Self-assembled Patterns Formed in Evaporating Droplets to Analyze Bi-component Homeopathic Preparations in the Low Dilution Range.

BACKGROUND: Homeopathic complex remedies, composed of several homeopathic medicines in the low potency range, are frequently used in the treatment of a number of common disorders. At the same time, they represent an almost unexplored area of research. Are complex remedies just additive mixtures of the components, or are there interactions between the latter leading to new properties of the complex? METHODS: In the present study, we analyzed as an example the simple bi-component complex, Luffa 4x - Mercurius bijodatus 9x, by means of patterns from evaporated droplets and tested what influences the complex's single compounds have upon the patterns and if there are any interactions. For this purpose, we compared in a series of five experiments patterns from evaporated droplets of the complex, Luffa 4x - Mercurius bijodatus 9x, and three comparison samples in which one or both of the complex's compounds were replaced by potentized solute. The patterns were photographed and evaluated for their gray-level distribution and texture using the software ImageJ. The experimental set-up's stability was tested by means of systematic control experiments. RESULTS: We found that Mercurius bijodatus 9x significantly influenced the patterns of Luffa 4x, increasing their homogeneity; at the same time, the patterns of Mercurius bijodatus 9x combined with solvent were more heterogeneous than those obtained from a control consisting of two pure solvents. CONCLUSION: In this phenomenological assay, the complex Luffa 4x - Mercurius bijodatus 9x does not correspond to a simple addition of the components. The exact nature of the underlying interaction needs to be elucidated in further investigations.

Deep learning applied to analyze patterns from evaporated droplets of Viscum album extracts.

This paper introduces a deep learning based methodology for analyzing the self-assembled, fractal-like structures formed in evaporated droplets. To this end, an extensive image database of such structures of the plant extract Viscum album Quercus [Formula: see text] was used, prepared by three different mixing procedures (turbulent, laminar, and diffusion based). The proposed pattern analysis approach is based on two stages: (1) automatic selection of patches that exhibit rich texture along the database; and (2) clustering of patches in accordance with prevalent texture by means of a Dense Convolutional Neural Network. The fractality of the patterns in each cluster is verified through Local Connected Fractal Dimension histograms. Experiments with Gray-Level Co-Occurrence matrices are performed to determine the benefit of the proposed approach in comparison with well established image analysis techniques. For the investigated plant extract, significant differences were found between the production modalities; whereas the patterns obtained by laminar flow showed the highest fractal structure, the patterns obtained by the application of turbulent mixture exhibited the lowest fractality. Our approach is the first to analyze, at the pure image level, the clustering properties of regions of interest within a database of evaporated droplets. This allows a greater description and differentiation of the patterns formed through different mixing procedures.

Proceedings of the XXXIV GIRI meeting and V International Workshop on Homeopathy [Editorial]

The XXXIV annual GIRI Meeting was held for the first time together with the V International Workshop on Homeopathy (IWH) in frames of an academic-scientific partnership between the Brazilian Public University (Universidade Federal do Rio de Janeiro, UFRJ) and the GIRI society (International Research Group on Very Low Dose and High Dilution Effects). This event occurred on December 2ndto 4th2021 as online conference. Thanks to the partnership as well as the use of the virtual platform, the GIRI meeting 2021 reached the highest visibility in the whole history of already 34 meetings (there were ca. 600 inscriptions and more than 14 000 page views on the last conference day). At the meeting 12 abstractswere presented as oral contributions and further 34 as e-posters. The 5thEdition of International Workshop on Homeopathy regarded "The scientific evidence of homeopathy in health care" and provided the possibilities to exchange scientific experiences, todiscuss many research details, and to meet other researchers involved into basic and clinical homeopathic research from all over the world. The scientific sections were dedicated to the following subjects: -Homeopathy teaching and practice; -Chemical and physical models; -In vivoand in vitromodels; -Clinical trials.We are convinced that the content of the present special issue of the International Journal High Dilution Research dedicated to the memories of XXXIV GIRI meeting will bring new insights to the homeopathic scientific community.© International Journal of High Dilution Research. Not for commercial purposes.

