Triacylglycerol, fatty acid, and phytochemical profiles in a new red sorghum variety (Ji Liang No. 1) and its antioxidant and anti‐inflammatory properties

Abstract In this study, a new red sorghum variety (Ji Liang No. 1) was investigated for its triacylglycerol (TAG) and fatty acid profiles, carotenoid and tocopherol compositions, total phenolic, total flavonoid and phenolic acid contents, and antioxidant and anti‐inflammatory properties. A total of 17 TAGs were identified in the red sorghum oil. Linoleic and oleic acids were the primary fatty acids, contributing more than 80% of the total fatty acids. β‐Carotene was the primary carotenoid at a level of 26.14 μg/g. α‐, γ‐, and δ‐tocopherols were at levels of 0.19, 4.08, and 0.10 μg/g, respectively. Moreover, acetone–water (60:40, v/v) extract of the red sorghum exhibited the greatest total phenolic content of 2.77 mg GAE/g and total flavonoid content of 5.44 mg RE/g. The extract had scavenging capacities against DPPH, ABTS +, and peroxyl radicals and suppressed LPS stimulated IL‐1β, IL‐6, and COX‐2 mRNA expressions in a dose‐dependent manner. Ferulic, p‐coumaric, isoferulic, and p‐hydroxybenzoic acids were found in the red sorghum, with ferulic acid as the predominant phenolic acid and mostly in an insoluble bound form. These data indicated a potential utilization of the red sorghum in health‐promoting functional food or supplemental products.

Fats and oils are important food ingredients, not only providing energy but also important for their nutritional values (Yang et al., 2018). Triacylglycerols (TAGs) are the major components and contribute to about 95% weights of fats and oils (Andrikopoulos, Chiou, & Mylona, 2004). Growing evidences show that the triacylglycerol and fatty acid compositions of fats and oils might significantly alter their flavor, stabilities, and physiological activities in food systems (Priore, Stanca, Gnoni, & Siculella, 2012;Yoshinaga et al., 2015). Some previous studies have shown that different sorghum varieties are additional vegetable oil sources due to the high concentrations of unsaturated fatty acids in their oil fractions (Mehmood, Orhan, Ahsan, Aslan, & Gulfraz, 2008;Pontieri et al., 2011). Meanwhile, carotenoids and tocopherols are also detected in sorghum oils, which are considered as two groups of wellrecognized natural antioxidants with potential health benefits Chung, Yong, Lee, & Kim, 2013). However, to the best of our knowledge, the triacylglycerol composition in sorghum oil is still limited.
Besides, it is reported that sorghums with different genotypes and growing environments differed in their appearances, phytochemicals, physiological activities, and nutritional applications (Shen et al., 2018). Therefore, it is important to get such information for a new sorghum variety to determine whether it is a competitive source of the phytonutrients and potentially applied in functional foods or supplemental products. In the present study, a new high-yielding red sorghum variety (Ji Liang No. 1) grown in saline-alkali soil of Shandong was investigated for the profiles of TAGs, fatty acids, carotenoids, and tocopherols in its oil fraction. The sorghum variety was also examined for its phenolic acids composition, total phenolic, total flavonoid contents, and antioxidant and anti-inflammatory capacities. The results from this study may be used to provide useful information for exploring the nutritional value of this red sorghum variety.

| Red sorghum sample
The dehulled red sorghum sample (Ji Liang No. 1) was kindly donated by Shandong Academy of Agricultural Sciences (Jinan, Shandong, China) and ground into flour using a pulverizer (IKA A11 basic, Wilmington, NC, USA) to pass through a 40 mesh sieve. Then, the red sorghum flour was kept in a −20°C freezer in airtight containers until later use.

| Extraction of red sorghum oil
About 800 mg of the red sorghum flour was extracted with approximately 100 ml of petroleum ether (boiling point of 30-60°C) for 4 hr via a Soxhlet apparatus. Then the petroleum ether was evaporated using a Buchi R215 rotary evaporator (Flawil, Switzerland) at a reduced pressure, and the remaining oil was weighed. The oil was stored at ambient temperature under nitrogen in the dark until further testing.

