Enhanced Antioxidant Effects of Naringenin Nanoparticles Synthesized using the High-Energy Ball Milling Method

Abstract

Naringenin, one of the flavonoid components, is majorly found in and obtained from grapefruits and oranges. Naringenin also acts as a potent antioxidant, which possesses hypolipidemic as well as anti-inflammatory potential. Naringenin reduces the expressions of several inflammatory mediators, viz., NF-κB, cycloxygenase-2, and other cytokine mediators. In spite of having various biological effects, the clinical application of naringenin is restricted due to its very poor aqueous solubility. In the present study, the high-energy ball milling method was employed for the preparation of naringenin nanoparticles without using any chemical with an aim to enhance the anti-oxidant potential of naringenin. The milled naringenin nanoparticles were characterized for their physicochemical properties using scanning electron microscopy (SEM) and X-ray diffraction. Additionally, the effects of milling time and temperature were further assessed on the solubility of crude and milled naringenin samples. The antioxidant potential of milled naringenin was evaluated with various assays such as DHE, DCFDA, and cleaved caspase-3 using SH-SY5Y human neuroblastoma cells. The nanoparticle size of naringenin after milling was confirmed using SEM analysis. Crystalline peaks for milled and crude samples of naringenin also established that both the naringenin forms were in the crystalline form. The solubility of naringenin was enhanced depending on the milling time and temperature. Moreover, crude and milled naringenin were found to be cytocompatible up to doses of 120 μM each for the duration of 24 and 48 h. It was also observed that milled naringenin at the doses of 1, 2, and 5 μM significantly reduced the levels of reactive oxygen species (ROS) generated by H₂O₂ and exhibited superior ROS scavenging effects as compared to those of crude or un-milled forms of naringenin. Furthermore, milled naringenin at the doses of 1 and 2 μM inhibited H₂O₂-induced cell death, as shown by immunofluorescence staining of cleaved caspase-3 and Annexin-V PI flow cytometry analysis. Conclusively, it could be suggested that the size reduction of naringenin using high-energy ball milling techniques substantially enhanced the antioxidant potential as compared to naïve or crude naringenin, which may be attributed to its enhanced solubility due to reduced size.

Figure 1

SEM images of naringenin at different milling times; here (a) unprocessed naringenin, (b) naringenin after 120 min of milling, (c) naringenin after 240 min of milling, (d) naringenin after 480 min, (e) naringenin after 600 min of milling, and (f) naringenin after 720 min of milling.

Figure 2

X-ray diffraction patterns of naringenin at milling time; Ncrude (not processed), N480 (after 480 min of milling), and at N720 (after 720 min of milling).

Figure 3

(a) Solubility values of crude and milled naringenin samples (black) without sonication and solubility values of crude and milled naringenin samples with sonication (black). (b) Effect of temperature on solubility of processed naringenin at different time points.

Figure 4

Dissolution study of naringenin at different milling times. In this graph, Ncrude, N120, N480, N600, and N720 show unprocessed naringenin, after 120 min of milling, after 480 min of milling, after 600 min of milling, after 720 min of milling, respectively.

Figure 5

Biocompatibility study of the (a) crude or naïve form and (b) nanosized form of naringenin as carried out in hTERT-BJ (human skin fibroblast) cell lines for a period of 24 and 48 h.

Figure 6

(a) DHE in the SH-SY5Y human neuroblastoma cell line and (b) its quantification.

Figure 7

(a) DCFDA in the SH-SY5Y human neuroblastoma cell line and (b) its quantification.

Figure 8

(a) Cleaved caspase-3 in the SH-SY5Y human neuroblastoma cell line and (b) its quantification.

Figure 9

(a) Annexin-V PI flow cytometry for cell death of the SH-SY5Y human neuroblastoma cell line and (b) its quantification.