Antioxidant, anti-inflammatory, anti-apoptotic, and skin regenerative properties of an Aloe vera-based extract of Nerium oleander leaves (nae-8(®))

Abstract

Objective: The goal for this study was to evaluate the effects of an Aloe vera-based Nerium oleander extract (NAE-8(®)), compared to an extract of A. vera gel alone (ALOE), and to an aqueous extract of N. oleander (AQ-NOE) in bioassays pertaining to dermatologic potential with respect to antioxidant protection, anti-inflammatory effects, and cytokine profiles in vitro.

Methods: Cellular antioxidant protection was evaluated in three separate bioassays: The cellular antioxidant protection of erythrocytes (CAP-e) assay, protection of cellular viability and prevention of apoptosis, and protection of intracellular reduced glutathione levels, where the last two assays were performed using human primary dermal fibroblasts. Reduction of intracellular formation of reactive oxygen species (ROS) was tested using polymorphonuclear cells in the absence and presence of oxidative stress. Changes to cytokine and chemokine profiles when whole blood cells and human primary dermal fibroblasts were exposed to test products were determined using a 40-plex Luminex array as a method for exploring the potential cross-talk between circulating and skin-resident cells.

Results: The NAE-8(®) provided significantly better antioxidant protection in the CAP-e bioassay than AQ-NOE. NAE-8(®) and AQ-NOE both protected cellular viability and intracellular reduced glutathione, and reduced the ROS formation significantly when compared to control cells, both under inflamed and neutral culture conditions. ALOE showed minimal effect in these bioassays. In contrast to the NAE-8(®), the AQ-NOE showed induction of inflammation in the whole blood cultures, as evidenced by the high induction of CD69 expression and secretion of a number of inflammatory cytokines. The treatment of dermal fibroblasts with NAE-8(®) resulted in selective secretion of cytokines involved in collagen and hyaluronan production as well as re-epithelialization during wound healing.

Conclusion: NAE-8(®), a novel component of a commercial cosmetic product, showed beneficial antioxidant protection in several cellular models, without the induction of leukocyte activation and secretion of inflammatory cytokines. The biological efficacy of NAE-8(®) was unique from both ALOE and AQ-NOE.

Keywords: CAP-e bioassay; ROS formation; dermal fibroblasts; oxidative damage; safety.

Figure 1

The concentration-dependent antioxidant capacity for the Aloe vera-based Nerium oleander extract (NAE-8®) and the aqueous N. oleander extract (AQ-NOE) are shown as gallic acid equivalents (GAE). The very minor antioxidant capacity of Aloe gel alone (ALOE) is shown as a control, since NAE-8® is extracted using this gel in the process. The antioxidant capacity of NAE-8® was higher than that of AQ-NOE. Notes: Levels of significance of data sets when comparing matching concentrations of NAE-8® to the AQ-NOE are indicated by asterisks. Significance P<0.05 is indicated by*, and a high level of significance P<0.01 is indicated by**. Samples were assayed in duplicate. Data are presented as the mean ± SD. Abbreviation: SD, standard deviation.

Figure 2

The cellular antioxidant protection provided in the CAP-e assay by the Aloe vera-based Nerium oleander extract (NAE-8®) and the aqueous N. oleander extract (AQ-NOE) is shown as percent (%) inhibition of intracellular oxidative damage. The cellular antioxidant protection of Aloe vera gel alone (ALOE) is shown as a control, since NAE-8® is extracted using this material in the process. The antioxidant capacity of NAE-8® was higher than that of AQ-NOE. ALOE did not contribute to this protective effect, demonstrating that the compounds in NAE-8® capable of entering into and protecting cells from oxidative damage were not derived from Aloe. Notes: Levels of significance of data sets when comparing matching concentrations of the NAE-8® to the AQ-NOE are indicated by asterisks: significance P<0.05 is indicated by *, and a high level of significance P<0.01 is indicated by **. Samples were assayed in duplicate. Data are presented as the mean ± SD. Abbreviations: SD, standard deviation; CAP-e, cellular antioxidant protection of erythrocytes.

Figure 3

The viability of dermal fibroblasts treated with H₂O₂ for 1 hour either alone or following a 30-minute incubation with test products. Results are represented as the % change compared to the H₂O₂ treated control. The three highest concentrations of both the aqueous Nerium oleander extract (AQ-NOE) and the Aloe vera-based N. oleander extract (NAE-8®) resulted in a 35%–55% protection from apoptosis. Conversely, pretreatment with serial dilutions of ALOE (aloe gel alone) did not lead to protection. Notes: Significance P<0.05 is indicated by *, and a high level of significance P<0.01 is indicated by **. Samples were assayed in duplicate. The data are representative of two different experiments with similar results. Data are presented as the mean ± SD. Abbreviation: SD, standard deviation.

