research article the extrusion process as an alternative

Research ArticleThe Extrusion Process as an Alternative for Improvingthe Biological Potential of Sorghum Bran PhenolicCompounds and Antiradical and Anti-Inflammatory Capacity

Norma Julieta Salazar Lopez1 Guadalupe Loarca-Pintildea2 Rociacuteo Campos-Vega2

Marcela Gaytaacuten Martiacutenez2 Eduardo Morales Saacutenchez3 J Marina Esquerra-Brauer1

Gustavo A Gonzalez-Aguilar4 and Maribel Robles Saacutenchez1

1Departamento de Investigacion y Posgrado en Alimentos Universidad de Sonora 83000 Hermosillo SON Mexico2Departamento de Investigacion y Posgrado en Alimentos Facultad de Quımica Universidad Autonoma de Queretaro76010 Santiago de Queretaro QRO Mexico3Instituto Politecnico Nacional Centro de Investigacion en Ciencia Aplicada y Tecnologıa Avanzada76090 Santiago de Queretaro QRO Mexico4Centro de Investigacion en Alimentacion y Desarrollo AC 83304 Hermosillo SON Mexico

Correspondence should be addressed to Maribel Robles Sanchez rsanchezguayacanusonmx

Received 27 May 2016 Revised 1 August 2016 Accepted 14 August 2016

Academic Editor Xiang Liu

Copyright copy 2016 Norma Julieta Salazar Lopez et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Approximately 80 of sorghum phenolic compounds are linked to arabinoxylans by ester bonds which are capable of resisting thedigestion process in the upper gastrointestinal tract compromising their bioaccessibility and biological potential The aim of thisstudy was to evaluate the effect of the extrusion process on the content of phenolic compounds in sorghum bran and its impact onphenolic compounds and antiradical and anti-inflammatory capacity Results revealed that the extrusion process increased totalphenol content in sorghum bran compared to nonextruded sorghum particularly for extrusion at 180∘Cwith 20moisture content(20222plusmn00157 versus 30729plusmn00187mgGAEg +52) which positively affected antiradical capacity measured by the DPPH andTEAC assaysThe percentage of inhibition of nitric oxide (NO) production by RAW cells due to the presence of extruded sorghumbran extract was significantly higher than that of nonextruded sorghum bran extract (902 plusmn 19 versus 762 plusmn 13) The resultssuggest that extruded sorghum bran could be used as a functional ingredient and provide advantages to consumers by reducingdiseases related to oxidative stress and inflammation

1 Introduction

Sorghum the fifthmost important cereal grown in the worldis resistant to semiarid climates gluten-free and a goodsource of phytochemical compounds that have been associ-ated with antioxidant anti-inflammatory and antiprolifera-tive capacities [1ndash4] The biological potential of sorghum hasbeen related to the presence of different hydroxycinnamicacids (HCAs) such as ferulic 120588-coumaric caffeic and sinapicacids

However much of the biological potential of sorghum isnot used by biological systems due to the structural propertiesof their phenolic acids Approximately 80 of these com-pounds are linked by ester bonds to arabinoxylans (ARAs)located mainly in the cell walls of the pericarp and thealeurone layer [5 6] The linkage between HCAs and ARAsrestricts their bioaccessibility and further bioavailabilitybecause ARAs are resistant to the digestion process inthe upper gastrointestinal tract which compromises theirabsorption Therefore it is necessary to find processes that

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 8387975 8 pageshttpdxdoiorg10115520168387975

2 Evidence-Based Complementary and Alternative Medicine

increase the bioaccessibility of the phenolic compounds priorto intake of this cereal

The structure of arabinoxylans can be hydrolyzed bychemical processes thermal processes fermentation enzy-matic action or a combination of these processes [7ndash11] Rosaet al [12] reported an increase in bioaccessibility of 86of ferulic acid from the aleurone layer in wheat with theuse of xylanase and ferulic esterase Bartolome and Gomez-Cordoves [13] found approximately 70 and 5 of ferulicacid and 120588-coumaric acid respectively released from barleyusing commercial enzyme preparations However enzymaticprocesses in sorghum can bemore complicated than in wheator barley because the sorghumarabinoxylan structure ismoresubstituted which is more difficult for the enzyme to gainaccess to the attached sites between ferulic acid and arabinosetherefore chaperone enzymes are necessary [14ndash17] Cardosoet al [11] and Afify et al [18] reported a loss of phenolic com-pound content and antioxidant capacity after the traditionalprocesses of wet cooking and soaking of sorghum On theother hand Zielinski et al [19] and Gumul and Korus [20]reported an increase in total phenolic content and hydrox-ycinnamic acids mainly ferulic acid in barley rice oatswheat and rye after extrusion processesThese studies revealthat extrusion is a promising process in the production offunctional foods based on cereals [19 20]

Extrusion consists of heat and mechanical treatmentsunder different conditions of low moisture shear and highpressure by producing structural alterations and changesin functional properties in a short time [21] The effect ofextrusion on the content of nutrients and nonnutritious com-ponents such as phenolic compounds depends on the processconditions and the foodmatrix [21]The aim of this study wasto evaluate the effect of the extrusion process under differenttemperature and moisture levels on the content of phenoliccompounds and antiradical and anti-inflammatory capacityin sorghum bran

2 Materials and Methods

21 Materials and Chemicals Antibiotic-antimycotic fetalbovine serum sodium pyruvate and Dulbeccorsquos modifiedEaglersquos medium (DMEM) were obtained from Gibco (GrandIsland NY USA) All other reagents were purchased fromSigma-Aldrich (Saint Louis Missouri USA)

