Multidimensional Outcome Parameters in a Cress Seedling-CuCl2 Crystallization Assay to Corroborate Specific Effects of Stannum metallicum 30x Compared to Lactose 30x.

BACKGROUND: Previously we developed a test system which yielded highly significant evidence for specific effects of a Stannum metallicum 30x preparation in a multi-center replication trial. This test system is based on cress seed germination in homeopathic or control samples, CuCl2 crystallization of the cress extract, and subsequent digital textural image analysis of the resulting crystallization patterns. OBJECTIVES: The current study aimed to investigate whether three novel outcome parameters could further corroborate and possibly characterize the specific effects of Stannum metallicum 30x. METHODS: To this end, (1) cress seedling length, (2) a second texture analysis parameter, entropy and (3) the local connected fractal dimension (LCFD) of crystallization patterns as a measure of complexity were considered. The stability of the experimental setup was monitored throughout the entire investigation with systematic negative control (SNC) experiments. RESULTS: Cress length and entropy revealed a time-modulated potency treatment effect, in the absence of a significant main treatment effect. This indicated that the effect of the potency treatment varied significantly across the different experimental days. LCFD yielded a highly significant potency treatment effect. In addition, a significant interaction of treatment with experimental day seems to indicate a modulation of this effect. No significant effects were observed in any of the evaluations of the SNC experiments, indicative of a stable experimental setup and a reliable and specific treatment effect. Neither significant nor strong correlations were found between the four parameters, indicating that they reflect different effects of Stannum metallicum 30x on the organism treated. CONCLUSION: This quadruple characterization of the biological effects of Stannum metallicum 30x provides an unprecedented opportunity for basic homeopathy research into, among others, the presumed specificity of homeopathic preparations.

Phase-Transition-Induced Pattern Formation Applied to Basic Research on Homeopathy: A Systematic Review.

BACKGROUND: Methods based on phase-transition-induced pattern formation (PTPF) are increasingly used in medical research. Frequent application fields are medical diagnosis and basic research in homeopathy. Here, we present a systematic review of experimental studies concerning PTPF-based methods applied to homeopathy research. We also aimed at categorizing the PTPF methods included in this review. MATERIALS AND METHODS: Experimental studies were collected from scientific databases (PubMed, Web of Science, Russian eLibrary) and from experts in the research field in question, following the PRISMA guidelines. The studies were rated according to pre-defined scientific criteria. RESULTS: The review included 15 experimental studies. We identified seven different PTPF methods applied in 12 experimental models. Among these methods, phase-transition was triggered through evaporation, freezing, or solution, and in most cases led to the formation of crystals. First experimental studies concerning the application of PTPF methods in homeopathic research were performed in the first half of the 20th century; however, they were not continued in the following years. Only in the last decade, different research groups re-launched the idea, introducing new experimental approaches and computerized pattern evaluation techniques. The here-identified PTPF methods are for the first time proposed to be classified as one group of methods based on the same basic physical phenomenon. CONCLUSIONS: Although the number of experimental studies in the area is still rather limited, the long tradition in the application of PTPF methods and the dynamics of the present developments point out the high potential of these methods and indicate that they might meet the demand for scientific methods to study potentized preparations.

Reproducibility of the effects of homeopathically potentised Argentum nitricum on the growth of Lemna gibba L. in a randomised and blinded bioassay.

BACKGROUND: A previous study reported a significant statistical interaction between experiment date and treatment effect of Argentum nitricum 14x-30x on the growth rate of duckweed (Lemna gibba L.). The aim of the present study was to investigate the stability of the test system and intra-laboratory reproducibility of the effects found. METHODS: Duckweed was treated with A. nitricum potencies (14x-30x) as well as succussed and unsuccussed water controls. The outcome parameter area-related growth rate for day 0-7 was determined by a computerised image analysis system in two series of independent randomised and blinded experiments. Systematic negative control (SNC) experiments were carried out to investigate test system stability. Statistical analysis was performed with full two-way analysis of variance (ANOVA) and protected Fisher's Least Significant Difference (LSD) test. RESULTS: In the first repetition series we found a significant treatment effect (p = 0.016), while in the second series no effect was observed. The negative control experiments showed that the experimental system was stable. An a posteriori subgroup analysis concerning gibbosity revealed the importance of this growth state of L. gibba for successful reproduction of the statistically significant interaction in the original study; flat: no interaction (p = 0.762); slight gibbosity: no interaction (p = 0.356); medium gibbosity: significant interaction (p = 0.031), high gibbosity: highly significant interaction (p = 0.005). CONCLUSIONS: With the original study design (disregarding gibbosity status of L. gibba) results of the original study could not be reproduced sensu stricto. We conclude that the growth state gibbosity is crucial for successful reproduction of the original study. Different physiological states of the test organisms used for bioassays for homeopathic basic research must carefully be considered.

