A homeopathic remedy from arnica, marigold, St. John's wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts

Abstract

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.

Figure 1

Effect of substances on fibroblasts migration. Migration of NIH 3T3 cells (30000/filter) by 0712-1(succussed solvent), 0712–2 (remedy) and 0712–3 (unsuccussed solvent) after 24 h was measured by chemotaxis using 10% FCS as chemoatractant and are expressed as percentages normalized to the untreated control value. As positive control 2 ng/ml EGF was used. Means ± SD from three independent experiments performed in duplicates are presented. All values with different letters are statistically different (p < 0.05).

Figure 2

Wound closure effect of substances. Effect of substances 0712-1(succussed solvent), 0712–2 (remedy) and 0712–3 (unsuccussed solvent) on the wound closure of NIH 3T3 fibroblasts (25000/well) after 24 h expressed in % of cells migrated into the wound area (A) and as percentages of wound closure (B). As positive control DMEM with 5% FCS was used. As 100% wound closure the density of cell without created wound was set. Means ± SD of three independent experiments are presented. All values with different letters are statistically different (p < 0.01).

Figure 3

Light microscope images of the wound closure in vitro using confluent monolayer of NIH3T3fibroblasts. Microphotographs showing one representative experiment of the cell migration into the created wound area in response to the treatment. (A) Wound area immediately after wounding and (B) after 24 h for the untreated control (medium only, set to 0%); (C) confluent area without wounding (set to 100%) as well as treated areas with substances at 1:100 dilution: succussed solvent 0712–1 (E), remedy 0712–2 (F) and unsuccussed solvent 0712–3 (G) after 24 h incubation. DMEM with 5% FCS (D) was used as positive control. Wound closure (indicated in%) was normalized to the untreated control (B) and the confluent area (C).