Polyphenol Containing Sorghum Brans Exhibit an Anti-Cancer Effect in Apc Min/+ Mice Treated with Dextran Sodium Sulfate.

Colon cancer (CC) is considered a high-risk cancer in developed countries. Its etiology is correlated with a high consumption of red meat and low consumption of plant-based foods, including whole grains. Sorghum bran is rich in polyphenols. This study aimed to determine whether different high-phenolic sorghum brans suppress tumor formation in a genetic CC rodent model and elucidate mechanisms. Tissue culture experiments used colorectal cancer cell lines SW480, HCT-116 and Caco-2 and measured protein expression, and protein activity. The animal model used in this study was APC Min+/mouse model combined with dextram sodium sulfate. High phenolic sorghum bran extract treatment resulted in the inhibition of proliferation and induced apoptosis in CC cell lines. Treatment with high phenolic sorghum bran extracts repressed TNF-α-stimulated NF-κB transactivation and IGF-1-stimulated PI3K/AKT pathway via the downregulation of ß-catenin transactivation. Furthermore, high-phenolic sorghum bran extracts activated AMPK and autophagy. Feeding with high-phenolic sorghum bran for 6 weeks significantly suppressed tumor formation in an APC Min/+ dextran sodium sulfate promoted CC mouse model. Our data demonstrates the potential application of high-phenolic sorghum bran as a functional food for the prevention of CC.

Genetic architecture of kernel composition in global sorghum germplasm.

BACKGROUND: Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop for dryland areas in the United States and for small-holder farmers in Africa. Natural variation of sorghum grain composition (protein, fat, and starch) between accessions can be used for crop improvement, but the genetic controls are still unresolved. The goals of this study were to quantify natural variation of sorghum grain composition and to identify single-nucleotide polymorphisms (SNPs) associated with variation in grain composition concentrations. RESULTS: In this study, we quantified protein, fat, and starch in a global sorghum diversity panel using near-infrared spectroscopy (NIRS). Protein content ranged from 8.1 to 18.8%, fat content ranged from 1.0 to 4.3%, and starch content ranged from 61.7 to 71.1%. Durra and bicolor-durra sorghum from Ethiopia and India had the highest protein and fat and the lowest starch content, while kafir sorghum from USA, India, and South Africa had the lowest protein and the highest starch content. Genome-wide association studies (GWAS) identified quantitative trait loci (QTL) for sorghum protein, fat, and starch. Previously published RNAseq data was used to identify candidate genes within a GWAS QTL region. A putative alpha-amylase 3 gene, which has previously been shown to be associated with grain composition traits, was identified as a strong candidate for protein and fat variation. CONCLUSIONS: We identified promising sources of genetic material for manipulation of grain composition traits, and several loci and candidate genes that may control sorghum grain composition. This survey of grain composition in sorghum germplasm and identification of protein, fat, and starch QTL contributes to our understanding of the genetic basis of natural variation in sorghum grain nutritional traits.

High-throughput micro-plate HCI-vanillin assay for screening tannin content in sorghum grain.

BACKGROUND: The HCI-vanillin assay is a well-accepted method for determining tannin content in sorghum but is limited to small sample sets due to the time-consuming nature of the method. The objective was to develop an accurate and repeatable high-throughput 96-well plate assay for breeders to screen large sample sets of sorghum for tannin content. Validation of the high-throughput assay was tested on 25 sorghums suspected to contain tannin. RESULTS: Approximately 30 measurements per day were completed using the conventional assay compared to 224 measurements using the 96-well platform. The correlation between the two tannin assays was 0.98. The coefficient of variation (CV) was 3.54% and 3.21% for the 96-well and conventional method, respectively. The 96-well assay exhibited good repeatability, with the inter-plate CV between 2.77% and 4.85%. CONCLUSION: The high-throughput 96-well HCI-vanillin assay exhibited an eightfold increase in the number of measurements completed and was as accurate as the conventional HCI-vanillin assay.

