Sorghum [<i>Sorghum bicolor</i> (L.) Moench] Genotypes with Contrasting Polyphenol Compositions Differentially Modulate Inflammatory Cytokines in Mouse Macrophages

Rhodes, Davina H.; Kresovich, Stephen

doi:https://doi.org/10.1155/2016/9640869

Journal of Chemistry

On this page

Abstract Introduction Materials and Methods Results Discussion Conclusions Abbreviations Disclosure Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2016 | Article ID 9640869 | https://doi.org/10.1155/2016/9640869

Sorghum [Sorghum bicolor (L.) Moench] Genotypes with Contrasting Polyphenol Compositions Differentially Modulate Inflammatory Cytokines in Mouse Macrophages

Davina H. Rhodes¹and Stephen Kresovich²

Academic Editor: Thaila C. Putarov

Received04 Mar 2016

Revised07 Jun 2016

Accepted21 Jun 2016

Published18 Jul 2016

Abstract

This study sought to characterize and compare anti-inflammatory effects of twenty sorghum accessions with contrasting grain polyphenol concentrations but similar genetic backgrounds (based on a genomewide estimate of relatedness). Cell viability, tumor necrosis factor- (TNF-) α, and interleukin- (IL-) 6 were measured in RAW 264.7 macrophages treated with increasing doses (0, 15, 30, and 60 μg/mL) of sorghum ethanol extracts and stimulated with lipopolysaccharide (LPS). Extract dose had a significant effect on TNF-α and IL-6, with a trend of cytokines decreasing between 0 μg/mL and 15 μg/mL of sorghum extract. Genotype also had a significant effect on the cytokines, with extracts from four accessions significantly decreasing TNF-α and/or IL-6. Cells treated with 3-deoxyanthocyanidin-containing accessions had less cytokine production than non-3-deoxyanthocyanidin accessions, whereas cells treated with proanthocyanidin-containing accessions had more cytokine production than cells treated with nonproanthocyanidin accessions. Additionally, there was a significant effect of the Tannin1 allele on TNF-α and IL-6. Our results demonstrate that sorghum genotypes differentially modulate induction of inflammatory cytokine production in RAW 264.7 macrophages and that specialty grain has the potential to be tailored by controlling traits at the nucleotide level. This study adds to our knowledge of sorghum health effects and contributes to efforts aimed at developing health-promoting sorghum grain.

1. Introduction

Cereal crops provide the majority of nutrients to the world’s population and thus have a significant impact on human health. However, most cereal breeding programs primarily focus on increasing grain yield and stress resistance because of the challenges involved in integrating human nutrition research [1, 2]. To breed cereal crops for human health, it is essential to understand the impact of different crop species, genotypes, and their associated composition on human nutrition by characterizing the health effects of their nutrient differences. Consumption of whole grain has been correlated with protective health effects related to several chronic inflammatory diseases, including obesity, type 2 diabetes, cancer, and cardiovascular disease [3–8]. Inflammation is known to be the underlying cause of many chronic diseases [9, 10], and consuming foods with anti-inflammatory properties may help to prevent or attenuate the damage caused by inflammation. A large body of research demonstrates the anti-inflammatory effects of a variety of fruits [11–13], but these foods are a small contribution to daily food intake compared to grain products. Consumption of cereal grain with anti-inflammatory constituents has the potential to improve health outcomes in chronic disease.

A key feature of inflammation is the activation of inflammatory cells, especially monocytes and macrophages, which produce proinflammatory cytokines, including TNF-α and IL-6. These cytokines play crucial roles in inflammation, and excessive activation of the pathway leading to their production can result in chronic inflammation [14, 15]. Much of the research on sorghum polyphenol health benefits has been on its high antioxidant activity compared to commonly consumed fruits [16, 17], but some studies suggest that sorghum grain has anti-inflammatory activity as well [18–22].

Many of the beneficial health effects of whole grain may be due to polyphenols in the bran [23–25]. Polyphenols are found in abundance in fruits, vegetables, tea, chocolate, red wine, and coffee, but also in certain grain crops, including varieties of wheat, rice, maize, and sorghum [24, 26–28]. Sorghum is one of the world’s major cereal crops, feeding millions of people in Asia and Sub-Saharan Africa [29]. In the United States, it is used primarily as livestock feed, but it is also used in many specialty grain products and gluten-free food products [30–33]. It is a genetically and phenotypically diverse cereal crop, with some varieties containing no measurable amounts of polyphenols and others containing high levels of multiple polyphenols [34, 35], particularly proanthocyanidins and 3-deoxyanthocyanidins [36].

Most studies have only explored the health benefits of a small number of sorghum accessions, but over 45,000 sorghum accessions are available from the US National Plant Germplasm System’s Germplasm Resources Information Network (GRIN) [37]. Using a large genetically diverse sorghum panel to explore the effects of natural variation of sorghum polyphenols on induction of inflammatory cytokine production will aid in discovering particularly beneficial accessions. Although several studies have been conducted comparing health effects between high and low polyphenol sorghum, none of them controlled for genetic background of the sorghum [19, 22]. Without adequate control of other genetic factors it may not be possible to attribute health effects to polyphenols per se. The goals of this study were to characterize and compare the induction of inflammatory cytokine production of twenty sorghum accessions with contrasting grain polyphenol concentrations but similar genetic backgrounds and to gain a broader understanding of the diversity of anti-inflammatory effects available in sorghum germplasm.

