Food Chemistry 152 (2014) 210–217

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Food Chemistry

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Effects of processing with dry heat and wet heat on the antioxidantprofile of sorghum

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.11.106

⇑ Corresponding author. Tel.: +55 (31) 3899 1684.E-mail address: lcardoso.nutricao@hotmail.com (Leandro de Morais Cardoso).

Leandro de Morais Cardoso a,⇑, Tatiana Aguiar Montini a, Soraia Silva Pinheiro a,Helena Maria Pinheiro-Sant’Ana a, Hércia Stampini Duarte Martino b, Ana Vládia Bandeira Moreira c

a Laboratory of Vitamins Analysis, Department of Nutrition and Health, Federal University of Viçosa, PH Rolfs Avenue, s/n, Viçosa, Minas Gerais, 36570-900, Brazilb Laboratory of Experimental Nutrition, Department of Nutrition and Health, Federal University of Viçosa, PH Rolfs Avenue, s/n, Viçosa, Minas Gerais, 36570-900, Brazilc Laboratory of Food Analysis, Department of Nutrition and Health, Federal University of Viçosa, PH Rolfs Avenue, s/n, Viçosa, Minas Gerais, 36570-900, Brazil

a r t i c l e i n f o

Article history:Received 1 July 2013Received in revised form 15 November 2013Accepted 19 November 2013Available online 27 November 2013

Keywords:Stability of bioactive compoundsVitamin E3-DeoxyanthocyanidinsCarotenoidsPhenolic compoundsAntioxidant activity

a b s t r a c t

The effects of domestic processing with dry heat (F2-oven/milling; F3-milling/oven; F4-microwave oven/milling; F5-milling/microwave oven; F6-popped grains/milling) and wet heat (F7-cooking in water/dry-ing/milling) on the antioxidant profile of sorghum flours (F1-raw flour) were evaluated. 3-Deoxyanthocy-anidins and total phenolic compounds were stable to dry heat (retention between 96.1% and 106.3%) andreduced with wet heat. All processing with dry heat increased the vitamin E content (2,201.9–3,112.1 lg/100 g) and its retention, and reduced the carotenoids (4.78–17.27 lg/100 g). The antioxidant activity inprocessed flours with dry heat remained constant (F3 and F6) or increased (F2, F4 and F5) and decreasedafter processing with wet heat. Overall, the grains milled before processing in oven and in microwaveoven retained more vitamin E and less carotenoids than those milled after these processing. In conclu-sion, dry heat did not affect the phenolic compounds and 3-deoxyanthocyanidins of sorghum, butincreased the vitamin E and antioxidant activity, and reduced the carotenoids. The wet heat processingreduced all antioxidant compounds except carotenoids, which increased.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Sorghum (Sorghum bicolor L.) is the fifth most cultivated cerealin the world (Waniska & Rooney, 2000). It is one of the mostdrought-resistant cereals and, due to the increasing world popula-tion and the decreasing availability of water, it represents animportant crop for future use (Taylor, Schober, & Bean, 2006). Incountries of Africa, Asia and Central America, sorghum is a staplefood of the human feeding (Taylor, Schober, & Bean, 2006; Waniska& Rooney, 2000). However, it is underutilized in countries like theUnited States, Australia and Brazil, and is used mainly for animalfeeding (Taylor, Schober, & Bean, 2006; Waniska & Rooney, 2000).

The sorghum grain is formed by the pericarp (outer covering),testa (layer between the pericarp and endosperm), the endosperm(storage tissue) and the germ (embryo) (Slavin, 2004; Waniska &Rooney, 2000). Overall, there are some pigments (carotenoidsand anthocyanins) and phenolic compounds (phenolic acids, flavo-noids and tannins) in the pericarp and testa of the grain (Slavin,2004; Waniska & Rooney, 2000). There are also starch, proteins,minerals and B-complex vitamins in the endosperm, and lipids

and some fat-soluble vitamins in the germ (Slavin, 2004; Waniska& Rooney, 2000).

Sorghum is an important source of bioactive compounds suchas 3-deoxyanthocyanidins, tannins, vitamin E, carotenoids, andother antioxidants (Awika & Rooney, 2004; Kean, Bordenave, Ejeta,Hamaker, & Ferruzzi, 2011; Martino et al., 2012). These compoundsreduce the action and damage caused by free radicals and thus pro-mote benefits to human health (Valko et al., 2007).

However, like other cereals, sorghum grains need to be pro-cessed before human consumption, which may modify their chem-ical composition, and functional and nutritional value. Studies havedemonstrated the effects of processing traditionally used in Africanand Asian countries (fermentation, germination and soaking) onsome antioxidant compounds present in sorghum (Afify, El-Beltagi,El-Salam, & Omran, 2012; Jood, Khetarpaul, & Goyal, 2012; Rah-man & Osman, 2011). However, few studies evaluated the effectsof domestic processing that reflect the Western culture, such asheat treatment in conventional oven, cooking in water, and poppedgrains (Dlamini, Taylor, & Rooney, 2007; Moraes, 2011; Wu, Huang,Qin, & Ren, 2013), and the effects of microwave oven processing isunknown. Moreover, studies conducted so far evaluated a reducednumber of bioactive compounds.

This study evaluated the effects of domestic processing with dryheat (cooking in conventional oven, microwave oven and popped

L.d.M. Cardodo et al. / Food Chemistry 152 (2014) 210–217 211

grains) and wet heat (cooking in water) on the content of phenoliccompounds, 3-deoxyanthocyanidins, vitamin E, carotenoids andantioxidant activity of sorghum.