| Triacylglycerols composition
The TAGs composition of red sorghum oil was identified using a Waters Xevo-G2 Q-TOF MS system combined with the Ultra Performance Convergence Chromatography (UPC 2 ) system (Milford, MA, USA) according to a laboratory protocol (Gao et al., 2017).

| Total phenolic contents
The red sorghum extract was analyzed for total phenolic contents (TPC) following a laboratory protocol (Moore, Liu, Zhou, & Yu, 2006). Briefly, 100 μl of sample was mixed with 500 μl of the Folin-Ciocalteu reagent, 1.5 ml of 20 wt% sodium carbonate, and 6 ml of ultrapure water. After 2 hr of reaction, the absorbance at 765 nm was measured to calculate the TPC in samples using gallic acid as a standard. Results are expressed as milligrams of gallic acid equivalents per gram of dried red sorghum (mg GAE/g).

| Total flavonoid contents
The red sorghum extract was analyzed for total flavonoid contents (TFC) following a previous procedure with some modifications (Frassinetti et al., 2018). Briefly, 250 μl of sample was mixed with 75 μl of 5 wt% sodium nitrite solution, 1 ml of ultrapure water and incubated at room temperature for 5 min. Then, 75 μl of 10 wt% aluminum nitrate solution was added and incubated for 6 min. Next, 500 μl of 1 M NaOH and 600 μl of ultrapure water were added. After 10 min of incubation, the absorbance at 510 nm was measured to calculate the TFC in samples using rutin as a standard. Results are expressed as milligrams of rutin equivalents per gram of dried red sorghum (mg RE/g).

| DPPH˙ scavenging capacity
The red sorghum extract was evaluated for its DPPH˙ scavenging capacity according to a laboratory protocol (Whent, Slavin, Kenworthy, & Yu, 2010). Briefly, Trolox was used as a standard. 100 μl of sample, standard or blank, was added to 100 μl of freshly made DPPH˙ solution (0.2 mM) to initiate the reaction. The absorbance of the reaction mixtures was recorded at 517 nm for 30 min. DPPH˙ scavenging capacity was calculated from area under the curve and expressed as milligrams of Trolox equivalents (TE) per gram of dried red sorghum.

| ABTS˙+ scavenging capacity
Radical scavenging capacity of the red sorghum extract against ABTS˙+ was measured according to a laboratory protocol (Moore, Cheng, Su, & Yu, 2006). Briefly, ABTS˙+ working solution was prepared by reacting ABTS with manganese oxide and diluted to an absorbance of 0.7 at 734 nm. Trolox was used as a standard. The final reaction mixture consisted of 80 μl of sample, standard or blank, and 1 ml of ABTS˙+ working solution. The absorbance was read at 734 nm after 90 s of reaction. ABTS˙+ scavenging capacity was calculated using a standard curve prepared with Trolox and expressed as milligrams of TE per gram of dried red sorghum.

| Oxygen radical absorbance capacity
Oxygen radical absorbance capacity (ORAC) assay was conducted to assess peroxyl radical scavenging ability of the red sorghum extract according to a laboratory protocol (Slavin, Lu, Kaplan, & Yu, 2013).
Briefly, Trolox was used as a standard. The initial reaction mixture consisted of 225 μl of freshly made 81.6 nM FL and 30 μl of sample, standard or blank. The mixtures were preheated in a 96-well plate at 37°C for 10 min. Then, 25 μl of freshly made 0.36 M AAPH was added to each well to start the reaction. The fluorescence of assay mixture was recorded every 2 min over 2 hr at 37°C (λ Ex = 485 nm and λ Em = 528 nm). The results were calculated from area under the curve and expressed as milligrams of TE per gram of dried red sorghum.