22 Sorghum Sample Preparation Sorghum grains (Sorghumbicolor L Moench) unpigmented variety (UDG110) wereprovided by the Produce Foundation Mexico The sorghumgrains were decorticated using abrasive discs for 6min andfurther ground using Pulvex 200 mill to pass through a04mm sieve The sorghum bran was stored at minus20∘C untilanalysis

23 Extrusion Procedure The sorghum bran was allowed tohydrate for 8 h (20and 30) before extrusion andprocessedin an extruder (prototype) with a single screw with lengthof 45 cm and two jackets with length of 15 and 10 cm Thetemperature of the first jacket was controlled to 60∘C whilethat of the second jacket was set to 110∘C or 180∘CThe screw

speed was 15 rpm and the die diameter was 5mmThe extru-dateswere dried in an oven at 60∘C for 6 hThedried productswere ground and sieved with a 04mm sieve and stored atminus20∘C until analysis

24 Preparation of Sorghum Bran Extracts Extruded sor-ghum bran (EB) or nonextruded sorghum bran (NEB)extracts were prepared as follows 1 g of each sample wasmixed with 15mL of 80 aqueousmethanol sonicated for 1 h(100W power output) and centrifuged at 1500timesg for 15min[6] The supernatants were separated and the residues wereextracted twice for 30min Extracts were filtered throughWhatman number 1 paper and evaporated to dryness ina rotary evaporator at 35∘C and samples were redissolvedin 5mL of 50 methanol for the analysis of total phenoliccontent phenolic acid content and antiradical capacity Theextracts were lyophilized and redissolved in DMSO for thecell culture tests

25 Quantification of Phenolic Acids by UHPLC-DAD Thephenolic acid content in EB and NEB extracts was quantifiedusing UHPLC system (Agilent Technologies Germany) witha diode array detector The separation was conducted on aZorbax Eclipse Plus C18 rapid resolution column (50mm times21mm id 18 120583m particle size) Column temperature wasset to 30∘C A binary phase solvent system was used A (01acetic acidwater) and B (01 acetic acidmethanol) at a flowrate of 07mLminThe solvent gradient was as follows initial91 of A and 9 of B 0ndash11min 9 to 14 B and 11ndash15min15 B Detection of the acids was performed at 280 nmand their quantitationwas performedwith curves establishedusing external standards of caffeic 120588-coumaric ferulic andsinapic acids The results were expressed as 120583g phenolic acidper gram of dry weight [22]

26 Determination of Total Phenolic Content The total phe-nolic content of the EB and NEB extracts was determined bythe colorimetricmethod at 765 nm using the FolinndashCiocalteureagent [23] The results were expressed as mg of gallic acidequivalents (GAE) per gram of dry weight

27 Antiradical Capacity

271 DPPH Assay This assay is based on the measurementof the scavenging ability of antioxidants towards the stableradical DPPH relative to the DPPH scavenging ability ofthe water-soluble vitamin E analogue Trolox Briefly 39mLaliquots of DPPH (00634mM) solution were added to thetest tubes and 01mL of sorghum bran extracts (EB or NEB)or Trolox standards (0 to 20120583M range) were added andshaken vigorouslyThe tubeswere allowed to stand at 25∘C for60min A control reactionwas prepared as abovewithout anyextract and methanol was used for the baseline correctionChanges in the absorbance of the samples were measuredat 515 nm Radical scavenging activity was expressed as thepercent inhibitionThe final DPPH values were calculated byusing a regression equation between the Trolox concentrationand the percent inhibition and were expressed as micromolesof Trolox equivalents per gram of dry weight [24]

Evidence-Based Complementary and Alternative Medicine 3

272 Trolox Equivalent Antioxidant Capacity (TEAC) Theantiradical potential was determined using 221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt(ABTS) [25]This assay is based on the ability of antioxidantsto scavenge the blue-green ABTS∙+ radical cation relative tothe ABTS∙+ scavenging ability of the water-soluble vitamin Eanalogue TroloxTheABTS∙+ radical cation was generated bythe interaction of 5mL of 7mM ABTS solution and 88120583L of140mMK

2S2O8solutionTheworking solutionwas prepared

with 1mL of the active radical and 88mL of ethanol for initialabsorbance of 070 plusmn 02 at 734 nm using a Cary 50 VarianSpectrophotometer After the addition of 29mL of ABTS∙+solution to 01mL of each extract or Trolox standards (0 to20120583M range) the absorbance was monitored exactly 1 and30min after the initialmixing until the absorbancewas stableThe percentage of absorbance inhibition at 734 nm wascalculated and plotted as a function of that obtained for theextracts and the standard reference (Trolox) The final TEACvalueswere calculated by using a regression equation betweenthe Trolox concentration and the percentage inhibition andexpressed as micromoles of Trolox equivalents per gram ofdry weight

28 Anti-Inflammatory Capacity

281 Cell Culture Mouse macrophage cell line RAW 2647was obtained from cryopreserved culture kindly providedby the Autonomous University of Queretaro Mexico whichwas originally from the ATCC (American Type CultureCollection) Macrophages were cultured in DMEM (Dul-beccorsquos modified Eaglersquos medium) supplemented with 10fetal bovine serum 1 antibiotic-antimycotic 15 gL sodiumbicarbonate and 1mLL sodiumpyruvate (1mM)on a 60mmplate and grown at 37∘C and 5 CO

2in a humidified

atmosphere [26]

282 Effect of Sorghum Bran Extract on Cell Viability RAWcells were cultured in 96-well plates at 1times 104 cellswell at 37∘Cand 5 CO

2for 24 hThen the culture mediumwas replaced

by fresh medium in the absence (control viability 100) andpresence of different concentrations of extruded sorghumextract and nonextruded sorghum extract (extract equivalentto 43ndash101mg sorghummL) previously lyophilized and dis-solved in lt1 DMSO The culture was incubated for 24 hand themediumwas removedThe adhered cells were treatedwith 200120583L of MTT dissolved in DMEM free of fetal bovineserum and incubated for 2 h The transformation of MTT toformazan by the action of the enzyme succinate dehydroge-nase was evaluated at 570 nm Cell viability was expressedas a percentage calculated by the following equation cellviability = (absorbance of the sampleabsorbance controlcells) times 100 [26]

