Neem oil nanoemulsions: characterisation and antioxidant activity

Abstract

The aim of the present work is to develop nanoemulsions (NEs), nanosized emulsions, manufactured for improving the delivery of active pharmaceutical ingredients. In particular, nanoemulsions composed of Neem seed oil, contain rich bioactive components, and Tween 20 as nonionic surfactant were prepared. A mean droplet size ranging from 10 to 100 nm was obtained by modulating the oil/surfactant ratio. Physicochemical characterisation was carried out evaluating size, ζ-potential, microviscosity, polarity and turbidity of the external shell and morphology, along with stability in simulated cerebrospinal fluid (CSF), activity of Neem oil alone and in NEs, HEp-2 cell interaction and cytotoxicity studies. This study confirms the formation of NEs by Tween 20 and Neem oil at different weight ratios with small and homogenous dimensions. The antioxidant activity of Neem oil alone and in NEs was comparable, whereas its cytotoxicity was strongly reduced when loaded in NEs after interaction with HEp-2 cells.

Keywords: Neem oil; antioxidant activity; cell interaction studies; nanoemulsions; physicochemical characterisation.

Graphical abstract

Figure 1.

Shelf-life stability of evaluated NEs. Hydrodynamic diameter and ζ-potential values of different NEs (A–C) prepared using water¹ or Hepes buffer² as aqueous phase up to 90 days stored at 25 and 4 °C temperatures. Results are expressed as means ± SD (n = 3).

Figure 2.

Stability in CSF. Size and ζ-potential measurements by DLS of different NEs (A–C) prepared using water (A) and Hepes buffer 10⁻²M, pH 7.4 (B) at 37 °C up to 3 h.

Figure 3.

SAXS intensity spectrum. Scattered radiation intensity as a function of momentum transfer q of A2 sample at room temperature. The flattening of the intensity for q < 0.1 nm⁻¹ shows that aggregates have a globular shape of finite size, of the order of 30 nm. At higher q (0.18 < q < 0.75 nm⁻¹), the intensity I(q)/q ^−1.66. This decay behaviour is characteristic for an internal core composed by connected substructures or sponge phases.

Figure 4.

Transmission electron photomicrographs of the Hepes nanoemulsion samples. Panel A: A2; Panel B: B2; Panel C: C2. Arrows indicate NE sizes corresponding to DLS measures.

Figure 5.

Exposure of HEp-2 cells to Neem oil-based NEs. HEp-2 cells were treated with NEs or free Neem oil as described (see Methods). The OD values obtained by MTT assay for treated cells were converted into numbers of cells on a standard curve and expressed as percentages of untreated controls. Bars represent the mean of three independent experiments ± SD. *p < .05 (Panel A). Fluorescence microscopy images of HEp-2 control cells exposed to free Nile red (Panel B) and HEp-2 cells exposed to Nile red-loaded NEs for 7 (Panel C) and 24 h (Panel D) obtained by fluorescence microscopy as described (see Methods). Scale bar: 10 μm.