2. Materials and Methods

2.1. Plant Materials

We selected 20 sorghum accessions from a panel of 381 accessions that we previously evaluated for polyphenol concentrations [34]. The 20 accessions were selected based on contrasting polyphenol content and genetic relatedness. The panel primarily consisted of the sorghum association panel (SAP) [38], which includes accessions from all major cultivated races and geographic centers of diversity in Sub-Saharan Africa and Asia, as well as important breeding lines from the United States. Also included were 73 accessions selected based on the presence of a pigmented testa (which indicates presence of proanthocyanidins) using the GRIN data query. Seeds for all the sorghum accessions were obtained from GRIN and are readily available through GRIN.

The grain samples used for this experiment have previously been described [34]. Briefly, the panel was planted in late April 2012 at Clemson University Pee Dee Research and Education Center in Florence, SC, in a twofold replicated (hereafter referred to as biological replicates) complete randomized block design. Panicles were collected at physiological maturity between September and October and mechanically threshed. Samples were phenotyped using near-infrared spectroscopy (NIRS) as previously described [34]. Total phenol, proanthocyanidin, and 3-deoxyanthocyanidin data were calculated based on dry seed and are expressed as mg gallic acid equivalent (GAE)/g, mg catechin equivalents (CE)/g, and absorbance (abs/mL/g), respectively. For the purposes of this study, we use a cutoff greater than 10 mg CE/g to define proanthocyanidin-containing varieties and greater than 50 abs/mL/g to define 3-deoxyanthocyanidin-containing varieties. Seed protein, fat, and starch are expressed as a percentage of dry seed weight. Seed weight is expressed as the total grain weight of 100 grains per accession.

2.2. Genomic Analysis

To select accessions with comparable genetic backgrounds, we used the genotypes of each accession to assess relatedness. Genotypes were available for the 381 accessions [34, 39]. Based on 404,628 SNP markers, the degree of genetic relatedness between accessions was calculated in a kinship matrix in a unified mixed linear model [40] using the statistical genetics package Genome Association and Prediction Integrated Tool (GAPIT) [41]. To validate proanthocyanidin effects on TNF-α and IL-6, we used a SNP (S4_61667908) on chromosome 4 that is tightly linked (~200 bp) to the causal loss-of-function mutation in the Tannin1 gene. The wildtype allele Tannin1, along with two nonfunctional alleles tan1-a and tan1-b, is responsible for sorghum grain proanthocyanidin production [42].

2.3. Preparation of Sorghum Bran Extracts

Extraction was done according to the methods of Burdette et al. [19], with minor modifications. A tangential abrasive dehulling device (TADD; Venables Machine Works, Saskatoon, Canada) equipped with an 80-grit abrasive disk was used to remove the bran from the grain. Bran was mixed with 50% ethanol [10% (w/v)] and placed on a shaker at room temperature for three hours. Samples were then centrifuged at 5,000 rpm for 15 minutes and supernatant was poured through a 0.2-micromolar filter into a sterile container. Samples were refrigerated and protected from light until being ready to use.

2.4. Cell Cultures

The mouse macrophage cell line RAW 264.7 (TIB-71 from American Type Culture Collection (ATCC)) was cultured on 100 mm culture dishes and maintained in Dulbecco’s modified Eagle’s medium (DMEM, ATCC), supplemented with 10% fetal bovine serum (ATCC) and 100 I.U./mL penicillin and 100 μg/mL streptomycin (ATCC) at 37°C in a humidified incubator with 5% CO₂.

2.5. Cell Viability Assay

Cell viability was measured using the MTT Cell Proliferation Assay (R&D Systems), an indirect method of measuring metabolically active cells. RAW 264.7 cells were seeded in a 96-well plate at 1 × 10⁵ cells/well and incubated overnight to allow cells to recover and adhere to the cell culture plate. Cells were pretreated for one hour with sorghum extracts (two biological replicates per accession) at concentrations of 60 μg/mL, 30 μg/mL, 15 μg/mL, and 0 μg/mL (negative control: 50% EtOH diluted with cell culture medium to the same concentrations) and then stimulated with LPS at 1 μg/mL (diluted with cell culture medium) for an additional 18 hours. The MTT reagent was added to each well and cells were incubated for an additional 2 hours until purple dye was visible under the microscope. Detergent reagent was added and the plates were left in the dark at room temperature for 4 hours. Absorbance was measured at 570 nm in a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). Results are expressed as the percentage of absorbance of extract-treated cells versus negative controls. Viability less than 70% compared to the control was considered potentially cytotoxic.

2.6. Cytokine Assays

Cells were seeded in 96-well plates at 2 × 10⁵ cells/well and incubated overnight to allow time to recover and adhere to the substrate. Cells were pretreated for one hour with sorghum bran extracts (two biological replicates per accession) at concentrations of 60 μg/mL, 30 μg/mL, 15 μg/mL, and 0 μg/mL (negative control: 50% EtOH diluted with cell culture medium to the same concentrations) and then stimulated with LPS at 1 μg/mL (diluted with cell culture medium) for an additional 18 hours. Cell culture medium was collected and tested using TNF-α and IL-6 ELISA Ready-Set-Go! kits (eBioscience). Assays were carried out according to manufacturer’s instructions. Absorbance was measured at 450 nm with wavelength subtraction at 570 nm in a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). Results are expressed as the percentage of cytokine levels in extract-treated cells versus negative controls (negative control: 50% EtOH diluted with cell culture medium to the same concentrations) and normalized with respect to cell viability for each extract.

2.7. Statistical Analysis

Statistical differences were assessed using analysis of covariance (ANCOVA) followed by Tukey’s test to detect significant differences between treatments. values of <0.05 were considered statistically significant. Data are expressed as the means ± standard deviation (SD) of results from three technical replicates and two biological replicates. All calculations were performed using R, a statistical computing language and environment [43].