283 Determination of Nitric Oxide Production Nitric oxideproduction was measured according to the method previ-ously reported byNguyen et al [26] with slightmodificationsBriefly RAW cells were cultured in 96-well plates at adensity of 25 times 105 cellswell at 37∘C and 5 CO

2for 24 h

Subsequently the production of nitric oxide (NO) in cells was

induced with lipopolysaccharide (LPS) (1 120583gmL) in thepresence and absence of EB and NEB extracts for 24 h NOproduction in the culture medium was assessed indirectlyas nitrite by the Griess reaction The supernatant medium(100 120583L)wasmixedwith 100120583L ofGriess reagent (1 sulfanil-amide and 01 naphthylethylenediamine dihydrochloride)and incubated for 10min Absorbance was measured at550 nm The concentration of nitric oxide in the culturemedium was determined based on a standard sodium nitritecurve

29 Statistical Analysis The effect of independent factors(moisture and temperature) and their interaction on theresponse variables was determined by ANOVA Tukeyrsquos testwas used for the comparison of themeans Statistical analyseswere performed with the program JMP 501 (USA SAS insti-tute Inc) Values of 119901 lt 005 were accepted as statisticallysignificant

3 Results and Discussion

31 Total Phenolic and Phenolic Acid Content Table 1 showsthe total phenols and phenolic acid content of extruded andnonextruded sorghum branThe extrusion process increasedtotal phenol content in sorghum bran compared to nonex-truded sorghum particularly those extruded at 180∘C and20 moisture content (20222 plusmn 00157 versus 30729 plusmn00187mgGAEg +52)

The total hydroxycinnamic acids content increased inall extruded sorghum bran samples evaluated in this studycompared to NEB The EB samples treated at 180∘C showeda higher total HCA content compared to the rest of theextruded samples The amount of caffeic acid 120588-coumaricacid ferulic acid and sinapic acid significantly increased afterthe extrusion process Ferulic acidwas themain phenolic acidfound in the sorghum bran before and after the extrusionprocess However its concentration increased 27-fold withrespect to NEB when heat treatment at 180∘C was used

The results obtained in this study applying extrusion tosorghum bran agree with those found by Zielinski et al [19]These authors observed an increase in the total phenolic andhydroxycinnamic acids mainly ferulic and coumaric acidsin barley rice oats and wheat as a result of extrusion at tem-peratures of 120∘C 160∘C and 200∘C and 20moistureTheysuggested that heat treatment of cereals enhances the releaseof phenolic acids and their products from the cell walls Thislast statement agrees with Ti et al [27] who reported anincrease of 126 in total phenolic content of rice bran as aconsequence of the extrusion process The release of phenoland other related compounds is a function of food matrixand extrusion conditions Therefore optimization of extru-sion processes has to be established depending on the foodmatrix to have the highest release of bioactive compounds

32 Antiradical Capacity The effects on the antiradicalcapacity of sorghum before and after the extrusion processesmeasured by the DPPH and TEAC assays are shown inFigure 1 We observed that the antiradical capacities in bothassays (DPPH and TEAC) were higher (119901 lt 005) for


Table 1 Phenolic acid content and total phenols in sorghum bran extract before and after extrusion processes

119879∘C Mlowastlowast Phenolic acid content (120583gg) Total HCAslowastlowastlowast Total phenols

(mg GAEg)Caffeic Coumaric Ferulic SinapicNonextruded 149 plusmn 03

clowast 87 plusmn 02e 198 plusmn 02c 34 plusmn 01d 468 plusmn 05d 20222 plusmn 00157d

110 20 288 plusmn 02a 215 plusmn 03a 300 plusmn 09b 50 plusmn 01c 850 plusmn 07b 24068 plusmn 01079c

110 30 196 plusmn 02b 197 plusmn 06b 286 plusmn 03b 46 plusmn 00c 707 plusmn 23c 21336 plusmn 00516d

180 20 199 plusmn 08b 173 plusmn 02c 539 plusmn 16a 77 plusmn 02a 989 plusmn 13a 30729 plusmn 00187a

180 30 201 plusmn 09b 149 plusmn 03d 538 plusmn 11a 65 plusmn 02b 953 plusmn 24a 26192 plusmn 00101blowastEach value represents the mean of three replicates plusmn standard error Different letters within each column indicate significant differences (119901 lt 005)lowastlowast MmoisturelowastlowastlowastTotal HCAs total hydroxycinnamic acids

B B B

A A

p lt 005

0

5

10

15

DPP

H (120583

mol

TE

g)

Control 30203020 ( M)180

∘C110∘C(a)

DC

D

A

B

Control 30203020 ( M)

p lt 005

0

5

10

15

TEAC

(120583m

ol T

Eg)

180∘C110

∘C(b)

Figure 1 Antiradical capacity of sorghum bran before (control) and after extrusion processes (a) DPPH and (b) TEAC Each bar representsthemeanof three replicatesplusmn standard errorDifferent letters on bars represent significant differences (119901 lt 005) between treatments includingcontrol

sorghum bran extruded at 180∘C with 95 plusmn 04 and 173 plusmn04 120583g TEg than for nonextruded sorghum bran with 77 plusmn07 and 113plusmn04 120583g TEg respectively (Figures 1(a) and 1(b))The antiradical capacity of extracts extruded at temperatureof 110∘C was lower (119901 lt 005) than that of those extractsextruded at 180∘C This could be explained by the fact thattemperatures above 170∘C are sufficient to break down thechemical bond of lignin and ferulic acid [28] which couldfragment the structure of arabinoxylans and consequentlyenhance the release of ferulic acid and increment the antirad-ical activity Our results agreed with those reported by Ti etal [27] where an increase of 197 in the antioxidant capacityof rice bran as a result of the extrusion process was observed

