Valpalf®: A New Nutraceutical Formulation Containing Bovine Lactoferrin That Exhibits Potentiated Biological Activity

Abstract

As a nutraceutical, bovine lactoferrin (bLf), an iron-binding glycoprotein involved in innate immunity, is gaining elevated attention for its ability to exert pleiotropic functions and to be exceptionally tolerated even at high dosages. Some of bLf's activities, including its anti-inflammatory and antioxidant, are tightly linked to its ability to both chelate iron and enter inside the cell nucleus. Here, we present data about Valpalf^®, a new formulation containing bLf, sodium citrate, and sodium bicarbonate at a molar ratio of 10^-3. In the present study, Valpalf^® exhibits superior iron-binding capacity, resistance to tryptic digestion, and a greater capacity to accumulate into the nucleus over time when compared to the native bLf alone. In agreement, Valpalf^® effectively reduces interleukin(IL)-6 levels in lipopolysaccharide-stimulated macrophages and modulates the expression of antioxidant enzymes, such as superoxide dismutase 1 and 2, in phorbol-12-myristate-13-acetate-stimulated monocytes. Of note, this potentiated bioactivity was corroborated in a retrospective study on the treatment of anemia of inflammation in hereditary thrombophilic pregnant and non-pregnant women, demonstrating that Valpalf^® improves hematological parameters and reduces serum IL-6 levels to a higher extent than bLf alone.

Keywords: Valpalf®; anemia of inflammation; anti-inflammatory activity; antioxidant activity; bovine lactoferrin; iron chelation; proteolysis resistance.

Figure 1

SDS-PAGE with Coomassie blue staining (A) and densitometry analysis (B) of nat-bLf (1), holo-bLf (2), and Valpalf^® (3) after different times of incubation with trypsin. For each lane, 6 µg of bLf were loaded. Densitometry analysis: the data are normalized on the 80 kDa band of nat-bLf. Statistical significance is indicated as follows: ***: p < 0.001 (one-way ANOVA with post hoc Tukey test).

Figure 2

Analysis of bLf internalization and subcellular localization in Caco-2 cells. Western blot and densitometric analysis of bLfs in cytosolic and nuclear fractions after 6 h (A) and 24 h (B) of treatment with 100 μg/mL nat-bLf, holo-bLf or Valpalf^®. The data, normalized to the internal housekeeping genes (α-Tubulin for cytosolic fraction and Lamin A for nuclear fraction), are presented as means ± SEM. Statistical significance is indicated as follows: ***: p < 0.001 (one-way ANOVA with post hoc Tukey test).

Figure 3

ELISA quantitation of IL-6 levels in THP-1 cells untreated or challenged with 1 µg/mL LPS in the absence or presence of 100 μg/mL nat-bLf, holo-bLf, or Valpalf^®. Error bars: standard error of the mean. Statistical significance is indicated as follows: **: p < 0.01; ***: p < 0.001 (one-way ANOVA with post hoc Tukey test).

Figure 4

Western blot and densitometry analysis of SOD-1 (A) and SOD-2 (B) levels in THP-1 cells undifferentiated or differentiated with 0.16 µM PMA and treated with 100 µg/mL of nat-bLf, holo-bLf, or Valpalf^®. Data were calculated relative to the housekeeping gene GAPDH. Error bars: standard error of the mean. Statistical significance is indicated as follows: *: p < 0.05; **: p < 0.01 (one-way ANOVA with post hoc Tukey test).

Figure 5

Flow diagrams of enrolled pregnant and non-pregnant women suffering from hereditary thrombophilia (HT) and treated with bLf alone or Valpalf^®.

Figure 6

Levels of red blood cells (RBCs) (A), hemoglobin (Hb) (B), total serum iron (TSI) (C), serum ferritin (sFtn) (D) and IL-6 (E) before (black dots) and after 30 days of treatment with 200 mg two times a day of bLf (light grey dots) in 35 HT pregnant women or 200 mg two times a day of Valpalf^® (blue dots) in 32 HT pregnant women. Dots represented the values for each patient pre- and post-treatments. Mean values ± standard deviation are also reported. Statistical significance is indicated as follows: ***: p < 0.001; ****: p < 0.0001 (one-way ANOVA with multiple comparisons).

Figure 7

Levels of red blood cells (RBCs) (A), hemoglobin (Hb) (B), total serum iron (TSI) (C), serum ferritin (sFtn) (D) and IL-6 (E) before (black dots) and after 30 days of treatment with 200 mg two times a day of bLf (light grey dots) in 37 HT non-pregnant women or 200 mg two times a day of Valpalf^® (blue dots) in 30 HT non-pregnant women. Dots represented the values for each patient pre- and post-treatments. Mean values ± standard deviation are also reported. Statistical significance is indicated as follows: **: p < 0.01; ***: p < 0.001; ****: p < 0.0001 (one-way ANOVA with multiple comparisons).