3. Results

3.1. Targeted Set of Germplasm for Cell Activation Studies

To identify candidates with anti-inflammatory potential, we screened 381 sorghum accessions for high concentrations of proanthocyanidins and/or 3-deoxyanthocyanidins and used a kinship matrix to identify a subset of accessions with similar genetic background (high kinship value) but with contrasting polyphenol content (Figure 1). The resulting set included eleven proanthocyanidin-containing accessions (based on NIRS values greater than 10 mg CE/g or presence of a pigmented testa), eight 3-deoxyanthocyanidin-containing accessions (based on NIRS values greater than 50 abs/mL/g), and five low polyphenol accessions that did not contain either of the polyphenols (Figure 2). Three of the accessions contained both polyphenols. Total polyphenol concentrations in the panel ranged from 0 to 24 GAE/g, proanthocyanidin concentrations ranged from 0 to 42 mg CE/g, and 3-deoxyanthocyanidin concentrations ranged from 0 to 110 abs/mL/g.

(a)

(b)

(c)

3.2. Sorghum Extracts Do Not Reduce Cell Viability

To assess the effects of sorghum bran extracts on cell viability we used the MTT assay. RAW 264.7 macrophages were pretreated with sorghum extracts for one hour, followed by LPS for 18 hours. To characterize the effect of sorghum extracts on cell viability, we conducted an ANCOVA. Extract dose did not have a significant effect on cell viability (; Figure 3(a)), but genotype did (). A post hoc analysis was conducted to determine the effects of individual accessions on cell viability, and PI655978 was found to significantly increase cell viability (), whereas no accessions significantly decreased cell viability or dropped below 70% viability (Figure 3(b)).

(a)

(b)

Figure 3

MTT cell viability assays of RAW 264.7 cells treated with sorghum bran extracts. Cells were seeded in 96-well plates, incubated overnight, and pretreated with sorghum bran extracts at concentrations of 60 μg/mL, 30 μg/mL, and 15 μg/mL for 1 hour before LPS stimulation (1 μg/mL) for 18 hours. Results are expressed as percentage of absorbance in extract-treated cells versus control cells (no extract added). (a) Mean viability value of all accessions and (b) value of each of the twenty accessions at extract concentrations of 15 μg/mL (light gray), 30 μg/mL (gray), and 60 μg/mL (dark gray). Error bars represent the means of triplicate experiments ±SD. Accessions are ordered from lowest total phenols to highest ones. .

3.3. Sorghum Accessions Differentially Modulate TNF-α and IL-6

To determine differences in cytokine response to increasing doses of sorghum extracts, RAW 264.7 macrophages were pretreated with sorghum bran extracts for one hour, followed by stimulation with LPS for 18 hours. We first analyzed the average effect across accessions for TNF-α and IL-6 production. Based on the mean value of all accessions, extract dose had a significant effect on both TNF-α () and IL-6 (). A post hoc analysis showed that TNF-α decreased between 0 μg/mL and 15 μg/mL of extract, although it was not significant (), and increased between 15 μg/mL and 30 μg/mL of extract (), as well as 15 μg/mL and 60 μg/mL of extract (; Figure 4(a)). Similarly, IL-6 decreased between 0 μg/mL and 15 μg/mL of extract, although it was not significant (), and increased between 15 μg/mL and 60 μg/mL of extract (), as well as 30 μg/mL and 60 μg/mL of extract (; Figure 4(b)).

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Figure 4

Sorghum bran extracts differentially modulate TNF-α and IL-6 production in RAW 264.7 cells. Cells were seeded in 96-well plates, incubated overnight, and pretreated with sorghum bran extracts at concentrations of 15 μg/mL, 30 μg/mL, and 60 μg/mL for 1 hour before LPS stimulation (1 μg/mL) for 18 hours. (a) Effect of extract dose on TNF-α, (b) effect of extract dose on IL-6, (c) effect of 3-deoxyanthocyanidin-containing accessions on TNF-α, (d) effect of 3-deoxyanthocyanidin-containing accessions on IL-6, (e) effect of proanthocyanidin-containing accessions on TNF-α, (f) effect of proanthocyanidin-containing accessions on IL-6, (g) effect of Tannin1 on TNF-α, and (h) effect of Tannin1 on IL-6. Results are expressed as percentage of extract-treated cells versus negative controls (no extract added). Error bars represent the means of triplicate experiments ±SD. For (a) and (b), concentrations share the same letter if they are not significantly different from each other.

Next, we wanted to determine if there were genotypic differences affecting TNF-α and IL-6 production. Based on the mean value of all accessions, genotype had a significant effect on both IL-6 () and TNF-α (). A post hoc analysis was conducted to determine the effect of individual accessions on cytokine modulation. TNF-α was significantly decreased in cells treated with extracts from PI329440 (), PI561072 (), and PI655978 () compared to control cells. IL-6 was significantly decreased in cells treated with extracts from PI297139 (), PI329440 (), and PI655978 () compared to control cells.

3.4. Sorghum Polyphenols Differentially Modulate TNF-α and IL-6

We hypothesized that sorghum accessions with high polyphenol content would decrease cytokine production in LPS-activated RAW 264.7 cells. To test this hypothesis, an ANCOVA was conducted. Surprisingly, there was only a marginally significant effect of 3-deoxyanthocyanidins on TNF-α (; Figure 4(c)) and no significant effect on IL-6 (; Figure 4(d)). However, there was a trend in which cells treated with 3-deoxyanthocyanidin-containing accessions had less cytokine production than cells treated with non-3-deoxyanthocyanidin accessions, so it may be that there was not enough statistical power to detect significant differences. There was a significant effect of proanthocyanidins on TNF-α (; Figure 4(e)) and IL-6 (; Figure 4(f)), with cells treated with proanthocyanidin-containing accessions producing more cytokines than cells treated with nonproanthocyanidin accessions.