| Phenolic acid composition
The red sorghum was analyzed for its soluble free, soluble conjugated, insoluble bound and total (soluble free, soluble conjugated, and insoluble bound) phenolic acid compositions using a laboratory protocol (Moore et al., 2005). Acetone/methanol/water (7:7:6, v/v/v) was used to extract the soluble free and conjugated phenolic acids, whereas the insoluble bound phenolic acids in the solid residue were released by 2.10 | Anti-inflammation effect of the red sorghum extract in RAW 264.7 mouse macrophage cells RAW 264.7 mouse macrophage cells were cultured in DMEM containing 10% FBS and 1% amphotericin B/streptomycin/penicillin at 37°C under 5% CO 2 in air to reach a confluence of 80%. The dried red sorghum extract re-dissolved in pure DMSO was added into cell cultures at 10, 100, and 1,000 μg/ml for 24 hr. After pretreatment, LPS was added at an initial concentration of 10 ng/ml. After induction for 4 hr, culture media were discarded and cells were collected to perform RNA isolation and real-time PCR according to a laboratory protocol . Briefly, cells were washed with 1× PBS and TRIzol reagent was added for total RNA isolation. iScriptTM Advanced cDNA Synthesis kit was used to reverse transcribe complementary DNA.

| Statistical analysis
Data are reported as the mean ± standard deviation (SD) for triplicate determinations. One-way ANOVA and Tukey's test were employed to identify differences in means. Statistics were analyzed using SPSS for Windows (version rel. 10.0.5; 1999, SPSS Inc., Chicago, IL, USA).
Statistical significance was declared at p < 0.05.

| Triacylglycerols composition
The red sorghum oil was first obtained by using a Soxhlet extraction method, and a crude oil content of 5-6 g per 100 g dried red sorghum was determined (data not shown). Then, the TAG profiles of red sorghum oil were identified by UPC 2 -Q-TOF MS. Based on the accurate elementary composition and MS 2 fragmentation patterns provided by Q-TOF MS, a total of 17 TAGs were detected and identified in red sorghum oil, and the relative concentration of each TAG was calculated using the peak normalization method and reported as g/100 g of TAGs (Table 1)
These data indicated that the red sorghum oil may serve as a potential dietary source for unsaturated fatty acids. It has been recognized that unsaturated fatty acids are of great importance to human health, because they play an important role for the structure and function of biological membranes (Taylor, Schober, & Bean, 2006). Moreover, unsaturated fatty acids may lower the risk of thrombosis and related cardiovascular diseases due to their anti-aggregating activity on blood lipoprotein particles (De Souza et al., 2015).
Besides, the GC-MS analysis of fatty acid composition detected a total of six fatty acids, which are the same with the eight fatty acids from the UPC 2 -Q-TOF-MS analysis of triacylglycerols composition.
Only linolenic acid and zoomaric acid were not detected in fatty acid composition, which might be due to their relatively low contents.

| Carotenoid and tocopherol contents
The carotenoid composition including β-carotene, lutein, and zeaxanthin was also examined for red sorghum oil (Table 3). The content of β-carotene was 26.14 μg/g of red sorghum, which was found to be the primary carotenoid in red sorghum. Lutein was also present in red sorghum with its content of 5.37 μg/g of red sorghum, whereas no zeaxanthin was detected under the experimental conditions. Therefore, the total carotenoid contents were 31.51 μg/g of red sorghum, which was much greater than those of 3.82-19.5 μg/g in thirteen modern sorghum hybrids reported by Moreau, Harron, Powell, and Hoyt (2016). The difference in total carotenoid contents may be due to the different genotypes and cultivation locations of sorghum varieties.

| Optimization of extraction conditions for polyphenols in red sorghum
Single-factor experiment was designed to evaluate the extraction solvent, solvent concentration, solvent-to-solid ratio, and ultrasonic time on the extraction yields of TPC from the defatted red sorghum. Generally, solvent mixtures containing methanol, ethanol or acetone combined with water in the concentration range of 40%-80% (v/v) were most commonly used to extract phenolic components from plant materials (Lv et al., 2013;Santos et al., 2018). Therefore, these three kinds of solvents with the concentration of 40%, 60%, and 80% (v/v) were studied, respectively. As shown in Figure 1a, the extraction yield for TPC significantly increased with the methanol concentration increasing from 40% to 60% (v/v) and peaked at 60% (v/v). Then it decreased with the methanol concentration further increasing to 80% (v/v) (p < 0.05). The similar trend was also observed when using ethanol or acetone as the extraction solvent. Moreover, acetone with the concentration of 60% (v/v) had the greatest absolute value of TPC, when comparing with those of methanol or ethanol with the same concentration. This result may be explained by the similar polarity nature between acetone/water mixtures (60:40, v/v) and phenolic components in red sorghum, and therefore a higher solubility of phenolic components was achieved (Rostagno & Prado, 2013).
The solvent-to-solid ratio determined the concentration gradient of extraction medium and phenolic components during the TA B L E 1 Identification and relative content of triacylglycerols (TAGs) in red sorghum oil a Peak no.