2. Materials and methods

2.1. Raw sorghum

Sorghum with red pericarp, white endosperm and withouttannin (BRS 310 genotype) developed by Brazilian Company ofAgricultural Research (EMBRAPA)-Maize and Sorghum (SeteLagoas, Minas Gerais, Brazil) was used in this study.

2.2. Standards and reagents

The standards of carotenoids (lutein and zeaxanthin), luteolini-din chloride, gallic acid and trolox were obtained from Sigma–Al-drich (St. Louis, MO, USA). The apigeninidin chloride wasobtained from Chromadex (Santa Ana, CA, USA) and vitamin Estandards (a, b, c and d-tocopherols and tocotrienols) were pur-chased from Calbiochem�, EMD Biosciences, Inc. (USA).

For the extraction of carotenoids, vitamin E, 3-deoxyanthocy-anidins (3-DXAs) and total phenolic compounds (TPC), it was usedanalytic grade reagents purchased from VETEC (São Paulo, Brazil).For analysis of these compounds and antioxidant activity, it wasused HPLC grade reagents (methyl alcohol, ethyl acetate, acetone,acetonitrile, hexane, isopropyl alcohol, acetic acid and formic acid)purchased from Tedia (São Paulo, Brazil) and analytical grade re-agents (2,2-diphenyl-1-picrylhydrazyl radical and Folin–Ciocalteu)obtained from Sigma–Aldrich (St. Louis, MO, USA).

2.3. Preparation of sorghum flours

Seven types of sorghum flours were obtained using processingbased on previous studies (Dlamini, Taylor, & Rooney, 2007;Moraes, 2011; Wu, Huang, Qin, & Ren, 2013). The processing variedaccording to the type of heat treatment (in conventional oven,microwave oven, popped grains and cooking in water) and the mo-ment of the milling of the grains (before or after the heat treat-ment), as presented below:

(F1) Raw flour: milling of the grains.(F2) Oven/milling: heat treatment of the grains in an conven-

tional oven (121 �C, 25 min) followed by milling.(F3) Milling/oven: previous milling of the grains followed by

heat treatment of the flour in an conventional oven (121 �C,25 min).

(F4) Microwave oven/milling: heat treatment of the grains in amicrowave oven (middle power, 4 min) followed by selection ofnot popped grains (90% of the total) and milling of the selectedgrains.

(F5) Milling/microwave oven: milling of the grains followed byheat treatment of the flour in a microwave oven (middle power,4 min).

(F6) Popped grains/milling: popped grains in a conventional pop-per followed by selection of popped grains (85% of total) and mill-ing of the selected grains.

(F7) Cooking in water/drying/milling: cooking of the grains in450 mL of water (100� C, 25 min) in a covered pot followed by dry-ing of the grains in an oven (50� C, 180 min) until they presentedmoisture lower than 15% (Brasil, 1996) and milling of the driedgrains.

The grains were milled in an analytical mill micro-rotor(Marconi, MA 090, Brazil) with the sieve of 850 mesh. Once ob-tained through the previously described processing, the moistureof the flours was determined by gravimetry after oven drying

(Nova Etica, 4000, Brazil) at 105 �C. Next, the fresh flours werepacked in polyethylene bags and stored in a freezer until analyzes.

2.4. Optimization of method for the extraction of antioxidantcompounds in sorghum

The extracts preparation for the determination of TPC and anti-oxidant activity in sorghum flours was optimized. For this step, itwas used only the raw sorghum flour.

2.4.1. Effect of extractor meanThe extractor mean able to extract the greatest possible amount

of antioxidant compounds was determined using a system ofsequential extraction composed by three extractor means of differ-ent polarities (ether mean: ethyl ether; alcoholic mean: ethanol;and aqueous mean: water) (Moreira & Mancini-Filho, 2003)(Fig. 1A). All the extracts were prepared by adding 1 part of milledsorghum to 20 parts of extractor (1:20, w/v). This suspension wasstirred (180 rpm, 2 h) and centrifuged (2,790g, 15 min). Then, thesupernatant (extract) was completed with the extractor to 25 mL,in a volumetric flask (Moreira & Mancini-Filho, 2003).

2.4.2. Effect of the extraction timeThe lowest time able to extract the greatest possible amount of

antioxidant compounds (ideal extraction time) was determinedusing the best extractor mean selected in the previous step (etha-nol) and other extractor means similar to it and cited in literature(methanol, hydro-methanol and hydro-ethanol). The tested extrac-tion times ranged from 1 to 8 h (Fig. 1B).

The extracts preparation was performed using the extractionprotocol described in the item 2.4.1 (Moreira & Mancini-Filho,2003). The TPC and antioxidant activity of the extracts obtainedin the optimization steps (2.4.1 and 2.4.2) were determined usingthe methodologies described in the items 2.5.1 and 2.5.2.

2.5. Total phenolic compounds and antioxidant activity of processedsorghum flours

2.5.1. Extracts preparationThe extracts preparation was performed using the extractor and

extraction time selected in item 2.4 and using the extraction pro-tocol described in the same item (Moreira & Mancini-Filho, 2003).

2.5.2. Determination of total phenolic compoundsThe TPC were determined using the Folin–Ciocalteu reagent

(Singleton, Orthofer, & Lamuela-Raventós, 1999). Aliquots of0.5 mL of the extract, added of 0.5 mL of sodium carbonate 7.5%and 0.5 mL of Folin–Ciocalteu (diluted to 20% in water) were stir-red in vortex and incubated at room temperature (30 min). Then,the absorbance was read in a spectrophotometer (Thermo scien-tific, Evolution 60S, USA) at 765 nm. The quantification was per-formed by standard curve obtained by the reading of theabsorbance of solutions of gallic acid with different concentrations(0.01–0.10 mg/mL; y = 24.888x + 0.0246; R2 = 0.996). The resultswere expressed in mg of gallic acid equivalents per gram of flour(mg GAE/g).