Variability in tannin content, chemistry and activity in a diverse group of tannin containing sorghum cultivars.

BACKGROUND: Tannins are large polyphenolic polymers and are known to bind proteins, limiting their digestibility, but are also excellent antioxidants. Numerous studies investigating the functional properties of sorghum tannin have been conducted by comparing grain samples from different sorghum lines without considering the other intrinsic characteristics of the grain. The purpose of this study was to remove the confounding intrinsic factors present in the endosperm so the effect of the tannins could be evaluated utilizing a unique decortication/reconstitution procedure. RESULTS: The tannin content of the 14 cultivars tested ranged from 2.3 to 67.2 catechin equivalents. The bran fractions were studied for their impact on protein binding and antioxidant capacity. Protein digestibility by pepsin ranged from 8% to 58% at the highest tannin level addition. Protein binding ranged from 3.11 to 16.33 g blue bovine serum albumin kg⁻¹ bran. Antioxidant capacity ranged from 81.33 to 1122.54 µmol Trolox equivalents g⁻¹ bran. High-performance size-exclusion chromatography detailed molecular size distributions of the tannin polymers and relationship to tannin functionality. CONCLUSION: The tannin content and composition play a significant role in determining tannin functionality. These differences will allow for selections of high-tannin sorghums with consideration of the biological activities of the tannins.

High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour.

BACKGROUND: Sorghum possesses phenolic compounds that are health-promoting constituents of the grain. There are approximately 40 000 sorghum accessions, many of which have not been evaluated for the grain's health-promoting potential. Conventional methods for measuring total phenolic content, flavonoid content and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging capacity are time-consuming and labour-intensive, resulting in low overall throughput. The objective of this study was to develop a high-throughput screening assay for large sorghum sample sets to determine flavonoid and phenolic content and to modify existing DPPH and total phenolic assays. RESULTS: The 96-well assays exhibited a correlation of > 0.9 with the conventional assays. The 96-well assays allowed for up to 64 samples to be run per day compared with 20-24 samples (depending on the test) for the conventional methods. The 96-well assays had excellent accuracy (97.65-106.16% recovery), precision (1.06-8.28% coefficient of variation (CV)) and reproducibility (1.32-2.13% CV inter-day and 1.36-2.09% CV intra-day). CONCLUSION: The high-throughput 96-well plate method proved to be as robust and reproducible as the conventional method for determining total phenolic content, flavonoid content and DPPH-scavenging capacity in either sorghum bran or flour.

Sorghum Phenolic Compounds Are Associated with Cell Growth Inhibition through Cell Cycle Arrest and Apoptosis in Human Hepatocarcinoma and Colorectal Adenocarcinoma Cells.

Phenolic compounds in some specialty sorghums have been associated with cancer prevention. However, direct evidence and the underlying mechanisms for this are mostly unknown. In this study, phenolics were extracted from 13 selected sorghum accessions with black pericarp while F10000 hybrid with white pericarp was used as a control, and cell growth inhibition was studied in hepatocarcinoma HepG2 and colorectal adenocarcinoma Caco-2 cells. Total phenolic contents of the 13 high phenolic grains, as determined by Folin-Ciocalteu, were 30-64 mg GAE/g DW in the phenolic extracts of various accessions compared with the control F10000 at 2 mg GAE/g DW. Treatment of HepG2 with the extracted phenolics at 0-200 µM GAE up to 72 h resulted in a dose- and time-dependent reduction in cell numbers. The values of IC50 varied from 85 to 221 mg DW/mL while the control of F10000 was 1275 mg DW/mL. The underlying mechanisms were further examined using the highest phenolic content of PI329694 and the lowest IC50 of PI570481, resulting in a non-cytotoxic decrease in cell number that was significantly correlated with increased cell cycle arrest at G2/M and apoptotic cells in both HepG2 and Caco-2 cells. Taken together, these results indicated, for the first time, that inhibition of either HepG2 or Caco-2 cell growth by phenolic extracts from 13 selected sorghum accessions was due to cytostatic and apoptotic but not cytotoxic mechanisms, suggesting some specialty sorghums are a valuable, functional food, providing sustainable phenolics for potential cancer prevention.