Calc. Mass ([M+NH 4 ] + )
TAGs composition (g/100 g TAGs) Notes. a Values were analyzed in triplicates and reported as mean ± standard deviation (SD). Fatty acids with lower concentration (<0.05%) are not listed above. "C" and "D" stands for Carbohydrate and Double bond, respectively; "C:D" is the ratio of the total amount of Carbon atoms of the fatty acid in relation to the number of double (unsaturated) bonds in it.

TA B L E 3 Carotenoid and tocopherol contents of red sorghum oil a
Sample β-Carotene (μg/g) Lutein (μg/g) Zeaxanthin (μg/g) α-Tocopherol (μg/g) γ-Tocopherol (μg/g) δ-Tocopherol (μg/g) Red sorghum oil 26.14 ± 1.32 5.37 ± 0.09 nd 0.19 ± 0.00 4.08 ± 0.05 0.10 ± 0.01 Notes. a Values were analyzed in triplicates and reported as mean ± standard deviation (SD). nd represents not detectable. extraction process. The higher the concentration gradient was, the faster mass transfer rate reached, and the more thorough the extraction gained when they reached the balance (Rostagno & Prado, 2013). As shown in Figure 1b, the extraction yield for TPC increased significantly (p < 0.05), when the solvent-to-solid ratio increased from 10:1 to 30:1 with the extraction medium of acetone/water mixture (60:40, v/v) fixed. With further increase of solvent-to-solid ratio from 30:1 to 40:1, the TPC remained statistically unchanged (p > 0.05). The similar tendency for this factor was also showed for the extraction of polyphenolic compounds from red sorghum bran (Luo et al., 2018).
Compared with the conventional extraction method, ultrasonicassisted extraction has been reported to have an enhanced extraction yield for phenolic compounds (Wang, Boussetta, Lebovka, & Vorobiev, 2018). In this study, the effect of ultrasonic time on the extraction yield of TPC was also investigated from 0 to 40 min with the extraction medium of acetone/water mixture (60:40, v/v) and solvent-to-solid ratio of 30:1 fixed (Figure 1c). However, the result indicated that the extraction yield of TPC was not significantly influenced by the ultrasonic time (p > 0.05), which might due to that the soluble phenolic components had already been extracted completely without ultrasonic assistance. Moreover, in most cases, ultrasound is insufficient to break ester or ether bonds and releases some insoluble bound phenolic components, such as insoluble bound phenolic acid, despite the likelihood that the shearing force generated during cavitation would enhance mass transfer by increasing solid solubility and diffusion of solubilized phenolic components into the solvent (Đurović et al., 2018).
Based on the single-factor experiment results, the optimized extraction conditions were determined as follows: the red sorghum was extracted with acetone/water mixture (60:40, v/v) at the solvent-to-solid ratio of 30:1. Under this extraction condition, the values of TPC and TFC of red sorghum extract were 2.77 mg GAE/g and 5.44 mg RE/g of red sorghum, respectively (Table 4).

| Antioxidant activity of red sorghum extract
In this study, DPPH radical, ABTS radical cation scavenging capacities, and ORAC assays were chosen by evaluating the antioxidant activity of red sorghum extract, respectively. As shown in Table 4, DPPH, ABTS, and ORAC values of the red sorghum extract were measured to be 1.97, 13.71, and 40.59 mg TE/g of red sorghum, respectively. It seemed that the ABTS value was 6-7 times higher than the DPPH value. It is known that anthocyanins are the major extractable phenols from red or black sorghums (Akogou, Kayodé, den Besten, & Linnemann, 2017), and they contribute a major portion  (Arnao, 2000). Moreover, compared with the DPPH or ABTS values, the red sorghum extract showed the greatest ORAC value of 40.59 mg TE/g of red sorghum, which indicated that phenolic compounds in red sorghum extract were more efficient at quenching peroxyl radical than DPPH and ABTS cation radicals. This result was consistent with our previous observations for soybean extracts Whent et al., 2010).

