Inflamyar™ Protects from Oxidative Stress

Introduction: Oxidative stress (OS) is associated with significant damage to cell structures leading to multiple disease patterns. Dietary supplementation or topical administrations of non-enzymatic antioxidants are described to prevent OS-related disease rates. Many of the most powerful and most discussed natural non-enzymatic antioxidants can be found in the plant kingdom. In this study, the antioxidant effect of a commercially available plant extract remedy was evaluated. Methods: Primary human cells (peripheral blood mononuclear cells [PBMC], erythrocytes and polymorphonuclear cells [PMN]) were pre-incubated with Inflamyar™ (PEKANA Naturheimittel GmbH, Germany). Cell viability (MTT-assay), cellular antioxidant protection (CAP-e assay) and reactive oxygen species (ROS)-formation assays were performed. Results: The MTT test showed a predominantly consistent to slightly increased cellular viability. A mild protection of oxidative damage in human erythrocytes at higher doses of the test substance was seen in the CAP-e assay. In the ROS assay, slightly reduced levels of ROS formation for all tested concentrations of the test substance were documented in PMN cells from two of three donors. Conclusion: This study documented stable vitality and protective capacities against cellular oxidative stress of cells treated with the test substance. Inflamyar™ Protects from Oxidative Stress.


Introduction
Oxygen is one of the most important elements for life, but in certain circumstances oxygen can also harm the organism.
Influences of the outside world such as redox chemicals, ionizing radiation, UV light, heavy metal ions, ozone exposure and cigarette smoke can readily generate reactive oxygen species (ROS), which have an oxidizing effect on other substances [1,2]. A large part of these reactive species are represented by free radicals, for example hydroxyl radicals, superoxide radicals, nitric oxide radicals and the lipid peroxyl radical, and have an excess of free electrons. These substances are unstable and have a highly reactive potential on other substances [2]. In addition to the environmental formation of ROS, these can also be found as by-products in the oxygen metabolism of aerobic organisms. ROS are generated in connection with the electron transport in mitochondrial energy generation, immune reactions against microorganisms and viruses [3,4] as well as processes of the cytochrome P-450 system [5,6].
The human antioxidant defense system consists out of two components, an enzyme component on the one hand, comprising enzymes such as glutathione peroxidase, catalase and superoxide dismutase and the small molecule antioxidant component on the other, which comprises vitamin A and E, ascorbate, glutathione and thioredoxin. These substances are the natural protection against endogenous ROS and other free radicals. Oxidative stress occurs when the production of ROS exceeds the natural antioxidant defense mechanisms of the body and damages biomolecules such as lipids, proteins and nucleic acids [1].
Several studies associated an additional supply of antioxidants with lower disease rates and preventive protection by dietary supplementation or topical administration [30][31][32]. Thus, the supplementation of non-enzymatic antioxidants could be a feasible way of restoring redox homeostasis and reduce ROS-associated diseases. Many of the most powerful and most discussed natural non-enzymatic antioxidants, for example the polyphenols, can be found in the plant kingdom. Polyphenols are naturally occurring compounds found in multiple plants, exhibiting largest antioxidant potential of secondary plant metabolites [33]. Five major groups of phytophenolics are classified, flavonoids, phenolic acids, stilbenes, lignans and tannins [34][35][36]. A special antioxidative activity has been described for substances from the flavonoid and phenolic acid classes both in vitro and in vivo. They also show a synergistic enhancement when interacting with other antioxidants such as tocopherol and ascorbate [33,37].
In this study, the antioxidant effect of a commercially available plant extract, consisting of extracts from Arnica montana, Bryonia cretica, Guajacum, Toxicodendron quercifolium, Bellis perennis, Ledum palustre, Ruta graveolens and Viscum album was evaluated.

