Comparison of in vitro tests for antioxidant and immunomodulatory capacities of compounds

Abstract

Oxidative stress is considered to be critically involved in the normal aging process but also in the development and progression of various human pathologies like cardiovascular and neurodegenerative diseases, as well as of infections and malignant tumors. These pathological conditions involve an overwhelming production of reactive oxygen species (ROS), which are released as part of an anti-proliferative strategy during pro-inflammatory immune responses. Moreover, ROS themselves are autocrine forward regulators of the immune response. Most of the beneficial effects of antioxidants are considered to derive from their influence on the immune system. Due to their antioxidant and/or radical scavenging nature, phytochemicals, botanicals and herbal preparations can be of great importance to prevent oxidation processes and to counteract the activation of redox-regulated signaling pathways. Antioxidants can antagonize the activation of T-cells and macrophages during the immune response and this anti-inflammatory activity could be of utmost importance for the treatment of above-mentioned disorders and for the development of immunotolerance. Herein, we provide an overview of in vitro assays for the measurement of antioxidant and anti-inflammatory activities of plant-derived substances and extracts, by discussing possibilities and limitations of these methods. To determine the capacity of antioxidants, the oxygen radical absorbance capacity (ORAC) assay and the cell-based antioxidant activity (CAA) assay are widely applied. To examine the influence of compounds on the human immune response more closely, the model of mitogen stimulated human peripheral blood mononuclear (PBMC) cells can be applied, and the production of the inflammatory marker neopterin as well as the breakdown of the amino acid tryptophan in culture supernatants can be used as readout to indicate an immunomodulatory potential of the tested compound. These two biomarkers of immune system activation are robust and correlate with the course of cardiovascular, neurodegenerative and malignant tumor diseases, but also with the normal aging process, and they are strongly predictive. Thus, while the simpler ORAC and CAA assays provide insight into one peculiar chemical aspect, namely the neutralization of peroxyl radicals, the more complex PBMC assay is closer to the in vivo conditions as the assay comprehensively enlights several properties of immunomodulatory test compounds.

Keywords: ARE; CAA; Cell-based antioxidant activity (CAA); GTP-CH1; GTP-cyclohydrolase I; IDO; IFN-γ; IL-2; Immunomodulation; In vitro test systems; Inflammation; Kyn/Trp; NO; Nf-κB; ORAC; Oxidative stress; Oxygen radical absorbance capacity (ORAC) assay; PBMC; Peripheral blood mononuclear cells; ROS; Reactive oxygen species; TNF-α; antioxidant response element; cell-based antioxidant activity; iNOS; indoleamine 2,3-dioxygenase; inducible nitric oxide synthase; interferon-gamma; interleukine-2; kynurenine to tryptophan ratio; nitric oxide; nuclear factor-κB; oxygen radical absorbance capacity; peripheral blood mononuclear cells; reactive oxygen species; tumor necrosis factor-alpha.