To validate that differences in cytokines were due to proanthocyanidins, we took advantage of a previously identified variant in a major gene regulating grain proanthocyanidins in sorghum, Tannin1 [42]. We used an ANCOVA to determine differences in cytokine production between cells treated with extracts from Tannin1 accessions (proanthocyanidin-containing) and cells treated with extracts from tan1-a or tan1-b accessions (nonproanthocyanidin-containing). For TNF-α, there was a significant effect of the Tannin1 allele () and extract concentration (), as well as an interaction between them (; Figure 4(g)). For IL-6, there was also a significant effect of the Tannin1 allele () and extract concentration (), but no significant interaction () (Figure 4(h)). As expected, cells treated with extracts from accessions containing Tannin1 had more cytokine production than cells treated with extracts from accessions containing tan1-a or tan1-b.

3.5. Accessions Differ in Nutritional Traits

In addition to ability to reduce induction of inflammatory cytokine production, other nutritional traits may influence the choice of accessions for use in the development of food-grade specialty varieties. Therefore grain protein, fat, starch, and weight were measured in the four accessions that inhibited both TNF-α and IL-6 production (Table 1). As a percentage of dry seed weight, protein ranged from 11.2 to 15.2%, fat ranged from 2.7 to 3.7%, and starch ranged from 65.2 to 68.5%. Seed weight ranged from 2.2 to 3.1 g. Since grain color influences end-user acceptance, we characterized the appearance of the outer layer of the grain: two accessions were red, one was yellow, and one was white.

4. Discussion

4.1. Crop Improvement for Human Health

Improving the nutritional value of crops will benefit from an integrative approach [1]—a combination of biochemistry, genomics, high-throughput phenotyping, and statistics. Also important is human nutrition research that uses cell cultures, animal models, and human clinical trials to characterize the health effects of a food plant. By combining crop improvement and human nutrition tools, we were able to screen hundreds of sorghum accessions relatively quickly and cost-effectively, permitting a targeted study on cell activation properties of sorghum grain (Figure 5). Additionally, utilizing accessions that are readily available from crop genebanks allows for authentication of the accessions and reproducibility of the experiments.

The primary goal of surveying a large number of diverse varieties is to identify those with a trait of interest. We identified four out of the twenty sorghum accessions in our diverse panel that significantly decreased TNF-α and/or IL-6 in RAW 264.7 macrophages, making them good candidates for use in developing specialty varieties. PI655978, which inhibited both TNF-α and IL-6 production, is a good source of high 3-deoxyanthocyanidin alleles for use in developing varieties that reduce induction of inflammatory cytokine production. Nutritional traits cannot be looked at in isolation but rather need to be considered in the context of other grain quality and agronomic traits, as well as the needs of growers and consumers. PI655978 is a yellow-grain sorghum with a slightly higher than average protein content (12.2%) and weight (2.8 g) compared to the entire SAP, a high concentration of 3-deoxyanthocyanidins (95.2 abs/mL/g), and no proanthocyanidins. These characteristics make this accession well suited for use in developing health-promoting specialty crop since light colored grain is generally more accepted by consumers than dark colored grain, proanthocyanidins can impart distasteful bitterness, and low grain weight is not desirable since grain weight is crucial to grain yield [44–46].

Interestingly, two low polyphenol accessions (PI329440 and PI561072) demonstrated ability to reduce induction of inflammatory cytokine production. Previous studies, conducted with a smaller number of sorghum varieties, did not find significant ability to reduce induction of inflammatory cytokine production in their low polyphenol controls [19, 22], which highlights the importance of exploring the cell activation effects of many genetically diverse sorghums in order to identify beneficial accessions. One strategy to reduce confounding factors in future studies is to control for differences in genetic background using near isogenic lines, pairs of lines developed through backcrossing that only differ in the genomic region of interest [47].

Considerable effort has been aimed at identifying genetic markers in crops, with the goal of using them in marker-assisted breeding [48]. We found a significant effect of the Tannin1 allele on TNF-α and IL-6, which demonstrates that marker-assisted breeding can be conducted down to the nucleotide level to develop health-promoting specialty crops.

4.2. 3-Deoxyanthocyanidins and Cell Activation

Sorghum 3-deoxyanthocyanidins are not widely found in nature, and sorghum is their only known dietary source [49–51]. They are a promising class of health-promoting polyphenols, with antioxidant and antiproliferative properties. Our results showed only a marginally significant effect of 3-deoxyanthocyanidins on TNF-α and no significant effect on IL-6 levels in LPS-activated cells. Our sample size of 3-deoxyanthocyanidin-containing accessions was small, though, so there was low statistical power to detect differences. We did, however, see a trend in which cells treated with 3-deoxyanthocyanidin-containing accessions had less cytokine production than cells treated with non-3-deoxyanthocyanidin accessions. Several studies have demonstrated anticancer properties of sorghum 3-deoxyanthocyanidins in vitro [52–55], so studying the effects of sorghum 3-deoxyanthocyanidins in in vivo cancer models will help to further our understanding of potential therapeutic properties of 3-deoxyanthocyanidins. Given that the effect of 3-deoxyanthocyanidin-containing accessions was only marginally significant, other constituents in the bran must also be contributing to the reduced induction of inflammatory cytokine production. For example, PI329440 is a low polyphenol sorghum, but addition of its extract significantly inhibited both TNF-α and IL-6 in cell cultures. In a recent study, phenolic acid derivatives isolated from sorghum grains decreased LPS-stimulated NO, iNOS, and COX-2 in RAW 264.7 cells [21]. Other phenolic compounds have been identified in sorghum bran, including flavones, flavanones, phlobaphenes, and anthocyanins [36], which may be contributing to the reduced induction of inflammatory cytokine production observed in this study. Follow-up studies that identify and test individual constituents in the bran will help to determine which constituents have health protective effects and thus could be targeted for specialty breeding.