Antimicrobial Activity of Sorghum Phenolic Extract on Bovine Foodborne and Mastitis-Causing Pathogens.

Antimicrobial resistance in bacterial pathogens associated with bovine mastitis and human foodborne illnesses from contaminated food and water have an impact on animal and human health. Phenolic compounds have antimicrobial properties and some specialty sorghum grains are high in phenolic compounds, and the grain extract may have the potential as a natural antimicrobial alternative. The study's objective was to determine antimicrobial effects of sorghum phenolic extract on bacterial pathogens that cause bovine mastitis and human foodborne illnesses. Bacterial pathogens tested included Escherichia coli, Salmonella Typhimurium, Campylobacter jejuni, Campylobacter coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella oxytoca, Staphylococcus aureus, and Enterococcus faecalis. Antibacterial activities of sorghum phenolic extracts were determined by agar-well diffusion assay. Sorghum phenolic extract was added to the wells in concentrations of 0, 100, 200, 500, 1000, or 4000 µg/mL. The control wells did not receive phenolic extract. Plates were incubated for 18-24 h, and the diameter of each zone of inhibition was measured. The results indicated that sorghum phenolic extract had inhibitory effects on Staphylococcus aureus, Enterococcus faecalis, Campylobacter jejuni, and Campylobacter coli.

Vitamin and mineral supplements have a nutritionally significant impact on micronutrient intakes of older adults attending senior centers.

Older adults frequently report use of vitamin and mineral (VM) supplements, although the impact of supplements on dietary adequacy remains largely unknown. The purpose of the current study was to evaluate micronutrient intakes of older adults with emphasis on identifying nutrients most improved by VM supplements, nutrients most likely to remain inadequate, and nutrients most likely consumed in excess. Community-based volunteers were recruited from senior centers and completed a questionnaire querying demographic data, current health status, and VM supplement use. Participants (n = 263) were then contacted by telephone to complete two 24-hour diet recalls and confirm VM supplement use. Dietary adequacy was determined by comparing the ratio of mean dietary intake to the Dietary Reference Intakes (DRI). Dietary consumption was lowest for vitamins D and E, calcium, and magnesium. VM supplementation most improved intakes of vitamins E, D, B(6), folic acid, and calcium. Participants were most likely to exceed the Tolerable Upper Limit with supplementation of niacin, folic acid, and vitamin A.

Anticancer Activity of a Novel High Phenolic Sorghum Bran in Human Colon Cancer Cells.

Human colon cancer is the third leading cause of mortality in the United States and worldwide. Chemoprevention using diet is widely accepted as a promising approach for cancer management. Numerous population studies indicate a negative correlation between the incidence of colon cancer and consumption of whole grains with a high content of bioactive phenolic compounds. In the current study, we evaluated the anticancer properties of a high phenolic sorghum bran extract prepared using 70% ethanol with 5% citric acid solvent at room temperature. A significant dose-dependent suppression of cell proliferation was observed in human colon cancer cells treated with the high phenolic sorghum bran extract. Apoptosis and S phase growth arrest were induced, while cell migration and invasion were inhibited by this treatment; these effects were accompanied by altered expression of apoptosis, cell cycle, and metastasis-regulating genes. We also found that the high phenolic sorghum bran extract stimulated DNA damage in association with induction of extracellular signal-regulated kinase (ERK) and c-Jun-NH2-terminal kinase (JNK) and subsequent expression of activating transcription factor 3 (ATF3). The present study expands our understanding of the potential use of high phenolic sorghum bran to prevent human colon cancer.

Advancing provitamin A biofortification in sorghum: Genome-wide association studies of grain carotenoids in global germplasm.