The use of plant-based bioassays in homeopathic basic research.

OBJECTIVES: The objective was to evaluate homeopathic basic research studies that use plant-based bioassays. With this in view, a compilation was made of the findings of three systematic literature reviews covering plant-based bioassays in the three fields of healthy, abiotically, or biotically stressed plants. This compilation focused on investigations using advanced experimental methods and detailed descriptions, also with the aim of supporting the design of future experiments. METHODS: Publications included had to report on studies into the effects of homeopathic preparations on whole plants, seeds, plant parts and cells. Outcomes had to be measured by established procedures and statistically evaluated. A Manuscript Information Score (MIS) was applied using predefined criteria to identify publications with sufficient information for adequate interpretation (MIS ≥ 5). Additional evaluation focused on the use of adequate controls to investigate specific effects of homeopathic preparations, and on the use of systematic negative control (SNC) experiments to ensure the stability of the bioassay. Only a fraction of the studies reported here were performed with 'ultra high' dilutions, whereas other studies were performed with moderate or high dilutions. RESULTS: A total of 157 publications were identified, describing a total of 167 experimental studies. 84 studies included statistics and 48 had a MIS ≥ 5, thus allowing adequate interpretation. 29 studies had adequate controls to identify specific effects of homeopathic preparations, and reported significant effects of decimal and centesimal homeopathic potencies, including dilution levels beyond Avogadro's number. 10 studies reported use of SNC experiments, yielding evidence for the stability of the experimental set-up. CONCLUSION: Plant models appear to be a useful approach for investigating basic research questions relating to homeopathic preparations, but more independent replication trials are needed in order to verify the results found in single experiments. Adequate controls and SNC experiments should be implemented on a routine basis to exclude false-positive results.

Reproducibility of effects of homeopathically potentised gibberellic acid on the growth of Lemna gibba L. in a randomised and blinded bioassay.

BACKGROUND: Reproducibility of basic research investigations in homeopathy is challenging. This study investigated if formerly observed effects of homeopathically potentised gibberellic acid (GA3) on growth of duckweed (Lemna gibba L.) were reproducible. METHODS: Duckweed was grown in potencies (14x-30x) of GA3 and one time succussed and unsuccussed water controls. Outcome parameter area-related growth rate was determined by a computerised image analysis system. Three series including five independent blinded and randomised potency experiments (PE) each were carried out. System stability was controlled by three series of five systematic negative control (SNC) experiments. Gibbosity (a specific growth state of L. gibba) was investigated as possibly essential factor for reactivity of L. gibba towards potentised GA3 in one series of potency and SNC experiments, respectively. RESULTS: Only in the third series with gibbous L. gibba L. we observed a significant effect (p = 0.009, F-test) of the homeopathic treatment. However, growth rate increased in contrast to the former study, and most biologically active potency levels differed. Variability in PE was lower than in SNC experiments. The stability of the experimental system was verified by the SNC experiments. CONCLUSIONS: Gibbosity seems to be a necessary condition for reactivity of L. gibba to potentised GA3. Further still unknown conditions seem to govern effect direction and the pattern of active and inactive potency levels. When designing new reproducibility studies, the physiological state of the test organism must be considered. Variability might be an interesting parameter to investigate effects of homeopathic remedies in basic research.

Systematic review of conceptual criticisms of homeopathy.

Homeopathy is the subject of frequent debates, especially in public media. This systematic review aims to give an overview of conceptual criticisms of homeopathy in the scientific literature. The literature search was conducted in four databases (EMBASE, PubMed, Web of Science, PhilPapers) on August 25, 2020. Included were peer reviewed articles in English or German criticising the basic concepts of homeopathy as main topic; excluded were articles criticising homeopathy primarily based on analysis of empirical clinical and/or preclinical data. The formal structure of publications included was evaluated regarding the recommended structure for scientific publications (IMRaD, acronym for 'Introduction', 'Methods', 'Results' and 'Discussion'). Arguments criticising the concepts of homeopathy were extracted and classified into groups. The literature search revealed 5139 articles, of which 15 articles (published between 1959 and 2020) met the inclusion criteria. These articles complied only partly with the IMRaD structure; just four articles considered with 8 or 9 IMRaD criteria the majority of the defined 11 IMRaD criteria. Extracted arguments against the concepts of homeopathy were classified into five groups: 'Conflict with current scientific principles and the foundations of modern medicine', 'Lack of a scientific basis', 'Arguments based on scientific theories', 'Ethical considerations and social consequences', 'Lack of empirical clinical evidence'. This classification is intended to provide a basis for future in-depth scientific analyses and discussions. Based on the number of articles found in the peer reviewed literature, it can be concluded that the on-going discussion about homeopathy in the public media is not reflected in a corresponding academic debate.