2.5.3. Determination of the antioxidant activityThe antioxidant activity was determined using the DPPH meth-

od (1,1-diphenyl-2-picrylhydrazil radical) (Bloor, 2001). Aliquotsof 0.1 mL of the extracts obtained in item 2.5.1 were added of1.5 mL of methanol solution of DPPH 100 lmol, followed by man-ual shaking for 1 min. After 30 min of rest, the absorbance was readin a spectrophotometer at 517 nm.

A standard curve was constructed using 50–100 lmol/L oftrolox solutions (R2 = 0.9975). The antiradical activity (%) was

Fig. 1. Flowchart of optimization method for the preparation of extracts for analysis of total phenolic compounds and antioxidant activity.

212 Leandro de Morais Cardoso et al. / Food Chemistry 152 (2014) 210–217

calculated using the equation ½1� ðAbssample � AbsblankÞ=ðAbscontrol � AbsblankÞ� � 100. The inhibited % DPPH for each samplewas plotted against the concentration of trolox standard, and theantioxidant activity of the samples was calculated from the linearequation. The results were expressed in lmol trolox equivalent(TE)/g sorghum flour.

2.6. 3-Deoxyanthocyanidins in sorghum flours

Approximately 1 g of flour was added of 10 mL of methanol andstirred (180 rpm, 2 h). Then, the extract was centrifuged (2,790g,10 min) and the supernatant was collected and stored in a freezer(�20 ± 1 �C) (Dykes, Seitz, Rooney, & Rooney, 2009).

The methods described by Yang, Allred, Geera, Allred, andAwika (2012) and Yang, Browning, and Awika (2009) were usedwith modifications to identify and quantify 3-DXAs (luteolinidin,apigeninidin, 7-methoxy-apigeninidin and 5-methoxy-luteolini-din) in the sorghum. 3-DXA analyses were performed in a high per-formance liquid chromatography (HPLC) system (Shimadzu, SCL10AT VP, Japan) equipped with high pressure pump (Shimadzu,LC-10AT VP, Japan), autosampler with loop of 500 lL (Shimadzu,SIL-10AF, Japan), diode array detector (DAD) (Shimadzu, SPD-M10A, Japan) and helium degassing system of the mobile phase(Shimadzu, DGU-2 A, Japan).

The chromatographic conditions used for analysis included aHPLC system, C-18 Kinetex column (150 � 4.6 mm i.d., 5 lm) fittedwith C-18 guard column (4 mm � 3 mm) (Phenomenex, Torrance,CA), column temperature at 35 �C, injection volume of 15 lL, scan-ning of the spectrum from 200 to 700 nm and detection at 480 nm.The mobile phase consisted of (A), 2% formic acid in ultrapurewater, and (B), 2% formic acid in acetonitrile. The elution gradientfor B was as follows: 0–3 min, 10% isocratic; 3–4 min, 10–12%;

4–5 min, 12% isocratic; 5–8 min, 12–18%; 8–10 min, 18% isocratic;10–12 min, 18–19%; 12–14 min, 19% isocratic; 14–18 min,19–21%; 18–22 min, 21–26%; 22–28 min, 26–28%; 28–32 min,28–40%; 32–34 min, 40–60%; 34–36 min, 60% isocratic;36–38 min, 60–10%; 38–45 min, 10% isocratic. To increase therepeatability, it was used the following flow gradient: 0–36 min,1.0 mL/min isocratic; 36–38 min, 1.0–2.0 mL/min; 38–44 min,2.0 mL/min isocratic; 44–45 min, 2.0–1.0 mL/min and the mobilephase was degassed with helium gas at 50 kPa during the runs.

Identification of luteolinidin and apigeninidin was based on thecommercial standards retention times and UV–Vis spectra. Thequantification was performed by comparing peak areas with thatobtained in the standard curve constructed from injection, induplicate, of six different concentrations of standard solutions(luteolinidin: 0.70–105.65 ng, y = 8,141.52 � �2,823.41, R2: 0.9989,LOD: 28.32 ng/mL and LOQ: 141.60 ng/mL; apigeninidin: 0.36–109.29 ng, y = 6,647.14� + 8,276.37, R2: 0.9992, LOD: 35.12 ng/mLand LOQ 175.60 ng/mL). Quantification of 5-methoxy-luteolinidinand 7-methoxy-apigeninidin was performed using the luteolinidinand apigeninidin standards, respectively, along with the appropriatemolecular weight correction factor (Dykes, Seitz, Rooney, & Rooney,2009) (5-methoxy-luteolinidin: 0.73–110.40 ng, y = 7,790.79x�2,823.41, R2: 0.9985; 7-methoxy-apigeninidin: 0.38–114.57 ng,y = 6,345.79x + 8276.37, R2: 0.9991). The 3-DXAs were expressedin lg/g of flour, as single compounds and as total 3-DXAs (sum of3-DXAs).

2.7. Determination of carotenoids and vitamin E in sorghum flours

During analyzes of carotenoids and vitamin E, the samples andthe extracts were protected from light (sunlight and artificial) and

L.d.M. Cardodo et al. / Food Chemistry 152 (2014) 210–217 213

oxygen using amber glassware, dark environment, and bottles withnitrogen gas environment and hermetically closed.