To evaluate a possible association between changes inantioxidant capacity and total phenolic content (TPC) acorrelation analysis was performed A significant correlationwas found between TPC and DPPH (1199032 = 0735 119901 lt 005)(Figure 2(a)) and between TPC and TEAC (1199032 = 0915 119901 lt005) (Figure 2(b))These findings suggest that total phenoliccontent is a good predictor of in vitro antiradical capacityShih et al [29] reported a concomitant relationship between

total phenolic content and antioxidant capacity (DPPH) insweet potatoes after the extrusion process

Additionally a concomitant increase in antioxidantcapacity and the content of phenolic acids mainly ferulicacid was previously reported in extruded rye [20] Theincrease of phenolic compounds and antiradical capacity dueto extrusion could be explained by the structural modifica-tion of the cell walls where phenolic acids such as ferulicand 120588-coumaric acids are covalently linked to arabinoxylansfavoring the release of these compounds The increase in theefficacy of the extraction process can be accomplished by themodification of the bran matrix under conditions of hightemperature pressure and shear [19 27]

Extrusion processing of cereals provides advantages interms of phenolic compound content and antioxidant capac-ity compared to the conventional wet cooking and soakingmethods [11 18]

In extruded corn flour Mora-Rochin et al [30] showedthat the extrusion process has some advantages over thenixtamalization process They evaluated the phenolic com-pound content and antioxidant capacity of corn tortillas and


22 24 26 28 3020Total phenols (mg GAEg)

7

8

9

10

11

DPP

H (120583

mol

TE

g)

p lt 005

r2= 0735

(a)

10

12

14

16

18

TEAC

(120583m

ol T

Eg)


p lt 005

r2= 0915

(b)

Figure 2 Correlations between the contents of total phenols in extruded sorghumbran and their antiradical capacity as determined byDPPH(a) and TEAC (b) assays

observed that tortillas made with extruded corn flour retaina higher content of total phenolic compounds ferulic acidand antioxidant capacity compared to tortillas prepared withnixtamalized corn flour in the traditional method Neverthe-less Dlamini et al [10] reported that porridge obtained fromAfrican sorghum by traditional methods had higher antioxi-dant capacity that that of products cooked by extrusion

The extrusion temperature of 180∘C was a determiningfactor in the achievement of a higher total phenol contentHowever further studies focusing on combined processesmay be necessary to increase the biological potential ofsorghum bran These differences in the content of phenoliccompounds and antioxidant capacity of extruded cereals andalso their nutritional value depend on the conditions used inthe extrusion process and the chemical composition of thefood matrix [21]

33 Nitric Oxide Production The inhibition of NO produc-tion by cell culture has been widely used as a biomarker toassess anti-inflammatory capacity because NO production isexacerbated by the action of the inducible nitric oxide syn-thase (iNOS) which is activated under conditions of oxida-tive stress the presence of polysaccharides in Gram-negativebacteria the tumor necrosis factor (TNF-120572) and interleukin-1120573which causes the activation of nuclear factor kappa B (NF-120581B) and the production of proinflammatory cytokines [31ndash34] NO production by RAW 2647 macrophages in the pres-ence of extruded and nonextruded sorghum bran extractswas evaluated For this assay the extrusion treatment withthe higher content of total phenols and antiradical capacitywas selected (180∘C20 moisture) Prior to nitric oxideevaluation the possible cytotoxic effects of sorghum branphenolic extracts weremeasured using theMTT assayWhenextruded or nonextruded sorghum bran extracts were addedto LPS-activated RAW 2647 no significant (119901 gt 005) effects

Bran concentration + LPS

43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

40

80

120

ce

ll vi

abili

ty

minus +

p gt 005

Figure 3 Cell viability () of RAW 2647 cells treated withextruded sorghum bran (180∘C20 moisture) and nonextrudedsorghumbran Control (minus) represents untreated cells and control (+)represents cells treated with LPS only Each bar represents the meanof five replicates from three independent experiments plusmn standarderror

on the cell viability () of the RAW 2647 cells at 43ndash101mgsorghum branmL were observed (Figure 3)

Figure 4 shows the effects of extruded or nonex-truded sorghum bran on the production of nitric oxide by


B

a

b

a

b

a

b a aa

b

A


43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

10

20

30

NO

pro

duct

ion

(120583M

)

minus +

p lt 005

Figure 4 Nitric oxide production of RAW 2647 cells treatedwith extruded sorghum bran at 180∘C and 20 moisture andnonextruded sorghum bran Control (minus) represents untreated celland control (+) represents cells treated with LPS only Each barrepresents the mean of five replicates from three independentexperiments plusmn standard error Bars with different letters in the sameconcentration are significantly different (119901 lt 005) Capital lettersrepresent significant differences (119901 lt 005) between nonextrudedand extruded treatments and positive control (LPS)

LPS-induced RAW 2647 mouse macrophages Accordingto the concentrations of both extruded and nonextrudedsorghum bran extracts selected for this study it was observedthat NO production was reduced significantly compared tothe positive control (LPS-activated RAW 2647) A dose-response effect on nitric oxide production in sorghum branextracts evaluated was also observed Using these results theconcentration of extract (extruded or nonextruded) was cal-culated and the concentration at which therewas 50 inhibi-tion of nitric oxide production (EC

50) was obtained showing

lower EC50

for extruded sorghum bran (523mgmL) thanthat of nonextruded sorghum bran (658mgmL)