4.3. Proanthocyanidins and Cell Activation

Proanthocyanidins are not commonly found in high concentrations in cereal crops, but many sorghum varieties are rich sources of this polyphenol [56]. They are also a promising class of health-promoting polyphenols but their effects are more complex. Cells treated with proanthocyanidin-containing accessions showed a trend towards lower production of TNF-α and IL-6 at 15 μg/mL of extract compared to 0 μg/mL but higher cytokine production compared to nonproanthocyanidin accessions. A study in 2010 found that high concentrations of a proanthocyanidin-containing sorghum accession slightly induced induction of inflammatory cytokine production in PBMC cells; however, the same study also found that this accession reduced induction of inflammatory cytokine production in a mouse model of ear inflammation, demonstrating the complexity of the effects on cytokine modulation and the need for disease specific and tissue specific research [19]. PI297139 is the only high proanthocyanidin accession in our study that significantly reduced induction of inflammatory cytokine production. This accession also contains a high concentration of 3-deoxyanthocyanidins, which may have been responsible for the reduced induction. It is also possible that there were differences in the proanthocyanidins in this accession compared to other accessions, which led to differing effects on induction of inflammatory cytokine production in vitro. Several structural variations of sorghum proanthocyanidins have been described, including differences in the monomer repeats, interflavan bonds, patterns of hydroxylation and glycosylation, and degree of polymerization [36, 57]. The degree of polymerization highly influences absorption of proanthocyanidins. Small proanthocyanidin compounds are absorbed in the small intestine, whereas large ones pass through the small intestine into the large intestine where they are catabolized by intestinal bacteria before they are absorbed [58]. For this reason, it has been suggested that health benefits derived from proanthocyanidins may be largely due to their effects on intestinal bacteria [59]. Several studies have found that proanthocyanidins from grape seeds attenuate inflammation in colitis animal models in part by improving the profile of beneficial gut bacteria [60, 61], and a recent study found that a high proanthocyanidin sorghum bran diet may protect colitis-induced rats by enriching colon bacteria [62]. Further study on proanthocyanidin structural variation in PI297139 and the effects of its grain extracts in colitis animal models could help clarify dietary effects of sorghum proanthocyanidins.

5. Conclusions

The RAW 264.7 cell activation model provides a good starting point for understanding the biological activity of a test compound and is a good high-throughput screening method to characterize the diversity of cell activation effects within a crop species. This can act as a guide in the selection of a subset of accessions for use in more complex disease models. Overall this study provides evidence that sorghum extracts significantly and differentially modulate induction of inflammatory cytokines TNF-α and IL-6, with 3-deoxyanthocyanidin and proanthocyanidin having a significant effect on their production. Additionally, in showing that the modulation of cytokines varies between accessions, this study demonstrates the importance of exploring diverse germplasm within a plant species to discover the full health-promoting potential of this crop. Finally, the effect of the Tannin1 allele on cytokines shows the potential of tailoring grain for beneficial end-uses by controlling traits at the nucleotide level. The results reported here can inform future sorghum studies, which should focus on testing with in vivo disease models such as a colitis mouse model, while reducing confounding factors through the use of near isogenic lines. This study contributes to the growing body of evidence that sorghum accessions differentially modulate induction of cytokine production and this difference is related to polyphenol profile and other active compounds.

Abbreviations

abs:	Absorbance
ANOVA:	Analysis of variance
CE:	Catechin equivalent
GAE:	Gallic acid equivalent
GAPIT:	Genome Association and Prediction Integrated Tool
GRIN:	Germplasm Resources Information Network
IL-6:	Interleukin-6
MLM:	Mixed linear model
NIRS:	Near-infrared spectroscopy
TNF-α:	Tumor necrosis factor alpha.

Disclosure

Davina H. Rhodes present address is as follows: USDA-ARS-Center for Grain and Animal Health Research, 1515 College Avenue, Manhattan, KS 66502, USA. The United Sorghum Checkoff Program only provided financial support and had no involvement in the study design; in the collection, analysis, and interpretation of the data; in the writing of the report; or in the decision to submit the paper for publication.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgments

The authors would like to thank Giamila Fantuzzi and Scott Bean for their helpful suggestions. This research was funded by the United Sorghum Checkoff Program.