2.7.1. CarotenoidsThe occurrence and content of lutein and zeaxanthin were

investigated in sorghum flours. Carotenoids were extracted accord-ing to Rodriguez-Amaya, Raymundo, Lee, Simpson, and Chichester(1976), with modifications. About 2.5 g of sorghum flour werehomogenized in 15.0 mL of cooled acetone using a mixer (IKA, UtraTurrax � T18 basic, Germany). The suspension obtained was vac-uum filtered in Büchner funnel and the residue was maintainedin the extraction tube. Then, the extraction procedure was re-peated by adding 15.0 mL of cooled acetone to the residue, withsubsequent homogenization and vacuum filtration.

Subsequently, the partition of carotenoids from acetone topetroleum ether was performed. The filtrate was transferred intotwo fractions to a separatory funnel containing 20.0 mL of cooledpetroleum ether. After the transfer of each fraction, distilled waterwas added for phase separation (carotenoids-petroleum ether andacetone–water), and the lower phase (acetone–water) was dis-carded. Anhydrous sodium sulphate was added to the extract inpetroleum ether to remove the residual water. Then, the extractvolume was completed with petroleum ether to 25.0 mL, in a vol-umetric flask.

For carotenoids analyzes, 25.0 mL of extract were evaporatedunder a flow of nitrogen gas, were dissolved in 1.0 mL of hexane:isopropanol (90:10, v/v) and were filtered in filter units with aporosity of 0.45 lm. The carotenoid analyzes were performed onthe same HPLC-DAD system used for analysis of 3-DXAs accordingto Panfili, Fratianni, and Irano (2004): HPLC-DAD system, scanningof the spectrum from 350 to 600 nm, detection at 450 nm, LunaSi100 column (250 � 4 mm i.d., 5 lm) fitted with Si100 guard col-umn (4 mm � 3 mm) (Phenomenex, Torrance, CA), column atroom temperature and injection volume of 50 lL. The mobilephase consisted of hexane: isopropanol (95:5, v/v). The mobilephase flow was 1.3 mL/min, isocratic.

Identification of the carotenoids was based on commercialstandards retention times and UV–Vis spectra. The quantificationwas performed by comparing peak areas with that obtained inthe standard curve constructed from injection, in duplicate, ofsix different concentrations of standard solutions (lutein:0.03�2.43 lg, y = 5,573,136.44x + 995.45, R2: 0.9984, LOD:6.86 lg/mL, LOQ: 34.30 lg/mL; zeaxanthin: 0.04–1.37 lg,y = 502,310.96x�3,485.95, R2: 0.9899, LOD: 4.79 lg/mL, LOQ:23.95 lg/mL). The carotenoids were expressed in lg/100 g offlour, as single compounds and as total of carotenoids (sum oflutein + zeaxanthin).

2.7.2. Vitamin EThe extraction and analysis of the components of vitamin E (a,

b, c and d-tocopherols and tocotrienols) in the sorghum flours were

True retentionð%Þ ¼ content of the compound in the processed flourðmg=100 gÞ � weight of the processed flourðgÞ � 100content of the compound in the raw flourðmg=100 gÞ �weight of the raw flourðgÞ ð1Þ

performed according to Pinheiro-Sant’Ana et al. (2011), with somemodifications. Approximately 5.0 g of flour were weighed andadded of 4.0 mL of heated ultrapure water (80 ± 1 �C), 10.0 mL ofisopropanol, 1.0 mL of hexane containing 0.05% BHT and 5.0 g ofsodium sulfate anhydrous. Slowly, 25.0 mL of mixture solvent ofextraction (hexane: ethyl acetate, 85:15, v/v) were added and the

suspension was homogenized using a mixer (1 min). The homoge-nized suspension was vacuum filtered using a Büchner funnel withfilter paper and the residue was maintained in the extraction tube.The extraction step was repeated adding 5.0 mL of isopropanol and30.0 mL of the mixture solvent of extraction to the residue, fol-lowed by homogenization and filtration. Then, the extract was con-centrated on a rotary evaporator (70 ± 1 �C, 2 min), and the volumewas completed with mixture solvent of extraction to 25.0 mL, in avolumetric flask.

After extraction, aliquots of 5.0 mL of the extract were dried innitrogen gas, dissolved in 2.0 mL of HPLC grade hexane and filteredin filter units with a porosity of 0.45 lm. The vitamin E compo-nents were analyzed in a HPLC system (Shimadzu, SCL 10AD VP, Ja-pan) composed of a high pressure pump with quaternary gradientvalve for low pressure (Shimadzu, LC-10AD VP), autosampler withloop of 50 lL (Shimadzu, SIL-10AF), helium degassing system ofthe mobile phase (Shimadzu, DGU-2 A), and fluorescence detector(Shimadzu, RF10AXL).

The chromatographic conditions used included HPLC system;fluorescence detection (290 nm excitation and 330 nm emission);Luna Si100 column (250 � 4 mm i.d., 5 lm) fitted Si100 guard col-umn (4 mm � 3 mm) (Phenomenex, Torrance, CA), column atroom temperature, injection volume of 20 lL. The mobile phasewas composed by hexane: isopropanol: glacial acetic acid(98.9:0.6:0.5, v/v/v) and mobile phase flow was 1.0 mL/min,isocratic.

