Evaluating the Antioxidant Properties of the Ancient-Crop Tef (Eragrostis tef) Grain Extracts in THP-1 Monocytes

Abstract

Tef (Eragrostis tef) is an orphan crop that is widely grown in East Africa, primarily in Ethiopia as a staple crop. It is becoming popular in the Western world owing to its nutritious and gluten-free grains and the forage quality of its biomass. Tef is also considered to have a high antioxidant capacity based on cell-free studies. However, the antioxidant activity of tef has never been validated using a physiologically relevant cell model. The purpose of this study was to investigate the antioxidant capacity of tef grain extracts using a mammalian cell model. We hypothesized that the tef grain extracts are capable of modulating the cellular antioxidant response via the modulation of glutathione (GSH) biosynthetic pathways. Therefore, we evaluated the antioxidant activity of purified tef grain extracts in the human acute monocytic leukemia (THP-1) cell line. Our findings revealed that the organic fraction of grain extracts increased the cellular GSH level, which was more evident for brown-colored tef than the ivory variety. Moreover, a brown-tef fraction increased the expressions of GSH-pathway genes, including γ-glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits and glutathione reductase (GR), an enzyme that plays a key role in GSH biosynthesis, suggesting that tef extracts may modulate GSH metabolism. Several compounds were uniquely identified via mass spectrometry (MS) in GSH-modulating brown-tef samples, including 4-oxo-β-apo-13-carotenone, γ-linolenic acid (methyl ester), 4,4'-(2,3-dimethyl-1,4-butanediyl)bis-phenol (also referred to as 8,8'-lignan-4,4'-diol), and (3β)-3-[[2-[4-(Acetylamino)phenoxy]acetyl]oxy]olean-12-en-28-oic acid. Tef possesses antioxidant activity due to the presence of phytochemicals that can act as direct antioxidants, as well as modulators of antioxidant-response genes, indicating its potential role in alleviating diseases triggered by oxidative stresses. To the best of our knowledge, this is the first report revealing the antioxidant ability of tef extracts in a physiologically relevant human cell model.

Keywords: Eragrostis tef; antioxidant activity; glutathione-pathway genes; grain extracts; phytochemicals.

Figure 1

Schematic diagram showing how tef grains were extracted to prepare fractions for the antioxidant assay. (A) Tef seeds to be used for extraction (Note: brown and ivory are the most common tef seed colors). (B) Crude tef fractions obtained via flash chromatography. (C) Microscopic images (10×) of Human cell line (THP-1 monocytes) used for GSH assay. IE, ivory extract; IFR, ivory fraction; BE, brown extract; BFR, brown fraction.

Figure 2

Cell viability of THP-1 monocytes in response to tef fractions. Cells were cultured without (V) or with DMSO, CDDO-Im, or 50 µg/mL of tef extracts dissolved in DMSO for 24 h, and their viability was determined by dividing the number of live cells by the total number of cells counted, as described in the Materials and Methods. Bars represent the mean and SE of three independent experiments, each replicated at least three times.

Figure 3

Cellular GSH in response to tef extracts and fractions. (A) GSH level as affected by ivory-tef grain in THP-1 monocytes. Cells were cultured without (V) or with DMSO, CDDO-Im, or 50 µg/mL of ivory-tef extract (IE) and fractions obtained via flash chromatography (IFR1, IFR2, IFR3, and IFR4). (B) GSH level as affected by brown-tef grain in THP-1 monocytes. Cells were cultured without (V) or with DMSO, CDDO-Im, or 50 µg/mL of brown-tef extract (BE) and fractions obtained via flash chromatography (BFR1, BFR2, BFR3, BFR4, and BFR5). Cellular GSH level was determined as described in Section 2. Bars represent the mean and SE of three independent experiments, each replicated at least three times. Bars bearing the same letter are not statistically different.

Figure 4

Comparison of ivory- and brown-tef extracts based on cellular GSH levels. Cells were cultured without (V) or with DMSO, CDDO-Im, and 50 µg/mL of brown-tef (BE) and ivory-tef (IE) extracts and the most active fractions of brown-tef (BFR3) and ivory-tef (IFR4) extracts obtained via flash chromatography. Cellular GSH level was determined as described in Section 2. Bars represent the mean and SE of three independent experiments, each replicated at least three times. Bars bearing the same letter are not statistically different.

Figure 5

Expressions of GSH-pathway genes in THP-1 monocytes in response to tef fractions. Cells were cultured without or with DMSO, CDDO-Im, or 50 µg/mL of brown-tef (BFR3) and ivory-tef (IFR4) extracts for 24 h, and gene expression was analyzed via qPCR as described in Section 2. Bars represent the mean and SE of three independent assays, each with four replicates. Bars bearing the same letter are not statistically different. (A), (B), and (C) represent the relative quantification (RQ) for GCLC, GCLM, and GR gene expression, respectively, after treatment with V, DMSO, CDDO-Im, BRF3, and IFR4.

Figure 6

Tef phytochemicals uniquely identified in active tef fractions: (A) (3β)-3-[[2-[4-(acetylamino)phenoxy]acetyl]oxy]olean-12-en-28-oic acid, (3β)); (B) 4,4′-(2,3-dimethyl-1,4-butanediyl)bis-phenol or 8,8′-lignan-4,4′-diol; (C) 4-oxo-β-apo-13-carotenone; (D) γ-linolenic acid (methyl ester). The THP-1 monocytes were treated using chromatographically purified tef grain extracts, and changes to intracellular GSH levels were observed. Tef grain fractions that increased GSH levels were deemed active and later analyzed via mass spectrometry. Thirty-eight unique features, not observed in the inactive fraction, were identified using an online database, and these were narrowed down to four candidate compounds.