Vitamin A deficiency is one of the most prevalent nutritional deficiencies worldwide. Sorghum [Sorghum bicolor L. (Moench)] is a major cereal crop consumed by millions of people in regions with high vitamin A deficiency. We quantified carotenoid concentrations in a diverse sorghum panel using high-performance liquid chromatography and conducted a genome-wide association study (GWAS) of grain carotenoids to identify genes underlying carotenoid variation. There was moderate variation for ß-carotene (00.8 µg g-1 ), lutein (0.3-9.4 µg g-1 ), and zeaxanthin (0.2-9.1 µg g-1 ), but ß-cryptoxanthin and α-carotene were nearly undetectable. Genotype had the largest effect size, at 81% for zeaxanthin, 62% for ß-carotene, and 53% for lutein. Using multiple models, GWAS identified several significant associations between carotenoids and single nucleotide polymorphisms (SNPs), some of which colocalized with known carotenoid genes that have not been previously implicated in carotenoid variation. Several of the candidate genes identified have also been identified in maize (Zea mays L.) and Arabidopsis (Arabidopsis thaliana) carotenoid GWAS studies. Notably, an SNP inside the putative ortholog of maize zeaxanthin epoxidase (ZEP) had the most significant association with zeaxanthin and with the ratio between lutein and zeaxanthin, suggesting that ZEP is a major gene controlling sorghum carotenoid variation. Overall findings suggest there is oligogenic inheritance for sorghum carotenoids and suitable variation for marker-assisted selection. The high carotenoid germplasm and significant associations identified in this study can be used in biofortification efforts to improve the nutritional quality of sorghum.

Evaluation of ethanol-based extraction conditions of sorghum bran bioactive compounds with downstream anti-proliferative properties in human cancer cells.

Certain foods such as turmeric and green tea have been extensively studied for anticancer properties, while high polyphenol sorghum has not received the same attention. Some bioactive compounds in Sorghum bicolor with anticancer activity have been identified, indicating the further need for research and screening methods of high polyphenol sorghum varieties. This study was aimed at improving the extraction of sorghum bioactive compounds by using food-grade solvents using ethanol and citric acid. We used three sorghum varieties and green tea (GT) as a control. The extraction methods were screened for anti-proliferative properties in HepG2 and HCT-15 cancer cell lines, using a cell viability assay. Extraction conditions were improved for anti-proliferative compounds from a high-phenolic sorghum variety (HP), sumac sorghum (CS), and GT. HP was more effective at inhibiting cell viability than CB, CS, and GT. The results demonstrate an efficient method for extracting sorghum bioactive compounds for future anticancer research using food approved ingredients.

High-Polyphenol Sorghum Bran Extract Inhibits Cancer Cell Growth Through ROS Induction, Cell Cycle Arrest, and Apoptosis.

As diet is one of the major controllable factors in cancer development, potentially chemopreventive foods are of significant interest to public health. One such food is sorghum (Sorghum bicolor), a cereal grain that contains varying concentrations of polyphenols. In a panel of 15 sorghum germplasm, we identified strains with higher polyphenol content than previously reported for this grain. Bran extracts from the germplasm with the highest and lowest polyphenol content were then tested against HepG2 and Caco2 cancer cells to assess effects on cancer cell viability, reactive oxygen species, apoptosis, DNA damage, cell cycle arrest, and protein expression patterns. High-polyphenol extracts, but not low-polyphenol extracts, reduced cell viability by inducing apoptosis and cell cycle arrest following production of reactive oxygen species and oxidative DNA damage. The results indicate that high-polyphenol sorghum bran extracts have potential anticancer properties and warrant further research, not only to test against specific cancers but also to elucidate underlying mechanisms of action.

Effect of sorghum flour composition and particle size on quality properties of gluten-free bread.