With respect to nitric oxide production it was found thatbran sorghum subjected to the extrusion process showed lessnitric oxide production (119901 lt 005) Considering the max-imum concentration of the sorghum extracts (101mgmL)the percentage of inhibition of NO production by RAW cellsdue to the presence of extruded sorghum bran extract wassignificantly higher (119901 lt 005) than that of nonextrudedsorghum bran extract (902plusmn 19 versus 762plusmn 13)Theseresults agree with those reported by Shim et al [35] whoevaluated the anti-inflammatory capacity of ethanol extractsof sorghummeasured as inhibition ofNOproduction in LPS-induced RAW 2647 cells

Hwang et al [36] reported that chloroform extracts ofsorghum showed a significantly higher inhibitory effect onthe production ofNO iNOS TNF120572 and IL-6 in LPS-inducedRAW cells compared to the inhibitory effects of corn andbarley extracts However these studies only evaluated anti-inflammatory capacity in sorghum grain without thermalprocesses As far as we know this is the first time that the anti-inflammatory capacity of extruded sorghum bran has beenevaluated in vitro

Several studies of sorghum grain have reported an asso-ciation between phenolic compounds and anti-inflammatorycapacity In this context Burdette et al [4] observed a cor-relation between the anti-inflammatory capacity of sorghumextracts and their phenolic compound content and antiox-idant capacity Hwang et al [36] established a relationshipbetween the anti-inflammatory capacity and the content offlavonoids Previous studies have shown that white sorghumvariety is poor in flavonoid content Therefore the contentof phenolic compounds and the antioxidant capacity appar-ently is provided mainly by the phenolic acid derivatives ofcinnamic acid [37 38]

Previous reports have indicated that the antioxidantcapacity of cereal extracts is due to the presence of cinnamicacids which are able to inhibit the pathway of nuclear factor-(NF-) 120581B Kim et al [39] evaluated the anti-inflammatorycapacity of hydroxycinnamic acids isolated from corn branin RAW 2647 macrophages and observed inhibition of iNOSandNOproduction in connection to theNF-120581Bpathway Yunet al [40] and Shin et al [41] evaluated the effect of sinapicacid (40 to 160 120583M) and caffeic acid and its derivatives (25ndash100 120583M) on anti-inflammatory capacity and reported that theinhibitory effects were due to the suppression of iNOS COX-2 TNF-120572 and IL-1120573 expression through the effect of the NF-120581B pathway on RAW 2647 macrophages Other phenoliccompounds such as quercetin and caffeic acid phenethyl esteralso have been able to block the activation of NF-120581B and as aconsequence inhibit the production of iNOS and NO [32]

4 Conclusions

Applying the extrusion process to sorghum bran increasedtotal phenol and cinnamic acid contents which positivelyaffected the antioxidant capacity and the inhibition of LPS-induced nitric oxide production in RAW macrophages Theextrusion process could be a good alternative for processingsorghum bran to increase its functionalityThis improvementof extruded sorghum bran can be beneficial for peoplewith diseases related to oxidative stress and inflammationAdditional studies examining the increase in bioaccessibilityof phenolic compounds of extruded sorghum bran are inprogress

Competing Interests

The authors declare that there are no competing interests


Acknowledgments

This work was performed with the support of PROINNOVAgrant (Project no 218169) Norma Julieta Salazar Lopezreceived scholarship from CONACyT (National Researchand Technology Council) Mexico

References

[1] F Barros JM Awika and LW Rooney ldquoInteraction of tanninsand other sorghumphenolic compounds with starch and effectson in vitro starch digestibilityrdquo Journal of Agricultural and FoodChemistry vol 60 no 46 pp 11609ndash11617 2012

[2] JM Awika L Yang J D Browning andA Faraj ldquoComparativeantioxidant antiproliferative and phase II enzyme inducingpotential of sorghum (Sorghum bicolor) varietiesrdquo LWTmdashFoodScience and Technology vol 42 no 6 pp 1041ndash1046 2009

[3] K F Benson J L Beaman B Ou A Okubena O Okubenaand G S Jensen ldquoWest African Sorghum bicolor leaf sheathshave anti-inflammatory and immune-modulating properties invitrordquo Journal ofMedicinal Food vol 16 no 3 pp 230ndash238 2013

[4] A Burdette P L Garner E P Mayer J L Hargrove D KHartle and P Greenspan ldquoAnti-inflammatory activity of selectsorghum (Sorghum bicolor) bransrdquo Journal of Medicinal Foodvol 13 no 4 pp 879ndash887 2010

[5] L Dykes and L W Rooney ldquoSorghum and millet phenols andantioxidantsrdquo Journal of Cereal Science vol 44 no 3 pp 236ndash251 2006

[6] C Chiremba J R N Taylor L W Rooney and T Beta ldquoPhe-nolic acid content of sorghum and maize cultivars varying inhardnessrdquo Food Chemistry vol 134 no 1 pp 81ndash88 2012

[7] B A Acosta-Estrada J A Gutierrez-Uribe and S O Serna-Saldıvar ldquoBound phenolics in foods a reviewrdquo Food Chemistryvol 152 pp 46ndash55 2014

[8] S D McClendon H-D Shin and R R Chen ldquoNovel bacterialferulic acid esterase from Cellvibrio japonicus and its applica-tion in ferulic acid release and xylan hydrolysisrdquo BiotechnologyLetters vol 33 no 1 pp 47ndash54 2011

[9] S Mathew and T E Abraham ldquoBioconversions of ferulic acidan hydroxycinnamic acidrdquoCritical Reviews inMicrobiology vol32 no 3 pp 115ndash125 2006

[10] N R Dlamini J R N Taylor and L W Rooney ldquoThe effect ofsorghum type and processing on the antioxidant properties ofAfrican sorghum-based foodsrdquo Food Chemistry vol 105 no 4pp 1412ndash1419 2007

