Inflamyar™ Possesses Anti-Inflammatory Effect on Human Immune Cells and Cytokine Expression In Vitro

Background: Acute and chronic inflammation can have serious effects on the body and cause severe pain reactions. In such situations, an application of anti-inflammatory substances would be useful. The active ingredients from classical medicinal plants are increasingly becoming the focus of research, as they were used in traditional medicine for many centuries and thus relatively safe. Plant metabolites have a wide range of pharmacological effects, such as high antioxidant, antiviral, anti-inflammatory and carcinogenic activity. In this study, the anti-inflammatory effect of Inflamyar™, a commercially available homeopathic spagyric product, was evaluated. Methods: Primary human immune cells were incubated with the test substance with / without a subsequent inflammatory stimulus (LPS). Consecutively, the expression of CD69 and cytokines was evaluated. Results: In the absence of an inflammatory stimulus, a reduced expression of, in particular, proinflammatory cytokines and no induction of CD69 expression were found in human immune cells. In the presence of an inflammatory stimulus, reduced CD69 expression and biphasic regulation of cytokine expression (reduction at lower and induction at higher concentrations) was detected. Conclusion: This study documented an anti-inflammatory effect of the test substance on human immune cells in vitro . Inflamyar™ Possesses Anti-Inflammatory Effect on Human Im mune Cells and Cytokine Expression In Vitro


Introduction
Since virtually all organisms are constantly exposed to the influences of the living environment, the immune system has a great importance for the physical integrity of humans. It protects against threatening external influences such as infestation by microorganisms and parasites, but also against threats from the inside of the body, like e.g. necrotic and apoptotic cell material as well as functionally degenerated cells [1,2]. The human immune system is made up of several components. A distinction is made between a cellular and a humoral part. The cellular immune system comprises highly specialized immune cells that are either mobile (e.g. in the blood) or located in various tissues, e.g. Monocytes, Granulocytes, B cells, T cells, NK cells. The humoral immune system is the part of the immune system based on plasma proteins (antibodies, complement factors and cytokines) [2,3]. Upon induction of an immune response, humoral components of the immune system are initially released by the cells located in the affected tissue. By secreting these factors, other immune cells are lured to the focus of infection [2,3]. The inflammatory response in the body is important for the resolution of the cause of the inflammation but is also the cause of the symptoms of the disease [4]. The immune response is not entirely specific to its cause; even healthy tissue is always damaged [5]. This is especially important when an acute inflammation becomes a chronic inflammation that does not succeed in elimination of the trigger. For example, this may be due to the presence of debris in the tissue or frequent overload of muscles and joints. This persisting inflammation can massively damage the surrounding, primarily healthy tissue [6,7]. The damage of healthy tissue leads to severe pain reactions in both acute and chronic inflammatory reactions [5,8]. Moreover, chronic inflammation can lead to neoplasms (carcinomas or lymphomas) in many organs and in the lymphatic tissue and promote their growth and vascularization. Causes include the chronic proliferation stimulus, the growth-promoting effects of cytokines and the genomic damage caused by reactive oxygen species produced by immigrated immune cells [9][10][11]. In such situations, use of anti-inflammatory agents to assist and reduce the side effects of inflammation is useful [12,13].
Among the most widely used anti-inflammatory drugs include i.e., synthetic substances such as cyclooxygenase inhibitors, steroids, immunosuppressants and cytokine inhibitors [14,15]. These substances are usually proven to be extremely effective -but in part also show a wide range of unwanted side effects [16][17][18]. For this reason, the active ingredients from classical medicinal plants are increasingly becoming the focus of research.
In particular, secondary plant metabolites are in the interest of science since these have been in use in traditional medicine for many centuries and thus relatively safe and side effects associated with their use often relatively low [19][20][21][22][23]. Plant metabolites have a wide range of pharmacological effects, such as high antioxidant, antiviral, anti-inflammatory and carcinogenic activity [24][25][26]. Antiinflammatory ingredients such as alkaloids, phenols, flavonoids, glycosides, terpenes, quinones, catechins and carbohydrates of aqueous extracts from various traditional herbs have been described in several studies [25,[27][28][29]. Besides of direct antiinflammatory actions, an interaction with immunological signal cascades, such as the reduction of proinflammatory mediators by inhibiting transcription factors of gene expression (Nuclear Factor κB, Inhibitor of κB), has already been demonstrated for some of these substances [30][31][32]. These modulatory effects of plant substances are particularly interesting for research. The individual effects of active ingredients from plant extracts are very diverse and have so far been described inadequately. For this reason further investigations are mandatory. In this study, the anti-inflammatory effect of Inflamyar™, a commercially available homeopathicspagyric product consisting of plant extracts from Arnica montana, Bryonia cretica, Guajacum, Toxicodendron quercifolium, Bellis perennis, Ledum palustre, Ruta graveolens and Viscum album was evaluated.