Investigation of arsenic-stressed yeast (Saccharomyces cerevisiae) as a bioassay in homeopathic basic research.

This study investigated the response of arsenic-stressed yeast (Saccharomyces cerevisiae) towards homeopathically potentized Arsenicum album, a duckweed nosode, and gibberellic acid. The three test substances were applied in five potency levels (17x, 18x, 24x, 28x, 30x) and compared to controls (unsuccussed and succussed water) with respect to influencing specific growth parameters. Five independent experiments were evaluated for each test substance. Additionally, five water control experiments were analyzed to investigate the stability of the experimental setup (systematic negative control experiments). All experiments were randomized and blinded. Yeast grew in microplates over a period of 38 h in either potentized substances or water controls with 250 mg/l arsenic(V) added over the entire cultivation period. Yeast's growth kinetics (slope, Et50, and yield) were measured photometrically. The test system exhibited a low coefficient of variation (slope 1.2%, Et50 0.3%, yield 2.7%). Succussed water did not induce any significant differences compared to unsuccussed water. Data from the control and treatment groups were both pooled to increase statistical power. In this study with yeast, no significant effects were found for any outcome parameter or any homeopathic treatment. Since in parallel experiments arsenic-stressed duckweed showed highly significant effects after application of potentized Arsenicum album and duckweed nosode preparations from the same batch as used in the present study, some specific properties of this experimental setup with yeast must be responsible for the lacking response.

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 Avogadro's 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.

Effects of homeopathic arsenicum album, nosode, and gibberellic acid preparations on the growth rate of arsenic-impaired duckweed (Lemna gibba L.).

This study evaluated the effects of homeopathically potentized Arsenicum album, nosode, and gibberellic acid in a bioassay with arsenic-stressed duckweed (Lemna gibba L.). The test substances were applied in nine potency levels (17x, 18x, 21x-24x, 28x, 30x, 33x) and compared with controls (unsuccussed and succussed water) regarding their influence on the plant's growth rate. Duckweed was stressed with arsenic(V) for 48 h. Afterwards, plants grew in either potentized substances or water controls for 6 days. Growth rates of frond (leaf) area and frond number were determined with a computerized image analysis system for different time intervals (days 0-2, 2-6, 0-6). Five independent experiments were evaluated for each test substance. Additionally, five water control experiments were analyzed to investigate the stability of the experimental setup (systematic negative control experiments). All experiments were randomized and blinded. The test system exhibited a low coefficient of variation (approximately equal to 1%). Unsuccussed and succussed water did not result in any significant differences in duckweed growth rate. Data from the control and treatment groups were pooled to increase statistical power. Growth rates for days 0-2 were not influenced by any homeopathic preparation. Growth rates for days 2-6 increased after application of potentized Arsenicum album regarding both frond area (p < 0.001) and frond number (p < 0.001), and by application of potentized nosode (frond area growth rate only, p < 0.01). Potencies of gibberellic acid did not influence duckweed growth rate. The systematic negative control experiments did not yield any significant effects. Thus, false-positive results can be excluded with high certainty. To conclude, the test system with L. gibba impaired by arsenic(V) was stable and reliable. It yielded evidence for specific effects of homeopathic Arsenicum album preparations and it will provide a valuable tool for future experiments that aim at revealing the mode of action of homeopathic preparations. It may also be useful to investigate the influence of external factors (e.g., heat, electromagnetic radiation) on the effects of homeopathic preparations.

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 Avogadro's 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.

Duckweed (Lemna gibba L.) as a test organism for homeopathic potencies.

OBJECTIVES: A bioassay with duckweed (Lemna gibba L.) was used to study the effects of homeopathic potencies on the plant's growth rate. Screening included 12 substances: argentum nitricum, copper sulfate, gibberellic acid, 3-indole acetic acid, kinetin, lactose, lemna minor, methyl jasmonate, metoxuron, phosphorus, potassium nitrate, and sulfur. Each substance was tested in the potency range 14x-30x. Controls were unsuccussed and succussed water. DESIGN: In randomized and blinded experiments, duckweed was grown in either potentized substances or water controls over 7 days. Frond (leaf) growth was measured regularly with a computerized image analysis system and growth rates were calculated for different time intervals (day 0-7, 0-3, 3-7). Additionally, a water control run with unsuccussed water as the only test substance was performed to determine the variability of the bioassay. RESULTS: For the water control run, the between-group coefficient of variance for groups of five replicates was 0.87% for the frond area-related average specific growth rate r(area) compared to 1.60% for the frond number-related average specific growth rate r(num). Thus, the former is the preferred parameter to be used. Of twelve tested substances, potentized argentum nitricum, phosphorus, and kinetin significantly (p<0.05, analysis of variance F-test) affected the main parameter: frond area-related average specific growth rate (day 0-7). Segmented area growth rates (day 0-3 or 3-7) were affected by potentized argentum nitricum, gibberellic acid, lactose, and phosphorus. CONCLUSIONS: The described experimental set-up with L. gibba as test organism appears to be a promising new model system to investigate effects of potentized substances. Yet larger sets of replication experiments with selected test substances and systematic negative controls are necessary to verify the effects found.