[11] L D M Cardoso T A Montini S S Pinheiro H M Pinheiro-SantrsquoAna H S D Martino and A V B Moreira ldquoEffects ofprocessing with dry heat and wet heat on the antioxidant profileof sorghumrdquo Food Chemistry vol 152 pp 210ndash217 2014

[12] N N Rosa C Dufour V Lullien-Pellerin and V MicardldquoExposure or release of ferulic acid fromwheat aleurone impacton its antioxidant capacityrdquo Food Chemistry vol 141 no 3 pp2355ndash2362 2013

[13] B Bartolome and C Gomez-Cordoves ldquoBarley spent grainrelease of hydroxycinnamic acids (ferulic and p-coumaricacids) by commercial enzyme preparationsrdquo Journal of theScience of Food and Agriculture vol 79 no 3 pp 435ndash439 1999

[14] M A Verbruggen B A Spronk H A Schols et al ldquoStruc-tures of enzymically derived oligosaccharides from sorghumglucuronoarabinoxylanrdquo Carbohydrate Research vol 306 no 1-2 pp 265ndash274 1998

[15] J Agger A Viksoslash-Nielsen and A S Meyer ldquoEnzymatic xyloserelease from pretreated corn bran arabinoxylan differentialeffects of deacetylation and deferuloylation on insoluble andsoluble substrate fractionsrdquo Journal of Agricultural and FoodChemistry vol 58 no 10 pp 6141ndash6148 2010

[16] J R N Taylor T J Schober and S R Bean ldquoNovel foodand non-food uses for sorghum and milletsrdquo Journal of CerealScience vol 44 no 3 pp 252ndash271 2006

[17] H-D Shin S McClendon T Le F Taylor and R R ChenldquoA complete enzymatic recovery of ferulic acid from cornresidues with extracellular enzymes from Neosartorya spinosaNRRL185rdquo Biotechnology and Bioengineering vol 95 no 6 pp1108ndash1115 2006

[18] A E-M M R Afify H S El-Beltagi S M A El-Salam andA A Omran ldquoBiochemical changes in phenols flavonoidstannins vitamin E 120573-carotene and antioxidant activity duringsoaking of three white sorghum varietiesrdquo Asian Pacific Journalof Tropical Biomedicine vol 2 no 3 pp 203ndash209 2012

[19] H Zielinski H Kozlowska and B Lewczuk ldquoBioactive com-pounds in the cereal grains before and after hydrothermalprocessingrdquo Innovative Food Science and Emerging Technologiesvol 2 no 3 pp 159ndash169 2001

[20] D Gumul and J Korus ldquoPolyphenol content and antioxidantactivity of rye bran extrudates produced at varying parametersof extrusion processrdquo Food Science and Technology vol 9 no 4pp 1ndash11 2006

[21] S Singh S Gamlath and L Wakeling ldquoNutritional aspects offood extrusion a reviewrdquo International Journal of Food Scienceand Technology vol 42 no 8 pp 916ndash929 2007

[22] W Guo and T Beta ldquoPhenolic acid composition and antiox-idant potential of insoluble and soluble dietary fibre extractsderived from select whole-grain cerealsrdquo Food Research Inter-national vol 51 no 2 pp 518ndash525 2013

[23] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquoAmeri-can Journal of Enology and Viticulture vol 16 pp 144ndash158 1965

[24] R A Ortız Cruz J L Cardenas Lopez G A Gonzalez Aguilaret al ldquoInfluence of sorghum kafirin on serum lipid profile andantioxidant activity in hyperlipidemic rats (in vitro and in vivostudies)rdquo BioMed Research International vol 2015 Article ID164725 8 pages 2015

[25] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[26] P-H Nguyen B T Zhao J H Lee Y H Kim B S Min andM HWoo ldquoIsolation of benzoic and cinnamic acid derivativesfrom the grains of Sorghum bicolor and their inhibition oflipopolysaccharide-induced nitric oxide production in RAW2647 cellsrdquo Food Chemistry vol 168 pp 512ndash519 2015

[27] H Ti R Zhang M Zhang et al ldquoEffect of extrusion onphytochemical profiles in milled fractions of black ricerdquo FoodChemistry vol 178 pp 186ndash194 2015

[28] C Chiremba L W Rooney and T Beta ldquoMicrowave-assistedextraction of bound phenolic acids in bran and flour fractionsfrom sorghum andmaize cultivars varying in hardnessrdquo Journalof Agricultural and Food Chemistry vol 60 no 18 pp 4735ndash4742 2012

[29] M-C Shih C-C Kuo and W Chiang ldquoEffects of drying andextrusion on colour chemical composition antioxidant activ-ities and mitogenic response of spleen lymphocytes of sweetpotatoesrdquo Food Chemistry vol 117 no 1 pp 114ndash121 2009


[30] S Mora-Rochin J A Gutierrez-Uribe S O Serna-Saldivar PSanchez-Pena C Reyes-Moreno and J Milan-Carrillo ldquoPhe-nolic content and antioxidant activity of tortillas produced frompigmented maize processed by conventional nixtamalization orextrusion cookingrdquo Journal of Cereal Science vol 52 no 3 pp502ndash508 2010

[31] F Aktan ldquoiNOS-mediated nitric oxide production and itsregulationrdquo Life Sciences vol 75 no 6 pp 639ndash653 2004

[32] J B Calixto M F Otuki and A R S Santos ldquoAnti-inflamma-tory compounds of plant origin part i action on arachidonicacid pathway nitric oxide and nuclear factor 120581B (NF-120581B)rdquoPlanta Medica vol 69 no 11 pp 973ndash983 2003

[33] T Lawrence ldquoThenuclear factorNF-kappaB pathway in inflam-mationrdquo Cold Spring Harbor Perspectives in Biology vol 1 no 6pp 1ndash10 2009

