Inflamyar™ Possesses Anti-Inflammatory Effect and Induces Growth Factor Expression on Human Dermal Fibroblasts In Vitro

With a surface of 2 m2 and a weight of 10-12 kg, the skin is the largest human organ. In addition to the protection against external mechanical and physical influences, the skin also plays an important role as an active immune organ against the infestation of microorganisms [1]. In the case of a skin structure injury, wound healing and thus the reconstitution of the barrier function is of central importance. Wound healing is a highly complex, dynamic process in which a variety of different cellular and humoral components work Abstract

together in different phases. The beginning of wound healing is called the inflammatory or exudation phase. If the blood vessels are damaged, blood coagulation begins and a fibrin net, which serves as a matrix for the newly formed granulation tissue, forms. Neutrophil granulocytes migrate through the capillaries into the tissue, secreting cytokines and protein-degrading proteases. Immigrated macrophages eliminate microorganisms through phagocytosis and secrete growth factors and cytokines. These stimulate the proliferation and migration of fibroblasts and vascular endothelial cells into the wound bed. Capillaries sprout into the wound area and precursors of extracellular matrix are synthesized.
The final closure of the wound area is achieved by contraction of the wound area and by immigration of fibroblasts and keratinocytes into the wound bed [2]. The search for remedies that support wound healing is one of the oldest challenges in medicine.
Since ancient times, the use of topically applied extracts from medicinal plants has been widespread throughout the world [3].
Many representatives of the plant kingdom show an enormous potential for the treatment of wounds [4,5]. Their natural active ingredients induce healing and regeneration of the injured tissue through different mechanisms [4]. These components include various groups of substances such as terpenoids, polyphenols, alkaloids and essential oils, polypeptides and lectins and other compounds [6].
Active ingredients of plant origin are not only inexpensive, but also have relatively low side effects compared to many synthetic substances [7,8]. This is not least due to the many years of experience in the use of these substances. Important factors for the effect of these substances are their exact composition, dose, processing and administration form. In particular, the dose of these drugs is an important factor in their effectiveness. This was already expressed in the 16 th century by the alchemist and physician Paracelsus ("All things are poison and nothing is without poison; just the dose makes sure that a thing is not poison.") [9]. Thus, different plant compounds show a toxic effect at high concentrations and a healing effect at low concentrations. In fact, the relevant literature does not distinguish between poisonous plants or medicinal plants.
Some studies have shown efficacy of plant extracts in different indications even at highly diluted concentrations [10][11][12][13]. Overall, the individual effects of active ingredients from plant extracts are very diverse and have so far been described only inadequately [14].
For this reason, further investigations are necessary.
In this study, the effect of Inflamyar™, a commercially available homeopathic spagyric product consisting of plant extracts from Arnica montana, Bryonia cretica, Guajacum, Toxicodendron quercifolium, Bellis perennis, Ledum palustre, Ruta graveolens and Viscum album, on wound healing was investigated in vitro.

Test Substance
The test substance, Flamyar™, is a homeopathic spagyric natural remedy manufactured by PEKANA Naturheilmittel GmbH Treatments of cells with the test substance were tested in duplicate.
Untreated and LPS alone cultures were tested in triplicate.

Statistical Analysis
Calculations and statistical analysis was performed using the two-tailed, independent t-test using Microsoft Excel.

Discussion
The analysis of cytokine expression after treatment of human dermal fibroblasts without a subsequent inflammatory stimulus possessed, except expression of IFN-γ, either no effect or an increased expression of cytokines and growth factors. Increased cytokines of the chemokine group, e.g. IL-8, MCP-1, MIP-1α, IP-10 and Eotaxin, were described to be involved in important aspects of early stages of wound healing [15,16] and re-epithelialization [17].
An up-regulation of anti-and pro-inflammatory cytokines (IL-6, -12p70, -7, -9, -1ra and -10) by topical application of natural products has been suggested to enhance normal immune surveillance of skin tissue and maintenance of homeostasis [18]. Interestingly, the treatment of skin cells with all four concentrations of the test substance resulted in a 20-30% increase in VEGF production. This regenerative growth factor has a role in tissue repair through its function as an angiogenic factor [19] and topical application of VEGF in a diabetic mouse model has been shown to promote wound healing [20]. Moreover, an induction of PDGF-BB was seen after cell stimulation with higher doses of the test substance. PDGF-BB also plays an important role in the late stage of wound healing [21].
In presence of inflammatory stimuli, most of the cytokines tested showed either no reaction or a reduction, especially at higher test substance concentrations. Interestingly, at higher doses of the test substance an increased expression in the anti-  [22][23][24][25]. In contrast, the moderately reduced anti-inflammatory IL-10 is a major player in inflammatory processes and inhibits the proinflammatory cytokine expression via interaction with the NFκB and the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway [26].
The growth factors bFGF, VEGF and GM-CSF have an important function and are upregulated in late stages of wound healing like angiogenesis [28], reepithelialization [29], granulation tissue formation [30,31], matrix formation and remodeling [32].  In comparison to previously published work, the data generated in this study is consistent with data from other workgroups. For example, Karow et al. showed reduced wound irritation after treatment of patients with A. montana extracts [33]. Moreover, a stimulation of expression of extracellular matrix genes in a wound-healing phenotype macrophage cell line by A. montana was described by Marzotto et al. [34]. In another study, A. montana showed an inhibition of carragenin-induced rat paw oedema [7].
A. montana extracts have also been reported to promote healing, as some of the modified genes are key factors of tissue remodeling, inflammation, and chemotaxis [35]. In addition, Mahajan et al.
show a significant reduction of IL-6, IL-1 and TNFα from human whole blood culture and RAW-264.7 cells after LPS induction and incubation with various homeopathic dilutions of A. montana and Bryonia species in vitro [36]. In a study of dos Santos et al. T.
quercifolium extracts have anti-inflammatory effects and appear to act on inflammatory processes with prostaglandins, histamine, and other inflammatory mediators [37].
In the circular excision wound model in rats, a wound healing potential of a topically applied ointment made from B. perennis flowers could also be shown [38]. Tolmacheva   Effects of the test product alone (-LPS) and effects of a test product pretreatment prior to the addition of the inflammatory compound LPS (+LPS) was observed and displayed in comparison to control cultures without the test substance (* p<0.05; ** p<0.005).

Figure 3:
Percent change in levels of cytokines with pro-and anti-inflammatory activity and growth factors in supernatants from dermal fibroblast cultures incubated with serial dilutions of the test product for 24 hours. Effects of the test product alone (-LPS) and effects of a test product pretreatment prior to the addition of the inflammatory compound LPS (+LPS) was observed and displayed in comparison to control cultures without the test substance (* p<0.05; ** p<0.005).