Irene Schlingensiepen and Maria Olga Kokornaczyk [Editorial]

The 2022 International Society for Low Dose and High Dilution Research -GIRI-meeting was a joint meeting held alongside the annual Science Meets Homeopathy -SMH -meeting organized by the International Academy of Science in Homeopathy and Integrative Medicine, founded by Irene Schingensiepen. The two meetings took place from June1st to 4th in Berlin/Germany. For the convenience of participants from other countries, the meetings also allowed online participation; while the live events gave the GIRI & SMH members, researchers from different Universities and hospitals from European countries and South America, and homeopathy practitionersthe opportunity to meet again for the first time after the pandemic restrictions. About 40 participants took part in the meetings.The quiet and beautiful location near the Havelsee enabled concentrated work in an inviting and friendly atmosphere.The scientific program included contributions on basic and clinical research, case reports, and theorieson homeopathy's mode of action. Furthermore, there was one presentation onthe Natura Academy [1] in Germany giving the possibility of obtaining the bachelor's degree in integrative medicine and another one ona new initiative for raising awareness for the potential offered byalternative and complementarymedicine. A total of 27 full oral contributions and eight short poster presentations were presented over the four days.This year the presentation of overview lectures summarizingthe main results archived by the different research groups or scientists was encouraged. Several such lectures focused on water research and reported, among other things, on the water structures and the fourth phase of water. There were also overviewpresentations on spectroscopic analysisof dilutions, application of the droplet evaporation method in homeopathyresearch, vitality of crops treated with homeopathic preparations,and veterinary biotypology. As with the last two GIRI meetings, this year there were several contributions dealing with the use of homeopathic treatments in COVID patients.Furthercontributionsintroduceda new experimental setup with immobilized solvatochromic dyes, electronic spectra and UV/VIS measurements of high dilutions, and theveterinary use of homeopathic remedies in snakes,catsanddogs. Important findings were presented on the role of the standard deviation in basic experiments with potentised substances.Regardingclinical research there were contributions on the methodology of drug prescription, long-time evaluation of case studies, and one on the periodic table and its importance in homeopathy. Finally, two lectures focused on homeopathy's possible mode of action and put it inrelationto anthroposophical conceptsand clinical research on hormesis.

Series of experiments performed with the droplet evaporation method on low potencies

Homeopathic preparations in low potencies, containing still measurable quantities of the starting substance, constitute a unique research field in homeopathic basic research. Here a series of experiments is presented carried out by means of the droplet evaporation method (DEM), investigating the specificity of the method, and presumed effects of the succussion procedure applied in the production of homeopathic preparations. Methods:DEM analysis consisted in the evaporation of droplets of the potencies perse placed on microscope slides. Resulting patterns were photographed. Images were evaluated by means of ImageJ software, by measuring grey level distribution, texture, and fractality. The experimentation consisted of four series: (i) screening (1x­6x potencies from 19 substances), (ii) differentiation experiments (2x­6x potencies of Echinacea, Baptisia, Luffa, and Spongia), (iii) differentiation between succussed (100 or 10 times) and unsuccussed samples (Echinacea 2x, Baptisia 3x, Baptisia 4x, Luffa 4x, and Spongia 6x). (iv) investigation of the influence upon the patterns of single compounds present in a remedy complex. The experimental set-up stability was examined by systematic positive control experiments. Results:(i) Homeopathic preparations of mineral origin showed the greatest form variety, whereas those of vegetal origin created fractal patterns in the potency range 2x­4x. (ii) Differentiation of potencies of different origin at the same dilution level was possible from 2x to 4x. (iii) In all potency levels, succussed (100 and 10 times) and unsuccussed variants could be significantly differentiated. Significant differences between all variants were found in some cases in potency levels 4x and higher. In general, application of succussion reduced size, homogeneity, and complexity of the DEM patterns. (iv) Patterns of a remedy complex Luffa 4x -Mercurius bijodatum 9x showed a clear predominance of the Luffa 4x; however also the second component, present in a much lower concentration, influenced significantly the pattern of the remedy complex as also differed significantly from the pattern of succussed water control. Conclusions:The results suggest that DEM is a suitable tool for scientific investigation of homeopathic preparations in the low potency range. DEM might be applied to assess further research questions, such different potentization procedures (vessel shape, overhead volume, material), storing time, and difference between batches.