| Phenolic acid compositions
As reported in our previous studies, phenolic acids existed in a variety of forms in grains, including soluble free, soluble conjugated, and insoluble bound form (Moore, Luther, Cheng, & Yu, 2009).
Therefore, soluble free, soluble conjugated, and insoluble bound phenolic acid compositions in red sorghum extract were also determined, respectively. As a result, ferulic, p-coumaric, isoferulic, and p-hydroxybenzoic acids were detected under the experimental conditions (Table 5). Similar to other sorghum varieties (Shen et al., 2018) and cereal samples (Moore et al., 2005), the total content of ferulic acid in red sorghum showed the highest level compared to other detected phenolic acids, accounting for 128.93 μg/g of red sorghum and over 47% of the total identified phenolic acids on a weight basis. Most of the ferulic acid in red sorghum was insoluble bound with the content of 88.49 μg/g of red sorghum, and this level accounted for 68.6% of the total ferulic acid. Also, each gram of red sorghum contained 11.21 μg of soluble free, 39.56 μg of soluble conjugated, 29.64 μg of insoluble bound p-coumaric acid, which was the second high content of phenolic acid in red sorghum. Actually, ferulic and p-coumaric acids are also the most commonly detected phenolic acids for sorghum samples according to some previous publications (Chiremba, Taylor, Rooney, & Beta, 2012;Luthria & Liu, 2013), which may be due to their relative high contents. Moreover, isoferulic and p-hydroxybenzoic acids were also detected in red sorghum with their total contents ranging from 45 to 49 μg/g of red sorghum.

| Anti-inflammatory effect of red sorghum extract in RAW 264.7 mouse macrophage cells
Chronic inflammation has been recognized as a risk factor for a number of health problems, including cardiovascular disease, cancer, and obesity (Väyrynen et al., 2018). Several cytokines, including interleukin-1β (IL-1β), IL-6, and cyclo-oxygenase (COX-2), are three critical proinflammation mediators involved in multiple inflammatory pathways. The inhibition of their mRNA expression may lead to alleviation of the inflammatory responses. In this study, the effect of red sorghum extract on IL-1β, IL-6, and COX-2 mRNA expressions was examined for their potential anti-inflammatory activities. Notes. a Values were analyzed in triplicates and reported as mean ± standard deviation (SD). nd represents not detectable.
TA B L E 5 Phenolic acid composition of red sorghum extract a As shown in Figure 2, the red sorghum extract significantly suppressed the LPS-induced IL-1β, IL-6, and COX-2 mRNA expressions at 100 or 1,000 μg/ml initial treatment concentration (p < 0.05), whereas 10 μg/ml of red sorghum extract did not have significant inhibitory effect. Moreover, at 100 or 1,000 μg/ml initial treatment concentration, the inhibitory effect of red sorghum extract was in a dose-dependent way. The higher initial treatment concentration of red sorghum extract was, the stronger inhibitory effect was observed. In this study, the red sorghum extract showed the strongest inhibitory effect in suppressing IL-6 mRNA expression at 1,000 μg/ ml initial treatment concentration, which inhibited 97.5% of IL-6 mRNA expression under the experimental conditions ( Figure 2b).
Taken together, these results indicated that the red sorghum extract might have an anti-inflammatory potential.

| CON CLUS ION
In summary, the present study demonstrated the triacylglycerol composition of red sorghum oil for the first time and indicated that the new red sorghum variety may serve as a potential dietary source for unsaturated fatty acids and other beneficial phytochemicals, such as tocopherols, carotenoids, and phenolic compounds, therefore contributing to its antioxidant and antiinflammatory properties. Additional studies with larger sample sizes and detailed growing conditions are necessary to further investigate the relationship between chemical compositions of red sorghum and its health-promoting or disease-preventing activities in humans.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflict of interests.