References

C. H. Diepenbrock and M. A. Gore, “Closing the divide between human nutrition and plant breeding,” Crop Science, vol. 55, no. 4, pp. 1437–1448, 2015.
View at: Publisher Site | Google Scholar
G. Acquaah, Principles of Plant Genetics and Breeding, Wiley-Blackwell, 2nd edition, 2012.
N. M. McKeown, J. B. Meigs, S. Liu, P. W. F. Wilson, and P. F. Jacques, “Whole-grain intake is favorably associated with metabolic risk factors for type 2 diabetes and cardiovascular disease in the Framingham Offspring Study,” The American Journal of Clinical Nutrition, vol. 76, no. 2, pp. 390–398, 2002.
View at: Google Scholar
T. T. Fung, F. B. Hu, M. A. Pereira et al., “Whole-grain intake and the risk of type 2 diabetes: a prospective study in men,” American Journal of Clinical Nutrition, vol. 76, no. 3, pp. 535–540, 2002.
View at: Google Scholar
S. Liu, W. C. Willett, J. E. Manson, F. B. Hu, B. Rosner, and G. Colditz, “Relation between changes in intakes of dietary fiber and grain products and changes in weight and development of obesity among middle-aged women,” The American Journal of Clinical Nutrition, vol. 78, no. 5, pp. 920–927, 2003.
View at: Google Scholar
M. He, R. M. van Dam, E. Rimm, F. B. Hu, and L. Qi, “Whole-grain, cereal fiber, bran, and germ intake and the risks of all-cause and cardiovascular disease-specific mortality among women with type 2 diabetes mellitus,” Circulation, vol. 121, no. 20, pp. 2162–2168, 2010.
View at: Publisher Site | Google Scholar
D. Aune, D. S. M. Chan, R. Lau et al., “Dietary fibre, whole grains, and risk of colorectal cancer: systematic review and dose-response meta-analysis of prospective studies,” BMJ, vol. 343, no. 7833, p. 1082, 2011.
View at: Publisher Site | Google Scholar
S. S. Cho, L. Qi, G. C. Fahey Jr., and D. M. Klurfeld, “Consumption of cereal fiber, mixtures of whole grains and bran, and whole grains and risk reduction in type 2 diabetes, obesity, and cardiovascular disease,” American Journal of Clinical Nutrition, vol. 98, no. 2, pp. 594–619, 2013.
View at: Publisher Site | Google Scholar
J. Couzin-Frankel, “Inflammation bares a dark side,” Science, vol. 330, no. 6011, p. 1621, 2010.
View at: Publisher Site | Google Scholar
I. Manabe, “Chronic inflammation links cardiovascular, metabolic and renal diseases,” Circulation Journal, vol. 75, no. 12, pp. 2739–2748, 2011.
View at: Publisher Site | Google Scholar
G. S. Jensen, X. Wu, K. M. Patterson et al., “In vitro and in vivo antioxidant and anti-inflammatory capacities of an antioxidant-rich fruit and berry juice blend. Results of a pilot and randomized, double-blinded, placebo-controlled, crossover study,” Journal of Agricultural and Food Chemistry, vol. 56, no. 18, pp. 8326–8333, 2008.
View at: Publisher Site | Google Scholar
A. Basu, M. Rhone, and T. J. Lyons, “Berries: emerging impact on cardiovascular health,” Nutrition Reviews, vol. 68, no. 3, pp. 168–177, 2010.
View at: Publisher Site | Google Scholar
A. Szajdek and E. J. Borowska, “Bioactive compounds and health-promoting properties of Berry fruits: a review,” Plant Foods for Human Nutrition, vol. 63, no. 4, pp. 147–153, 2008.
View at: Publisher Site | Google Scholar
P. J. Barnes and M. Karin, “Nuclear factor-κB—a pivotal transcription factor in chronic inflammatory diseases,” The New England Journal of Medicine, vol. 336, no. 15, pp. 1066–1071, 1997.
View at: Publisher Site | Google Scholar
P. P. Tak and G. S. Firestein, “NF-κB: a key role in inflammatory diseases,” Journal of Clinical Investigation, vol. 107, no. 1, pp. 7–11, 2001.
View at: Publisher Site | Google Scholar
J. M. Awika, L. W. Rooney, X. Wu, R. L. Prior, and L. Cisneros-Zevallos, “Screening methods to measure antioxidant activity of sorghum (sorghum bicolor) and sorghum products,” Journal of Agricultural and Food Chemistry, vol. 51, no. 23, pp. 6657–6662, 2003.
View at: Publisher Site | Google Scholar
M.-A. Carbonneau, M. Cisse, N. Mora-Soumille et al., “Antioxidant properties of 3-deoxyanthocyanidins and polyphenolic extracts from Côte d'Ivoire's red and white sorghums assessed by ORAC and in vitro LDL oxidisability tests,” Food Chemistry, vol. 145, pp. 701–709, 2014.
View at: Publisher Site | Google Scholar
E. Bralley, P. Greenspan, J. L. Hargrove, and D. K. Hartle, “Inhibition of hyaluronidase activity by select sorghum brans,” Journal of Medicinal Food, vol. 11, no. 2, pp. 307–312, 2008.
View at: Publisher Site | Google Scholar
A. Burdette, P. L. Garner, E. P. Mayer, J. L. Hargrove, D. K. Hartle, and P. Greenspan, “Anti-inflammatory activity of select sorghum (Sorghum bicolor) brans,” Journal of Medicinal Food, vol. 13, no. 4, pp. 879–887, 2010.
View at: Publisher Site | Google Scholar
T.-J. Shim, T. M. Kim, K. C. Jang, J.-Y. Ko, and D. J. Kim, “Toxicological evaluation and anti-inflammatory activity of a golden gelatinous sorghum bran extract,” Bioscience, Biotechnology and Biochemistry, vol. 77, no. 4, pp. 697–705, 2013.
View at: Publisher Site | Google Scholar
P.-H. Nguyen, B. T. Zhao, J. H. Lee, Y. H. Kim, B. S. Min, and M. H. Woo, “Isolation of benzoic and cinnamic acid derivatives from the grains of Sorghum bicolor and their inhibition of lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells,” Food Chemistry, vol. 168, pp. 512–519, 2015.
