Sorghum Grain Polyphenolic Extracts Demonstrate Neuroprotective Effects Related to Alzheimer's Disease in Cellular Assays

Abstract

Sorghum grain contains high levels and a diverse profile of polyphenols (PPs), which are antioxidants known to reduce oxidative stress when consumed in the diet. Oxidative stress leading to amyloid-β (Aβ) aggregation, neurotoxicity, and mitochondrial dysfunction is implicated in the pathogenesis of Alzheimer's disease (AD). Thus, PPs have gained attention as possible therapeutic agents for combating AD. This study aimed to (a) quantify the phenolic compounds (PP) and antioxidant capacities in extracts from six different varieties of sorghum grain and (b) investigate whether these PP extracts exhibit any protective effects on human neuroblastoma (BE(2)-M17) cells against Aβ- and tau-induced toxicity, Aβ aggregation, mitochondrial dysfunction, and reactive oxygen species (ROS) induced by Aβ and tert-butyl hydroperoxide (TBHP). PP and antioxidant capacity were quantified using chemical assays. Aβ- and tau-induced toxicity was determined using the 3-(4,5-dimenthylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTS) assay. The thioflavin T (Th-T) assay assessed anti-Aβ aggregation. The dichlorodihydrofluorescein diacetate (DCFDA) assay determined the levels of general ROS and the MitoSOX assay determined the levels of mitochondrial superoxide. Sorghum varieties Shawaya short black-1 and IS1311C possessed the highest levels of total phenolics, total flavonoids, and antioxidant capacity, and sorghum varieties differed significantly in their profile of individual PPs. All extracts significantly increased cell viability compared to the control (minus extract). Variety QL33 (at 2000 µg sorghum flour equivalents/mL) showed the strongest protective effect with a 28% reduction in Aβ-toxicity cell death. The extracts of all sorghum varieties significantly reduced Aβ aggregation. All extracts except that from variety B923296 demonstrated a significant (p ≤ 0.05) downregulation of Aβ-induced and TBHP-induced ROS and mitochondrial superoxide relative to the control (minus extract) in a dose- and variety-dependent manner. We have demonstrated for the first time that sorghum polyphenolic extracts show promising neuroprotective effects against AD, which indicates the potential of sorghum foods to exert a similar beneficial property in the human diet. However, further analysis in other cellular models and in vivo is needed to confirm these effects.

Keywords: Alzheimer’s disease; amyloid-β; antioxidants; mitochondrial function; neuroprotection; polyphenols; reactive oxygen species; sorghum.

Figure 1

Protective effects of the six different varieties of sorghum extracts dissolved in two different solvents on the BE (2)-M17 cells against the Aβ-induced neurotoxicity of the BE (2)-M17 cells using the MTS assay. All the samples were compared to the negative control set at 100% viability. The concentrations C1 (C2) of the extract of each sorghum variety calculated in terms of the sorghum flour equivalents were Shawaya short black-1, 750 µg/mL (375 µg/mL); IS1311C, 750 µg/mL (375 µg/mL); QL33/QL36, 1000 µg/mL (500 µg/mL); B923296, 1000 µg/mL (500 µg/mL); QL12, 2000 µg/mL (1000 µg/mL); and QL33, 2000 µg/mL (1000 µg/mL). The half dosages were tested for the DMSO extracts only. The data are expressed as mean ± SD (n = three to four independent assays). The statistically significant differences in comparison to the control are marked (*) for (p ≤ 0.05) and marked (**) for (p ≤ 0.01).

Figure 2

Total fluorescence quantification (23 h) as a measure of self-induced aggregation kinetics using the Thioflavin-T assay. The statistically significant differences are marked (*) for (p ≤ 0.05) and marked (**) for (p ≤ 0.01). Negative control = sorghum extracts only; Aβ42 = positive control (20 µM Aβ42 only); six different varieties were assessed over a 23 h period by the (Th-T) fluorescence assay. Th-T fluorescence is represented as arbitrary units (AUs).

Figure 3

The effects of PP-rich extracts from the six different varieties of sorghum on the Aβ-induced oxidative stress in the BE (2)-M17 cells. The ROS level is expressed as a fold change in the treatments compared to the positive control. All the values are presented as mean ± SD of four independent experiments (n = 4). Each experiment included three replicates. The statistically significant differences in comparison to the positive control are marked (*) for (p ≤ 0.05) and marked (**) for (p ≤ 0.01). NC = negative control, PC = positive control.

Figure 4

The neuroprotective effects of the PP-rich extracts from the six different varieties of sorghum on the TBHP-induced oxidative stress in the BE (2)-M17 cells. The initial cellular ROS level was obtained from a fluorescence microplate reader using a DCFDA kit. The ROS level is expressed as a fold change in PC after background subtraction. All the values presented correspond to mean ± SD of two to four independent experiments. Each experiment included two replicates. The statistically significant differences in comparison to the PC are marked (*) for (p ≤ 0.05) and marked (**) for (p ≤ 0.01). NC = negative control, PC = positive control.

Figure 5

The neuroprotective effects of the PP-rich extracts from the six different varieties of sorghum on the Aβ-induced mitochondrial superoxide in the BE (2)-M17 cells. The relative MitoSOX fluorescence intensity was analyzed by the processing of images taken from the fluorescence microscopy. The mean ± SD of three independent experiments is presented. The statistically significant differences in comparison to the PC are marked **, where p ≤ 0.01. NC = negative control, PC = positive control.

Figure 6

The neuroprotective effects of the PP-rich extracts from the six different varieties of sorghum on the TBHP-induced mitochondrial superoxide in the BE (2)-M17 cells. The relative MitoSOX fluorescence intensity was analyzed by the processing of images taken from fluorescence microscopy. The mean ± SD of three independent experiments is presented. The statistically significant differences in comparison to the PC are marked **, where p ≤ 0.01. NC = negative control, PC = positive control.