Identification of the vitamin E components was based on com-mercial standards retention times and co-chromatography. Thequantification was performed by comparing peak areas with thatobtained in the standard curve constructed from injection, induplicate, of six different concentrations of standard solutions(a-tocopherol: 1.02–104.21 ng, y = 76,030,901.90x � 66,102.66;R2 = 0.999; a-tocotrienol: 3.21–157.6 ng, y = 28,452,328.82x �105,303.68; R2 = 0.997; c-tocopherol: 2.2–107.6 ng, y = 93,182,765.60x + 170,331.40, R2 = 0.989; d-tocopherol: 2.08–13.6 ng,y = 119,134,728.50x � 279,396.64, R2 = 0.999 and d-tocotrienol:2.70–12.76 ng, y = 143,447,824.17x � 256,481.66, R2 = 0.998). Thelimits of detection and quantification of the components of vitaminE were 0.02–0.07 lg/mL and 0.11–0.36 lg/mL, respectively. Thevitamin E content was expressed in lg/100 g of flour, as singlecompounds and as total vitamin E (sum of the vitamin Ecomponents).

2.8. True retention of antioxidant compounds

Considering the possible losses during milling of the grains,for the calculation of retention of antioxidant compounds, allprocessed flours were weighed on an analytical balance(GEHAKA, AG 200) before and after heat treatment. The trueretention was calculated by the equation of Murphy, Criner,and Gray (1975):

2.9. Experimental design and statistical analysis

The best extractor of antioxidant compounds in sorghum wasdetermined using a completely randomized design (CRD), with 3solvents. The ideal time of extraction was evaluated using a CRD,in a factorial scheme 4 � 8 (4 types of solvent and 8 extraction

214 Leandro de Morais Cardoso et al. / Food Chemistry 152 (2014) 210–217

times). The effect of the processing on the antioxidant profile of theflour was evaluated using a CRD, with 7 types of processing. Alltests were performed in three repetitions.

Data normality was assessed using the Shapiro–Wilk test anddifferences between treatments were evaluated by ANOVA. Dun-can test was used to compare the treatment averages. The associ-ation between antioxidant activity and other variables wasassessed by Pearson’s correlation test. Statistical analyzes wereperformed using the SAS package (Statistical Analysis System), ver-sion 9.2 (2008), licensed for the UFV, at 5% probability.

3. Results and discussion

3.1. Methods for extraction of antioxidant compounds in sorghumflours

3.1.1. Effect of the type of solventThe alcoholic mean solubilized the higher content of phenolic

compounds (0.20 mg GAE/g; 50.4% of total), followed by aqueous(0.15 mg GAE/g; 38.8% of total) and ether means (0.06 mg GAE/g,10.8% of total) (p < 0.05) (see supplementary material). The antiox-idant activity and the TPC content of the extracts correlated posi-tively (R2 = 0.955, p < 0.05). Thus, the alcoholic extract showedantioxidant activity (13.0 lmol TE/g) significantly higher than thatobserved in the aqueous and ether extracts (5.3 and 2.0 lmol TE/g,respectively) (see supplementary material).

The presence of antioxidant compounds in the three extractormeans demonstrated that there is not a solvent that is able to iso-late all or a specific class of antioxidants. The results of this studysuggest that the use of sequential extraction or a hydro-alcoholicmixture of solvent may be more appropriate to extract the antiox-idant compounds from sorghum. Thus, to confirm this hypothesis,we assessed the ideal extraction time using two types of 100% alco-hol (ethanol and methanol) and their respective hydro-alcoholicmeans (40:60, v/v).

3.1.2. Effect of the extraction timeDetailed results about the lower extraction time required for

the extraction of antioxidant compounds in alcoholic means (eth-anol and methanol) and their respective hydro-alcoholic meansare presented in supplementary material. The extraction of pheno-lic compounds was influenced by the extractor (p < 0.001). Alco-holic means solubilized more phenolic compounds than theirrespective hydro-alcoholic means (p < 0.05). Methanol was themost effective extractor, followed by ethanol, and by hydro-alco-holic means. The shortest time necessary for extraction in metha-nol was 2 h and in ethanol was 3 h.

Antioxidant activity was influenced by the interaction betweenthe type and concentration of alcohol, and extraction time(p < 0001). The hydro-alcoholic means were more effective thantheir respective alcoholic means. The antioxidant activity washigher in the hydro-methanolic means, followed by hydro-etha-nolic, methanolic and ethanolic means (p < 0.05). The shortest timeof extraction for both hydro-alcoholic extracts was 3 h.

The TPC content and antioxidant activity of alcoholic and hy-dro-alcoholic extracts were inversely correlated (R2 = �0.897,p < 0.05). Despite the alcoholic extracts have shown the highestcontent of phenolic compounds, they exhibited the lowest antiox-idant activity. These results may be explained by diferences be-tween the compositions of the extracts and the differentmechanisms involved in the radical–antioxidant reactions. Thehighest antioxidant activity exhibited by hydroalcoholic extractsmay be due to the presence of higher content and synergistic ac-tion of antioxidant compounds with hydrophilic and hydrophobicnature. This suggests that, despite phenolics are considered as a

major group of antioxidant compounds of sorghum, the non-phe-nolic compounds can also significantly contribute to this antioxi-dant activity of this cereal.

3.2. 3-Deoxyanthocyanidins and total phenolic compounds

The genotype BRS 310 presented the four 3-DXAs investigated,similar to that observed by other authors in red sorghum (Table 1)(Dykes, Seitz, Rooney, & Rooney, 2009). The profile and content of3-DXAs remained constant after all processing with dry heat (oven,microwave oven and popped grains). The flour obtained from thesorghum cooked in water (F7) showed lower 3-DXA content, whichcan be partially attributed to the dilution of the compounds in thefood matrix. Its moisture (F7: 14.9%) was significantly higher thanthe control (F1: 12.8%) and dry heat flours (F2: 12.2%; F3: 12.0%; F4:12.2%; F5: 11.9%; F6: 12.0%) (Data not presented), which were sig-nificantly equals. In addition, a fraction of the 3-DXAs may havebeen leached during cooking.