[34] J Ruland ldquoReturn to homeostasis downregulation of NF-120581BresponsesrdquoNature Immunology vol 12 no 8 pp 709ndash714 2011

[35] T-J Shim T M Kim K C Jang J-Y Ko and D J Kim ldquoToxi-cological evaluation and anti-inflammatory activity of a goldengelatinous sorghum bran extractrdquo Bioscience Biotechnology andBiochemistry vol 77 no 4 pp 697ndash705 2013

[36] J-M Hwang K-C Choi S-J Bang et al ldquoAnti-oxidant andanti-inflammatory properties of methanol extracts from vari-ous cropsrdquo Food Science and Biotechnology vol 22 no 1 pp265ndash272 2013

[37] J M Awika and L W Rooney ldquoSorghum phytochemicals andtheir potential impact on human healthrdquo Phytochemistry vol65 no 9 pp 1199ndash1221 2004

[38] K K Adom and R H Liu ldquoAntioxidant activity of grainsrdquoJournal of Agricultural and Food Chemistry vol 50 no 21 pp6182ndash6187 2002

[39] E O Kim K J Min T K Kwon B H Um R A Moreau andS W Choi ldquoAnti-inflammatory activity of hydroxycinnamicacid derivatives isolated from corn bran in lipopolysaccharide-stimulated Raw 2647 macrophagesrdquo Food and Chemical Toxi-cology vol 50 no 5 pp 1309ndash1316 2012

[40] K-J Yun D-J Koh S-H Kim et al ldquoAnti-inflammatory effectsof sinapic acid through the suppression of inducible nitric oxidesynthase cyclooxygase-2 and proinflammatory cytokinesexpressions via nuclear factor-120581B inactivationrdquo Journal ofAgricultural and Food Chemistry vol 56 no 21 pp 10265ndash10272 2008

[41] K-M Shin I-T Kim Y-M Park et al ldquoAnti-inflammatoryeffect of caffeic acidmethyl ester and its mode of action throughthe inhibition of prostaglandin E

2

nitric oxide and tumornecrosis factor-120572 productionrdquo Biochemical Pharmacology vol68 no 12 pp 2327ndash2336 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


increase the bioaccessibility of the phenolic compounds priorto intake of this cereal

The structure of arabinoxylans can be hydrolyzed bychemical processes thermal processes fermentation enzy-matic action or a combination of these processes [7ndash11] Rosaet al [12] reported an increase in bioaccessibility of 86of ferulic acid from the aleurone layer in wheat with theuse of xylanase and ferulic esterase Bartolome and Gomez-Cordoves [13] found approximately 70 and 5 of ferulicacid and 120588-coumaric acid respectively released from barleyusing commercial enzyme preparations However enzymaticprocesses in sorghum can bemore complicated than in wheator barley because the sorghumarabinoxylan structure ismoresubstituted which is more difficult for the enzyme to gainaccess to the attached sites between ferulic acid and arabinosetherefore chaperone enzymes are necessary [14ndash17] Cardosoet al [11] and Afify et al [18] reported a loss of phenolic com-pound content and antioxidant capacity after the traditionalprocesses of wet cooking and soaking of sorghum On theother hand Zielinski et al [19] and Gumul and Korus [20]reported an increase in total phenolic content and hydrox-ycinnamic acids mainly ferulic acid in barley rice oatswheat and rye after extrusion processesThese studies revealthat extrusion is a promising process in the production offunctional foods based on cereals [19 20]

Extrusion consists of heat and mechanical treatmentsunder different conditions of low moisture shear and highpressure by producing structural alterations and changesin functional properties in a short time [21] The effect ofextrusion on the content of nutrients and nonnutritious com-ponents such as phenolic compounds depends on the processconditions and the foodmatrix [21]The aim of this study wasto evaluate the effect of the extrusion process under differenttemperature and moisture levels on the content of phenoliccompounds and antiradical and anti-inflammatory capacityin sorghum bran

2 Materials and Methods

21 Materials and Chemicals Antibiotic-antimycotic fetalbovine serum sodium pyruvate and Dulbeccorsquos modifiedEaglersquos medium (DMEM) were obtained from Gibco (GrandIsland NY USA) All other reagents were purchased fromSigma-Aldrich (Saint Louis Missouri USA)

22 Sorghum Sample Preparation Sorghum grains (Sorghumbicolor L Moench) unpigmented variety (UDG110) wereprovided by the Produce Foundation Mexico The sorghumgrains were decorticated using abrasive discs for 6min andfurther ground using Pulvex 200 mill to pass through a04mm sieve The sorghum bran was stored at minus20∘C untilanalysis

23 Extrusion Procedure The sorghum bran was allowed tohydrate for 8 h (20and 30) before extrusion andprocessedin an extruder (prototype) with a single screw with lengthof 45 cm and two jackets with length of 15 and 10 cm Thetemperature of the first jacket was controlled to 60∘C whilethat of the second jacket was set to 110∘C or 180∘CThe screw

speed was 15 rpm and the die diameter was 5mmThe extru-dateswere dried in an oven at 60∘C for 6 hThedried productswere ground and sieved with a 04mm sieve and stored atminus20∘C until analysis

24 Preparation of Sorghum Bran Extracts Extruded sor-ghum bran (EB) or nonextruded sorghum bran (NEB)extracts were prepared as follows 1 g of each sample wasmixed with 15mL of 80 aqueousmethanol sonicated for 1 h(100W power output) and centrifuged at 1500timesg for 15min[6] The supernatants were separated and the residues wereextracted twice for 30min Extracts were filtered throughWhatman number 1 paper and evaporated to dryness ina rotary evaporator at 35∘C and samples were redissolvedin 5mL of 50 methanol for the analysis of total phenoliccontent phenolic acid content and antiradical capacity Theextracts were lyophilized and redissolved in DMSO for thecell culture tests

