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

Abstract

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.

Keywords

CuCl₂ crystallization - fractal dimension - bio-assay - systematic negative control experiments - potentized substances

• References

• 1 Sehon S, Stanley D. Evidence and simplicity: why we should reject homeopathy. J Eval Clin Pract 2010; 16: 276-281
• 2 Ücker A, Baumgartner S, Sokol A, Huber R, Doesburg P, Jäger T. Systematic review of plant-based homeopathic basic research: an update. Homeopathy 2018; 107: 115-129
• 3 Baumgartner S. The state of basic research on homeopathy. In: Henning A, Witt C. eds. New Directions in Homeopathy Research: Advice From an Interdisciplinary Conference. KVC Verlag; 2009
• 4 Ücker A, Reif M, Doesburg P, Martin D, Baumgartner S. Standard deviation as outcome parameter in plant-based test systems investigating homeopathic preparations in high potency levels. Homeopathy 2024; 113: A12
• 5 Baumgartner S, Doesburg P, Scherr C, Andersen J-O. Development of a biocrystallisation assay for examining effects of homeopathic preparations using cress seedlings. Evid Based Complement Alternat Med 2012; 2012: 125945
• 6 Doesburg P, Andersen J-O, Scherr C, Baumgartner S. Empirical investigation of preparations produced according to the European Pharmacopoeia monograph 1038. Eur J Pharm Sci 2019; 137: 104987
• 7 Baumgartner S, Heusser P, Thurneysen S. Methodological standards and problems in preclinical homoeopathic potency research. Forsch Komplementarmed 1998; 5: 27-32
• 8 Gallinet JP, Gauthier-Manuel B. Wetting of a glass surface by protein adsorption induces the crystallization of an aqueous cupric chloride solution. J Colloid Interface Sci 1992; 148: 155-159
• 9 Schweizer F. Beobachtungen bei der Biokristallisation von Glykogen. Der Einfluss des Verhältnisses Zusatz/Kupferchlorid auf die Ausbildung der dendritischen Nadeln. Elem Nat 2007; 87: 76-89
• 10 Kahl J, Busscher N, Doesburg P, Mergardt G, Huber M, Ploeger A. First tests of standardized biocrystallization on milk and milk products. Eur Food Res Technol 2009; 229: 175-178
• 11 Busscher N, Doesburg P, Mergardt G, Sokol A, Kahl J, Ploeger A. Crystallization patterns of an aqueous dihydrate cupric chloride solution in the presence of different amounts of bovine serum albumin. J Cryst Growth 2020; 529: 125272
• 12 Geier U. Pflanzenorganbildtypen in Kupferchloridkristallisation und Steigbild. Leb Erde 2005; 5: 42-45
• 13 Busscher N, Kahl J, Doesburg P, Mergardt G, Ploeger A. Evaporation influences on the crystallization of an aqueous dihydrate cupric chloride solution with additives. J Colloid Interface Sci 2010; 344: 556-562
• 14 Busscher N, Kahl J, Ploeger A. From needles to pattern in food quality determination. J Sci Food Agric 2014; 94: 2578-2581
• 15 Huber M, Andersen J-O, Kahl J. et al. Standardization and validation of the visual evaluation of biocrystallizations. Biol Agric Hortic 2010; 27: 25-40
• 16 Doesburg P, Huber M, Andersen J-O. et al. Standardization and performance of a visual Gestalt evaluation of biocrystallization patterns reflecting ripening and decomposition processes in food samples. Biol Agric Hortic 2015; 31: 128-145
• 17 Doesburg P, Fritz J, Athmann M. et al. Kinesthetic engagement in Gestalt evaluation outscores analytical ‘atomic feature’ evaluation in perceiving aging in crystallization images of agricultural products. PLoS One 2021; 16: e0248124
• 18 Fritz J, Athmann M, Bornhütter R. et al. Analytical perception and kinesthetic engagement in evaluation of copper chloride crystallization patterns of wheat, grape juice and rocket samples from conventional, organic and biodynamic cultivation. Chem Biol Technol Agric 2022; 9: 103
• 19 Andersen J-O, Henriksen CB, Laursen J, Nielsen AA. Computerised image analysis of biocrystallograms originating from agricultural products. Comput Electron Agric 1999; 22: 51-69
• 20 Doesburg P, Nierop AF. Development of a structure analysis algorithm on structures from CuCl2.2H2O crystallization with agricultural products. Comput Electron Agric 2013; 90: 63-67