Sorghum 3-DXAs were stable to all processing with dry heat(F2-F6) (retention greater than 97.4%) and reduced in wet heat(retention of 85.8%). The stability of 3-DXAs observed in this studydiffered from that observed in other classes of anthocyanins pres-ent in foods which are unstable to heat treatment. The lack of neg-ative influence of the processing on sorghum 3-DXAs may be dueto the use of a binomial temperature/time less aggressive thanthose tested in other studies. The degree of instability and the deg-radation products depend on the severity (temperature and time)and heating nature (Ni et al., 2012; Patras, Brunton, O’Donnell, &Tiwari, 2010).

In addition, the different chemical structure of the sorghum 3-DXAs, which contribute to their greater chemical stability com-pared to other classes of anthocyanins, may have increased thethermal stability by mechanisms not studied yet. It is believed thatthe high stability of sorghum 3-DXAs results from the absence ofan oxygen atom at position C3 (Shih et al., 2006). The 3-DXAs areespecially promising ingredients for food applications due to theirexceptional stability pigment when compared to anthocyanins(Awika, Rooney, & Waniska, 2004).

The TPC content increased in flour F4 (microwave oven/milling).In the others processed flours with dry heat (F2, F3 and F5) thephenolic content remained constant. The increase of the TPC inthe flour F4 can be attributed to chemical reactions that increasedtheir extractability (Xu & Chang, 2008) resulting in a retentiongreater than 100%. TPC were stable to dry heat processing and pre-sented retention between 99.1 and 106.3%. The cooking in waterreduced the content and retention of phenolic compounds in flourF7 (p < 0.05). These reductions may be due to the loss of the com-pounds in the cooking water (Afify, El-Beltagi, El-Salam, & Omran,2012) and higher moisture of this flour when compared to otherflours.

3.3. Carotenoids

The raw flour of sorghum BRS 301 presented total of carote-noids similar to that observed by other authors in sorghum withwhite endosperm (Fernandez et al., 2008) and 7–18 times smallerthan the range observed in 8 sorghum varieties with yellow endo-sperm (Kean, Bordenave, Ejeta, Hamaker, & Ferruzzi, 2011)(Table 2). As observed by other authors (Kean, Bordenave, Ejeta,Hamaker, & Ferruzzi, 2011), the zeaxanthin was the major caroten-oid of the sorghum flours (73 to 81% of total content).

Treatments with dry heat (F2–F6) differently decreased the con-tent and retention of carotenoids (p < 0.05). The sensitivity ofcarotenoids to processing with dry heat, including oven and themicrowave oven was also observed in other food matrices (Fratian-ni, Cinquanta, & Panfili, 2010; Mayer-Miebach, Behsnilian, Regier,

Table 1Content and retention of 3-deoxyanthocyanidins and phenolic compounds in sorghum flours.A,B

Sorghum flours LUT APIG 5-MeO-LUT 7-MeO-APIG Total 3-DXAs TPC

Content (lg/g) Content (lg/g) Retention(%)

Content (mg/g)

Retention(%)

F1–Raw 57.18 ± 1.65a 26.23 ± 0.83a 12.54 ± 1.02a 4.32 ± 0.81a 97.27 ± 7.03a – 0.58 ± 0.03a –F2–Oven/milling 58.26 ± 1.92a 25.98 ± 1.64a 12.72 ± 0.93a 4.10 ± 0.87a 94.06 ± 3.98a 96.1 0.59 ± 0.01b 101.1F3–Milling/oven 57.77 ± 2.77a 25.56 ± 1.74 a 13.43 ± 0.72a 3.98 ± 0.69a, b 94.74 ± 5.56a 96.6 0.58 ± 0.02b 99.2F4–Microwave oven/milling 56.39 ± 1.56a 28.73 ± 1.55a 12.56 ± 0.91a 3.78 ± 0.50a 97.46 ± 3.14a 99.6 0.62 ± 0.02a 106.3F5–Milling/microwave oven 56.18 ± 3.88a 29.23 ± 1.65a 9.35 ± 0.98a 4.09 ± 0.63a 96.85 ± 5.47a 98.7 0.58 ± 0.03b 99.1F6–Popped grains/milling 57.19 ± 2.82a 27.12 ± 0.94a 8.45 ± 0.64a 3.97 ± 0.51a, b 96.73 ± 4.85a 98.6 0.58 ± 0.02b 99.2F7–Cooking in water/drying/

milling50.13 ± 0.69b 23.98 ± 0.94b 7.23 ± 0.75b 2.07 ± 0.15b 83.41 ± 3.21b 87.6 0.42 ± 0.06c 73.9

LUT: luteolinidin; APIG: apigeninidin; 7-MeO-APIG: 7-methoxy-apigeninidin; 5-MeO-LUT: 5-methoxy-luteolinidin; 3-DXAs: 3-deoxyanthocyanidins; TPC: total phenoliccompounds. Flour moisture: F1: 12.8%; F2: 12.2%; F3: 12.0%; F4: 12.2%; F5: 11.9%; F6: 12.0% and F7: 14.9%.

A Results are the mean ± standard deviation of three repetitions and were expressed in fresh basis.B Means followed by the same letter in the columns are not statistically different at 5% probability by Duncan test.