Figure 4

Intracellular reduced glutathione levels in dermal fibroblasts treated with H₂O₂ for 1 hour either alone or following a 30-minute incubation with test products. Results are shown as the mean fluorescence intensity of the ThiolTracker™ Violet indicator dye divided by a factor of 1,000. The two highest concentrations of the Aloe vera-based Nerium oleander extract (NAE-8®) and the highest concentration of the ALOE (Aloe gel alone) and aqueous N. oleander extract (AQ-NOE) protected intracellular reduced glutathione levels in dermal fibroblasts exposed to oxidative stress. Notes: Statistical significance was calculated by comparing to cells treated with H₂O₂ in the absence of test products, and is indicated by *P<0.05, and **P<0.01. Untreated (UT) cell cultures are shown as a control. Samples were assayed in duplicate. The data are representative of three different experiments. Data are presented as the mean ±SD. Abbreviation: SD, standard deviation.

Figure 5

The inflammation-induced intracellular formation of reactive oxygen species (ROS) in polymorphonuclear (PMN) cells is shown as the percent (%) change relative to untreated PMN cells. Samples were assayed in triplicate and the data shown are representative of three separate experiments using PMN cells from three different healthy adult donors. Both the Aloe vera-based Nerium oleander extract (NAE-8®) and the aqueous N. Oleander extract (AQ-NOE) inhibited ROS formation across a similar concentration range, and the inhibition at concentrations between 0.002–0.2 mL/L was statistically significant when compared to PMN cells not exposed to test product (*P<0.05, **P<0.01). At the concentration of 0.2 mL/L, the NAE-8® performed significantly better than the AQ-NOE (*P<0.05). In comparison, Aloe gel alone (ALOE) did not contribute to this anti-inflammatory effect, suggesting that the compounds in NAE-8® responsible for the reduced ROS production were not derived from the A. vera used during extraction. Note: Data are presented as the mean ± SD. Abbreviations: SD, standard deviation; UT, untreated.

Figure 6

The levels of intracellular formation of reactive oxygen species (ROS) in non-inflamed polymorphonuclear (PMN) cells (in the absence of induced oxidative stress) is shown as the percent (%) change, relative to untreated PMN cells. Samples were assayed in triplicate, and the data shown are representative of three similar experiments using PMN cells from three different healthy adult donors. Both the Aloe vera-based Nerium oleander extract (NAE-8®) and the aqueous N. Oleander extract (AQ-NOE) inhibited ROS formation across a similar concentration range, and the inhibition at concentrations between 2–20 mL/L was statistically significant when compared to PMN cells not exposed to test product (*P<0.05, **P<0.01). In comparison, Aloe gel alone (ALOE) did not contribute to this anti-inflammatory effect, suggesting that the compounds in NAE-8® responsible for the reduced ROS production were not derived from the ALOE used during extraction, but were derived from N. oleander. Note: Data are presented as the mean ± SD. Abbreviations: SD, standard deviation; UT, untreated.

Figure 7

CD69 expression on lymphocyte (A), monocyte (B), and polymorphonuclear (PMN) cell (C) populations in 24-hour whole blood cultures. Samples were assayed in triplicate, and mean fluorescence intensity data are shown and are representative of three separate experiments using whole blood from three different healthy human donors. In all three cell types, exposure to the highest dose of the aqueous Nerium oleander extract (AQ-NOE) resulted in an increase in CD69 expression. Lipopolysaccharide (LPS) was used as a positive control (10 ng/mL) and resulted in an increase in CD69 expression on all three cell types. In the case of monocytes, the 0.2 mL/L concentration of AQ-NOE activated cells better than LPS. Monocytes were also activated by the 0.2 mL/L concentration of Aloe vera gel alone (ALOE). Notes: Statistical significance is indicated by asterisks with * indicating P<0.05 and ** indicating P<0.01. Data are presented as the mean ± SD. Abbreviations: SD, standard deviation; UT, untreated; NAE-8®, Aloe vera-based Nerium oleander extract.

Figure 8

Changes in cytokine levels in cultures of dermal fibroblasts exposed to either Aloe vera gel alone (ALOE), A. vera-based Nerium oleander extract (NAE-8®), or aqueous N. oleander extract (AQ-NOE). Results are shown as percent (%) change from untreated control cultures. Of the 40 cytokines and chemokines tested, only seven reached detectable levels in the culture supernatants. The strong induction of CXCLI 1 by the NAE-8® extract was not seen for either ALOE or AQ-NOE. Samples were assayed in duplicate. Note: Data are presented as the mean ± SD. Abbreviation: SD, standard deviation.