• 21 Unluturk S, Pelvan M, Unluturk MS. The discrimination of raw and UHT milk samples contaminated with penicillin G and ampicillin using image processing neural network and biocrystallization methods. J Food Compos Analysis 2013; 32: 12-19
• 22 Piva MT, Lumbroso S, Sieso V. et al. Cupric chloride crystallization with human blood–study of pictures obtained in different pathologies. Elem Nat 1994; 61: 25-39
• 23 Shibata T, Matsumoto S, Kogure M. et al. Effects of diabetic human blood addition on morphology of cupric chloride dendrites grown from aqueous solutions. J Cryst Growth 2000; 219: 423-433
• 24 Kahl J, Busscher N, Mergardt G, Mäder P, Torp T, Ploeger A. Differentiation of organic and non-organic winter wheat cultivars from a controlled field trial by crystallization patterns. J Sci Food Agric 2015; 95: 53-58
• 25 Fritz J, Athmann M, Meissner G. et al. Quality assessment of grape juice from integrated, organic and biodynamic viticulture using image forming methods. OENO One 2020; 54: 373-391
• 26 Baars E, Baars T. Towards a philosophical underpinning of the holistic concept of integrity of organisms within organic agriculture. NJAS Wagening J Life Sci 2007; 54: 463-477
• 27 Carstensen JM. Description and simulation of visual texture. . PhD Thesis, Institute of Mathematical Statistics and Operations Research, Technical University of Denmark; Lyngby, 1992
• 28 Andersen J-O, Laursen J, Koelster P. A refined biocrystallization method applied in a pictomorphological investigation of a polymer. Elem Nat 1998; 68: 1-20
• 29 Kahl J. Entwicklung, in-house Validierung und Anwendung des ganzheitlichen Verfahrens Biokristallisation für die Unterscheidung von Weizen-, Möhren- und Apfelproben aus unterschiedlichem Anbau und Verarbeitungsschritten. Habilitation: University of Kassel; 2007
• 30 Baumgartner SM, Flückiger H. Biologische Wirksamkeit des Iscador-spezifischen Mischprozesses von Winter- und Sommermistelsaft. In: Alban S, Becker H, Holzgrabe U. et al. Die Mistel in der Tumortherapie. KVC Verlag; 2001
• 31 Haralick RM, Shanmugam K, Dinstein IH. Textural features for image classification. IEEE Trans Syst Man Cybern 1973; SMC-3: 610-621
• 32 Karperien A. FracLac for ImageJ-FracLac advanced user's manual. 2007 . Available at: http://rsb.info.nih.gov/ij/plugins/fraclac/fraclac-manual.pdf
• 33 Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Meth 2012; 9: 671-675
• 34 Mandelbrot B. The fractal geometry of nature. Volume 1. WH Freeman; New York: 1982
• 35 Team R Development Core. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2009. . Available at: http://www.R-project.org
• 36 Wickham H. ggplot2—Elegant Graphics for Data Analysis. Springer; 2009
• 37 Shri M, Kumar S, Chakrabarty D. et al. Effect of arsenic on growth, oxidative stress, and antioxidant system in rice seedlings. Ecotoxicol Environ Saf 2009; 72: 1102-1110
• 38 Hasanuzzaman M, Nahar K, Alam MM, Roychowdhury R, Fujita M. Physiological, biochemical, and molecular mechanisms of heat stress tolerance in plants. Int J Mol Sci 2013; 14: 9643-9684
• 39 Ampilova N, Soloviev I, Barth J-G. Application of fractal analysis methods to images obtained by crystallization modified by an additive. J Meas Eng 2019; 7: 48-57
• 40 Vermeulen F. Konkordanz der Materia Medica. Haarlem (NL), Emryss bv Publishers; 2000
• 41 Möllinger H, Schneider R, Walach H. Homeopathic pathogenetic trials produce specific symptoms different from placebo. Forsch Komplement Med 2009; 16: 105-110
• 42 Scherr C, Baumgartner S, Spranger J, Simon M. Effects of potentised substances on growth kinetics of Saccharomyces cerevisiae and Schizosaccharomyces pombe . Forsch Komplement Med 2006; 13: 298-306
• 43 Scherr C, Simon M, Spranger J, Baumgartner S. Effects of potentised substances on growth rate of the water plant Lemna gibba L. Complement Ther Med 2009; 17: 63-70
• 44 Jäger T, Scherr C, Simon M, Heusser P, Baumgartner S. Effects of homeopathic arsenicum album, nosode, and gibberellic acid preparations on the growth rate of arsenic-impaired duckweed (Lemna gibba L.). ScientificWorldJournal 2010; 10: 2112-2129