Table 2Content and retention of carotenoids in sorghum flours.A,B

Sorghum flours Lutein Zeaxanthin Total of carotenoids (Lutein + Zeaxanthin)

Content (lg/100 g) Content (lg/100 g) True retention (%)

F1–Raw 3.49 ± 0.51a 13.78 ± 0.58a 17.27 ± 1.07a –F2–Oven/milling 2.19 ± 0.37b 9.06 ± 0.56c 11.28 ± 0.83d 64.6F3–Milling/oven 3.86 ± 0.06a 10.22 ± 0.54b 14.09 ± 0.59b,c 80.5F4–Microwave oven/milling 3.28 ± 0.81a 9.68 ± 0.26c 12.96 ± 0.57c 74.0F5–Milling/microwave oven 3.69 ± 0.58a 11.14 ± 0.60b 14.84 ± 1.00b 84.9F6–Popped grains/milling 1.17 ± 0.15c 3.61 ± 0.58d 4.78 ± 0.31e 27.3F7–Cooking in water/drying/milling 3.24 ± 0.30b 13.50 ± 1.07a 16.74 ± 0.47a 98.8

Flour moisture: F1: 12.8%; F2: 12.2%; F3: 12.0%; F4: 12.2%; F5: 11.9%; F6: 12.0% and F7: 14.9%A Results are the mean ± standard deviation of three repetitions and were expressed in fresh basis.B Means followed by the same letter in the columns are not statistically different at 5% probability by Duncan test.

L.d.M. Cardodo et al. / Food Chemistry 152 (2014) 210–217 215

& Schuchmann, 2005). However, this reduction can be accompa-nied and compensated by a possible increase in the accessibilityof carotenoids (Maiani et al., 2009). The retention and content inthe flour of popped grains (F6) was from 3 to 4 times lower thanin other flours processed with dry heat. This result indicates thatthe increase of internal pressure that occurred within the grain be-fore bursting increased losses in carotenoids. The mechanisms in-volved in the effects need to be studied.

The flours in which the grains were milled before the processingwith dry heat in conventional oven and microwave oven (F3 andF5) showed content and retention of carotenoids greater than thoseones where the grains were milled after these heat treatments (F2and F4). These results can be attributed to the location of carote-noids in the sorghum grain, which are found in pericarp and testaof the grain (Waniska & Rooney, 2000). The previous milling of thegrains increased the exposure of the endosperm and germ and,consequently, reduced the direct contact of the pericarp and testawith the heat.

Table 3Content and retention of vitamin E in sorghum flours.A,B

Sorghum flours a-Tocopherol a-Tocotrienol c-Tocophero

Content (lg/100 g)

F1–Raw 299.2 ± 1.3e 241.7 ± 1.1b 1,370.5 ± 50F2–Oven/milling 538.0 ± 14.1c 218.5 ± 0.9c 1,683.5 ± 15F3–Milling/oven 389.5 ± 2.5d 172.7 ± 2.1d 1,517.3 ± 40F4–Microwave oven/milling 556.3 ± 4.6b 217.7 ± 2.5c 2,192.6 ± 31F5–Milling/microwave oven 675.6 ± 7.9a 256.3 ± 1.6a 1,990.3 ± 50F6–Popped grains/milling 541.0 ± 15.6c 136.8 ± 3.6f 1,713.1 ± 32F7–Cooking in water/drying/milling 339.5 ± 4.6d 153.4 ± 1.9e 1,156.6 ± 23

Flour moisture: F1: 12.8%; F2: 12.2%; F3: 12.0%; F4: 12.2%; F5: 11.9%; F6: 12.0% and F7:A Results are the mean ± standard deviation of three repetitions and were expressed iB Means followed by the same letter in the columns are not statistically different at 5

On the other hand, the wet heat processing (F7) was less severeto carotenoids than the dry heat treatments. In F7, content andretention of carotenoids were not affected. This result suggeststhat, unlike the observed for 3-DXAs and TPC, the cooking in waterwas not able to significantly leach the carotenoids of sorghum.Moreover, this treatment may have caused a greater increase inthe accessibility of carotenoids which offset the small losses dueto leaching and dilution due to the higher moisture of this flour.

3.4. Vitamin E

The raw flour of sorghum BRS 301 showed 5 components ofvitamin E (a, d and c-tocopherols, a and d-tocotrienols). Thec-tocopherol was the major component corresponding to 67.5%of the total content (Table 3). The content of a-tocopherol in rawsorghum flour varied within the range observed by other authors(35.7 a 520 lg/100 g) (Afify, El-Beltagi, El-Salam, & Omran, 2012;Martino et al., 2012). However, after processing, the a-tocopherol

l d-Tocopherol d-Tocotrienol Total vitamin E

Content (lg/100 g) True retention (%)

.8e 79.3 ± 0.1e 37.6 ± 0.4b 2,029.0 ± 49.0d

.4c 77.1 ± 0.2f 36.3 ± 0.9c 2,554.1 ± 44.4b 124.3

.0d 83.6 ± 0.4c 38.1 ± 0.6b 2,201.9 ± 39.6c 107.5

.7a 94.7 ± 0.7a 50.5 ± 0.4a 3,112.1 ± 23.7a 150.8

.3b 88.6 ± 2.7b 51.5 ± 0.5a 3,062.3 ± 41.6a 148.8

.4c 72.3 ± 0.4g 38.1 ± 0.6b 2,501.9 ± 31.6b 122.0

.2f 81.3 ± 0.5d 36.3 ± 0.9c 1,767.2 ± 18.8e 86.3

14.9%.n fresh basis.% probability by Duncan test.

