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Objectives: From a pharmaceutical point of view, we see a need to develop stable preclinical test systems to identify and investigate effects of potentized remedies as used in Anthroposophic Medicine and Homeopathy. We evaluated a plant bioassay regarding its capacity to distinguish homeopathic remedies from placebo, applied as sucrose pillules., Methods: Pea seed (Pisum sativum L) was soaked for 24 hours in water with dissolved homeopathic or placebo pillules, or in water only. Shoot length was measured 14 days after planting and treatment groups were compared by analysis of variance (ANOVA). The stability of the system was validated by systematic negative control experiments., Results: The system is suitable to test a common application form - sucrose pillules - of a potentized preparation without influence of the pharmaceutical carrier substance. A screening of 13 potentized preparations revealed Calcium carbonicum to affect pea shoot growth (p < 0.05). Three independent series of main experiments were performed with potentized Calcium carbonicum to assess reproducibility. Meta-analysis of all data revealed significant effects of Calcium carbonicum 12c and 30c on pea shoot growth (p < 0.05), which were however dependent on the date of experiment and/or the experimental series., Conclusions: Potentized Calcium carbonicum, applied as sucrose pillules, influenced pea shoot growth in the assay investigated. However, due to the small effect size and due to the modulation of the effects by still unknown external factors, further optimization of this bioassay is necessary to be used in pharmaceutical quality control or in investigating the biological or pharmaceutical mode of action of potentized preparations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.)

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.

Background: In ecotoxicological and environmental studies Lemna spp. are used as test organisms due to their small size, rapid predominantly vegetative reproduction, easy handling and high sensitivity to various chemicals. However, there is not much information available concerning spatial and temporal stability of experimental set-ups used for Lemna bioassays, though this is essential for interpretation and reliability of results. We therefore investigated stability and natural variability of a Lemna gibba bioassay assessing area-related and frond number-related growth rates under controlled laboratory conditions over about one year., Methodology/principal Findings: Lemna gibba L. was grown in beakers with Steinberg medium for one week. Area-related and frond number-related growth rates (r(area) and r(num)) were determined with a non-destructive image processing system. To assess inter-experimental stability, 35 independent experiments were performed with 10 beakers each in the course of one year. We observed changes in growth rates by a factor of two over time. These did not correlate well with temperature or relative humidity in the growth chamber. In order to assess intra-experimental stability, we analysed six systematic negative control experiments (nontoxicant tests) with 96 replicate beakers each. Evaluation showed that the chosen experimental set-up was stable and did not produce false positive results. The coefficient of variation was lower for r(area) (2.99%) than for r(num) (4.27%)., Conclusions/significance: It is hypothesised that the variations in growth rates over time under controlled conditions are partly due to endogenic periodicities in Lemna gibba. The relevance of these variations for toxicity investigations should be investigated more closely. Area-related growth rate seems to be more precise as non-destructive calculation parameter than number-related growth rate. Furthermore, we propose two new validity criteria for Lemna gibba bioassays: variability of average specific and section-by-section segmented growth rate, complementary to average specific growth rate as the only validity criterion existing in guidelines for duckweed bioassays.

European Pharmacopoeia monograph 2371 describes the production of homeopathic preparations. A specific efficacy of these preparations in high dilution levels is questionable in view of basic scientific principles. There is empirical evidence for such effects, for example in a Lemna-intoxication bioassay published 2010. To test the replicability and robustness of this bioassay, we conducted two experimental series (five independent blinded and randomised experiments each). The specimen of Lemna gibba L., clone-number 9352, were stressed in arsenic solution for 48 h (158 mg/L AsNa2HO4 (250 mg/L in series 2)), then grew in either As2O3 preparations produced according to Eu. Pharm. Monogr. 2371 or control solution. Comparing the area-related relative growth rate of day 3–9 (rgr 3–9) between treatment and control groups for each series showed differences that were not significant in series 1 (p = 0.10), significant in series 2 (p = 0.04) and significant in the pooled data of both series (p < 0.01). The effect direction (rgr 3–9 increase) was comparable to experiments of 2010, but the effect size was smaller, likely due to a changed light cycle. These results are not compatible with the hypothesis that the application of European Pharmacopoeia monograph 2371 results in pharmaceutical preparations without specific effects. Further studies are needed to investigate a potential mode of action explaining these effects. [ABSTRACT FROM AUTHOR]