Formation of Self-organized Structures in Drying Droplets: a Promising Analytical Tool for Homeopathy Basic Research

The droplet evaporation method (DEM) is based on the evaporation-induced pattern formation in droplets and is applied mainly for medical diagnosis[1].Here, we present aseries of experiments performed by our team showing DEMs potential also forhomeopathy basic research, in particular, for the investigation of(i) low potencies, (ii) low potency complexes (physical model), and (iii) the action of high potencies (plant-based model).Methods:(i) DEM differentiated significantly between Luffa, Baptisia, Echinacea, and Spongiauntil 4x[2]. Furthermore, the patterns varied in function of the numberof succussion strokes (0, 10, or 100) applied during potentization[3]. The performance of chaotic succussions vs. laminar flow vs. slight mixing during the potentization of Viscum album quercus3x influenced the DEM patterns; the chaotic succussions reduced, whereas laminar flow enhanced the patterns complexity vs. the unsuccussed control.(ii) The addition of Mercurius bijodatus9x to Luffa4x changed significantly the DEM patterns, even if the material quantity present in the 9x potency lied far beyond that of ultrapure water.(iii) Leakages obtained by placing healthy or arsenic-damaged wheat-seeds into Arsenicum album45x orheat-damaged intoZincum metallicum30c vs. water created significantly different DEM structures [4, 5]. Results:The damaged seeds put into the potency created structures characterized by a higher complexity than those obtained from damaged seeds put into control water. Furthermore, the potency action seemed to increase with rising numbers ofsuccussion strokes applied during potentization,ascould be shown by means of DEM patterns and germination rate using the same wheat-seed model[6].In all our studies, the pattern evaluation was computerized (texture and fractal analysis performed by means of ImageJ) or based on deep-learning algorithms and the robustness of the experimental system was checked by means of systematic control experiments.Conclusion:DEM together with other similarmethods has also been reviewed by our team for what concerns theapplication in homeopathy basic research[7].

Diagnostic tests based on pattern formation in drying body fluids - A mapping review.

There are numerous diagnostic tests based on pattern formation in desiccating body fluids, where the pattern or some of its characteristics constitute the diagnostic test outcome. However, partially due to the development in different time periods, and partially due to publications in languages different from English, most of these diagnostic tests exist as separate approaches and have never been grouped, systematized, nor compared with each other. In the present mapping review, we performed a wide literature search with the aim to collect all diagnostic tests based on pattern formation in desiccating body fluids. Furthermore, we grouped the identified diagnostic tests according to their experimental protocols, type of body fluids investigated, and target conditions, and propose so for the first time a classification of different diagnostic tests based on pattern formation in desiccating body fluids. The literature search revealed 1603 publications, out of which 141 were included into the review. Following three main classification criteria (way of deposition of the fluid for desiccation, addition of reagents, and spatial restrictions during evaporation), we identified six different methods; following a further classification concerning the analyzed body fluid we identified 30 different diagnostic tests based on pattern formation in evaporating body fluids. Amongst these tests are well-known procedures such as ferning tests (tear ferning for the assessment of tear film quality, saliva and cervical mucus ferning for the detection of the fertile period, and amniotic fluid ferning for the diagnosis of fetal membrane rupture), whereas other tests are less well-established. In the latter group, the most frequently investigated body fluids were serum, saliva, and blood; the most frequently addressed target conditions were cancer, inflammation, and benign tumors. We recommend conducting further systematic reviews and meta-analyses concerning groups of methods addressing the same target condition.

Impact of succussion on pharmaceutical preparations analyzed by means of patterns from evaporated droplets.

The aim of the present study was to investigate if patterns obtained from evaporating droplets of pharmaceutical preparations reveal the impact of succussion on such medicinal products. For this purpose, five pharmaceutical preparations (Echinacea 10-2, Baptisia 10-3, Baptisia 10-4, Luffa 10-4, and Spongia 10-6) were prepared according to the European Pharmacopoeia guidelines for the production of homeopathic remedies, in three variants each: with varying numbers of succussion strokes (i) 100, (ii) 10 (succussed samples), and (iii) zero (gently mixed, unsuccussed sample). System stability was studied by means of systematic positive control experiments. Patterns were evaluated by means of computerized image analysis regarding grey level distribution, texture, and fractality. For all investigated pharmaceutical preparations, significant differences were found between the succussed and gently mixed samples; whereas, all three samples (prepared with 100, 10 and zero succussion strokes) could be significantly differentiated for Luffa 10-4 and Spongia 10-6 for one image evaluation parameter each. Control experiments showed a reasonable stability of the experimental set-up.

