*2.2. Reactive Oxygen Species (ROS)-Inducing Activity of the CRG and the Ech on the Human Blood Cells*

The ROS induction in human neutrophils, in the presence of the Ech and its complexes with CRGs, was determined with the Ech concentration varying from 1 to 10 μg mL−1. To assess the effect of the content of the polysaccharide in the complex with the Ech, on the formation of ROS, we used CRG concentrations in the range of 5 to 200 μg mL−1. Thus, complexes with different CRGs/Ech ratios (5:1, 10:1, and 20:1) were prepared.

The corresponding effects were detected using a fluorescent probe and measured by means of flow cytometry (Figure 2). Lipopolysaccharide (LPS) from *E. coli* was used as a reference immunomodulator, in the current test, and the ROS production induced by the LPS, as a positive control, was approximately twice as much as the negative control (the vehicle). At low concentrations, the activity of Ech towards the ROS formation was comparable to the negative control, whereas at high concentrations its effect was lower than that of the control by 20%. The influence of the CRGs on the activation of ROS, at lower concentrations, was not significant except for the λ-CRG. In contrast, the CRGs at a concentration of 100 μg mL−<sup>1</sup> intensified the induction of the ROS by up to 25–55%, relative to the negative control. The addition of the Ech to the CRGs, especially at high concentrations, resulted in significant diminishment of the ROS formation induced by the CRGs alone. The action of the complexes was compared to the negative control, where at higher concentrations, the effect of the samples was more noticeable.

**Figure 2.** Neutrophils reactive oxygen species (ROS) formation in the presence of the Ech, CRGs, and their complexes. The concentration of the lipopolysaccharide (LPS) was 10 μg mL−<sup>1</sup> and of the Ech was 1.0, 5.0, or 10.0 μg mL<sup>−</sup>1, final value. The concentrations of the CRGs alone and in complexes with the Ech changed in the following ratios row: = 5:1; = 10:1; = 20:1. The results are expressed as % change in ROS, relative to the control (100%), \* *p* < 0.05.