View at: Publisher Site | Google Scholar
É. A. Moraes, D. I. G. Natal, V. A. V. Queiroz et al., “Sorghum genotype may reduce low-grade inflammatory response and oxidative stress and maintains jejunum morphology of rats fed a hyperlipidic diet,” Food Research International, vol. 49, no. 1, pp. 553–559, 2012.
View at: Publisher Site | Google Scholar
A. Fardet, “New hypotheses for the health-protective mechanisms of whole-grain cereals: what is beyond fibre?” Nutrition Research Reviews, vol. 23, no. 1, pp. 65–134, 2010.
View at: Publisher Site | Google Scholar
R. H. Liu, “Whole grain phytochemicals and health,” Journal of Cereal Science, vol. 46, no. 3, pp. 207–219, 2007.
View at: Publisher Site | Google Scholar
R. Borneo and A. E. León, “Whole grain cereals: functional components and health benefits,” Food & Function, vol. 3, no. 2, pp. 110–119, 2012.
View at: Publisher Site | Google Scholar
B. Winkel-Shirley, “Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology,” Plant Physiology, vol. 126, no. 2, pp. 485–493, 2001.
View at: Publisher Site | Google Scholar
E. Himi and K. Noda, “Red grain colour gene (R) of wheat is a Myb-type transcription factor,” Euphytica, vol. 143, no. 3, pp. 239–242, 2005.
View at: Publisher Site | Google Scholar
E.-S. M. Abdel-Aal, J. C. Young, and I. Rabalski, “Anthocyanin composition in black, blue, pink, purple, and red cereal grains,” Journal of Agricultural and Food Chemistry, vol. 54, no. 13, pp. 4696–4704, 2006.
View at: Publisher Site | Google Scholar
FAO, “Sorghum and millets in human nutrition,” 1995, http://www.fao.org/docrep/T0818E/T0818E04.htm.
View at: Google Scholar
E. L. Janzen and W. W. Wilson, “Cooperative marketing in specialty grains and identity preserved grain markets,” Agribusiness & Applied Economics Report 500, North Dakota State University, Department of Agribusiness and Applied Economics, 2002.
View at: Google Scholar
J. R. N. Taylor, T. J. Schober, and S. R. Bean, “Novel food and non-food uses for sorghum and millets,” Journal of Cereal Science, vol. 44, no. 3, pp. 252–271, 2006.
View at: Publisher Site | Google Scholar
US Department of Agriculture and Center for Nutrition Policy and Promotion, “Trends in dietary fiber in the U.S. food supply; sales of grain products,” CNPP Fact Sheet 2, 2007, http://www.cnpp.usda.gov/Publications/FoodSupply/FiberFactSheet.pdf.
View at: Google Scholar
P. Cureton and A. Fasano, “The increasing incidence of celiac disease and the range of gluten-free products in the marketplace,” in Gluten-Free Food Science and Technology, E. Gallagher, Ed., pp. 1–15, Wiley-Blackwell, Oxford, UK, 2009.
View at: Google Scholar
D. H. Rhodes, L. Hoffmann, W. L. Rooney, P. Ramu, G. P. Morris, and S. Kresovich, “Genome-wide association study of grain polyphenol concentrations in global sorghum [Sorghum bicolor (L.) Moench] germplasm,” Journal of Agricultural and Food Chemistry, vol. 62, no. 45, pp. 10916–10927, 2014.
View at: Publisher Site | Google Scholar
J. R. Taylor and K. G. Duodu, “Effects of processing sorghum and millets on their phenolic phytochemicals and the implications of this to the health-enhancing properties of sorghum and millet food and beverage products,” Journal of the Science of Food and Agriculture, vol. 95, no. 2, pp. 225–237, 2015.
View at: Publisher Site | Google Scholar
J. M. Awika and L. W. Rooney, “Sorghum phytochemicals and their potential impact on human health,” Phytochemistry, vol. 65, no. 9, pp. 1199–1221, 2004.
View at: Publisher Site | Google Scholar
USDA, GRIN National Genetic Resources Program, 2014, http://www.ars-grin.gov.
A. M. Casa, G. Pressoir, P. J. Brown et al., “Community resources and strategies for association mapping in sorghum,” Crop Science, vol. 48, no. 1, pp. 30–40, 2008.
View at: Publisher Site | Google Scholar
G. P. Morris, P. Ramu, S. P. Deshpande et al., “Population genomic and genome-wide association studies of agroclimatic traits in sorghum,” Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 2, pp. 453–458, 2013.
View at: Publisher Site | Google Scholar
J. Yu, G. Pressoir, W. H. Briggs et al., “A unified mixed-model method for association mapping that accounts for multiple levels of relatedness,” Nature Genetics, vol. 38, no. 2, pp. 203–208, 2006.
View at: Publisher Site | Google Scholar
A. E. Lipka, F. Tian, Q. Wang et al., “GAPIT: genome association and prediction integrated tool,” Bioinformatics, vol. 28, no. 18, Article ID bts444, pp. 2397–2399, 2012.
View at: Publisher Site | Google Scholar
Y. Wu, X. Li, W. Xiang et al., “Presence of tannins in sorghum grains is conditioned by different natural alleles of Tannin1,” Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 26, pp. 10281–10286, 2012.
View at: Publisher Site | Google Scholar
R Core Team, R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria, 2015, http://www.R-project.org/.
A. Aboubacar, A. W. Kirleis, and M. Oumarou, “Important sensory attributes affecting consumer acceptance of sorghum porridge in West Africa as related to quality tests,” Journal of Cereal Science, vol. 30, no. 3, pp. 217–225, 1999.
View at: Publisher Site | Google Scholar