Fig. 2. Antioxidant activity of sorghum flours A, B. AResults are the mean ± standard deviation of three repetitions and were expressed in fresh basis; BColumns followed by thesame letter are not significantly different at 5% probability by Duncan test. Flour moisture: F1: 12.8%; F2: 12.2%; F3: 12.0; F4: 12.2%; F5: 11.9%; F6: 12.0% and F7: 14.9%.

Table 4Correlation between the antioxidant activity and antioxidant compounds in sorghum flours.

Compounds Correlation coefficient P Value Compounds Correlation coefficient P Value

TPC 0.823 0.031* Lutein 0.231 0.453Total 3-DXAs 0.771 0.035* Zeaxanthin 0.168 0.346LUT 0.823 0.033* Total vitamin E 0.686 0.047*

5-MeO-LUT 0.678 0.057 a-Tocopherol 0.658 0.045*

APIG 0.776 0.038* a-Tocotrienol 0.232 0.3217-MeO-APIG 0.697 0.059 c- Tocopherol 0.543 0.049*

Total of carotenoids 0.208 0.273 d- Tocopherol 0.246 0.189d-Tocotrienol 0.136 0.427

* Significant by Pearson’s correlation test, at 5% probability; LUT: luteolinidin; APIG: apigeninidin; 7-MeO-APIG: 7-methoxy-apigeninidin; 5-MeO-LUT: 5-methoxy-luteo-linidin; Total 3-DXAs: 3-deoxyanthocyanidins; TPC: total phenolic compounds.

216 Leandro de Morais Cardoso et al. / Food Chemistry 152 (2014) 210–217

content increased up to 2 times. Therefore, the intake of 100 g ofraw sorghum flour can supply 2% of the daily recommendation ofvitamin E for adult men aged between 19 and 30 years (15 mg ofa-tocopherol/day) (U. S. Institute of Medicine, 2000) while pro-cessed flours can supply between 2.5% (F7) and 4.5% (F5) of thisrecommendation.

The retention and content of vitamin E in the flours increasedafter the dry heat (p < 0.05). These increases were also observedin solid food matrices subjected to heat treatment and may beresult of an increased extractability of this vitamin due to therelease of their components of its binding sites (Hwang,Stacewicz-Sapuntzakis, & Bowen, 2012; Seybold, Fröhlich, Bitsch,Otto, & Böhm, 2004). Flours processed in microwave oven (F4and F5) showed the highest contents and retentions of vitamin E,followed by the flour F2 and F6 (p > 0.05), F3, F1 (control) and F7(p > 0.05). The wet heat processing reduced the retention and con-tent of vitamin E.

The retention and content of vitamin E in the flour oven/milling(F2) were higher than that obtained by the inverse processing (F3).As for carotenoids, this difference can be attributed to the exposureof this vitamin to heat. Vitamin E is located in the germ of the sor-ghum grain, which is covered and protected by the pericarp andtesta (Waniska and Rooney, 2000). The previous milling of thegrains became the germ and consequently the vitamin E more sus-ceptible to the action of the heat, which contributed to a lowerretention. The absence of influence of the processing order in theretention of vitamin E of the flours processed in microwave ovenmay result from the shorter heat exposure, when compared withtreatment in oven (5 vs 25 min) and the lowest cooking effectivetemperature.

3.5. Antioxidant activity

The antioxidant activity of flours processed in microwave oven(F4: 22.40 lmol TE/g and F5: 23.19 lmol TE/g) (p < 0.05), followedby the flour oven/milling (F2: 21.79 lmol TE/g) was significantlygreater than the control flour (F1: 20.18 lmol TE/g). Cooking in

water reduced the antioxidant activity of sorghum flour (F7:18.04 lmol TE/g) (p < 0.05) (Fig. 2).

The antioxidant activity of flours correlated positively with thecontent of a e c-tocopherols, total vitamin E, TPC, luteolinidin,apigeninidin; and total 3-DXAs (Table 4). The correlation of antiox-idant activity with TPC, including 3-DXAs, was also observed byother authors (Dlamini, Taylor, & Rooney, 2007). This correlationindicated that the highest antioxidant activity of the flourmicrowave oven/milling (F4) may have resulted from a concomi-tant increase in the content of total vitamin E, a e c-tocopherolsand TPC. Increased antioxidant activity in the flour oven/milling(F2) and milling/microwave oven (F5) resulted only from the in-creased content of vitamin E and a and c-tocopherols. In contrast,the reduction in antioxidant activity in the flour subjected to wetcooking (F7) resulted from the decrease in TPC, total 3-DXAs andtotal vitamin E.

4. Conclusion

Overall, the processing with dry heat (oven/milling; milling/oven; microwave oven/milling; milling/microwave oven; poppedgrains/milling) did not affect the content and retention of 3-deoxy-anthocyanidins, total phenolic compounds and antioxidantactivity; increased the vitamin E; and decreased the carotenoidsof the sorghum flours (BRS 301 genotype). In general, the grainsmilled before processing in oven and in microwave oven resultedin greater vitamin E retention and lower carotenoids retentionthan that milled after these processing. On the other hand, thewet heat (cooking in water/drying/milling) decreased 3-deoxy-anthocyanidins, total phenolic compounds, vitamin E, and antioxi-dant activity; and increased the carotenoids of the flour.

Acknowledgements

The authors acknowledge the EMBRAPA - Maize and Sorghum(Brazil) by providing the sorghum grains.

L.d.M. Cardodo et al. / Food Chemistry 152 (2014) 210–217 217

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2013.11.106.

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