25 Quantification of Phenolic Acids by UHPLC-DAD Thephenolic acid content in EB and NEB extracts was quantifiedusing UHPLC system (Agilent Technologies Germany) witha diode array detector The separation was conducted on aZorbax Eclipse Plus C18 rapid resolution column (50mm times21mm id 18 120583m particle size) Column temperature wasset to 30∘C A binary phase solvent system was used A (01acetic acidwater) and B (01 acetic acidmethanol) at a flowrate of 07mLminThe solvent gradient was as follows initial91 of A and 9 of B 0ndash11min 9 to 14 B and 11ndash15min15 B Detection of the acids was performed at 280 nmand their quantitationwas performedwith curves establishedusing external standards of caffeic 120588-coumaric ferulic andsinapic acids The results were expressed as 120583g phenolic acidper gram of dry weight [22]

26 Determination of Total Phenolic Content The total phe-nolic content of the EB and NEB extracts was determined bythe colorimetricmethod at 765 nm using the FolinndashCiocalteureagent [23] The results were expressed as mg of gallic acidequivalents (GAE) per gram of dry weight

27 Antiradical Capacity

271 DPPH Assay This assay is based on the measurementof the scavenging ability of antioxidants towards the stableradical DPPH relative to the DPPH scavenging ability ofthe water-soluble vitamin E analogue Trolox Briefly 39mLaliquots of DPPH (00634mM) solution were added to thetest tubes and 01mL of sorghum bran extracts (EB or NEB)or Trolox standards (0 to 20120583M range) were added andshaken vigorouslyThe tubeswere allowed to stand at 25∘C for60min A control reactionwas prepared as abovewithout anyextract and methanol was used for the baseline correctionChanges in the absorbance of the samples were measuredat 515 nm Radical scavenging activity was expressed as thepercent inhibitionThe final DPPH values were calculated byusing a regression equation between the Trolox concentrationand the percent inhibition and were expressed as micromolesof Trolox equivalents per gram of dry weight [24]







2in a humidified

atmosphere [26]





2for 24 h


















B B B

A A

p lt 005

0

5

10

15

DPP

H (120583

mol

TE

g)

Control 30203020 ( M)180

∘C110∘C(a)

DC

D

A

B

Control 30203020 ( M)

p lt 005

0

5

10

15

TEAC

(120583m

ol T

Eg)

180∘C110

∘C(b)










7

8

9

10

11

DPP

H (120583

mol

TE

g)

p lt 005

r2= 0735

(a)

10

12

14

16

18

TEAC

(120583m

ol T

Eg)


p lt 005

r2= 0915

(b)






43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

40

80

120

ce

ll vi

abili

ty

minus +

p gt 005





B

a

b

a

b

a

b a aa

b

A


43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

10

20

30

NO

pro

duct

ion

(120583M

)

minus +

p lt 005




lower EC50






4 Conclusions


Competing Interests



Acknowledgments


References











































2







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2in a humidified

atmosphere [26]





2for 24 h


















B B B

A A

p lt 005

0

5

10

15

DPP

H (120583

mol

TE

g)

Control 30203020 ( M)180

∘C110∘C(a)

DC

D

A

B

Control 30203020 ( M)

p lt 005

0

5

10

15

TEAC

(120583m

ol T

Eg)

180∘C110

∘C(b)










7

8

9

10

11

DPP

H (120583

mol

TE

g)

p lt 005

r2= 0735

(a)

10

12

14

16

18

TEAC

(120583m

ol T

Eg)


p lt 005

r2= 0915

(b)






43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

40

80

120

ce

ll vi

abili

ty

minus +

p gt 005





B

a

b

a

b

a

b a aa

b

A


43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

10

20

30

NO

pro

duct

ion

(120583M

)

minus +

p lt 005




lower EC50






4 Conclusions


Competing Interests



Acknowledgments


References











































2







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























B B B

A A

p lt 005

0

5

10

15

DPP

H (120583

mol

TE

g)

Control 30203020 ( M)180

∘C110∘C(a)

DC

D

A

B

Control 30203020 ( M)

p lt 005

0

5

10

15

TEAC

(120583m

ol T

Eg)

180∘C110

∘C(b)










7

8

9

10

11

DPP

H (120583

mol

TE

g)

p lt 005

r2= 0735

(a)

10

12

14

16

18

TEAC

(120583m

ol T

Eg)


p lt 005

r2= 0915

(b)






43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

40

80

120

ce

ll vi

abili

ty

minus +

p gt 005





B

a

b

a

b

a

b a aa

b

A


43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

10

20

30

NO

pro

duct

ion

(120583M

)

minus +

p lt 005




lower EC50






4 Conclusions


Competing Interests



Acknowledgments


References











































2







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















7

8

9

10

11

DPP

H (120583

mol

TE

g)

p lt 005

r2= 0735

(a)

10

12

14

16

18

TEAC

(120583m

ol T

Eg)


p lt 005

r2= 0915

(b)






43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

40

80

120

ce

ll vi

abili

ty

minus +

p gt 005





B

a

b

a

b

a

b a aa

b

A


43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

10

20

30

NO

pro

duct

ion

(120583M

)

minus +

p lt 005




lower EC50






4 Conclusions


Competing Interests



Acknowledgments


References











































2







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















B

a

b

a

b

a

b a aa

b

A


43 59 69 80 101

NonextrudedExtruded

Control

(mgmL)

0

10

20

30

NO

pro

duct

ion

(120583M

)

minus +

p lt 005




lower EC50






4 Conclusions


Competing Interests



Acknowledgments


References











































2







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References











































2







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























2







of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article the extrusion process as an alternative

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