Phenomenological Characterization of Low-Potency Homeopathic Preparations by Means of Pattern Formation in Evaporating Droplets.

BACKGROUND: Evaporation-induced pattern formation in droplets has been applied to test effects of high potencies. Here we propose for the first time the droplet evaporation method (DEM) as a tool to characterize low potencies on a qualitative and quantitative basis. MATERIALS AND METHODS: The present investigation consisted of: (1) screening of 18 different substances of mineral, vegetal, and animal origin in the 1x to 6x potency range; choice of four substances with characteristic pattern-forming properties; (2) replication experiments aiming at the differentiation of four homeopathic preparations at the same potency levels (2x-6x); and (3) control experiments performed on three preparations. The DEM experimental protocol consisted of the evaporation of droplets of the analyzed potency per se, placed on microscope slides and in controlled conditions. The resulting patterns were photographed and subjected to computerized image analysis. RESULTS: The screening experiments yielded a wide variety of patterns. Homeopathic preparations of mineral origin showed the largest variety of forms, whereas potencies of vegetal origin mostly created dendritic patterns, probably due to diffusion-limited aggregation. The here-analyzed image analysis variables (gray-level distribution, entropy, and inverse difference moment) allowed a highly significant differentiation of patterns prepared from four substances (Echinacea, Baptisia, Luffa, and Spongia) at the same potency levels in the range 2x to 4x, whereas patterns obtained from potencies 5x and 6x could no longer be differentiated and resembled the pattern of pure solute (purified water). The control experiments showed reasonable experimental model stability. CONCLUSIONS: DEM seems to be a promising tool for qualitative phenomenological characterization of homeopathic preparations in low potency. We propose the application of the current experimental model for investigating further research topics in this field, such as the comparison of potencies versus simple dilutions or the contribution of component remedies to the patterns formed by homeopathic combination medicines.

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.

HRI Malta 2017-Cutting Edge Research in Homeopathy: HRI's Third International Research Conference in Malta.

The third international conference on "Cutting Edge Research in Homeopathy" organised by the Homeopathy Research Institute (HRI) was held on the inspiring and historic island of Malta from 9th to 11th of June, 2017. One hundred and two abstracts underwent peer review by the HRI Scientific Advisory Committee and external experts to produce the programme of 36 oral presentations and 37 posters, presented by researchers from 19 countries. The 2.5-day programme covered a diverse range of topics, including quantitative and qualitative clinical research, basic research, veterinary research, and provings. These intensive plenary and parallel sessions were interspersed with multiple opportunities for delegates to discuss and exchange ideas, in particular through interactive panel discussions and a pre-conference workshop. The continuing commitment of the homeopathy research community to generate high-quality studies in this rapidly evolving field was clear. In this conference report, we present highlights from this memorable event.

Number of succussion strokes affects effectiveness of ultra-high-diluted arsenic on in vitro wheat germination and polycrystalline structures obtained by droplet evaporation method.

OBJECTIVES: The aim of this study is to investigate whether the number of succussion strokes applied after each dilution step when preparing the homeopathic treatments influences the effectiveness of ultra-high-diluted (UHD) arsenic trioxide at the 45th decimal dilution/dynamization (As2O3 45x). DESIGN: Wheat seeds, previously stressed with ponderal As2O3, were treated with: As2O3 45x, H2O 45x (dynamized control), or pure water (negative control). The succussion was done manually, and various succussion durations (numbers of strokes) were tested for each treatment. Treatment effectiveness was tested blind using the in vitro germination test and the droplet evaporation method (DEM). Data were processed by the Poisson test (germination test) and by two-way analysis of variance (DEM). MAIN OUTCOME MEASURES: We evaluated both the in vitro germination rate, by counting the non-germinated seeds, and the complexity of polycrystalline structures (PCS) (local connected fractal dimension (LCFD)) obtained by evaporating leakage droplets from stressed seeds that had been watered with the different treatments. RESULTS: We observed a highly significant increase in germination rate when the number of strokes (NS) was ≥32 for both As2O3 45x and H2O 45x, and a significant increase in the LCFD of PCS for As2O3 45x when the NS was ≥32 and for H2O 45x when it was 70. CONCLUSIONS: Both experimental approaches showed increased effectiveness for treatments prepared with a higher number of succussion strokes. These results indicate that succussion may have an important influence on treatment effectiveness, and so highlight the need for further research.