White, food-grade sorghum was milled to flour of varying extraction rates (60%, 80%, and 100%) and pin-milled at different speeds (no pin-milling, low-speed, and high-speed) to create flours of both variable composition and particle size. Flours were characterized for flour composition, total starch content, particle size distribution, color, damaged starch, and water absorption. Bread was characterized for specific volume, crumb structure properties, and crumb firmness. Significant differences were found (P < 0.05) in the composition of sorghum flours of varying extraction rate, most notably for fiber and total starch contents. Flour particle size and starch damage were significantly impacted by extraction rate and speed of pin-milling. Water absorption increased significantly with increasing extraction rate and pin-milling speed. Breads produced from 60% extraction flour had significantly higher specific volumes, better crumb properties, and lower crumb firmness when compared with all other extractions and flour types. The specific volume of bread slices ranged from 2.01 mL/g (100% extraction, no pin-milling) to 2.54 mL/g (60% extraction, low-speed pin-milling), whereas the firmness ranged from 553.28 g (60% extraction, high-speed pin-milling) to 1096.26 g (commercial flour, no pin-milling). The bread characteristics were significantly impacted by flour properties, specifically particle size, starch damage, and fiber content (P < 0.05).

Wheat bran particle size influence on phytochemical extractability and antioxidant properties.

It is unknown if particle size plays a role in extracting health promoting compounds in wheat bran because the extraction of antioxidant and phenolic compounds with particle size reduction has not been well documented. In this study, unmilled whole bran (coarse treatment) was compared to whole bran milled to medium and fine treatments from the same wheat bran. Antioxidant properties (capacity, ability, power), carotenoids and phenolic compounds (phenolic acids, flavonoids, anthocyanins) were measured and compared. The ability of whole bran fractions of differing particle size distributions to inhibit free radicals was assessed using four in vitro models, namely, diphenylpicrylhydrazyl radical-scavenging activity, ferric reducing/antioxidant power (FRAP) assay, oxygen radical absorbance capacity (ORAC), and total antioxidant capacity. Significant differences in phytochemical concentrations and antioxidant properties were observed between whole bran fractions of reduced particle size distribution for some assays. The coarse treatment exhibited significantly higher antioxidant properties compared to the fine treatment; except for the ORAC value, in which coarse was significantly lower. For soluble and bound extractions, the coarse treatment was comparatively higher in total antioxidant capacity (426.72 mg ascorbic acid eq./g) and FRAP value (53.04 µmol FeSO4/g) than bran milled to the finer treatment (314.55 ascorbic acid eq./g and 40.84 µmol FeSO4/g, respectively). Likewise, the fine treatment was higher in phenolic acid (7.36 mg FAE/g), flavonoid (206.74 µg catechin/g), anthocyanin (63.0 µg/g), and carotenoid contents (beta carotene, 14.25 µg/100 g; zeaxanthin, 35.21 µg/100 g; lutein 174.59 µg/100 g) as compared to the coarse treatment. An increase of surface area to mass increased the ORAC value by over 80%. With reduction in particle size, there was a significant increase in extracted anthocyanins, carotenoids and ORAC value. Particle size does effect the extraction of phytochemicals.

Composition and molecular weight distribution of carob germ protein fractions.

Biochemical properties of carob germ proteins were analyzed using a combination of selective extraction, reversed-phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC) coupled with multiangle laser light scattering (SEC-MALS), and electrophoretic analysis. Using a modified Osborne extraction procedure, carob germ flour proteins were found to contain approximately 32% albumin and globulin and approximately 68% glutelin with no prolamins detected. The albumin and globulin fraction was found to contain low amounts of disulfide-bonded polymers with relatively low M(w) ranging up to 5 x 10(6) Da. The glutelin fraction, however, was found to contain large amounts of high molecular weight disulfide-bonded polymers with M(w) up to 8 x 10(7) Da. When extracted under nonreducing conditions and divided into soluble and insoluble proteins as typically done for wheat gluten, carob germ proteins were found to be almost entirely ( approximately 95%) in the soluble fraction with only ( approximately 5%) in the insoluble fraction. As in wheat, SEC-MALS analysis showed that the insoluble proteins had a greater M(w) than the soluble proteins and ranged up to 8 x 10(7) Da. The lower M(w) distribution of the polymeric proteins of carob germ flour may account for differences in functionality between wheat and carob germ flour.