Materials and Methods
All experiments were conducted by NIS Labs, Klamath Falls, USA.

Test Substance
The test substance, Flamyar™, is a homeopathic spagyric natural remedy manufactured by PEKANA Naturheilmittel GmbH

CD69 Activation Marker Expression on Human Leukocyte Subsets
Peripheral whole blood from human healthy adult donors

Cytokine Production in Peripheral Blood Mononuclear Cell Cultures
Cell culture supernatants were obtained from 24-hour culture setup described above. Expression levels of the following cytokines

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

Results
Immune cells harvested from human peripheral blood were used as a model for potential activities on immune activating and modulating. On the one hand, the direct activation of immune cells by the test substance, and on the other hand, the priming of immune cells to respond differently to a subsequent inflammatory insult was analyzed. Therefore, two sets of cell cultures were examined in parallel, on the one hand the highly inflammatory bacterial LPS from Escherichia coli and on the other hand the recombinant Interleukin-2 (IL-2) for activation was used as a positive control, and LPS was additionally used in one of the two cultures to induce inflammation after treating the immune cells with the test substance. As indicator for immune cell activation, the activation marker CD69 (cluster of differentiation 69) was chosen.
In the case of lymphoid activation, CD69 is the earliest inducible surface glycoprotein and leads to lymphocyte proliferation and signal transmission at the cellular level [33,34]. Incubation of the cell culture with the test product (Figure 1 Figure 2: Percent change in proinflammatory cytokine levels in PBMC culture supernatants of three donors treated with serial dilutions of the test product in absence (column "product") and presence (column "product + LPS") of a subsequent inflammatory stimulus. Samples without inflammatory stimulus are compared to untreated control cultures; samples with inflammatory stimulus are compared to LPS-control (*p<0.05; **p<0.005).  Unfortunately, not all donors in this study showed an equal response to the test substance. In view of the individual variations in the immune status of the donors (e. g. genetic and epigenetic aspects, potential pre-existing conditions), high deviations in the data from primary cells of different donors are not unexpected [35][36][37]. From this point of view, the results of this study are relatively consistent. The observed dose-dependent regulation of the CD69 and cytokine expression of individual groups clearly underlines the validity of the data. In addition, the data generated in this study is consistent with previously published data from other workgroups.  [39]. In another study, T. quercifolium in dilutions of 6CH, 12CH, 30CH and 200CH appeared to interfere with an histamine, prostaglandins and other inflammatory mediators driven inflammatory processes [40]. Anti-inflammatory actions due to inhibition of both lipoxygenase and cyclooxygenase metabolic pathways were also seen in homeopathic remedy containing A. montana and T. quercifolium [41]. Porozov et al. described reduced IL-1β, TNFα and IL-8 secretion without an effect on human T cell and monocyte proliferation by a homeopathic remedy containing A. montana and B. perennis [42]. In addition, various studies showed anti-inflammatory properties of various extracts out of A. montana [43][44][45][46][47][48], Bryonia species [48][49][50], T. quercifolium [40,51,52], B.
In summary, the data of the present study possesses a clear antiinflammatory effect and thus a potential for the test substance for the treatment of acute or chronic inflammatory reactions.

Ethics Approval and Consent to Participate
Not applicable.

Consent for Publication
Not applicable.

Availability of Data and Materials
All data described in the manuscript are available from the corresponding author on reasonable request.

Competing Interests
The authors declare that they have no competing interests.

Funding
This work was financially supported by PEKANA Naturheilmittel GmbH (Kißlegg, Germany).