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

Test Substance
The test substance, Flamyar™, is a natural plant extract

Isolation of Peripheral Blood Mononuclear and Polymorphonuclear Cells and Erythrocytes
Upon written informed consent from healthy human donors, and as approved by the Sky Lakes Medical Center Institutional Review Board (Federalwide Assurance 2603) for ethical standards, peripheral blood was drawn into heparinized vacutainer tubes.
Blood was layered onto Lympholyte-Poly (Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged at 1800 rpm for 35 minutes.

Reactive Oxygen Species Assay
Since they are known to produce high levels of ROS, freshly purified PMN cells were used for the testing of effects of the test substance on reactive oxygen species (ROS) formation [39]. PMN cells (2x10 5

Statistical Analysis
Averages and standard deviations for each data set were calculated using Microsoft Excel. Statistical analysis was performed using the two-tailed, dependent t-test. Statistical significance was designated as p<0.05, and a high level of significance was designated as p<0.01.

Results
In order to exclude any cytotoxic or cytostatic effects of the test substance, the impact of serial dilutions of the test substance on cell viability of PBMCs were evaluated using MTT Cell-Viability Assay. The data obtained showed no negative effects on cellular viability or mitochondrial function at any of the doses of the test substance; only at the highest concentration of 100ml/l a slightly, but not significantly reduced cell viability was seen (93.95±3.42%, to -10.38±10.01% (6.25ml/l; p=0.2608). Data from donor 3 were evaluated from -1.30±9.95% (0.39ml/l; p=0.8578) to -6.00±2.45% (100ml/l; p=0.1811).

Discussion
The application of plant extract remedies is widespread among the people. However, the effect of these products is controversially discussed in the scientific community. In the past years, plant extract agents have come more and more into the focus of research and a rising number of studies, showing a positive effect of these remedies can be found in the databases. In contrast to this, different studies conclude that these preparations are not significantly different from placebo treatment [40]. In this study, a plant extract remedy from A. montana, B. cretica, Guajacum, T. quercifolium, B.
perennis, L. palustre, R. graveolens and V. album was evaluated in vitro regarding its anti-oxidative capacity on human cells. At the beginning of this study, a MTT cell viability assay was performed on PBMCs in order to get an insight in effects of the test substance on cellular mitochondrial activity, also serving as a baseline for the concentrations used in further experiments. Given the dilution (D4 -D12) of the ingredients, we expected to see little or no cellular stress, even at higher doses, as we typically do with many traditional botanical extracts [41][42][43][44]. As expected, the test substance showed no significant reduction of cell viability or mitochondrial function ( Figure 1).
In the next step, the antioxidant potential of the test product was assessed by a Cellular Antioxidant Protection (CAP-e) assay [45,46]. This test allows assessment of antioxidant potential in a method that is comparable to the Oxygen Radical Absorbance  Figure 2). Combining data from CAP-e assay with results from ROS assay, some interesting conclusions for the test substance could be made. A large number of antioxidative natural substances are also able to reduce the ROS release in immune cells [27,33]. In contrast, ROS formation is increased in part despite the initial antioxidative effect of a substance through the interaction with the inflammatory signaling pathways of cells. The following three effects of natural substances on the ROS formation of PMN cells are therefore possible:  (1) Direct inhibitory effect on the ROS formation; (2) Indirect inhibitory effect through activation of an antiinflammatory signaling pathway or (3) Activation of an inflammatory cascade, which results in an increased release of ROS [27,47].
In this study, three patients were analysed, since due to the In this context, Gaspar, et al. described no cytotoxicity for isolates from A. montana at low concentrations [42]. Other studies also described no or only low cytotoxicity for Arnica-species [43,44]. For other plant species enclosed in the test substance, the information of cytotoxicity is very poor. No data regarding mitochondrial activity was found. Interestingly, an antioxidant capacity was described for each plant enclosed in the test substance: A. montana [43,48], B. cretica [49], Guajacum [50,51], T.

Conclusion
This study documented stable vitality and protective capacities against cellular oxidative stress of cells treated with the test substance.