Development of a test system for homeopathic preparations using impaired duckweed (Lemna gibba L.).

OBJECTIVES: A bioassay with arsenic-stressed duckweed (Lemna gibba L.) was developed to study potentially regulative effects of homeopathic preparations. We compared potentized substances (nine different potency levels between 17 x and 33 x ) with two controls (unsuccussed and succussed water) regarding their influence on number- and area-related growth rate and color of fronds (leaves). Screening included 11 potentized substances: Arsenicum album, gibberellic acid, nosode, arsenic(V), phosphorus, Conchae, Acidum picrinicum, Argentum nitricum, Crotalus horridus, Hepar sulfuris, and Mercurius vivus naturalis. DESIGN: Duckweed was stressed with arsenic(V) for 48 hours. Afterwards, plants grew in either potentized substances or water controls for 6 days. Growth rate and color of fronds were determined with a computerized image analysis system for different time intervals (days 0-2, 2-6, 0-6). A systematic negative control experiment with unsuccussed water was used to investigate the stability of the bioassay. All experiments were randomized and blinded. RESULTS: Arsenicum album and nosode potencies increased frond number-related growth rate compared to controls (succussed water controls or pooled water controls [succussed and unsuccussed], p < 0.05, t test). Regarding color classification, no effects were observed. CONCLUSIONS: The experimental setup with L. gibba stressed by arsenic(V) provides a valuable tool to investigate regulative effects of potentized substances. In order to verify the effects of Arsenicum album and nosode potencies, further independent replication experiments are necessary.

Effects of potentised substances on growth rate of the water plant Lemna gibba L.

OBJECTIVES: This study investigated, whether the growth rate of Lemna gibba L. (duckweed) can be influenced by the application of homeopathic potencies of gibberellic acid, kinetin, argentum nitricum, and lemna minor. METHODS: Duckweed was grown in either potencies (14x-30x, decimal steps) or water controls (unsuccussed and succussed) over seven days. Frond (leaf-like structure) growth was measured using a non-destructive image analysis system. Growth rates were calculated for three time intervals (0-7, 0-3, 3-7 days). Five to six independent, randomized and blinded experiments were analysed for each of the four tested substances. Water control experiments were performed repeatedly to test the reliability of the experimental set-up (systematic negative controls). RESULTS: The systematic negative control experiments did not yield any significant effects. Hence, false positive results could be excluded. The test system had a low coefficient of variation (1.5%). Out of the four tested substances gibberellic acid had the most pronounced effect (p=0.0002, F-test) on the main outcome parameter frond growth rate (r(area) day 0-7). Potency levels 15x, 17x, 18x, 23x and 24x reduced growth rate of Lemna gibba (p<0.05 against the pooled water control, LSD test). CONCLUSIONS: Lemna gibba may be considered as a suitable test organism for further studies on the efficacy of homeopathic potencies. Evidence accumulates, that adjacent potency levels may strongly differ in their biological activity. Potential consequences for therapeutical application might be worth investigating.

Effects of potentised substances on growth kinetics of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

BACKGROUND: Homeopathic potencies are used as specific remedies in complementary medicine. Since the mode of action is unknown, the presumed specificity is discussed controversially. OBJECTIVE: This study investigated the effects of potentised substances on two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe, in a stable and reliable test system with systematic negative controls. MATERIALS AND METHODS: Yeast cells were cultivated in either potentised substances or water controls in microplates and their growth kinetics were measured photometrically. Water control runs were performed repeatedly to investigate the stability of the experimental set-up (systematic negative controls). RESULTS: 4 out of 14 screened substances seem to have affected the growth curve parameters slope or yield. Out of these substances, azoxystrobin and phosphorus were chosen for 8 further replication experiments, which partly confirmed the results of the screening. On the average of all experiments, azoxystrobin affected the slope of the growth curve of Saccharomyces cerevisiae (p < 0.05), and phosphorus affected the slope of the growth curve of Schizosaccharomyces pombe (p < 0.05). No effects were seen in the water control runs. In addition, significant interactions between treatment with potentised substances and experiment number were observed in all experiments with potentised substances (p < 0.01), but not in the water control runs. CONCLUSIONS: Both yeast species reacted to certain potentised substances by changing their growth kinetics. However, the interactions found point to additional factors of still unknown nature, that modulate the effects of potentised substances. This stable test system with yeasts may be suitable for further studies regarding the efficacy of homeopathic potencies.