## 4 Discussion  
IMMUNEPOTENT CRP(r) is a bovine dialyzable leukocyte extract. Our research group has published extensive research about IMMUNEPOTENT CRP's antioxidant, antitumor and anti-inflammatory properties at in vitro, preclinical and clinical levels [12; 13; 14; 15]. However, the classical preparation process of the bDLE IMMUNEPOTENT CRP(r) presents some challenges for the establishment of a production line. Our main challenge is that fresh bovine spleen is difficult to formulate; therefore, we added a drying step in the production process of the bDLE. Dried spleen can be turned into powder and this powder is our active ingredient. This is the first study to use dry, powdered bovine spleen as a functional compound to elaborate IMMUNEPOTENT CRP(r). We designed the present study to evaluate if this modification in the preparation method would affect the physical, chemical and biological properties of the final product. For wet bDLE, we observed vibration modes related to radical groups CC at 1330 cm\({}^{-1}\), CH at 1350, and 1400 cm\({}^{-1}\) and NH\({}_{2}\)+ at 1516 cm\({}^{-1}\). These wavelengths were not observed in dry bDLE, showing that the structures of wet and dry bDLE present clear differences in the interaction levels of the aforementioned groups. This result could be related to the percentage of humidity, given that the bandwidth observed at 1300 and 1350 cm\({}^{-1}\) that corresponds to CH and CC bonds was greater for wet bDLE [16]. We noted a difference in the diffraction pattern of the bDLEs, with the dry bDLE being more crystalline. The higher quantity of radical groups of wet bDLE correlates with a tendency to generate hydrogen bonds with its environment [17], which produces a modification of crystallinity in the structure, which was confirmed by the X-ray diffraction. Wet bDLE possibly presents an \(\alpha\)-helix structure due to the presence of a greater number of CH bonds. Dry bDLE, on the other hand, does not present these bands and thus we presume a \(\beta\)-folded structure. The positions of the hydrogens are important for the \begin{table}
\begin{tabular}{c c c c c c} \hline  & & \begin{tabular}{c} **Wet bDLE** \\ **(100 mg/mL)** \\ \end{tabular} & \begin{tabular}{c} **Wet bDLE** \\ **(0.4 mg/mL)** \\ \end{tabular} & \begin{tabular}{c} **Dry bDLE** \\ **(0.4 mg/mL)** \\ \end{tabular} & \
\begin{tabular}{c} **Dry bDLE** \\ **(0.4 mg/mL)** \\ \end{tabular} &
\begin{tabular}{c} **Dry bDLE** \\ **(0.4 mg/mL)** \\ \end{tabular} \\ \hline IL-2 (pg/mL) & 3 & 4 & 4 & 3 & 4 & 4 \\ IL-4 (pg/mL) & 7.2 & 320 * & 67.8 & 14.8 & 78.3 & 17.4 \\ TNF-\(\alpha\) (pg/mL) & 19.3 & 1389.4 * & 359.5 & 27.3 & 27.3 & 32.4 \\ IFN-\(\gamma\) (pg/mL) & 10.8 & 1000 * & 101 & 100 & 25.3 & 34.2 \\ IL-17A (pg/mL) & 39.9 & 598.5 * & 129.7 & 39.1 & 45.6 & 43.7 \\ IL-10 (pg/mL) & 23.3 & 703.3 * & 253.9 & 43.2 & 89.3 & 59.4 \\ \hline \end{tabular} Human PBMC (\(5\times 10^{5}\)) were stimulated with LPS (100 ng/mL) and simultaneously treated with wet and dry bDLE. Cytokine levels were determined in the cell culture supernatant with the cytometric bead array human Th1/Th2/Th17 kit by flow cytometry. Statistical significance was determined by one-way ANOVA (* \(p<0.05\)).  
\end{table} 
Table 6: Cytokine levels in LPS stimulated human PBMC treated with wet and dry bDLE.