R. I. Kobue-Lekalake, J. R. N. Taylor, and H. L. De Kock, “Effects of phenolics in sorghum grain on its bitterness, astringency and other sensory properties,” Journal of the Science of Food and Agriculture, vol. 87, no. 10, pp. 1940–1948, 2007.
View at: Publisher Site | Google Scholar
R. I. Kobue-Lekalake, J. R. N. Taylor, and H. L. de Kock, “Influence of PROP taster status on the consumer acceptability of food made from tannin sorghums,” Journal of the Science of Food and Agriculture, vol. 89, no. 11, pp. 1809–1814, 2009.
View at: Publisher Site | Google Scholar
J. J. B. Keurentjes, L. Bentsink, C. Alonso-Blanco et al., “Development of a near-isogenic line population of Arabidopsis thaliana and comparison of mapping power with a recombinant inbred line population,” Genetics, vol. 175, no. 2, pp. 891–905, 2007.
View at: Publisher Site | Google Scholar
B. C. Collard and D. J. Mackill, “Marker-assisted selection: an approach for precision plant breeding in the twenty-first century,” Philosophical Transactions of the Royal Society of London B: Biological Sciences, vol. 363, no. 1491, pp. 557–572, 2008.
View at: Publisher Site | Google Scholar
C. S. Winefield, D. H. Lewis, E. E. Swinny et al., “Investigation of the biosynthesis of 3-deoxyanthocyanins in Sinningia cardinalis,” Physiologia Plantarum, vol. 124, no. 4, pp. 419–430, 2005.
View at: Publisher Site | Google Scholar
M. Sharma, C. Chai, K. Morohashi, E. Grotewold, M. E. Snook, and S. Chopra, “Expression of flavonoid 3′-hydroxylase is controlled by P1, the regulator of 3-deoxyflavonoid biosynthesis in maize,” BMC Plant Biology, vol. 12, article 196, pp. 1–13, 2012.
View at: Publisher Site | Google Scholar
P. Malathi, R. Viswanathan, P. Padmanaban, D. Mohanraj, V. Ganesh Kumar, and K. P. Salin, “Differential accumulation of 3-deoxy anthocyanidin phytoalexins in sugarcane varieties varying in red rot resistance in response to Colletotrichum falcatum infection,” Sugar Tech, vol. 10, no. 2, pp. 154–157, 2008.
View at: Publisher Site | Google Scholar
C. Gómez-Cordovés, B. Bartolomé, W. Vieira, and V. M. Virador, “Effects of wine phenolics and sorghum tannins on tyrosinase activity and growth of melanoma cells,” Journal of Agricultural and Food Chemistry, vol. 49, no. 3, pp. 1620–1624, 2001.
View at: Publisher Site | Google Scholar
C.-H. Shih, S.-O. Siu, R. Ng et al., “Quantitative analysis of anticancer 3-deoxyanthocyanidins in infected sorghum seedlings,” Journal of Agricultural and Food Chemistry, vol. 55, no. 2, pp. 254–259, 2007.
View at: Publisher Site | Google Scholar
H. J. Woo, I. T. Oh, J. Y. Lee et al., “Apigeninidin induces apoptosis through activation of Bak and Bax and subsequent mediation of mitochondrial damage in human promyelocytic leukemia HL-60 cells,” Process Biochemistry, vol. 47, no. 12, pp. 1861–1871, 2012.
View at: Publisher Site | Google Scholar
P. Suganyadevi, K. M. Saravanakumar, and S. Mohandas, “The antiproliferative activity of 3-deoxyanthocyanins extracted from red sorghum (Sorghum bicolor) bran through P53-dependent and Bcl-2 gene expression in breast cancer cell line,” Life Sciences, vol. 92, no. 6-7, pp. 379–382, 2013.
View at: Publisher Site | Google Scholar
L. Gu, M. A. Kelm, J. F. Hammerstone et al., “Concentrations of proanthocyanidins in common foods and estimations of normal consumption,” Journal of Nutrition, vol. 134, no. 3, pp. 613–617, 2004.
View at: Google Scholar
C. G. Krueger, M. M. Vestling, and J. D. Reed, “Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of heteropolyflavan-3-ols and glucosylated heteropolyflavans in sorghum [Sorghum bicolor (L.) Moench],” Journal of Agricultural and Food Chemistry, vol. 51, no. 3, pp. 538–543, 2003.
View at: Publisher Site | Google Scholar
S. Deprez, I. Mila, J.-F. Huneau, D. Tome, and A. Scalbert, “Transport of proanthocyanidin dimer, trimer, and polymer across monolayers of human intestinal epithelial Caco-2 cells,” Antioxidants & Redox Signaling, vol. 3, no. 6, pp. 957–967, 2001.
View at: Publisher Site | Google Scholar
K. Ou and L. Gu, “Absorption and metabolism of proanthocyanidins,” Journal of Functional Foods, vol. 7, pp. 43–53, 2014.
View at: Publisher Site | Google Scholar
H. Wang, Y. Xue, H. Zhang et al., “Dietary grape seed extract ameliorates symptoms of inflammatory bowel disease in IL10-deficient mice,” Molecular Nutrition and Food Research, vol. 57, no. 12, pp. 2253–2257, 2013.
View at: Publisher Site | Google Scholar
X. Li, X. Yang, Y. Cai et al., “Proanthocyanidins from grape seeds modulate the NF-κB signal transduction pathways in rats with tnbs-induced ulcerative colitis,” Molecules, vol. 16, no. 8, pp. 6721–6731, 2011.
View at: Publisher Site | Google Scholar
L. E. Ritchie, J. M. Sturino, R. J. Carroll, L. W. Rooney, M. A. Azcarate-Peril, and N. D. Turner, “Polyphenol-rich sorghum brans alter colon microbiota and impact species diversity and species richness after multiple bouts of dextran sodium sulfate-induced colitis,” FEMS Microbiology Ecology, vol. 91, no. 3, Article ID fiv008, 2015.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2016 Davina H. Rhodes and Stephen Kresovich. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

2571

Downloads

1156

Citations