Gold Nanoparticles Synthesized Using Extracts of Cyclopia intermedia, Commonly Known as Honeybush, Amplify the Cytotoxic Effects of Doxorubicin

Abstract

Cyclopia intermedia (C. intermedia) is an indigenous South African shrub used to prepare the popular medicinal honeybush (HB) tea. This plant contains high levels of mangiferin (MGF), a xanthonoid that was reported to have numerous biological activities, including anti-tumor activity. MGF and extracts that contain high concentrations of MGF, such as extracts from Mangifera indica L. or mango have been used to synthesize gold nanoparticles (AuNPs) using green nanotechnology. It has previously been shown that when AuNPs synthesized from M. indica L. extracts are used in combination with doxorubicin (DOX) and Ayurvedic medicine, the anti-tumor effects appear to be augmented. It has also been demonstrated that MGF used in combination with DOX resulted in enhanced anti-tumor effects. In this study, C. intermedia (HB) and MGF were used to synthesize HB-AuNPs and MGF-AuNPs, respectively. The physicochemical properties of the AuNPs were characterized by the UV-Visible Spectroscopy (UV-Vis), dynamic light scattering (DLS), Fourier transform infra-red spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD) and high-resolution transmission electron microscopy (HR-TEM). The cytotoxicity of HB-AuNPs and MGF-AuNPs were assessed on human colon (Caco-2), prostate (PC-3) and glioblastoma (U87) cancer cells; as well as normal breast epithelial (MCF-12A) cells using the MTT assay. Both HB-AuNPs and MGF-AuNPs demonstrated relatively low cytotoxicity in these cells. However, when these nanoparticles were used in combination with DOX, the cytotoxicity of DOX was significantly augmented.

Keywords: Cyclopia intermedia; gold nanoparticles; green synthesis; honeybush; mangiferin; nanotechnology.

Scheme 1

Possible mechanism for green synthesis of biogenic MGF-gold nanoparticles (AuNPs) and HB-AuNPs.

Figure 1

UV-Visible absorption of HB-AuNPs and MGF-AuNPs.

Figure 2

Morphology and energy dispersive X-ray spectroscopy (EDX) profiling of the biogenic AuNPs. High-resolution transmission electron microscopy (HR-TEM) micrographs of HB-AuNPs (a) and MGF-AuNPs (b); particle size distribution of HB-AuNPs (c) and MGF-AuNPs (d); EDX profiles of HB-AuNPs (e) and MGF-AuNPs (f) for their chemical composition.

Figure 3

XRD and (SAED) patterns of the biogenic AuNPs as determined by XRD Spectroscopy and HR-TEM. XRD patterns of HB-AuNPs (a) and MGF-AuNPs (b); SAED patterns of HB-AuNPs (c) and MGF-AuNPs (d).

Figure 4

FTIR spectra of the HB and MGF extracts and their respective biogenic AuNPs. (a) HB-Extract and HB-AuNPs; (b) MGF-Extract and MGF-AuNPs; (c) HB-AuNPs and MGF-AuNPs.

Figure 5

Stability of AuNPs in biological media at 0 and 24 h incubation at 37 °C. UV-Vis spectra of HB-AuNPs (a–e) and MGF-AuNPs (f–j) in DMEM (a,f), RPMI (b,g), BSA (c,h), FBS (d,i) and DPBS (e,j).

Figure 6

Investigation of the cytotoxic effects of HB-AuNPs and MGF-AuNPs in human cell lines. The in vitro effects of HB-AuNPs and MGF-AuNPs were tested in MCF-12A (a) U87 (b), Caco-2 (c) and PC-3 (d) cells. Data represented as the mean from three independent experiments. Error bars represent the standard error mean (± SEM). Data were considered to be statistically significant if p < 0.05, *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 7

Co-treatment of Caco-2 cells with AuNPs and DOX enhanced cell death, compared to individual treatments. (a) shows the viability of Caco-2 cells following treatment with increasing concentrations (1.56–100 µg/mL) of DOX for 24 hrs. (b) shows the viability of Caco-2 cells following treatment with MGF (1000 µg/mL), HBE (1000 µg/mL), MGF-AuNPs (1000 µg/mL), HB-AuNPs (1000 µg/mL) and DOX (1.56 µg/mL) in combination with either MGF-AuNPs (MGF-AuNPs-DOX) or HB-AuNPs (HB-AuNPs-DOX). Data represented as the mean from three independent experiments. Error bars represent the standard error mean (± SEM). Data were considered to be statistically significant if p <0.05. *** p < 0.001, ** p < 0.01, * p < 0.05, # < 0.05 HB-AuNPs vs. MGF-AuNPs.