Background: Macrophages are highly versatile cells that play an important role in tumour microenvironment. Tumour associated macrophages (TAMs) have been linked to both, good or bad prognosis of several cancer types depending on their number, composition and polarization. Viscum album lipophilic extract (VALE) contains several pentacyclic triterpenes known to modulate the activity of monocytes and other immune cells and to exhibit anticancer properties. In our in vitro study, we investigated the effect of tumour cell lines on macrophage polarization and monocyte chemotactic transmigration and examined the modulatory potential of VALE and its predominant triterpene oleanolic acid (OA). Methods: Human peripheral blood monocytes were differentiated into monocyte derived macrophages (MDM) using M-CSF and polarized into M1 by IFN-γ and LPS and into M2 macrophages by IL-4 and IL-13 or by co-culture with two different tumour cell lines. Polarized macrophages were subsequently treated with VALE or OA. Phenotypic markers and cytokines were assessed by flow cytometry and immunoanalysis. Migration of human peripheral blood monocytes induced by monocyte chemotactic protein-1 (MCP-1) or supernatants of different tumour cell lines under the influence of VALE or OA was measured in a chemotaxis transmigration assay. Results: In vitro polarized M1 and M2 type macrophages revealed specific phenotypic patterns and tumour cell co-cultured MDM displayed ambiguous phenotypes with M1 as well as M2 associated markers. VALE and OA showed modest influence on cell surface marker profile and cytokine expression of tumour cell co-cultured macrophages. All tumour cell supernatants markedly enhanced the migratory activity of monocytes. VALE and OA significantly inhibited MCP-1 induced monocyte transmigration, whereas monocyte migration initiated by tumour cell derived supernatants was not affected. Conclusions: In our study we reconfirmed that co-culture with different tumour cell lines can result in a mixed macrophage phenotype with M1 as well as M2 patterns, a finding that is important for a better understanding of tumour microenvironment functions. Moreover, we demonstrated that VALE shows slight immunomodulatory effects on tumour cell co-cultured macrophages and modulates monocyte chemotactic transmigration in vitro, indicating promising possibilities of triterpenes from Viscum album L. to contribute in a multimodal concept of anti-cancer therapy in future. Our data contribute to an understanding of monocyte function and macrophage polarization in vitro and of the possibility to influence their behaviour by triterpene containing mistletoe extracts. [ABSTRACT FROM AUTHOR]

Background: Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. Methods: We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712-2), its succussed hydroalcoholic solvent (0712-1) and unsuccussed solvent (0712-3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined "wound field". All assays were performed in three independent controlled experiments. Results: None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712-2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712-1) at 1:100 dilutions (p < 0.001). Unsuccussed solvent (0712-3) had no influence on cell migration (6.3%; p > 0.05). Preparation (0712-2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712-1), which caused 22.1% wound closure. Conclusion: Results of this study showed that the low potency homeopathic remedy (0712-2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis. [ABSTRACT FROM AUTHOR]

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 14x30x. 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 07, 03, 37). 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 arearelated average specific growth rate r(area) compared to 1.60 for the frond numberrelated 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 arearelated average specific growth rate (day 07). Segmented area growth rates (day 03 or 37) 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. [ABSTRACT FROM AUTHOR]

The effect of six different homeopathic remedies on cress (Lepidium sativum L.) has been examined by CuCl2-biocrystallization. The method was based on the protocols of [1] and a follow up study (not yet published). Cress seeds germinated and grew for four days in vitro in a 30x potentization of Stannum metallicum, Arsenic Album, Mercurius metallicum, Sulphur, Silicea or Lactose (control) in a blinded and fully randomized assignment. Each remedy was prepared sterile and divided into 20 bottles used in 10 experiments. CuCl2-biocrystallization of seedlings extracted in the homeopathic preparations was performed on circular glass plates. Resulting biocrystallograms were analysed by digital textural image analysis. Texture analysis variables analysed yielded significant results between the control and some of the homeopathic treatments, as well as between a number of the different treatments. As the texture of the biocrystallograms of homeopathically treated cress exhibited effects depending on the remedies, the CuCl2-biocrystallization method will be further applied to a larger study investigating specific effects of different homeopathic preparations. [ABSTRACT FROM AUTHOR]