz-frequency, with infrared (IR) carried to 635-950 nm, and with an impedance (IPD) of 10(-21) W/Hz cm(2) and a power supply from 0 to 20 mW. OUTCOME MEASUREMENTS Pollen-tube emergence was expressed as a percent of grains producing a tube and tube elongation was measured at 4 hours of incubation by a turbidimetric assay (A(500)) of cultures, expressed as the net absorbance increase over time 0. RESULTS At days 2 and 4 during aging, both percent of germination and tube growth significantly and consistently improved over controls in kiwifruit pollen grains irradiated for 30 minutes at day 0 at 10 Hz frequency with the CromoStim 2000. Highly significant effects, either stimulant or inhibitory, were also observed on kiwifruit pollen (stressed or not) growing in a medium prepared with water previously irradiated either cw or modulated. Irradiated water affected pollen germination immediately and even after several days following EHF treatment. CONCLUSIONS Either direct or indirect EHF irradiation performed by the CromoStim 2000 is effective on pollen growth processes. In both cases, water seemed to play a primary role. According to the quantum electrodynamical coherence theory, our work could also have implications for homeopathy, suggesting a key to explain the efficacy of high dilutions and succussion procedures.