Cell-Type-Specific Modulation of Hydrogen Peroxide Cytotoxicity and 4-Hydroxynonenal Binding to Human Cellular Proteins In Vitro by Antioxidant Aloe vera Extract

Abstract

Although Aloe vera contains numerous bioactive components, the activity principles of widely used A. vera extracts are uncertain. Therefore, we analyzed the effects of genuine A. vera aqueous extract (AV) on human cells with respect to the effects of hydrogen peroxide (H₂O₂) and 4-hydroxynonenal (HNE). Fully developed A. vera leaves were harvested and analyzed for vitamin C, carotenoids, total soluble phenolic content, and antioxidant capacity. Furthermore, human cervical cancer (HeLa), human microvascular endothelial cells (HMEC), human keratinocytes (HaCat), and human osteosarcoma (HOS) cell cultures were treated with AV extract for one hour after treatment with H₂O₂ or HNE. The cell number and viability were determined using Trypan Blue, and endogenous reactive oxygen species (ROS) production was determined by fluorescence, while intracellular HNE⁻protein adducts were measured for the first time ever by genuine cell-based HNE⁻His ELISA. The AV extract expressed strong antioxidant capacities (1.1 mmol of Trolox eq/g fresh weight) and cell-type-specific influence on the cytotoxicity of H₂O₂, as well as on endogenous production of ROS and HNE⁻protein adducts induced by HNE treatment, while AV itself did not induce production of ROS or HNE⁻protein adducts at all. This study, for the first time, revealed the importance of HNE for the activity principles of AV. Since HMEC cells were the most sensitive to AV, the effects of AV on microvascular endothelia could be of particular importance for the activity principles of Aloe vera extracts.

Keywords: 4-hydroxynonenal (HNE); Aloe vera; HNE–protein adducts; antioxidants; cell growth; cell-based ELISA; hydrogen peroxide; lipid peroxidation; microvascular endothelium; oxidative stress; plant extract; reactive oxygen species (ROS).

Figure 1

Partial characterization of the antioxidant levels of Aloe vera leaves. Values are given as mean values for triplicates.

Figure 2

The effects of Aloe vera (AV) extract (1 or 10%) on the human cervical cancer (HeLa) cells with respect to H₂O₂ treatment (0.0025% or 0.05%). The cell count values (viability determined using Trypan blue) were obtained 24 h after treatment (H₂O₂ followed by AV extract 1 h later) and are given as mean values for triplicates. * significant difference to untreated control; ** significant difference to H₂O₂ treatment alone.

Figure 3

The effects of AV extract (1 or 10%) on the human microvascular endothelial cells (HMEC) with respect to H₂O₂ treatment (0.0025% or 0.05%). The cell count values (viability determined using Trypan blue) were obtained 24 h after treatment (H₂O₂ followed by AV extract 1 h later) and are given as mean values for triplicates. * significant difference to untreated control.

Figure 4

The effects of AV extract (1 or 10%) on the human keratinocyte (HaCaT) cells with respect to H₂O₂ treatment (0.0025% or 0.05%). The cell count values (viability determined using Trypan blue) were obtained 24 h after treatment (H₂O₂ followed by AV extract 1 h later) and are given as mean values for triplicates. * significant difference to untreated control; ** significant difference to respective H₂O₂ treatment alone.

Figure 5

The effects of AV extract (1 or 10%) on the human osteosarcoma (HOS) cells with respect to H₂O₂ treatment (0.0025% or 0.05%). The cell count values (viability determined using Trypan blue) were obtained 24 h after treatment (H₂O₂ followed by AV extract 1 h later) and are given as mean values for triplicates. * significant difference to untreated control.

Figure 6

The effects of AV extract (1%) on the cellular generation of 4-hydroxynonenal (HNE)–protein adducts induced by HNE treatment (50 µM). The amounts of HNE–protein adducts were determined by cell-based HNE–His ELISA and are given as mean values for triplicates. * significant difference to respective untreated control (ctrl); ** significant difference to respective HNE-treated control.

Figure 7

The effects of AV extract (1%) on the cellular generation of reactive oxygen species (ROS; mostly H₂O₂) induced by HNE treatment (50 µM). The amounts of the HNE–protein adducts were determined using luminescence and are given as mean values for triplicates. * significant difference to respective untreated control.