Water-soluble egg membrane enhances the immunoactivating properties of an Aloe vera-based extract of Nerium oleander leaves

Abstract

Objective: To evaluate a blend of two natural ingredients on immune parameters relevant for their current topical use and potential support of microcirculation in skin tissue.

Materials and methods: A blend (BL) of Aloe vera-based Nerium oleander extract (NAE-8i, oleandrin-free) and hydrolyzed water-soluble egg membrane (WSEM) was applied to human whole-blood cultures for 24 hours, with each separate ingredient serving as a control. Immune-cell subsets were analyzed for expression levels of the activation markers CD69 and CD25. Culture supernatants were analyzed for cytokines, chemokines, and immunoregulating peptides.

Results: BL increased CD69 expression on lymphocytes, monocytes, and CD3^-CD56⁺ natural killer cells, and CD25 expression on natural killer cells. The number of CD69⁺CD25⁺ lymphocytes increased in cultures treated with BL and the separate ingredients. BL triggered production of multiple cytokines and chemokines, where CC chemokines MIP1α and MIP3α, as well as cytokines involved in wound healing - Groα, Groβ, ENA78, and fractalkine - reached levels manyfold above treatment with either NAE-8i or WSEM alone.

Conclusion: Data on BL showed that WSEM strongly enhanced NAE-8i's effects on immunoactivation in vitro. This has potential relevance for support of immunity in skin tissue, including antibacterial and antiviral defense mechanisms, wrinkle reduction, and wound care.

Keywords: chemokines; cytokines; leukocyte activation.

Figure 1

Expression of the CD69 cellular activation marker on leukocyte subsets. Notes: *P<0.05; **P<0.01. CD69 expression on lymphocytes (A), monocytes (B), and polymorphonuclear (PMN) cells (C) in human whole-blood cultures treated for 24 hours with serial dilutions of an Aloe vera-based Nerium oleander extract (Nae-8i), water-soluble egg membrane (WSEM), or a blend of both (BL). Mean fluorescence intensity for CD69 expression is shown. Lipopolysaccharide (LPS) was used as a positive control (10 ng/mL) and resulted in an increase in CD69 expression on all three cell types. For all three cell types, exposure to the highest dose of BL resulted in an increase in CD69 expression. In the case of monocytes, the 20 mg/mL concentration of Nae-8i and BL activated cells better than LPS. For PMN cells, a reduction in CD69 expression was seen when compared to untreated PMN cells for the two higher doses of WSEM, as well as the intermediate dose of Nae-8i and BL. For the highest dose of BL, CD69 expression was induced in PMN cells. Data presented as mean ± standard deviation from triplicate cultures and are representative of three separate experiments using whole blood from three different healthy human donors. Abbreviation: UT, untreated.

Figure 2

Expression of CD69 and CD25 on natural killer cells and CD69⁺CD25⁺ lymphocytes. Notes: *P<0.05; **P<0.01. CD69 (A) and CD25 (B) expression on CD3⁻CD56⁺ natural killer (NK) cells and percentage of lymphocytes positive for both CD69 and CD25 (C) in human whole-blood cultures treated for 24 hours with serial dilutions of an Aloe vera-based Nerium oleander extract (NAE-8i), water-soluble egg membrane (WSEM), or a blend of both (BL). The mean fluorescence intensity of CD69 expression (A) and CD25 expression (B) on NK cells and the percentage of CD69⁺CD25⁺ lymphocytes (C) are shown. Lipopolysaccharide (LPS) was used as a positive control (10 ng/mL) and resulted in an increase in CD69 and CD25 expression on NK cells and an increase in the percentage of CD69⁺CD25⁺ double-positive lymphocytes. Even though both NAE-8i and WSEM induced CD69 and CD25 expression on NK cells, the BL blend triggered more robust and statistically significant increases. BL showed the highest level of activation of NK cells as well, as an increase in CD69⁺CD25⁺ double-positive lymphocytes. Data presented as mean ± standard deviation from triplicate cultures and are representative of three separate experiments using whole blood from three different healthy human donors. Abbreviation: UT, untreated.