Polycrystalline structures formed in evaporating droplets as a parameter to test the action of Zincum metallicum 30c in a wheat seed model.

BACKGROUND: Polycrystalline structures formed inside evaporating droplets of different biological fluids have been shown sensitive towards various influences, including ultra high dilutions (UHDs), representing so a new approach potentially useful for basic research in homeopathy. In the present study we tested on a wheat seed model Zincum metallicum 30c efficacy versus lactose 30c and water. MATERIALS AND METHODS: Stressed and non-stressed wheat seeds were watered with the three treatments. Seed-leakage droplets were evaporated and the polycrystalline structures formed inside the droplet residues were analyzed for their local connected fractal dimensions (LCFDs) (measure of complexity) using the software ImageJ. RESULTS: We have found significant differences in LCFD values of polycrystalline structures obtained from stressed seeds following the treatments (p<0.0001); Zincum metallicum 30c lowered the structures' complexity compared to lactose 30c and water. In non-stressed seeds no significant differences were found. CONCLUSIONS: The droplet evaporation method (DEM) might represent a potentially useful tool in basic research in homeopathy. Furthermore our results suggest a sensitization of the stressed model towards the treatment action, which is conforming to previous findings.

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.

Status quo and future research challenges on organic food quality determination with focus on laboratory methods.

Organic food quality determination needs multi-dimensional evaluation tools. The main focus is on the authentication as an analytical verification of the certification process. New fingerprinting approaches such as ultra-performance liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, direct analysis in real time-high-resolution mass spectrometry as well as crystallization with and without the presence of additives seem to be promising methods in terms of time of analysis and detecting organic system-related parameters. For further methodological development, a system approach is recommended, which also takes into account food structure aspects. Furthermore, the authentication of processed organic samples needs more consciousness, hence most of organic food is complex and processed.

Approximate bilateral symmetry in evaporation-induced polycrystalline structures from droplets of wheat grain leakages and fluctuating asymmetry as quality indicator.

The present paper reports on an observation that dendrite-like polycrystalline structures from evaporating droplets of wheat grain leakages exhibit bilateral symmetry. The exactness of this symmetry, measured by means of fluctuating asymmetry, varies depending on the cultivar and stress factor influence, and seems to correspond to the seed germination rate. In the bodies of plants, animals, and humans, the exactness of bilateral symmetry is known to reflect the environmental conditions of an organism's growth, its health, and its success in sexual selection. In polycrystalline structures, formed under the same conditions, the symmetry exactness depends on the properties of the crystallizing solution such as the composition and viscosity; however, it has never been associated with sample quality. We hypothesize here that, as in living nature, the exactness of approximate bilateral symmetry might be considered a quality indicator also in crystallographic methods applied to food quality analysis.

Self-organized crystallization patterns from evaporating droplets of common wheat grain leakages as a potential tool for quality analysis.

We studied the evaporation-induced pattern formation in droplets of common wheat kernel leakages prepared out of ancient and modern wheat cultivars as a possible tool for wheat quality analysis. The experiments showed that the substances which passed into the water during the soaking of the kernels created crystalline structures with different degrees of complexity while the droplets were evaporating. The forms ranged from spots and simple structures with single ramifications, through dendrites, up to highly organized hexagonal shapes and fractal-like structures. The patterns were observed and photographed using dark field microscopy in small magnifications. The evaluation of the patterns was performed both visually and by means of the fractal dimension analysis. From the results, it can be inferred that the wheat cultivars differed in their pattern-forming capacities. Two of the analyzed wheat cultivars showed poor pattern formation, whereas another two created well-formed and complex patterns. Additionally, the wheat cultivars were analyzed for their vigor by means of the germination test and measurement of the electrical conductivity of the grain leakages. The results showed that the more vigorous cultivars also created more complex patterns, whereas the weaker cultivars created predominantly poor forms. This observation suggests a correlation between the wheat seed quality and droplet evaporation patterns.