Figure 3

Changes in proinflammatory cytokine levels in human whole-blood cultures. Notes: Cultures were treated for 24 hours with an Aloe vera-based Nerium oleander extract (NAE-8i; 4 mg/mL), water-soluble egg membrane (WSEM; 1 mg/mL), or a blend of both (BL). All three treatments led to statistically significant increases in cytokine production of IL-1β (A), IL-2 (B), IL-6 (C), IL-8 (D), IFNγ (E), and TNFα (F). Treatment with BL led to the greatest increases, and these were statistically significant from control cultures (UT), as well as from cultures treated with NAE-8i or WSEM alone. For all six cytokines, the effects of BL were manyfold higher than additive contributions from NAE-8i and WSEM. The increased production of IL-2 and IL-8 following treatment with BL was similar to levels resulting from lipopolysaccharide (LPS) treatment, while IL-6 production following treatment with BL was much greater than LPS treatment. With the exception of IL-2, data are plotted using a logarithmic scale on the y-axis. The table inserts contain statistical comparisons between treatments and control (UT), as well as comparisons of BL to each of its ingredients: NAE-8i and WSEM. Samples were tested in duplicate, and data shown are representative of three separate experiments using three different blood donors. Data presented as mean ± standard deviation. Numbers above each data bar indicate cytokine concentration in pg/mL. Abbreviations: IL, interleukin, IFNγ, interferon gamma; TNFα, tumor necrosis factor alpha.

Figure 4

Changes in anti-inflammatory cytokine levels in human whole-blood cultures treated for 24 hours. Notes: Cultures were treated with an Aloe vera-based Nerium oleander extract (NAE-8i; 4 mg/mL), water-soluble egg membrane (WSEM; 1 mg/mL), or a blend of both (BL). All three treatments led to statistically significant increases in cytokine production of IL-4 (A), IL-10 (B), and GM-CSF (C). Treatment with NAE-8i and WSEM had similar effects on increasing the production of IL-4, IL-10, and GM-CSF, while treatment with BL led to larger increases that were additive when compared to NAE-8i and WSEM alone. The table inserts contain statistical comparisons between treatments and control (UT), as well as a comparison of BL to each of its ingredients: NAE-8i and WSEM. Samples were tested in duplicate, and data shown are representative of three separate experiments using three different blood donors. Data presented as mean ± standard deviation. Numbers above each data bar indicate cytokine concentrations in pg/mL. Abbreviations: LPS, lipopolysaccharide; IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor.

Figure 5

Changes in levels of cytokines that signal through the CXCR2 receptor and/or are involved in wound healing in human whole-blood cultures. Notes: Cultures were treated for 24 hours with an Aloe vera-based Nerium oleander extract (NAE-8i; 4mg/mL), water-soluble egg membrane (WSEM; 1 mg/mL), or a blend of both (BL). The effects of BL were manyfold higher than additive contributions from NAE-8i and WSEM, showing robust increases in the production of Groα (A), Groβ (B), ENA78 (C), and fractalkine (D) that were much greater than the lipopolysaccharide (LPS) control. Groα data were plotted using a logarithmic scale on the y-axis. The table inserts contain statistical comparisons between treatments and control (UT), as well as a comparison of BL to each of its ingredients: NAE-8i and WSEM. Samples were tested in duplicate, and data shown are representative of three separate experiments using three different blood donors. Data presented as mean ± standard deviation. Numbers directly above each data bar indicate cytokine concentrations in pg/mL.

Figure 6

Changes in levels of cytokines involved in natural killer-cell activation and antiviral protection in human whole-blood cultures. Notes: Cultures were treated for 24 hours with an Aloe vera-based Nerium oleander extract (NAE-8i; 4 mg/mL), water-soluble egg membrane (WSEM; 1 mg/mL), or a blend of both (BL). Treatment with BL led to the greatest increases for all four cytokines MIP1α (A), MIP3α (B), SDF1α (C), and IP10 (D), and these were statistically significant from control cultures (UT), as well as from cultures treated with NAE-8i or WSEM alone. The effects of NAE-8i and WSEM were enhanced in BL, leading to synergistic effects on the production of MIP1α and MIP3α. The increase in MIP3α following exposure to BL was tenfold greater than the increase elicited by lipopolysaccharide (LPS). MIP1α, MIP3α, and IP10 data were plotted using a logarithmic scale for the y-axis. The table inserts contain statistical comparisons between treatments and control (UT), as well as a comparison of BL to each of its ingredients: NAE-8i and WSEM. Samples were tested in duplicate, and data shown are representative of three separate experiments using three different blood donors. Data presented as mean ± standard deviation. Numbers above each data bar indicate cytokine concentrations in pg/mL.